「RNA メタボロームによる細胞内小胞体の解明に基づいた
革新的がん治療法の開発」

平成 27 年度～令和元年度
私立大学戦略的研究基盤形成支援事業
研究成果報告書

令和 2 年 3 月
学 校 法 人 名 東 京 医 科 大 学 
大学名 東 京 医 科 大 学 

研 究 組 織 名 分子病理学分野
研 究 代 表 者 黒 田  雅 彦
（東京医科大学医学部医学科主任教授）

目

刊行にあたって次･･･････････････････････････････････････････････････････

1

プロジェクトの概要
・ 学校法人名 ･･･････････････････････････････････････････････････････

3

・ 大学名 ･･･････････････････････････････････････････････････････････

3

・ 研究組織名

3･･････････････････････････････････････････････････････････････････････････････････････････････････････

3

・ 研究プロジェクト名･････････････････････････････････････････････････

3

・ 研究観点 ･････････････････････････････････････････････････････････

3

・ 研究代表者･･･････････････････････････････････････････････････････

3

・ プロジェクト参加研究者数 ･････････････････････････････････････････

3

・ 該当審査区分･････････････････････････････････････････････････････

3

・ 研究プロジェクトに参加する主な研究者 ･････････････････････････････

3

・ 研究の概要･･･････････････････････････････････････････････････････

4

・ キーワード･･･････････････････････････････････････････････････････

9

・ 研究発表の状況 ･･･････････････････････････････････････････････････

9

・ プロジェクト所在地

・ その他の研究成果等･･･････････････････････････････････････････････ 42

・ 「選定時」及び「中間評価時」に付された留意事項及び対応･････････････ 43
・ 施設・装置・設備・研究費の支出状況 ･･･････････････････････････････ 44
・ 施設・装置・設備の整備状況
・ 研究費の支出状況･･･････････････････････････････････････ 45･････････････････････････････････････････････････ 45

プロジェクトの成果
48

・

主要論文

･･･････････････････････････････････････････････････････

・

その他･････････････････････････････････････････････････････････ 602

私立大学戦略的研究基盤形成支援事業｢RNAメタボロームによる細胞内小胞体の解明に基づいた革新的がん治療法の開発｣
研究成果報告書刊行にあたって
本私立大学戦略的研究基盤形成支援事業が採択されて5年間が経過し、研究成果をまとめる時期がまいりました。本事業は、RNA特にnon-coding RNAをキーワードにして、学内の4つの研究グループが集結し立案したものです。特に基礎分野の研究班においては、常に臨床応用を目指して挑戦的な研究をしていくことを目指しました。また、本事業を遂行するうえで、人材育成も大切な事と考え、今後の医療を担うResearch PhysicianそしてResearch
Scientistの育成を目指しました。さて、この5年間の研究を振り返ると改めて班員の先生方の成果に感銘を受けます。まだ、完結していない成果もありますが、本研究事業が種となり実を結ぶことを確信致します。最後になりますが、これまでのご支援ご協力を賜りました文部科学省、
学校法人東京医科大学、多くの共同研究者の皆様に心より厚く御礼申し上げます。
東京医科大学 分子病理学分野
主任教授 黒田雅彦-1-

プロジェクトの概要-2-

（様式 2）
法人番号プロジェクト番号131054
S1511011

平成 27 年度～令和元年度「私立大学戦略的研究基盤形成支援事業」
研究成果報告書概要
１ 学校法人名

東京医科大学３ 研究組織名

２ 大学名

東京医科大学

小胞体創薬研究センター

４ プロジェクト所在地

東京都新宿区新宿６−１−１

５ 研究プロジェクト名 RNA メタボロームによる細胞内小胞体の解明に基づいた革新的がん治療法の開発

６ 研究観点研究拠点を形成する研究７ 研究代表者
研究代表者名

所属部局名

職名

黒田 雅彦

分子病理学分野

主任教授

８ プロジェクト参加研究者数
９ 該当審査区分

39 名

理工・情報

生物・医歯

人文・社会

１０ 研究プロジェクトに参加する主な研究者
研究者名

黒田 雅彦

大屋敷一馬

池田 徳彦

横須賀忠

梅津 知宏

所属・職名

分子病理
学分野・主
任教授
血液内科
学分野・主
任教授
呼吸器・甲
状腺外科
学分野・主
任教授
免疫学分
野・主任教
授
血液内科
学分野・講
師

プロジェクトでの研究課題

プロジェクトでの役割

Non-codingRNA の単離と診断、治療法の開発

プロジェクトの立案と総括

microRNA-細胞内分泌小胞を応用した医薬品の開発

個別化医療の確立

RNA メタボロームを基盤としたがん個別化・最適化治療
法の開発

個別化医療の確立

分泌小胞イメージングによるがん免疫チェックポイント機
構の解明
分泌小胞による薬耐性がん（がん幹細胞）の微小環境の解明（共同研究機関等）-3-

免疫チェック機構の解明

がん微小循環の解明

（様式 2）
法人番号プロジェクト番号131054
S1511011＜研究者の変更状況（研究代表者を含む）＞
旧
変更前の所属・職名変更（就任）後の所属・職名

先端分子探索寄附血液内科学分野・講師
講座・助教（変更の時期：平成 31 年 3 月 31 日）

研究者氏名

プロジェクトでの役割

梅津 知宏

がん微小循環の解明

新変更前の所属・職名変更（就任）後の所属・職名

研究者氏名

プロジェクトでの役割

先端分子探索寄附
講座・助教

先端核酸医療応用講座・准
教授

梅津 知宏

がん微小循環の解明

１１ 研究の概要（※ 項目全体を１０枚以内で作成）（１）研究プロジェクトの目的・意義及び計画の概要
我が国のがんによる死亡者数は年間 30 万人を超え、死亡原因の第１位を占めている。また、未曾有の高齢化社会を迎え、がん死が増加し、社会保障費が増大する中で、豊かな社
会を今後も継続し発展していくためには、革新的なアプローチが必要であり、新たなイノベー
ションが大学に求められている。そのために、我々は(1)現在 20％に過ぎないがん検診率を上げ、(2)安価で効果が高い次世代医薬品の実用化を目指す。そのことが、健康寿命をのばし、医療経済的にも患者 QOL の改善にも即効性が高いと考える。そこで我々は、本プロジェ
クトで、RNA メタボロームによる細胞内小胞体の解明とその臨床応用の研究を行う。近年
miRNA をはじめとする non-coding RNA が、様々な生命活動に関与し、多くの疾患で、これら
RNA の異常が明らかになり、診断・治療標的分子としてクローズアップされている。また、細胞の分泌小胞は、多彩な生理作用を有しているが、この分泌小胞内の 1 つであるエクソソー
ムには miRNA やペプチドが含有されている。近年腫瘍特異的に分泌される、miRNA 含有エク
ソソームが、腫瘍の微小環境や幹細胞機能の獲得、腫瘍免疫、薬剤耐性に重要な役割を果たすことが示された。これらの複合的な RNA メタボロームの制御は、診断・治療に大きな可能性を有しており、本研究では、基礎医学分野、医学総合研究所、臨床医学分野、および製薬
企業（BONAC、東レ）と連携を密にして、臨床応用を目指していく体制を構築する。
（２）研究組織本プロジェクトでは、基礎医学分野 2
部門（分子病理学分野、免疫学分野）、
臨床医学２部門（血液内科学分野、呼吸器・甲状腺外科学分野）の連携を行
い、基礎と臨床医学の融合とともに、産学連携による人的交流、技術支援などを積極的に行い、図に示した通り研究基盤構築を行う。

（３）研究施設・設備等
世界に先駆けて、細胞内小胞の可視化のために、1 細胞 1 分子イメージングを可能とする構-4-

（様式 2）
法人番号プロジェクト番号131054
S1511011

造化照明法 N-SIM と全反射蛍光顕微鏡の融合システムを構築した。本システムをもとに、新宿キャンパス、中央校舎 4 階に小胞体創薬研究センターを整備した。
（４）研究成果の概要 ※下記、１３及び１４に対応する成果には下線及び＊を付すこと。
＜優れた成果が上がった点＞
本事業により研究基盤が強化された共同研究の成果を中心に研究成果を記載する。それ
ぞれのテーマは縦割りでなく密接に関係しているが、役割分担と共同研究体制を明確化する
ため、研究組織に示した各分野も付記し、研究機器整備に伴う共同研究の進展状況についても報告する。( )内は対応する 13,14 の番号で、それぞれ論文（*）、学会発表（**）、特許（***）を付した。
【新規の non-codingRNA の解明と新たな miRNA の発現機構の解明】分子病理学分野①新規の non-codingRNA の単離
microRNA や lincRNA などの non-coding RNA(ncRNA)の異常は疾患の発生に深く関与する。これらの RNA を標的とする治療戦略は、次世代の分子標的治療薬として期待されている。このような背景の中、本事業では、次世代シークエンサーを用いて、肺がん細胞株 H1299
より網羅的 RNA 解析を行った。その結果、幾つかの、安定なヘアピン構造を示す RNA の転
写産物の単離に成功した。さらにこれらの転写産物から seed 配列を目印に標的遺伝子を予測したところ、がん遺伝子、がん抑制遺伝子を始め、多数の遺伝子を制御する事が判明した。我々は、これらの ncRNA を endogenous guide hairpin RNA(eghRNA)と命名した（19*、74**、103**、133**、144**）。これらの新規の ncRNA である eghRNA の生理機能解析や疾患における検討は、新たな疾患の分子メカニズムを切り開くと共に、創薬が困難であった各種疾患に対して治療を提供することを可能にする。さらに、これらの eghRNA を欠失した動物を作製し、その生理的機能を解明する事を試みている。（達成率 80%）
②DICER1 症候群発症機構の解析
DICER1 症候群は、DICER1 遺伝子の変異を原因とし、小児期から成人期において様々な臓
器で悪性腫瘍を発生する遺伝性疾患である。2009 年に胸膜肺芽腫患者の DICER1 遺伝子に高頻度に変異が発見され、本遺伝子変異が原因であることが明らかになった。その後、嚢胞性腎腫、ウィルムス腫瘍、肝細胞癌など、多くの難病で DICER1 遺伝子変異が見つかり、
DICER1 症候群と呼ばれるに至っている。DICER1 は miRNA のプロセッシングに必須であることから、miRNA の発現異常が原因と予想されているが、動物モデルが無いこともあり、発症機構は明らかでない。そこで我々は、世界に先駆けてコンディショナルに DICER1 変異体を発現するマウスを作成した。肝細胞特異的に DICER1 変異体を発現するマウスで肝癌が発症したことから、世界初の DICER1 症候群モデル動物として解析中である(134**、205**、212**、229**、245**)。（達成率 70%）
③新規 miRNA の機能解析
我々は、強力ながん抑制 microRNA (miRNA)として知られる miR-34a を導入した肺がん細胞において、多くの遺伝子の発現が誘導されることに注目し、miR-34a による遺伝子発現誘導機構の解析を行った。その結果、miR-34a は肺がん細胞において、がん抑制遺伝子として知られる BLU (ZMYND10)の転写を誘導していた。その際、miR-34a は AGO1 および AGO2 と複合体を形成し、BLU プロモーターから発現する新規の lncRNA に結合することで、BLU
mRNA の転写を誘導していることを明らかにした(136**、163**、211**、226**、228**、
247**)。これまでは、miRNA は、一般的に遺伝子の発現を抑制するだけと考えられていたしかし、今回の研究成果により、miRNA が、lncRNA の発現と制御によって、特定の遺伝子発現-5-

（様式 2）
法人番号プロジェクト番号131054
S1511011を誘導する機構が明らかになった。この発見は、革新的ながん治療の標的として期待出来る。（達成率 90%）
【microRNA-細胞内分泌小胞を応用した診断法の開発】①造血器腫瘍にける成果：血液内科学分野
造血器腫瘍における細胞内および細胞外小胞体 miRNA の臨床的意義を明らかにし、診断および治療に結び付く情報を得ることを目的とした。本事業として慢性骨髄性白血病の治療
反応性を規定する末梢血単核球 miRNA-148b の低下および細胞外小胞体 miRNA-215（
56*, 67**、80**）の意義を報告すると共に、TKI 治療薬中止後にみられる副作用と関連する
血清中の細胞外小胞体 miR-140-3p を明らかにした（45*、 60*）。また、造血器腫瘍のがん
微小環境における細胞間相互作用に細胞外小胞体が重要な役割を担うことに注目し、骨髄
間質細胞由来の細胞外小胞体 miR-101 が骨髄異形成症候群の進展に関与していることを明らかにした（125*）。現在は移植片対宿主病（GvHD）の発症に関わる小胞体 miRNA の同定を行い（97*）、GvHD の発症予測、重症度判定への臨床応用を試みている（達成率 80%）。
②肺がんにおける成果：呼吸器・甲状腺外科学分野
肺癌患者におけるリキッドバイオプシーの有用性を検討することを目的とした。肺癌の診断で手術が行われた手術症例を対象に、次世代シーケンサー(NGS)およびデジタル PCR を用いて血清と手術検体での検体種別の体細胞変異検査結果を比較した。IA-IIIA 期の手術切除標本とマッチした患者血清 150 検体を収集し、NGS による targeted sequencing を実施した。手術標本での結果と血清を用いた結果を比較検討した。腫瘍組織において検出された遺伝子変異とその検出頻度は、EGFR (37%), TP53 (39%), KRAS (10%)であった。血清を用いた検討では、150 検体中 149 検体で評価可能であり、検出された遺伝子変異およびその頻度
は、EGFR (3/150), TP53 (5/150), PIK3CA (1/150)であった。droplet digital PCR を用いて再解析を行い、NGS による検出率と比較した結果、98.8%の高い一致率を得た。同症例における血清中の体細胞変異検出率を規定する臨床病理学的因子を検索した結果、腫瘍の体積が規定因子であることが示された。手術の対象となる比較的早期の非小細胞肺癌患者においては、血清中の体細胞変異検出率は低く、腫瘍体積が血清を用いた体細胞変異検出の規定因子になると考えられた。手術適応をなる早期肺癌を対象とした研究では検出力が低い
ことが判明し、リキッドバイオプシーが有効であるかを検討するために進行した肺癌症例を対象に研究を行った。肺癌が疑われる症例に対して経気管支鏡下生検が施行された症例を対象に同意を得て、血液、組織検体を同時に採取した。それぞれの検体で Cobas EGFR 変異検出キット v2.0 を用いて EGFR 遺伝変異を測定した。測定可能であった 77 例を対象に比較した。組織、血液の EGFR 遺伝子変異はそれぞれ 37.7, 16.9%であった。血液を用いた検査では標準的な検査法である組織を用いた検査と比較して、陽性一致率は 42.1%、陰性一致率は 97.5%で、全体で 79.7％であった。血液を用いた検査でも有効であるが陽性一致率が十分でないことを理解した上で検査を行うことが重要であることが示された。
（達成率 80%）【分泌小胞イメージングによるがん免疫チェックポイント機構の解明】免疫学分野
免疫チェックポイント阻害療法は発明から瞬く間にがん臨床における標準療法となったが、一方でその分子機構は未解決な点が多く残されている。本プロジェクトでは免疫チェックポイ
ント分子の細胞内局在とシグナル伝達に関して、1)細胞骨格、2)糖鎖修飾、3)細胞内小胞輸
送の三つの視点から研究を遂行すると共に、免疫チェックポイント分子 4)LAG3 と 5)TIGIT の-6-

（様式 2）
法人番号プロジェクト番号131054
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PD-1 とは異なる免疫抑制機能を解明し、また次世代がん治療法としても期待される 6)キメラ
抗原受容体 CAR と免疫チェックポイント分子とのクロストーク機構を明らかにした。1)ではこれまで我々が解明してきた T 細胞受容体シグナロソーム「TCR マイクロクラスター」が、接着分子やアクチンを動員した新たな細胞骨格構造「マイクロシナプス」として T 細胞の活性化を制御することを明らかにした。2)では PD-1 の細胞表面発現に、小胞内での PD-1 の N-グリカンのフコシル化が重要なことを明らかにした。3)では本研究支援の整備機器として、1 細胞
1 分子イメージング構造化照明法 N-SIM と全反射蛍光顕微鏡の融合システムを構築し、ビデオレートでのエクソソームの 1 分子観察に成功した。4)では LAG3 のトロゴサイトーシスによる抗原提示細胞からの MHC クラス II の取り込みおよび小胞輸送の促進と免疫抑制機能を明らかにした。5)では TIGIT の分子高による標的活性化受容体の選択と免疫抑制の質の相違を解明している。6)では CAR の細胞外ドメインのデザインにより PD-1 抑制の受けやすさや、エンドサイトーシスと小胞輸送にも差があることを明らかしつつある。これらの結果は、免疫チェックポイント分子の細胞内局在とシグナル伝達機構を理解し、新たなメカニズムに基づいた
免疫チェックポイント阻害療法の進展を期待させる結果である。（達成率 80%）【分泌小胞による薬耐性がん（がん幹細胞）の微小環境の解明】血液内科学分野・先端核酸
医療応用講座本プロジェクトでは微小環境側、特に骨髄間質細胞が放出する細胞外小胞（EV）の生物学的意義を明らかにするとともに、それらを標的または応用した新規の EV-based therapy の開発を目指した。若年健常者（19 歳、20 歳）、高齢健常者（68 歳、72 歳）由来の骨髄間質細胞の培養上清から EV 分画を単離してその機能を解析した結果、高齢健常者由来と比較して、
若年健常者由来骨髄間質細胞が放出する EVs では多発性骨髄腫細胞が誘導する血管新生を抑制する能力（抗腫瘍血管新生能）が高かった。さらに、EV 内 miRNA のプロファイリングを行った結果、若年健常者由来骨髄間質細胞に特異的な４種の EV 内 miRNA（miR-340,
miR-365, miR-374a, miR-411）を見出した。これらの miRNA のうち、miR-340 と miR-365 の合成 miRNA を高齢健常者由来骨髄間質細胞が放出した EVs に直接的に導入すると、若年健常者由来骨髄間質細胞が放出する EVs と同等の抗腫瘍血管新生効果を有していた。本研究では、EV 治療のソースである骨髄間質細胞の加齢性変化に着目し、新規技術を用いて「EVの若返り」による機能強化を実現し、その作用機序を明らかにした（46*）。また、多発性骨髄腫をモデルに、がん患者由来骨髄間質細胞およびそれらが放出する EV の機能に関する研究も進め、多発性骨髄腫患者由来骨髄間質細胞（CAF）が特異的に miR-10a を EV に封入して排出していること、さらには miR-10a の排出を阻害すると CAF がアポトーシスを起こすことを見出した（118*）。このことから、CAF を含むがん微小環境側を標的とした soil-targeted
therapy への応用が考えられ、今後は EV のデリバリーに関する問題点を解決しながらがんの
次世代 EV-based therapy へと発展させていく。（達成率 80%）
【革新的な miRNA 補充療法の開発】 分子病理学分野
miRNA 補充療法はがん化の過程で発現が低下したがん抑制性の miRNA を細胞外から補充することで、その miRNA が標的とする複数のがん遺伝子の発現を抑制する革新的な治療法であるが、実用化には核酸に対する免疫応答や標的臓器に伝達するための技術の開発が必須である。この点に関して、本事業では、ヘアピン型やマッチ型の miRNA の開発に成功した（1*、4＊、14＊、15＊、 40＊、 42＊、111*、特許 2***、特許 3***、4***、5***）＜課題となった点＞
本プロジェクトにおいて、いずれの研究項目においても達成度は高いと評価された。しかし、-7-

（様式 2）
法人番号プロジェクト番号131054
S1511011

最終的な出口戦略が遅れている点は指摘された。特に新規のリキッドバイオプシーとして期待された、血小板由来の miRNA の解析が期待された成果を得る事が出来なかった。今後は、ゲノム情報を含めた NGS のデータの効果的な解析に注力を注ぐ。また、新規のリキッド
バイオプシーとして、non-codingRNA の解析を今後行っていく。

＜自己評価の実施結果と対応状況＞
毎月１回の研究進捗会議で進捗状況を報告し、達成率、費用対効果について審議し、必要に応じて研究計画の軌道修正を行ってきた。

＜外部（第三者）評価の実施結果と対応状況＞
平成 30 年 1 月 31 日に外部評価委員２名を招いて研究成果の中間報告会（公開）を開催し、外部評価委員からは A 評価を得た。また、令和 2 年 3 月 11 日に予定された最終報告会（公開）は、COVID-19 ウイルスの感染の拡大により中止された。今後、書面での評価を行い、可能であれば、公開シンポジウムを開催する予定である。

＜研究期間終了後の展望＞EV や non-codingRNA に関する基盤研究を発展させ、核酸医薬品や EV を用いた医薬品開発
およびリキッドバイオプシーの応用が最も期待される。

＜研究成果の副次的効果＞
1. 特許出願：「がんの罹患の可能性の試験方法およびそれに用いる試験試薬」発明者:大屋敷純子、梅津知宏 出願人: 東京医科大学 出願番号: 特願 2017-083898 国内外の別: PCT
2. 特許出願：「遺伝子発現制御のための人工マッチ型 miRNA およびその用途」 出願番
号：特願 2015-555078 （国内書面差出日：2016(H28)年 6 月 23 日） ※PCT/JP2014/084724 の日本国内移行に係る出願
3. 特許出願：「遺伝子発現制御のための発現制御核酸分子およびその用途」 発明者:黒田雅彦ほか
出願人: 東京医科大学 出願番号： 特願 2016-168499
4. 特許出願：「プロレニン遺伝子またはプロレニン受容体遺伝子の発現を抑制する一本鎖
核酸分子およびその用途」 発明者：黒田雅彦 出願日：2016/12/29 出願番号：
PCT/JP2016/089216
5. 特許出願：「核酸用担体および核酸の投与方法」 発明者：黒田雅彦、梅津知宏、大野慎一郎 出願日：2019 年 7 月 5 日 特願 2019-126172-8-

（様式 2）
法人番号プロジェクト番号131054
S1511011

１２ キーワード（当該研究内容をよく表していると思われるものを８項目以内で記載してください。）（１） miRNA（２） エクソソーム（３） 核酸医薬品（４） リキッドバイオプシー
（５）
（６）
（７）
（８）
１３ 研究発表の状況（研究論文等公表状況。印刷中も含む。）
上記、１１(４)に記載した研究成果に対応するものには＊を付すこと。
＜雑誌論文＞
1.

2.

3.

4.

5.

6.
7.

*Toyono T, Usui T, Yokoo S, Taketani Y, Nakagawa S, Kuroda M, Yamagami S, Amano S.
Angiopoietin-like 7 is an anti-angiogenic protein required to prevent vascularization of thecornea. PLoS One. 2015 Jan 26;10(1):e0116838. doi: 10.1371/journal.pone.0116838.
eCollection 2015.
Nakama T, Yoshida S, Ishikawa K, Kobayashi Y, Zhou Y, Nakao S, Sassa Y, Oshima Y, Takao
K, Shimahara A, Yoshikawa K, Hamasaki T, Ohgi T, Hayashi H, Matsuda A, Kudo A, Nozaki
M, Ogura Y, Kuroda M and Ishibashi T. Inhibition of choroidal fibrovascular membrane
formation by new class of RNA interference therapeutic agent targeting periostin. Gene
Ther. 2015 22(2):127-37. doi: 10.1038/gt.2014.112.
Yoon JH, Sudo K, Kuroda M, Taniguchi T, Kato M, Lee I, Han JS, Nakae S, Imamura T, Kim J,
Ji Ju JH, Kim DK, Matsuzaki K, Weinstein M, Nakao A, Matsumoto* I, Sumida T, Mamura M..
Phosphorylation status determines the opposing functions of Smad2 and Smad3 as STAT3
cofactors in TH17 differentiation. Nature communications. 2015 21;6:7600. doi:
10.1038/ncomms8600.
*Takanashi M, Sudo K, Ueda S, Ohno S, Yamada Y, Osakabe Y, Goto H, Matsunaga Y,
Ishikawa A, Usui Y, Kuroda M. Novel Types of Small RNA Exhibit Sequence- andTarget-dependent Angiogenesis Suppression Without Activation of Toll-like Receptor 3 inan Age-related Macular Degeneration (AMD) Mouse Model. Mol Ther Nucleic Acids. 2015
Oct 20;4:e258. doi: 10.1038/mtna.2015.34.
Braulke F, Platzbecker U, Müller-Thomas C, Götze K, Gotze K, Germing U, Brümmendorf
TH, Nolte F, Hofmann W-K, Giagounidis AN, Lübbert M, Greenberg PL, Bennett JM, Solé F,
Mallo M, Slovak ML, Ohyashiki K, Le Beau MM, Tüchler H, Pfeilstöcker M, Nösslinger T,
Hildebrandt B, Hindebrandt B, Shirmeshan K, Aul C, Stauder *, Sperr WR, Valent P,
Fonatsch C, Trümper L, Haase D, Schanz J: Validation of cytogenetic risk groups according
to International Prognostic Scoring Systems by peripheral blood CD34 + FISH: Results froma German diagnostic study in comparison with an international control group.
Haematologica, 2015, 100(2): 205-213.
Saito Y, Gotoh M, Katagiri S, Ohyashiki K: Diffuse large B-cell lymphoma resembling ametastatic liver tumor. British Journal of Haematology, 2015, 168(1):1.
Braulke F, Platzbecker U, Müller-Thomas C, Götze K, Gotze K, Germing U, Brümmendorf
TH, Nolte F, Hofmann W-K, Giagounidis AN, Lübbert M, Greenberg PL, Bennett JM, Solé F,-9-（様式 2）
法人番号プロジェクト番号131054
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Mallo M, Slovak ML, Ohyashiki K, Le Beau MM, Tüchler H, Pfeilstöcker M, Nösslinger T,
Hildebrandt B, Hindebrandt B, Shirmeshan K, Aul C, Stauder R, Sperr WR, Valent P,
Fonatsch C, Trümper L, Haase D, Schanz J: Frequency of del(12p) is commonly
undetermined in myelodysplastic syndromes: Results from a German diagnostic study incomparison with an international control group. Genes Chromosomes and Cancer, 2015,
54(12): 809-817.
8. Imanishi S, Takahashi R, Ohsuga M, Ohyashiki K, Ohyashiki JH: In vitro effect of deferasirox
with 5-azacytidine in human leukemia cells. Annals of Hematology, 2015, 90(9):1601-1602.
9. Katagiri S, Tauchi T, Tadokoro K, Ohyashiki JH, Ohyashiki K: BCL2L11 (BIM) deletion
polymorphism, rather than BIM SNP, is an effective predictor of early molecular relapse
after ABL tyrosine kinase inhibitor discontinuation in patients with chronic myeloid
leukemia. Integrative Cancer Science and Therapeutics, 2015, 2(5): 242-244.
10. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Efficacy of the polo-like kinase inhibitor
rigosertib, alone or in combination with Abelson tyrosine kinase inhibitors, against
breakpoint cluster region-c-Abelson- positive leukemia cells. Oncotarget, 2015, 6(24):20231-20240.
11. Okabe S, Tauchi T, Tanaka Y, Sakuta J, Ohyashiki K: Anti-leukemic activity of axitinib
against cells harboring the BCR-ABL T315I point mutation. Journal of Hematology andOncology, 2015, 8(1):97.
12. Katagiri S, Tauchi T, Umezu T, Saito Y, Suguro T, Asano M, Yoshizawa S, Kitahara T,
Akahane D, Tanaka Y, Fujimoto H, Okabe S, Gotoh M, Ito Y, Ohyashiki JH, Ohyashiki K.
Treatment-free molecular remission achieved by combination therapy with imatinib andIFNα in CML with BIM deletion polymorphism relapsed after stop imatinib. Rinsho
Ketsueki. 2015 Feb;56(2):216-219.
13. Kanekura K, Yagi T, Cammack AJ, Mahadevan J, Kuroda M, Harms MB, Miller TM, Urano F.

Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation. Hum
Mol Genet. 2016 Feb 29. pii: ddw052.
14. *Ohno S, Drummen GP, Kuroda M.Focus on Extracellular Vesicles: Development ofExtracellular Vesicle-Based Therapeutic Systems. Int J Mol Sci. 2016 Feb 6;17(2). pii: E172.
doi: 10.3390/ijms17020172. Review.
15. *Taketani Y, Usui T, Toyono T, Shima N, Yokoo S, Kimakura M, Yamagami S, Ohno S,
Onodera R, Tahara K, Takeuchi H, Kuroda M. Topical Use of Angiopoietin-like Protein 2
RNAi-loaded Lipid Nanoparticles Suppresses Corneal Neovascularization. Mol Ther Nucleic
Acids. 2016 Mar 8;5:e292. doi: 10.1038/mtna.2016.1.
16. Hata K, Yanase N, Sudo K, Kiyonari H, Mukumoto Y, Mizuguchi J, Yokosuka T. Differential
regulation of T-cell dependent and T-cell independent antibody responses througharginine methyltransferase PRMT1 in vivo. FEBS Lett. 2016 Apr;590(8):1200-10. doi:
10.1002/1873-3468.12161.
17. Kanekura K, Nishi H, Isaka K, Kuroda M. MicroRNA and gynecologic cancers. J Obstet

Gynaecol Res. 2016 Apr 20. doi: 10.1111/jog.12995.
18. Fujinaga H, Fujinaga H, Watanabe N, Kato T, Tamano M, Terao M, Takada S, Ito Y,-10-（様式 2）
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Umezawa A, Kuroda M. Cord blood-derived endothelial colony-forming cell function is
disrupted in congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol. 2016 Jun
1;310(11):L1143-54. doi: 10.1152/ajplung.00357.2015. Epub 2016 Apr 29.
19. *Ohno S, Itano K, Harada Y, Asada K, Oikawa K, Kashiwazako M, Okuyama H, Kumagai K,
Takanashi M, Sudo K, Ikeda N, Kuroda M. Development of Novel Small Hairpin RNAs That
do not Require Processing by Dicer or AGO2. Mol Ther. 2016 Aug;24(7):1278-89. doi:
10.1038/mt.2016.81
20. *Hashimoto-Tane A, Sakuma M, Ike H, Yokosuka T, Kimura Y, Ohara O, Saito T.
Micro-adhesion rings surrounding TCR microclusters are essential for T cell activation. J
Exp Med. 2016 Jul;213(8):1609-25. 10.1084/jem.20151088.
21. Mori A, Nishi H, Sasaki T, Nagamitsu Y, Kawaguchi R, Okamoto A, Kuroda M, Isaka K.
HLA-G expression is regulated by miR-365 in trophoblasts under hypoxic conditions.
Placenta. 2016 Sep;45:37-41. doi: 10.1016/j.placenta.2016.07.004. Epub 2016 Jul 21.
22. Matsumoto Y, Itami S, Kuroda M, Yoshizato K, Kawada N, Murakami Y. MiR-29a Assists in
Preventing the Activation of Human Stellate Cells and Promotes Recovery From Liver
Fibrosis in Mice. Mol Ther. 2016 Oct;24(10):1848-1859. doi: 10.1038/mt.2016.127.
23. Nakaya M, Watari K, Tajima M, Nakaya T, Matsuda S, Ohara H, Nishihara H, Yamaguchi H,
Hashimoto A, Nishida M, Nagasaka A, Horii Y, Ono H, Iribe G, Inoue R, Tsuda M, Inoue K,
Tanaka A, Kuroda M, Nagata S, Kurose H. Cardiac myofibroblast engulfment of dead cells
facilitates recovery after myocardial infarction. J Clin Invest. 2017 Jan 3;127(1):383-401.
doi: 10.1172/JCI83822. Epub 2016 Dec 5.
24. Katagiri S, Gotoh M, Tone K, Akahane D, Ito Y, Ohyashiki K, Makimura K: Fatal
cyberlindnera fabianii in a patient with mixed phenotype acute leukemia after anumbilical cord blood transplantation. International Journal of Hematology, 2016, 103(5):592-595.
25. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Combination therapy with copanlisib and ABL
tyrosine kinase inhibitors against Philadelphia chromosome-positive resistant cells.
Oncotarget, 2016, 7(33): 53116-53126.
26. Ohyashiki JH, Umezu T, Ohyashiki K: Exosomes promote bone marrow angiogenesis inhematologic neoplasia: The role of hypoxia. Current Opinion in Hematology, 2016, 23(3):268-273.
27. 大屋敷一馬：慢性骨髄性白血病 2016 今日の治療指針（監修：山口 徹、北原光夫：医学書院）. 2016：702−704.
28. 赤羽大悟、大屋敷一馬：細菌関連血球貪食症候群 別冊 日本臨床、新領域別症候群シリーズ No.35、免疫症候群（第 2 版）II（日本臨床社）. 2016：794−797.
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cell lung cancer. Oncol Lett. 2019, 17(6):5125-5131.
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105. Matsubayashi J, Miyake S, Kudo Y, Shimada Y, Maeda J, Saji H, Kakihana M, Park J,

Kajiwara N, Inoue S, Saito K, Ohira T, Ikeda N, Tokuue K, Nagao T: Cytological differences
between invasive and noninvasive or minimally invasive lung adenocarcinomas diagnosed
in Japanese patients using needle biopsy specimens of pulmonary lesions ≤3 cm indiameter. Diagnostic Cytopathology. 2019, 47(7):688-694.
106. Omori T, Aokage K, Nakamura H, Katsumata S, Miyoshi T, Sugano M, Kojima M, Fujii S,
Kuwata T, Ochiai A, Ikeda N, Tsuboi M, Ishii G: Growth patterns of small peripheralsquamous cell carcinoma of the lung and their impacts on pathological and biologicalcharacteristics of tumor cells. J Cancer Res Clin Oncol. 2019, 145(7):1773-1783.
107. Setojima Y, Shimada Y, Tanaka T, Shigefuku S, Makino Y, Maehara S, Hagiwara M, Masuno
R, Yamada T, Kakihana M, Kajiwara N, Ohira T, Ikeda N: Prognostic impact of solid-parttumour volume doubling time in patients with radiological part-solid or solid lung cancer.
Eur J Cardiothorac Surg. 2019, ezz305.
108. Kawaguchi Y, Okano T, Imai K, Maehara S, Maeda J, Yoshida K, Hagiwara M, Kakihana M,

Kajiwara N, Ohira T, Matsubayashi J, Ikeda N: Epidermal growth factor receptor mutation
subtype has differential effects on adjuvant chemotherapy for resected adenocarcinoma
pathological stages II-III. Oncol Lett. 2019, 18(6):6451-6458.
109. Okabe S, Tanaka Y, Tauchi T, Ohyashiki K: A novel phosphoinositide 3-kinase inhibitor.
copanlisib, and in combination with carfilzomib, inhibits multiple myeloma cells. Annals of
Hematology, 2019 Mar;98(3):723-733.
110. Umehara R, Kurata A, Takanashi M, Hashimoto H, Fujita K, Nagao T, Kuroda M: Fascin asa useful marker for identifying neural components in immature teratomas of human ovary
and those derived from murine embryonic stem cells. Int J Gynecol Pathol. 2019,38(4):377-385.
111. *Saito S, Ohno SI, Harada Y, Oikawa K, Fujita K, Mineo S, Gondo A, Kanno Y, Kuroda M:
rAAV6-mediated miR-29b delivery suppresses renal fibrosis. Clin Exp Nephrol. 2019
Dec;23(12):1345-1356. doi: 10.1007/s10157-019-01783-w. Epub 2019 Sep 3.
112. Kurata A, Saito A, Hashimoto H, Fujita K, Ohno SI, Kamma H, Nagao T, Kobayashi S,
Yamashina A, Kuroda M: Difference in immunohistochemical characteristics between
Takayasu arteritis and giant cell arteritis: it may be better to distinguish them in the sameage. Mod Rheumatol. 2019, 21:1-10.
113. Ueda A, Oikawa K, Fujita K, Ishikawa A, Sato E, Ishikawa T, Kuroda M, Kanekura K:

Therapeutic potential of PLK1 inhibition in triple-negative breast cancer. Lab Invest. 201999:1275-1286.
114. Katagiri S, Umezu T, Azuma K, Kobayashi C, Akahane D, Suguro T, Furuya N, Fujimoto H,
Nakamura N, Ohyashiki JH, Ohyashiki K: Maintenance 5-azacytidine therapy by MRD
monitoring after allogeneic HSCT in myeloid/lymphoid neoplasms with FGFR1
rearrangement. Bone Marrow Transplant. 2019 Jul;54(7):1148-1150.
115. Kubota S, Tokunaga K, Umezu T, Yokomizo-Nakano T, Sun Y, Oshima M, Tan KT, Yang H,-18-

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Kanai A, Iwanaga E, Asou N, Maeda T, Nakagata N, Iwama A, Ohyashiki K, Osato M,
Sashida G: Lineage-specific RUNX2 super-enhancer activates MYC and promotes thedevelopment of blastic plasmacytoid dendritic cell neoplasm. Nat Commun. 2019 Apr
10;10(1):1653.
116. Asano M, Ohyashiki JH, Kobayashi-Kawana C, Umezu T, Imanishi S, Azuma K, Akahane D,
Fujimoto H, Ito Y, Ohyashiki K: A novel non-invasive monitoring assay of 5-azacitidine
efficacy using global DNA methylation of peripheral blood in myelodysplastic syndrome.
Drug Des Devel Ther. 2019 May 30;13:1821-1833.
117. Katagiri S, Azuma K, Akahane D, Saito K, Ohyashiki K: A unique breast involvement
detected by MRI-DWIBS in T cell acute lymphoblastic leukemia. Breast J. 2019Nov;25(6):1276-1277.
118. *Umezu T, Imanishi S, Yoshizawa S, Kawana C, Ohyashiki JH, Ohyashiki K: Induction ofmultiple myeloma bone marrow stromal cell apoptosis by inhibiting extracellular vesicle
miR-10a secretion. Blood Adv. 2019 Nov 12;3(21):3228-3240.
119. Katagiri S, Makishima H, Azuma K, Nannya Y, Saitoh Y, Yoshizawa S, Akahane D, Fujimoto
H, Ito Y, Velaga R, Umezu T, Ohyashiki JH, Ogawa S, Ohyashiki K: Predisposed genomic
instability in pre-treatment bone marrow evolves to therapy-related myeloid neoplasms
in malignant lymphoma. Haematologica. 2019 Nov 7.
120. *Kong MS, Hashimoto-Tane A, Kawashima Y, Sakuma M, Yokosuka T, Kometani K5, Onishi
R, Carpino N, Ohara O, Kurosaki T, Phua KK, Saito T: Inhibition of T cell activation andfunction by the adaptor protein CIN85. Sci Signal. 2019 Feb 5;12:567. pii: eaav4373. doi:
10.1126/scisignal.aav4373.
121. 横須賀忠、若松英、秦喜久美、竹原朋宏、西航、矢那瀬紀子、町山裕亮: 免疫チェック
ポイント分子の分子作用機序. 実験医学増刊号「新・腫瘍免疫学」. 2019 年 第 37 巻 15
号 P.2469−2477.
122. Ueda S, Takanashi M, Sudo K, Kanekura K, Kuroda M: miR-27a ameliorates

chemoresistance of breast cancer cells by disruption of reactive oxygen species
homeostasis and impairment of autophagy. Lab Invest. 2020 Feb 17. doi:
10.1038/s41374-020-0409-4
123. Kitamoto K, Taketani Y, Fujii W, Inamochi A, Toyono T, Miyai T, Yamagami S, Kuroda M,
Usui T, Ouchi Y: Generation of mouse model of TGFBI-R124C corneal dystrophy using
CRISPR/Cas9-mediated homology-directed repair. Sci Rep. 2020; 10: 2000. Published
online 2020 Feb 6. doi: 10.1038/s41598-020-58876-w
124. Katagiri S, Gotoh A, Ohyashiki K: Very late relapse with rapid BCR-ABL1 elevation after
more than seven years of treatment-free remission with undetectable molecular residual
disease in chronic myeloid leukaemia. Br J Haematol. 2020 Jan;188(2):332-334.
125. *Saitoh Y, Umezu T, Imanishi S, Asano M, Yoshizawa S, Katagiri S, Suguro T, Fujimoto H,
Akahane D, Kobayashi-Kawana C, Ohyashiki JH, Ohyashiki K: Downregulation of
extracellular vesicle microRNA-101 derived from bone marrow mesenchymal stromal cells
in myelodysplastic syndrome with disease progression. Oncol Lett. 2020-19-

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Mar;19(3):2053-2061.

＜図書＞
1. 大野 慎一郎、黒田 雅彦：第 4 章 6 エクソソームを用いた DDS キャリア. P149-156. DDS
キャリア作製プロトコル集(The Prorocols for the Preparation of DDS Carriers) 監修:丸山一雄 シーエムシー出版 2015.8.25.
2. Ohno S, Kuroda M. Exosome-mediated targeted delivery of miRNAs. Federico, Maurizio(Ed.) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Meth Mol Biol.
vol.1448 doi:10.1007/978-1-4939-3753-0 IF 1.29.
3. 上田しのぶ、黒田雅彦: グリオーマ治療の Decesion Marking 脳神経外科診療プラクティ
ス. 文光堂. 総ページ数 304(P.260-261)、2016.
4. 黒田 雅彦、髙梨 正勝: 第 2 編 機能研究 第 6 章 創薬～DDS／新規バイオマーカー～ 第 2 節 エクソソームの臨床応用について. P195-216. パラダイムシフトをもたらす
エクソソーム機能研究最前線 シグナル伝達からがん、免疫、神経疾患との関わり、創薬利用まで. 編者 落谷 孝広 株式会社 エヌ・ティー・エス 2017.3.8 初版.
5. 上田 しのぶ、髙梨 正勝、黒田 雅彦: 14 章 血中 miRNA 研究の現状と創薬への期待.
P145-154. miRNA の最新知識～基礎領域から診断・治療応用まで～. 編者 落谷 孝広
医薬ジャーナル社（総ページ数 195）2017.6.20 初版.
6. 福本 学、黒田雅彦 編集: 特集 計量 (デジタル) 病理学 : 画像の数値化から補助診断まで 病理と臨床 文光堂 総ページ数 90 (P.9-99) 35 巻 1 号, 2017.
7. 横須賀忠: 第７章 免疫受容体とシグナル伝達 「アバスーリックマンーピレ 分子細胞免疫学 第９版」 P.147-183 総ページ 577 頁 ELSEVIER 2018 年 3 月
8. 横須賀忠: 第７章 リンパ球レセプターシグナル 「JANEWAY’S 免疫生物学 第９版」
P.257-293 総ページ 884 頁 南江堂 2019 年 3 月
9. 横須賀忠、若松英、町山裕亮: 第７章 第２節 免疫チェックポイント受容体 PD-1 とCTLA-4 「膜タンパク質工学ハンドブック」 株 NTS 総ページ 624 頁 2020 年 4 月 ISBN
978-4-86043-537-0
10. 横須賀忠: 第４章 認識のメカニズム H.TCR による認識 「標準免疫学」 2020 年発刊予定＜学会発表＞
1.

2.
3.

**Ohno S, Itano K, Harada Y, Asada K, Oikawa K, Kashiwazako M, Takanashi M, Sudo K,

Ikeda N, Kuroda M: Development of novel form of mimic microRNA for lung cancer
therapy. AACR Annual Meeting 2015. Philadelphia, Pennsylvania, USA. April. 18-22, 2015
老川桂生、浅田浩太朗、大野慎一郎、黒田雅彦: microRNA 生成経路を欠損する肺がん細胞株の解析．第 104 回日本病理学会総会 （名古屋） 2015 年 4 月 30 日- 5 月 2 日
浅田浩太朗、老川桂生、大野慎一郎、黒田雅彦: 肺がんにおける Argonaute ファミリーの発現・機能解析．第 104 回日本病理学会総会 （名古屋） 2015 年 4 月 30 日- 5 月 2日-20-（様式 2）
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山田正俊、齋藤 彰、山本陽一郎、立石文子、黒田雅彦. 核形態測定に基づく乳管内増殖病変診断補助の試み. 第 104 回日本病理学会総会 （名古屋） 2015 年 4 月 30 日- 5
月2日
4. 橋本浩次、倉田 厚、名城珠希、牛島友則、藤田浩司、堀内 啓、黒田雅彦: 内頚動脈
内膜における平滑筋細胞の分化度と、動脈硬化性疾患の合併の有無の相関. 第 104 回
日本病理学会総会 （名古屋） 2015 年 4 月 30 日- 5 月 2 日
5. 大野慎一郎、板野華蓮、原田裕一郎、浅田浩太朗、老川桂生、柏迫幹絵、高梨正勝、須藤カツ子、池田徳彦、黒田雅彦: 肺がん治療を想定した副作用の少ない短鎖型新規核酸医薬の開発. 第 104 回日本病理学会総会 （名古屋） 2015 年 4 月 30 日- 5 月 2 日
6. 倉田 厚、山田正俊、大野慎一郎、橋本浩次、藤田浩司、高梨正勝、黒田雅彦: 胃癌細胞株の形態変化における、上皮間葉系移行に関わるマイクロ RNA200c とその標的の検出．第 104 回日本病理学会総会 （名古屋） 2015 年 4 月 30 日- 5 月 2 日
7.

倉田 厚、真村瑞子、藤田浩司、平野和彦、長尾俊孝、黒田雅彦: 高安大動脈炎の進展に関わる病理像の変化．第 104 回日本病理学会総会 （名古屋） 2015 年 4 月 30 日- 5
月2日
8. 山田侑子、高梨正勝、須藤カツ子、上田しのぶ、黒田雅彦: 肺線維症に対する miR-29b
補充療法．第 104 回日本病理学会総会 （名古屋） 2015 年 4 月 30 日- 5 月 2 日
9. 藤田浩司、山田正俊、高梨正勝、黒田雅彦: 浸潤性乳癌に出現する let-7d 発現細胞の検討．第 104 回日本病理学会総会 （名古屋） 2015 年 4 月 30 日 - 5 月 2 日
10. 大木 拓也、梅津 知宏、大屋敷純子、福岡 豊： miRNA と mRNA の統合解析によるがん細胞の低酸素適応のメカニズム解析．第５４回日本生体医工学会大会、 名古屋国際会議場 （名古屋） 2015 年 5 月 7 日- 9 日
11. Yokosuka T, Hashimoto-Tane A, Saito T： Cytoskeletal regulation of the clustering of a
Ras-GEF, RasGRP1, upon T cell activation．第 25 回京都 T 細胞カンファレンス （京都）2015 年 5 月 15 - 16 日
12. Asano M, Kobayashi C, Umezu T, Imanishi S, Azauma K, Akahane D, Fujimoto H, Ohyashiki
K, Ohyashiki JH: The genome-wide DNA methylation status as a predictor of 5-azacytidine
in clinical responses. 6th JSH International symposium. 2015, May 22-23, 2015, Karuizawa,
Nagano.
13. Umezu T, Imanishi S, Yoshizawa S, Saitoh Y, Yamamoto Y, Ohyashiki K, Ohyashiki JH:
Age-related alterations in exosomal miRNAs derived from bone marrow mesenchymal
stromal cells. 6th JSH International Symposium, May 22-23, 2015, Karuizawa Prince Hotel
West, Karuizawa, Japan.
14. Azuma K, Umezu T, Imanishi S, Kobayashi C, Asano M, Katagiri S, Akahane D, Ohyashiki K,
Ohyashiki JH: Telomere repeat-containing RNA (TERRA), a possible epigenetic biomarker
of DNA damage phenotype in 5-azacytidine-resistant leukemia cells. 6th JSH International
Symposium, May 22-23, 2015, Karuizawa Prince Hotel West, Karuizawa, Japan.
15. Imanishi S, Ohsuga M, Takahashi R, Umezu T, Kobayashi C, Katagiri S, Asano M, Saitoh Y,
Ohyashiki K, Ohyashiki JH: BET protein inhibition interferes DNA integrity in azacytidine
resistant cells through ATM signaling. 6th JSH International symposium. 2015, May 22-23,
2015, Karuizawa, Nagano.
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16. 東 剣紅、今西 哲、大屋敷純子、梅津知宏、大屋敷一馬: 次世代シーケンサーによる

クリニカルシーケンスの実際：骨髄系腫瘍の体細胞変異解析.第 175 回東京医科大学医学会総会 東京医科大学病院 （東京） 2015 年 6 月 6 日
17. 藤本博昭、片桐誠一朗、大屋敷一馬、溝口 出、善本隆之、梅津知宏、大屋敷純子: 本
邦健常者における BCL2L11(BIM)遺伝的多型の頻度と免疫細胞との関係. 第 175 回東京
医科大学医学会総会 東京医科大学病院 （東京） 2015 年 6 月 6 日
18. 梅津 知宏、吉澤成一郎、東 剣虹、大屋敷一馬、大屋敷純子: がん微小環境を制御す
る新たな機構の解明 –細胞外小胞エクソソームを介した骨髄腫細胞—骨髄内間質細胞
間相互作用−．第１７５回東京医科大学医学会総会 東京医科大学病院 （東京） 2015
年6 月6 日19. 大屋敷純子、梅津知宏：エピジェネティックな変化およびテロメア動態を指標とした体細胞初期化品質の評価系の確立．第１７５回東京医科大学医学会総会 東京医科大学病院 （東京） 2015 年 6 月 6 日
20. Yoshizawa S, Umezu T, Saitoh Y, Gotoh M, Akahane D, Kobayashi C, Ohyashiki JH,
Ohyashiki K: Distinct exosomal miRNA expression of late onset graft-versus-host disease
in allogeneic hematopoietic stem cell transplantation. 20 th European Hematology
Association Congress. June 11-14, 2015, Wien, Austria.
21. Tauchi T, Okabe S, Ohyashiki K: The stemness inhibitor, BB1608, reduces the self-renewalof BCR-ABL positive leukemia cells. 20th European Hematology Association Congress. June
11-14, 2015, Wien, Austria.
22. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH. Bonemarrowmesenchymal stromal cells from healthy donors secrete exosomes that inhibit invivo tumor growth and angiogenesis in multiple myeloma. 20th Congress of EHA, June
11-14, 2015, Messe Vien, Vienna, Austria.
23. Yoshizawa S, Umezu T, Saitoh Y, Gotoh M, Akahane D, Kobayashi C, Ohyashiki JH,
Ohyashiki K. Distinct exosomal miRNA expression of late onset graft-versus-host diseasein
allogeneic hematopoietic stem cell transplantation. 20th Congress of EHA, June 11-14,
2015, Messe Vien, Vienna, Austria.
24. 関野 知聖、梅津 知宏、今西 哲、東 剣虹、武内 健、福岡 豊、大屋敷一馬、大屋敷
純子： 骨髄間質細胞由来の老化関連エクソソーム miRNA の解析．第４回医薬工３大学
包括連携推進シンポジウム 工学院大学 （東京） 2015 年 6 月 20 日
25. 大木 拓也、梅津 知宏、大屋敷純子、福岡 豊： 異種データの統合による多発性骨髄腫の低酸素耐性機序の解析．第４回医薬工３大学包括連携推進シンポジウム 工学院
大学 （東京） 2015 年 6 月 20 日
26. 横須賀忠（招待講演）： 分子イメージングが拓く T 細胞活性化の時空間的制御機構．第14 回四国免疫フォーラム 特別講演 （松山） 2015 年 6 月 20 日
27. 横須賀忠：ユビキチンが形成する T 細胞抑制性シグナルソームの分子イメージング解析．新学術領域「ユビキチンネオバイオロジー」第１回領域班会議 （京都） 2015 年 7 月
2日
28. 大野慎一郎、板野華蓮、浅田浩太朗、老川桂生、柏迫幹絵、高梨正勝、黒田雅彦．副作

用を軽減した短鎖型核酸医薬の開発．第 12 回日本病理学会カンファレンス 神戸六甲山ホテル （神戸） 2015 年７月 24 - 25 日.
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29. 老川桂生、浅田浩太朗、大野慎一郎、黒田雅彦．microRNA の生成経路を欠損する肺がん細胞株の解析．第 12 回日本病理学会カンファレンス 神戸六甲山ホテル （神戸）2015 年 7 月 24 - 25 日.
30. 浅田浩太朗、老川桂生、大野慎一郎、黒田雅彦．肺がんにおける Argonaute ﾌｧﾐﾘｰの発現・機能解析．第 12 回日本病理学会カンファレンス 神戸六甲山ホテル （神戸） 2015
年 7 月 24 - 25 日
31. 山田侑子、高梨正勝、須藤カツ子、上田しのぶ、黒田雅彦．肺線維症に対する miR-29b
補充療法．第 12 回日本病理学会カンファレンス 神戸六甲山ホテル （神戸） 2015 年 7
月 24 - 25 日
32. 柏迫幹絵、高梨正勝、黒田雅彦．ダニ抗原で誘発させたアトピー性皮膚炎モデルマウスの作成．第 12 回日本病理学会カンファレンス 神戸六甲山ホテル （神戸） 2015 年７月
24 - 25 日
33. 藤田浩司、倉田 厚、別所泰子、峯尾松一郎、黒田雅彦．子宮頸部上皮腫瘍（cervicalintraepithelial neoplasia; CIN）の抗 MUC1 抗体における染色性の検討．第 12 回日本病理学会カンファレンス 神戸六甲山ホテル （神戸） 2015 年 7 月 24 - 25 日
34. 黒田雅彦: 計測画像情報の病理学への応用．第 12 回日本病理学会カンファレンス 神
戸六甲山ホテル （神戸） 2015 年 7 月 24 - 25 日
35. Ohki T, Umezu T, Ohyashiki JH, Fukuoka Y. Integrative analysis of expression levels of
miRNA and mRNA: Investigation of hypoxic tolerance in leukemia. 37th International
Annual Conference of IEEE Engineering in Medicine and Biology Society, August 25-29,
2015, Milano Conference Center, Milan, Italy.
36. 黒田雅彦、エクソソームの臨床応用、近未来医療フォーラム （東京） 2015 年 9 月 1 日
37. Ohyashiki K, Umezu T, Imanishi S, Ohyashiki JH. Leukemia cells under persistent hypoxia
secrete exosomes enhancing angiogenesis in mice. ISEH 44th Annual Scientific Meeting,
September 17, 2015, Kyoto International Conference Center, Kyoto, Japan.
38. 大屋敷一馬、溝口 出、善本隆之、田内哲三、岡部聖一、片桐誠一朗、梅津知宏、大屋敷純子： Stop-imatinib CML 患者末梢血単核球における免疫関連 miRNA の意義.第 7 回
血液疾患免疫療法研究会 （東京） 2015 年 9 月 26 日
39. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH. Exosome-based
therapeutic approach using exosomal miRNAs derived from bone marrow mesenchymal
stromal cells. 第７４回日本癌学会学術総会 名古屋国際会議場 （名古屋） 2015 年 10
月 8 日- 10 日
40. Azuma K, Takeuchi K, Umezu T, Ohyashiki K, Ohyashiki JH. Establishment andcharacterization of a novel chronic hypoxia-resistant multiple myeloma cell line. 第７４回
日本癌学会学術総会 名古屋国際会議場 （名古屋） 2015 年 10 月 8 日- 10 日
41. 及川恒輔、高梨正勝、佐藤冬樹、黒田雅彦、村垣泰光.TLS-CHOP を発現する粘液型脂肪肉腫細胞の増殖に必須な新規分子経路の探索．第 74 回日本癌学会学術総会 名古屋国際会議場 （名古屋） 2015 年 10 月 8 日- 10 日
42. Saitoh Y, Imanishi S, Umezu T, Yoshizawa S, Asano M, Fujimoto H, Akahane D, Yamamoto
Y, Kobayashi C, Ohyashiki JH, Ohyashiki K: Exosomal miRNA signature of bone marrowmesenchymal stromal cells derived from MDS patients. 第 77 回日本血液学会学術集会-23-

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43. Imanishi S, Takahashi R, Miho Ohsuga, Umezu T, Kobayashi C, Katagiri S, Asano M, Saitoh
Y, Ohyashiki K, Ohyashiki JH: Targeting bromodomain protein in azacytidine resistantleukemia cells. 第 77 回日本血液学会学術集会 （金沢） 2015 年 10 月 16 日- 18 日
44. Ito Y, Umezu T, Tadokoro K, Saito Y, Katagiri S, Yoshizawa S, Fujimoto H, Akahane D,
Suguro T, Asano M, Tanaka Y, Ohyashiki JH, Ohyashiki K: BIM deletion polymorphism may
be associated with resistance to chemotherapy for follicular lymphoma. 第 77 回日本血液学会学術集会 （金沢） 2015 年 10 月 16 日- 18 日
45. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Phosphoinositide 3-kinase inhibitor, copanlisib
for the treatment of Ph positive leukemia cells. 第 77 回日本血液学会学術集会 （金沢）2015 年 10 月 16 日- 18 日
46. Yoshizawa S, Umezu T, Saito Y, Gotoh M, Akahane D, Kobayashi C, Ohyashiki JH, Ohyashiki
K: Exosomal miRNA signature of late onset acute GVHD in allogeneic HSCT. 第 77 回日本
血液学会学術集会 （金沢） 2015 年 10 月 16 日- 18 日
47. Akahane D, Asano M, Gotoh M. Saitoh Y, Katagiri S, Suguro T, Yoshizawa S, Sakuta J,
Tanaka Y, Furuya N, Fujimoto H, Okabe S, Tauchi T, Ito Y, Ohyashiki K: Successfultreatment with azacitidine after CBT for primary refractory AML with monosomal
karyotype. 第 77 回日本血液学会学術集会 （金沢） 2015 年 10 月 16 日- 18 日
48. Katagiri S, Tauchi T, Umezu T, Tadokoro K, Mizoguchi I, Yoshimoto T, Ohyashiki JH,
Ohyashiki K: Predictive factors for successful treatment discontinuation of CML patients.
第 77 回日本血液学会学術集会 （金沢） 2015 年 10 月 16 日- 18 日
49. Tanaka Y, Okabe S, Tauchi T, Ito Y, Ohyashiki K: Antitumor activity of phosphoinositide
3-kinase inhibitor, copanisib in multiple myeloma. 第 77 回日本血液学会学術集会 （金沢） 2015 年 10 月 16 日- 18 日
50. Tauchi T, Okabe S, Katagiri S, Tanaka Y, Ohyashiki K: Activity of stemness inhibitor, BBI608,
on the self-renewal of BCR-ABL1 positive leukemia cells. 第 77 回日本血液学会学術集会（金沢） 2015 年 10 月 16 日- 18 日
51. Yoshizawa S, Umezu T, Saitoh Y, Gotoh M, Akahane D, Kobayashi C, Ohyashiki JH,
Ohyashiki K. Exosomal miRNA signature of late onset acute graft-versus-host disease inallogenic hematopoietic stem cell transplantation. 第７７回日本血液学会学術集会 （金沢） 2015 年 10 月 16 日- 18 日
52. Saitoh Y, Imanishi S, Umezu T, Yoshizawa S, Asano M, Fujimoto H, Akahane D, Yamamoto
Y, Kobayashi C, Ohyashiki JH, Ohyashiki K. Exosomal miRNA signature of bone marrowmesenchymal stromal cells derived from MDS patients. 第７７回日本血液学会学術集会（金沢） 2015 年 10 月 16 日- 18 日
53. 武内 健、梅津知宏、小林千晶、大屋敷一馬、東 剣紅、大屋敷純子：低酸素耐性骨髄腫細胞株 IM-9-HR の樹立と性状.第 176 回東京医科大学医学会総会 （東京） 2015 年
11 月 7 日
54. 大須賀美穂、今西 哲、高橋諒子、大屋敷純子、梅津知宏、小林千晶、片桐誠一朗、大屋敷一馬：アザシチジン耐性白血病細胞株における BET ファミリー阻害剤の分子機構の解明.第 176 回東京医科大学医学会総会 （東京） 2015 年 11 月 7 日
55. 武内 健、梅津 知宏、東 剣虹、小林 千晶、大屋敷一馬、大屋敷純子：低酸素耐性骨髄腫細胞株 IM-9-HR の樹立と性状解析、第１７６回医学会総会 （東京） 2015 年 11 月
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7日
56. Yokosuka T, Hashimoto-Tane A, Furuhata M, Toyoda H, Saito T：Cytoskeletal regulation of
the clustering of a Ras-GEF, RasGRP1, upon T cell activation．第 44 回日本免疫学会総会
学術集会（札幌） 2015 年 11 月 18 - 20 日
57. 及川恒輔、田中正視、尾崎 敬、黒田雅彦、村垣泰光．粘液型脂肪肉腫特異的キメラ遺伝子 TLS-CHOP に制御される新規分子メカニズムの検討．第 38 回日本分子生物学会第88 回日本生化学学会大会 合同大会、神戸、2015 年 12 月 1 - 4 日
58. Schanz J, Bennett JM, Slovak ML, Fenaux P, Malcovati L, Cazzola M, Valent P, Ohyashiki
K, Levis A, Sekeres MA, Tauro A, Magalhaes S, an de Loosdrecht AA: Prognostic impact
of rare single abnormalities in myelodysplastic syndromes. 57th Annual Meeting ofAmerican Society of Hematology. Dec 5-8, 2015, Florida, USA.
59. Suzuki T, Kizaki M, Uike N, Abe Y, Hidaka M, Tagashira S, Tsuchiya S, Ohyashiki K, Miyazaki
M: A Phase 1/2 study of WT1 peptide cancer vaccine WT4869 in patients withmyelodysplastic syndromes (MDS). 57th Annual Meeting of American Society of
Hematology. Dec 5-8, 2015, Florida, USA.
60. Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, Ohyashiki K: Targeting the hedgehog
signaling pathway by PF-04449913 limits the self-renewal of MDS-derived induced potent
stem cells (iPSC). 57th Annual Meeting of American Society of Hematology. Dec 5-8, 2015,
Florida, USA.
61. Okabe S, Tauchi T, Tanaka Y, Sakuta J, Ohyashiki K: Dual HDAC and PI3K inhibitor,
CUDC-907 alone or in combination with ABL tyrosine kinase inhibitor against ABL tyrosine
kinase inhibitor resistant leukemia cells. 57th Annual Meeting of American Society of
Hematology. Dec 5-8, 2015, Florida, USA.
62. Okabe S, Tauchi T, Tanaka Y, Sakuta J, Ohyashiki K: Anti-leukemic activity of axitinib
against cells harboring the BCR-ABL point mutation. 57th Annual Meeting of American
Society of Hematology. Dec 5-8, 2015, Florida, USA.
63. 黒田雅彦: エクソソームが開く新しい医療．特別講演（招待講演）．第 19 回日本統合医療学会 （山口） 2015 年 12 月 12 - 13 日.
64. 横須賀忠：ユビキチンが形成する T 細胞抑制性シグナルソームの分子イメージング解析．新学術領域「ユビキチンネオバイオロジー」第 2 回領域班会議（富浦）2015 年 12 月
18 日
65. 横須賀忠（招待講演）：T 細胞シグナルソーム「TCR マイクロクラスター」による T 細胞活性化の時空間的制御機構．岡山大学第 10 回免疫学セミナー （岡山） 2016 年 2 月 4 日
66. 横須賀忠（招待講演）：イメージングが拓く免疫チェックポイント分子による時空間的 T 細
胞制御メカニズム．第 15 回新宿肺癌サミットミーティング 特別講演 （東京） 2016 年 2
月 17 日67. **Ohyashiki K, Umezu T, Katagiri S, Kobayashi C, Azuma K, Tauchi T. Okabe S, Fukuoka Y,
Ohyashiki JH: Down-regulation of plasma miR-215 in chronic myeloid leukemia patientswith successful imatinib discontinuation, AACR-JCA joint symposium. Feb 16-20, 2016,
Maui, USA.
68. Umezu T, Imanishi S, Azuma K, Kobayashi C, Ohyashiki K, Ohyashiki JH: Bone marrowstromal cells from young healthy donors secrete exosomes that inhibit in vivo
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angiogenesis in multiple myeloma. 10th AACR-JCA Joint Conference, February 16-20,
2016, Maui, Hawaii.
69. Yokosuka T : Balance of T cell activation and suppression controlled by Cbl-b-mediatedubiquitination of TCR microclusters. Environment controlling normal and diseased
hematopoietic and immune systems MEXT Research Project Symposium（横浜） 2016 年 3月2日
70. 大木 拓也、福岡 豊、梅津 知宏、大屋敷純子：microRNA と遺伝子の統計的統合解
析法の検討．電子情報通信学会 ME とバイオサイバネティクス研究会 （東京） 2016 年 3月 13 日71. 齋藤 優、浅野倫代、吉澤成一郎、藤本博昭、後藤守孝、伊藤良和、田内哲三、大屋敷
一馬： 白血球減少で発症した慢性骨髄性白血病.第 4 回日本血液学会関東甲信越地方
会 （東京） 2016 年 3 月 19 日
72. 大木 拓也、福岡 豊、梅津 知宏、大屋敷純子： miRNA と mRNA の統合解析における発現変化の閾値の最適化．第５５回日本生体医工学会大 （富山） 2016 年 4 月 26 28 日
73. Umezu T, Imanishi S, Azuma K, Kobayashi C, Ohyashiki K, Ohyashiki JH. Towards
exosome-based cancer therapy: rejuvenation of exosomes derived from normal bonemarrow stromal cells. 5th Annual Meeting of ISEV, May 4-7, 2016, Rotterdam.
74. ** Ohno S, Itano K, Harada Y, Asada K, Oikawa K, Kashiwazako M, Okuyama H,

Kumagai
K, Takanashi M, Sudo K, Ikeda N, Kuroda M: Development of novel Dicer- andAgo2-independent small hairpin RNAs. American Society of Gene & Cell Therapy(ASGCT)
19th Annual Meeting. (第 19 回米国遺伝子治療学会), Washington,DC, USA. May 4-9

2016.
75. 大平 達夫, 今井健太郎，前原 幸夫，大谷 圭志，前田 純一，吉田 浩一，萩原優，垣花 昌俊，梶原 直央，池田 徳彦: 分子生物学研究が呼吸器外科の治療にもたらすもの 第 33 回日本呼吸器外科学会総会 (京都) 2016 年 5 月 12 日
76. 小濱政子、倉田 厚、藤田浩司、長尾俊孝、黒田雅彦．卵巣奇形種の悪性度とその腫瘍

血管の未熟度との相関についての免疫組織科学的検討．第 105 回日本病理学会総会、
仙台国際センター （仙台） 2016 年 5 月 12 - 14 日
77. 上田しのぶ、黒田雅彦 他．乳がん幹細胞の酸化ストレス抵抗性における miR-27a の作

用．第 105 回日本病理学会総会、仙台国際センター （仙台） 2016 年 5 月 12 - 14 日
78. 大野慎一郎、黒田雅彦 他．肺がんにおける Argonaute ファミリーの機能解析．第 105 回
日本病理学会総会、仙台国際センター （仙台） 2016 年 5 月 12 - 14 日
79. 老川桂生、黒田雅彦 他．miRNA 生成経路を欠損する肺がん細胞株の解析．第 105 回

日本病理学会総会、仙台国際センター （仙台） 2016 年 5 月 12 - 14 日
80. **Katagiri S, Umezu T, Kobayashi C, Azuma K, Tauchi T, Okabe S, Ohyashiki JH, Ohyashiki
K: Clinical relevance of plasma miR-215 level in CML patients treated with imatinib 7 th JSH
International Symposium. May 13-14, 2016. Awajishima, Hyogo.
81. 秦喜久美、矢那瀬紀子、水口純一郎、横須賀忠： B 細胞の分化・機能におけるアルギニ
ンメチル化酵素 PRMT1 の役割の解析．第 26 回京都 T 細胞カンファレンス （京都） 2016
年 5 月 20 - 21 日
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82. 黒田雅彦: miRNA 補充療法の開発、第 12 回日本臨床プロテオーム研究会、東京大学山

上会館 （東京） 2016 年 5 月 21 日
83. 梅津 知宏、大屋敷一馬、大屋敷純子： 多発性骨髄腫における骨髄内微小環境でのエクソソームの役割．第４１回日本骨髄腫学会学術集会 （徳島） 2016 年 5 月 28 - 29 日
84. Imanishi S, Takahashi R, Katagiri S, Ohsuga M, Umezu T, Ohyashiki K, Ohyashiki JH:
Teriflunomide restores 5-azacytidine sensitivity via inhibition of DNA methyltransferase.
7th JSH International Symposium. May 13-14, 2016. Awajishima, Hyogo.
85. 東 剣紅、今西 哲、大屋敷純子、梅津知宏、浅野倫代、吉澤成一郎、大屋敷一馬： 次
世代シーケンサーを用いた骨髄系腫瘍における骨髄間質細胞の体細胞変異解析.第177 回東京医科大学医学会総会 （東京） 2016 年 6 月４日
86. 許 文聰、永川裕一、粕谷和彦、土田明彦、上田しのぶ、金蔵孝介、黒田雅彦: 膵臓癌

患者血清中 microRNA の網羅的解析による新規 Biomarker の探索. 第 177 回東京医科大学医学会総会 （東京） 2016 年 6 月 4 日
87. 山田侑子、高梨正勝、上田しのぶ、黒田雅彦、須藤カツ子: 肺線維症に対する miR-29b
補充療法. (医学会奨励賞受賞講演）第 177 回東京医科大学医学会総会 （東京） 2016
年6月4日
88. Tauchi T, Okabe S, Ohyashiki K: Hedgehog signaling pathyway inhibitor PF-04449913 limits
the self-renewal of MDS-derived induced potent stem cells (iPS): molecular mechanism.
21th Congress of European Hematology Association. June, 9-13, 2016, Copenhagen,Denmark.
89. Okabe S, Tauchi T, Tanaka Y, Sakuta J, Ohyashiki K： PPARγ is a novel therapeutic targets
in Ph-positive leukemia cells. 21th Congress of European Hematology Association. June,
9-13, 2016, Copenhagen, Denmark.
90. 小澤ひとみ、梅津 知宏、関野 知聖、吉澤成一郎、大屋敷一馬、大屋敷純子： 多発性骨髄腫細胞の浸潤様式解明を目的とした in vitro モデル実験系の構築．第５回医薬工３
大学包括連携推進シンポジウム （東京） 2016 年 6 月 18 日
91. 大木 拓也、松井 一馬、梅津 知宏、大屋敷純子、福岡 豊： 生体分子情報の網羅的解析法の開発．第５回医薬工３大学包括連携推進シンポジウム （東京） 2016 年 6 月 18日
92. 雨宮文惠、伊藤良和、齋藤 優、片桐誠一朗、勝呂多光子、浅野倫代、吉澤成一郎、田中裕子、藤本博昭、岡部聖一、後藤守孝、田内哲三、大屋敷一馬：JAK2-V617F 変異を有する MPN に発症した BCR-ABL1 陽性 CML。日本血液第 5 回関東甲信越学地方会（東京）2016 年 7 月 2 日
93. 梅津 知宏、大屋敷一馬、大屋敷純子：がん環境を標的とした治療戦略：骨髄間質細胞
由来エクソソームおよびその内包型 miRNA の役割．第８回日本 RNAi 研究会・第３回日
本細胞外小胞学会 （広島） 2016 年 8 月 31 日- 9 月 2 日
94. 大野慎一郎、原田裕一郎、村上善基、黒田雅彦. 新規ヘアピン型 RNA 干渉核酸の探と機能解析. 第 8 回日本 RNAi 研究会、第 3 回日本細胞外小胞体学会 （広島） 2016 年 8月 31 日- 9 月 2 日
95. **Ohno S, Harada Y, Murakami Y, Kuroda M. Searching and functional analysis of novel

hairpin-forming small RNA. The 8th JARI Anuual meeting. The 3rd JSEV Annual Meeting.
Hiroshima, Aug31-Sep.2 2016.
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96. 及川恒輔、高梨正勝、佐野冬樹、黒田雅彦、村垣泰光. TLS-CHOP を発現する粘液型脂肪肉腫細胞における腫瘍関連分子経路の解析. 第 75 回日本癌学会学術総会 （横浜）2016 年 10 月 6 - 8 日
97. Oikawa K, Takanashi M, Sano F, Kuroda M, Muragaki Y. Analysis of tumor-related

molecular pathways in TLS-CHOP-expressing myxoidliposarcoma cells. The 75th Annual
Meeting of the Japanese Cancer Association. Yokohama, Oct 6-8 2016.
98. Amemiya A, Akahane D, Ito Y, Katagiri S, Asano M, Suguro T, Tanaka Y, Furuya N,
Ohyashiki K: Management of splenectomy in patients with thrombopoietin receptor
agonist-dependent immune thrombocytopenia. 第 78 回日本血液学会総会 （横浜）2016 年 10 月 13 -15 日
99. Azuma K, Umezu T, Imanishi S, Asano M, Yoshizawa S, Katagiri S, Ohyashiki K, Ohyashiki
JH: Mutation analysis of bone marrow stromal cells derived from AML and MDS. 第 78 回
日本血液学会総会（横浜） 2016 年 10 月 13 -15 日
100. Imanishi S, Takahashi R, Umezu T, Kobayashi C, Asano M, Ohyashiki K, Ohyashiki JH:
Distinct histone modification patterns in 5-azacitidine sensitive human leukemia cells. 第78 回日本血液学会総会 （横浜） 2016 年 10 月 13 -15 日
101. Katagiri S, Azuma K, Umezu T, Akahane D, Wataya Y, Yoshida K, Makishima H, Ogawa S,
Ohyashiki JH, Ohyashiki K: Clonal evolution in AML which relapsed 15 years after BMT. 第78 回日本血液学会総会 （横浜） 2016 年 10 月 13 -15 日
102. Okabe S, Tauchi T, Tanaka Y, Sakuta J, Ohyashiki K: Activity of dual HDAC and PI3K
inhibitor, CUDC-907 against BCR-ABL-positive leukemia cells. 第 78 回日本血液学会総会（横浜） 2016 年 10 月 13 -15 日
103. Katagiri S, Tauchi T, Saito Y, Suguro T, Asano M, Yoshizawa S, Sakuta J, Akahane D, Tanaka
Y, Furuya N, Ando K, Fujimoto H, Okabe S, Gotoh M, Ito Y, Ohyashiki K: Assessment ofmusculoskeletal pain after tyrosine kinase inhibitor discontinuation in chronic myeloid
leukemia patients. 第 78 回日本血液学会総会 （横浜） 2016 年 10 月 13 -15 日
104. Tauchi T, Okabe S, Katagiri S, Tanaka Y, Tohyama K, Ohyashiki K: Hedgehog Signaling
Pathway Inhibitor, PF-04449913 Limits the Self-Renewal of MDS-Derived iPSC. 第 78 回日本血液学会総会 （横浜） 2016 年 10 月 13 -15 日
105. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH. Anti-angiogenic
effect of normal bone marrow stromal cell-derived exosomes rejuvenated by miR-340. The
78th Annual Meeting of JSH, October 13-15, 2016, Yokohama.
106. 黒田雅彦: エクソソームの臨床応用、エクソソーム 2016－エクソソーム研究は医療をどう変えるか?－、中央大学駿河台記念館 （東京）、2016 年 10 月 26 日
＊ ＊黒田雅彦: RNA ワールドと肺がん発がん機構. (教育講演Ⅲ) 第 108 回 ACCP 日本部会
定期教育講演会. 東京医科大学病院 6 階臨床講堂. （東京） 2016 年 10 月 29 日
107. 髙橋諒子、今西 哲、梅津知宏、小林千晶、大屋敷一馬、大屋敷純子： アザシチジン耐性白血病細胞におけるヒストン化学修飾の役割の解明.第 178 回東京医科大学医学会総会 （東京） 2016 年 11 月 5 日
108. 浅野倫代、梅津知宏、片桐誠一朗、小林千晶、田内哲三、岡部聖一、大屋敷純子、大屋敷一馬： 慢性骨髄性白血病におけるチロシンキナーゼ阻害薬中止後の筋骨格痛ではエクソソーム miR-140-3p の上昇がみられる.第 178 回東京医科大学医学会総会 （東京）-28-

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2016 年 11 月 5 日
109. 片桐誠一朗、田内哲三、雨宮文惠、齋藤 優、勝呂多光子、浅野倫代、吉澤成一郎、作田寿理、赤羽大悟、田中裕子、古屋奈穂子、安藤恵子、藤本博昭、岡部聖一、後藤守
孝、伊藤良和、大屋敷一馬： 慢性骨髄性白血病におけるチロシンキナーゼ阻害剤中止
後の筋骨格痛についての検討.第 178 回東京医科大学医学会総会 （東京） 2016 年 11月5日
110. 片桐誠一朗、田内哲三、雨宮文惠、齋藤 優、勝呂多光子、浅野倫代、吉澤成一郎、作田寿理、赤羽大悟、田中裕子、古屋奈穂子、安藤恵子、藤本博昭、岡部聖一、後藤守
孝、伊藤良和、大屋敷一馬： 慢性骨髄性白血病におけるチロシンキナーゼ阻害剤中止
後の筋骨格筋痛についての検討.第 178 回東京医科大学医学会総会 （東京） 2016 年
11 月 5 日
111. 浅野倫代、梅津知宏、片桐誠一朗、小林千晶、田内哲三、岡部聖一、大屋敷純子、大屋敷一馬：慢性骨髄性白血病におけるチロシンキナーゼ阻害薬中止後の筋骨格痛ではエクソソーム miR-140-3p の上昇がみられる.第 178 回東京医科大学医学会総会 （東京）2016 年 11 月 5 日
112. 浅野 倫代、梅津 知宏、片桐誠一朗、小林 千晶、田内 哲三、岡部 聖一、大屋敷純子、大屋敷一馬：慢性骨髄性白血病におけるチロシンキナーゼ阻害薬中止後の筋骨格痛ではエクソソーム miR-140-3p の上昇がみられる．第１７８回東京医科大学医学会総会（東京） 2016 年 11 月 5 日
113. 関野 知聖、梅津 知宏、吉澤成一郎、大屋敷一馬、大屋敷純子：骨髄間質細胞が放出するエクソソーム内加齢関連 miRNA の同定と機能解析．第１７８回東京医科大学医学会
総会 （東京） 2016 年 11 月 5 日
114. 梅津 知宏、大屋敷一馬、大屋敷純子：多発性骨髄腫における骨髄内微小環境でのエクソソームの役割．第１９回癌と骨病変研究会 （東京） 2016 年 11 月 11 日
115. 横須賀忠：ユビキチンが形成する T 細胞抑制性シグナルソームの分子イメージング解析．新学術領域「ユビキチンネオバイオロジー」第 3 回領域班会議 （札幌） 2016 年 11
月 17 日116. Yokosuka T（招待講演）, Hata K, Yanase N, Hashimoto-Tane A, Saito T : Microclusters as asignaling unit for T cell receptor endocytosis. 第 54 回日本生物物理学会大会 （つくば）2016 年 11 月 25 日
117. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Anti-leukemic effects of venetoclax on
Philadelphia chromosome positive leukemia cells. 90th Annual Meeting for American
Hematology society, Dec 3-5, 2016, SanDiego, USA.
118. Yokosuka T, Hata K, Yanase N, Hashimoto-Tane A, Siato T. Microclusters as a functional
unit for the endocytosis of T cell receptors 第 45 回日本免疫学会総会学術集会（沖縄）2016 年 12 月 5-7 日
119. Ohira T, Sakai K, Maehara S, Maeda J, Yoshida K, Hagiwara M, Kakihana M, Okano T,
Kajiwara N, Nishio K, Ikeda N: The Feasibility of Cell-Free DNA Sequencing for Mutation
Detection in Non–Small Cell Lung Cancer Was Detemined by Tumor Volume. IASLC 17th
World Conference on Lung Cancer, December, 5, 2016, Vienna, Austria.
120. Makino Y, Bae E, Yoon J, Mamura M, Ohira T, Kuroda M, Ikeda N. Linker-Phosphorylated
Smad2 and STAT3 Induce Resistance to Tyrosine Kinase Inhibition in Lung Cancer. IASLCWorld Conference on Lung Cancer, December, 6, 2016, Vienna, Austria.
-29-

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121. 大平 達夫, 坂井 和子，前原 幸夫，前田 純一，吉田 浩一，萩原優，垣花 昌俊，岡野 哲也，松林 純，梶原 直央，長尾 俊孝，西尾 和人，池田 徳彦: 肺癌手術患者に対するリキッドバイオプシーの有用性と限界 第 57 回日本肺癌学会学術集会(福岡) 2016 年 12 月 19 日
122. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH: Replenishing
exosomes from older bone marrow stromal cells by miR-340 inhibits myeloma-related
angiogenesis. The Royal Society Scientific Meeting 2017 “Extracellular vesicles and thetumor microenvironment”, January 23-24, London.
123. 横須賀忠、末永忠広、荒瀬尚： ネオ･セルフとしてのミスフォールド蛋白質解析．新学術
領域「ネオ･セルフ」キックオフシンポジウム （東京） 2017 年 1 月 31 日
124. 横須賀忠（招待講演）：イメージングが拓く免疫チェックポイント分子による T 細胞活性化の時空間的制御機構．第 14 回日本免疫治療学研究会学術集会 教育講演（東京）2017 年 2 月 11 日
125. Machiyama H, Hata K, Yanase N, Hashimoto-Tane A, Siato T, Yokosuka T: Microclusters asa signaling unit for T cell receptor endocytosis. 第 27 回京都 T 細胞カンファレンス （京都）2017 年 3 月 13 -14 日
126. 池田 徳彦: がんの低侵襲手術：次の breakthrough は何か. 次世代がん治療推進専門
家養成プランシンポジウム （東京） 2017 年 3 月 4 日
127. Makino Y, Yoom JH, Bae E, Kato M, Miyazawa K, Ohira T, Ikeda N, Kuroda M, Mamura M:
Repression of Smad3 by Stat3 and s-Ski/SnoN induces gefitinib resistance in lungadenocarcinoma. American Association for Cancer Research Annual Meeting, April 2,
2017, Washington DC, USA.
128. 片桐誠一朗、田内哲三、安藤恵子、岡部聖一、後藤守孝、大屋敷一馬： 慢性骨髄性白血病において body mass index 低値は imatinib 中止後筋骨格痛と関連する.第 114 回日本内科学会総会・講演会 （東京） 2017 年 4 月 14 - 16 日
129. 齋藤 彰、藤田浩司、山本陽一郎、硲 彰一、坂本和彦、永野浩昭、黒田雅彦: Digital

Pethology を用いた肝細胞がんの形態学解析による再発予測の検討. (ワークショップ６)
第 106 回日本病理学会総会 京王プラザホテル (東京) 2017 年 4 月 27 日
130. 藤田浩司、大野慎一郎、西 洋孝、井坂恵一、黒田雅彦. 子宮頸部上皮内腫瘍病変に対する新規モノクロナール抗体の開発. (Development of Novel monoclonal antibody forcervical intraepithelial neoplasma.) 第 106 回日本病理学会総会 京王プラザホテル (東京) 2017 年 4 月 27 日
131. 池畑直樹、高梨正勝、上田しのぶ、里見貴史、渡辺正人、長谷川温、河野通秀、榎本愛、黒田雅彦、近津大地: Activation of TLR2 regulates CARD10 gene expression by
miR-146a-5p in oral squamous cell carcinoma. 第 106 回日本病理学会総会 京王プラザ
ホテル (東京) 2017 年 4 月 27 日
132. ＊＊大野慎一郎、原田裕一郎、村上善基、黒田雅彦: RNA 干渉核酸に働く短鎖ヘアピン
RNA の探索と機能解析.(Search and characterization of novel hairpin RNAs acting on RNA
interference. 第 106 回日本病理学会総会 京王プラザホテル (東京) 2017 年 4 月 28 日
133. ＊＊老川桂生、淺田浩太朗、大野慎一郎、黒田雅彦: miRNA 生成経路を欠損する肺がん細胞株の解析(Analysis of microRNA-defict lung cancer cell line.) 第 106 回日本病理学-30-

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会総会 京王プラザホテル (東京) 2017 年 4 月 28 日
134. 橋本浩次、倉田 厚、藤原正親、原 啓、名城珠希、増田芳雄、鈴木良夫、菅間 博、黒田 雅 彦 、 堀 内 啓 . Clinicopathogic Characteristics of solitary pulmonary capillaryhemangioma. 第 106 回日本病理学会総会 京王プラザホテル (東京) 2017 年 4 月 28日
135. ＊＊三部芳裕、大野慎一郎、黒田雅彦: Mir-34a によるがん抑制遺伝子 BLU の発現制御機構.(Up-regulation of expression of BLU tumor suppressor by miR-32a.) 第 106 回日本病理学会総会 京王プラザホテル (東京) 2017 年 4 月 29 日
136. 鶴井敏光、藤田浩司、大野慎一郎、黒田雅彦: 浸潤性乳管癌に出現する let-7 発現細胞の検討.(let-7 induced invasive phenotype of breast cancer cells.) 第 106 回日本病理学会
総会 京王プラザホテル (東京) 2017 年 4 月 29 日
137. 大平 達夫, 濱中和嘉子，松浦 久美，今井健太郎，前原 幸夫，前田 純一，吉田 浩
一，萩原 優，垣花 昌俊，梶原 直央，池田 徳彦: 肺癌に対する網羅的遺伝子解析
第 117 回日本外科学会定期学術集会 (東京) 2017 年 4 月 29 日
138. Imanishi S, Takahashi R, Katagiri S, Umezu T, Kobayashi C, Ohyashiki K, Ohyashiki JH:
Teriflunomide restores 5-azacytidine sensitivity via activation of pyrimidine salvage in
5-azacytidine-resistant leukemia cells. MDS symposium. May 3-6, 2017 Valencia, Spain.
139. Ohyashiki K, Saitoh Y, Imanishi S, Umezu T, Yoshizawa S, Asano M, Fujimoto H, Akahane D,
Kobayashi C, Ohyashiki JH: Extracellular vesicles (EVs) released by bone marrow stromalcells show a distinct miRNA profile in high-risk MDS patients. The 14th International
Symposium on MDS2017, May 3-6, 2017, Valencia.
140. Ohyashiki JH, Umezu T, Kobayashi C, Imanishi S, Asano M, Ohyashiki K: Telomericrepeat-containing RNA (TERRA) in MDS: Utility of cell-free TERRA packaged in extracellular
vesicles. The 14th International Symposium on MDS2017, May 3-6, 2017, Valencia.
141. Umezu T, Imanishi S, Yoshizawa S, Ohyashiki K, Ohyashiki JH: A role of exosomal miR-10a
in bone marrow stromal cells obtained from patients with multiple myeloma. 6th Annual
Meeting of ISEV, May 18-21, 2017, Toronto.
142. 横須賀忠（招待講演）：イメージンブが拓く T 細胞活性化の時空間的制御機構 −シグナル
ソームの視点から免疫チェックポイントを解釈する− Expert Seminar of Immunology 特
別講演 （東京） 2017 年 5 月 22 日
143. ＊＊黒田雅彦: 核酸医薬の臨床応用、第 384 回 CBI 学会講演会－核酸医薬－、東京工業大学キャンパスイノベーションセンター （東京） 2017 年 5 月 25 日
144. 片桐誠一朗、田内哲三、大西文恵、齋藤 優、勝呂多光子、浅野倫代、吉澤成一郎、作田寿理、赤羽大悟、田中裕子、古屋奈穂子、安藤恵子、藤本博昭、岡部聖一、後藤守
孝、伊藤良和、大屋敷一馬： 慢性骨髄性白血病において患者体重および body massindex は imatinib 中止後筋骨格痛の出現と関連する.第 179 回東京医科大学医学会総会（東京） 2017 年 6 月 3 日
145. 齋藤 優、片桐 誠一朗、勝呂 多光子、吉澤 成一郎、赤羽 大悟、藤本 博昭、後藤 守
孝、伊藤 良和、大屋敷 一馬、東 剣紅、今西 哲、大屋敷 純子：節外性 NK/T 細胞リンパ腫・鼻型における骨髄 T 細胞受容体再構成の意義.第 179 回東京医科大学医学会総会（東京） 2017 年 6 月 3 日
146. 今西 哲、大屋敷 純子、梅津知宏、小林千晶、大屋敷 一馬：クロマチン制御因子 HP1
-31-

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S1511011のアザシチジン耐性白血病細胞における役割の解明.第 179 回東京医科大学医学会総会 （東京） 2017 年 6 月 3 日
147. 横須賀忠：イメージングからのアプローチによるネオ･セルフの解明．新学術領域「ネオ･セルフ」第 1 回領域班会議 （大阪） 2017 年 6 月 20 日
148. Tauchi T, Okabe S, Katagiri S, Tanaka Y, Ohyashiki K: Combining effects of the SMO
inhibitor and JAK1 inhibitor in MDS-derived induced potent stem cells (iPS). 22th European
Hematology association. June 23-26, 2017. Madrid, Spain.
149. Ohyashiki K, Asano M, Umezu T, Katagiri S, Kobayashi C, Tauchi T, Gotoh M, Ando K, Okabe
S, Ohyashiki JH: miRMA profiling of circulating extracellular vesicles in CML patients withmusculoskeletal pain associated with discontinuation of tyrosine kinase inhibitors. 22th
European Hematology association. June 23-26, 2017. Madrid, Spain.
150. Katagiri S, Tauchi T, Tanaka Y, Ando K, Okabe S, Gotoh M, Ito Y, Umezu T, Tadokoro K,
Ohyashiki JH, Ohyashiki K: Association of BCLL11 (BIM) deletion polymorphism withmolecular relapse after tyrosine kinase inhibitor cessation in chronic myeloid leukemia
patients with deep molecular response. 22th European Hematology association. June
23-26, 2017. Madrid, Spain.
151. Nanya Y, Takeda J, Shinozawa Y, Shiraishi Y, Okuno Y, Kataoka K, Chiba K, Tanaka H, Sanada
M, Chiba S, Asou N, Kiyoi H, Imai K, Hirase C, Dobashi N, Kiguchi T, Nakao S, Ohyashiki K,
Miyazaki Y, Naoe T, Makishima H, Miyanl S, Ogawa S: Molecular markers predicting
response to azacytidine treatment for myelodysplastic syndromes. 22th European
Hematology association. June 23-26, 2017. Madrid, Spain.
152. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Targeted strategy for ABL tyrosine kinase
inhibitor resistant Philadelphia chromosome positive leukemia cells. 22th European
Hematology association. June 23-26, 2017. Madrid, Spain.
153. Ohyashiki K, Asano M, Umezu T, Katagiri S, Kobayashi C, Tauchi T, Gotoh M, Ando K, Okabe
S, Ohyashiki JH: miRNA profiling of circulating extracellular vesicles in CML patients withmusculoskeletal pain associated with discontinuation of tyrosine kinase inhibitors. 22 ndCongress of EHA, June 22-25, 2017, Madrid.
154. Umezu T, Imanishi S, Yoshizawa S, Ohyahsiki K, Ohyashiki JH: Inhibition of extracellular
vesicle secretion induces apoptosis of bone marrow stromal cells: towards soil-targeted
therapy in multiple myeloma. 22nd Congress of EHA, June 22-25, 2017, Madrid.
155. 横須賀忠（招待講演）：イメージンブが拓く免疫チェックポイント分子による細胞活性化の時空間的制御機構．第 32 回日本肺癌学会ワークショップ 教育講演 （旭川） 2017 年 7月1日
156. 黒田雅彦: エクソソームの臨床応用 第 33 回日本 DDS 学会学術集会 招待講演 京都

市勧業館 みやこめっせ （京都） 2017 年 7 月 7 日.
157. Ohki T, Umezu T, Ohyahsiki JH, Fukuoka Y: A statistical integrative analysis method forsmall size expression data of microRNAs and genes. 39th International Conference of theIEEE Engineering in Medicine and Biology Society, July 11-15, 2017, Korea.
158. 横須賀忠：エンドサイトーシスの障害による TCR シグナルソームの持続的形成と T 細胞制
御機構の破綻．JST さきがけ「慢性炎症」継続領域会議 （修善寺） 2017 年 7 月 23 - 24 日
159. Katagiri S, Tauchi T, Tanaka Y, Ando K, Okabe S, Gotoh M, Ito Y, Ohyashiki K: Long term
follow-up after tyrosine kinase inhibitors cessation in chronic myeloid leukemia with deep-32-

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molecular response. 日本臨床腫瘍学会 （神戸） 2017 年 7 月 27 - 29 日
160. Umezu T, Imanishi S, Yoshizawa S, Ohyahsiki K, Ohyashiki JH: Cross-talk mediated by bonemarrow stromal cells-derived extracellular vesicles. Tokyo Medical University, Institute ofMedical Science, 1st International Symposium “Role of Aging and Cancer”, August 28,
2017, Tokyo.
161. Saito S, Ohno S, Harada Y, Oikawa K, Kanno Y, Kuroda M: AAV-mediated miRNA-29b

delivary suppuress renal fibrosis. 第 9 回日本 RNAi 研究会 (広島) 2017 年 9 月 1 日
162. ** Sanbu Y, Ohno S, Kuroda M: Up-regulation of BLU tumor suppuressor by miR-34a 第 9

回日本 RNAi 研究会 (広島) 2017 年 9 月 1 日
163. 横須賀忠（招待講演）：イメージングが拓く免疫チェックポイント分子による T 細胞活性化の時空間的制御機構 第 2 回肺癌バイオカンファレンス 特別講演 （東京） 2017 年 9 月
16 日
164. 横須賀忠（招待講演）：イメージンブが拓く免疫チェックポイント分子による T 細胞活性化の時空間的制御機構．上総イムノオンコロジーセミナー 特別講演 （君津） 2017 年 9 月
21 日
165. Umezu T, Imanishi S, Azuma K, Yoshizawa S, Ohyashiki K, Ohyashiki JH: Inhibition of
extracellular vesicle secretion induces apoptosis of bone marrow stromal cells derived
from multiple myeloma patients. The 76th Annual Meeting of JCA, September 28-30, 2017,
Yokohama.
166. 横須賀忠（招待講演）：イメージンブが拓く T 細胞活性化の時空間的制御機構 –TCR マイ
クロクラスターと免疫チェックポイント分子による T 細胞制御メカニズムの解明− 順天堂
大学アトピー疾患研究センター学術セミナー （東京） 2017 年 10 月 12 日
167. Ikeda N, Ohira T, Kajiwara N: Management of early stage lung cancer. IASLC 18th World
Conference on Lung Cancer, October 17, 2017, Yokohama.
168. Shimada Y, Yenerall P, Huffman K, Avila K, Park H, Timmons B, Gao B, Deb D, Ikeda N,
Minna J: The transfer of exogenous UCHL-1 via mesenchymal lung cancer exosomes tomediate phenotypic alterations of recipients. IASLC 18th World Conference on Lung
Cancer, October 17, 2017, Yokohama.
169. Nakajima E, Sugita M, Furukawa K, Takahashi H, Kawaguchi Y, Ohira T, Ikeda N, Hirsch F.R,
Franklin W.A: IASLC 18th World Conference on Lung Cancer, October 17, 2017, Yokohama.
170. Shigefuku S, Kudo Y, Yunaiyama D, Matsubayashi J, Park J, Nagao T, Shimada Y, Hagiwara
M, Saji H*, Okano T, Kakihana M, Kajiwara N, Ohira T, Ikeda N: Prognostic factors for
surgically resected non-small cell lung cancer with cavity formation. IASLC 18th World
Conference on Lung Cancer, October 17, 2017, Yokohama.
171. OhiraT, Matsubayashi J, Maehara S, Maeda J, Yoshida K, Hagiwara M, Kakihana M, Okano
T, Kajiwara N, Nagao T, Ikeda N: Liquid based cytology (LBC) specimens were useful for
EGFR mutation test. IASLC 18th World Conference on Lung Cancer, October 18, 2017,
Yokohama.
172. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Targeting peroxisome proliferator-activatedreceptors: a novel strategy for BCR-ABL positive cells. 79th Annual Meeting of the JapaneseSociety of Hematology. Oct 22-22, 2017, Tokyo.
173. Saito Y, Azuma K, Imanishi S, Katagiri S, Suguro T, Yoshizawa S, Akahane D, Fujimoto H,-33-

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Gotoh M, Itoh Y, Ohyashiki JH, Ohyashiki K: Significance of T cell receptor rearrangementof bone marrow in extranodal NK/T cell lymphoma, nasal type. 79 th Annual Meeting of theJapanese Society of Hematology. Oct 22-22, 2017, Tokyo.
174. Nannya Y, Takeda J, Sato S, Shinozawa Y, Shiraishi Y, Kataoka K, Chiba K, Tanaka H, Sanada
M, Chiba S, Asou N, Kiyoi H, Nakao S, Ohyashiki K, et al: Efficacy of azacitidine for
TP53-mutated myelodysplastic syndromes (MDS). 79th Annual Meeting of the JapaneseSociety of Hematology. Oct 22-22, 2017, Tokyo.
175. Tanaka Y, Okabe S, Tauchi T, Ohyashiki K: A novel dual inhibitor (PI3K/HDAC), CUDC-907
and combination with carfilzomib inhibits myeloma cells. 79 th Annual Meeting of theJapanese Society of Hematology. Oct 22-22, 2017, Tokyo.
176. Kubota S, Tokunaga K, Oshima M, Umezu T, Kanai A, Ton Tan K, Yang H, Iwanaga E, Asou
N, Maeda T, Iwama A, Ohyashiki K, Osato M, Sashida G: RUNX2 super enhancer promotes
the development of blastic plasmacytoid dendritic cell neoplasia. 79th Annual Meeting of
the Japanese Society of Hematology. Oct 22-22, 2017, Tokyo.
177. Katagiri S, Tauchi T, Ohnishi A, Saito Y, Suguro T, Asano M, Yoshizawa S, Sakuta J, Akahane
D, Tanaka Y, Furuya N, Ando K, Fujimoto H, Okabe S, Gotoh M, Ito Y, Ohyashiki K:
Retrospective analysis of CML patients receiving second TKIs as initial therapy. 79 th Annual
Meeting of the Japanese Society of Hematology. Oct 22-22, 2017, Tokyo.
178. Yoshizawa S, Umezu T, Saitoh Y, Gotoh M, Akahane D, Kobayashi C, Ohyashiki JH,
Ohyashiki K: Altered exosomal miRNA expression of late onset acute graft-versus-hostdisease in allogenic hematopietic stem cell transplantation. The 79 th Annual Meeting of
JSH, October 20-22, 2017, Tokyo.
179. 横須賀忠（招待講演）： イメージングが拓く T 細胞活性化の時空間的制御機構 −シグナ
ルソームの視点から免疫チェックポイントを解釈する− 国立がん研究センターがん免疫セミナー （東京） 2017 年 10 月 23 日
180. 小澤ひとみ、梅津 知宏、小林 千晶、東 剣虹、今西 哲、大屋敷純子：多発性骨髄腫
細胞の浸潤様式の解明を目的とした in vitro および in vivo モデル系の構築．第１７９回東京医科大学医学会総会 （東京） 2017 年 11 月 4 日
181. 内山のぞみ、梅津 知宏、小林 千晶、東 剣虹、今西 哲、大屋敷純子：多発性骨髄腫
細胞の低酸素応答性に関与する lncRNA の解析．第１７９回東京医科大学医学会総会（東京） 2017 年 11 月 4 日
182. 鈴木 未来、梅津 知宏、吉澤成一郎、赤羽 大悟、田中 裕子、小林 千晶、東 剣虹、
今西 哲、大屋敷一馬、大屋敷純子： 多発性骨髄腫細胞の増殖に影響を与える骨髄間質由来因子の同定．第１７９回東京医科大学医学会総会 （東京） 2017 年 11 月 4 日
183. 横須賀忠（招待講演）： 分子イメージングが明らかにする T 細胞の活性化制御機構− がんの免疫チェックポイント療法はなぜ効くのか？ − 第 71 回東邦医学会総会 特別講演（東京） 2017 年 11 月 9 日
184. 横須賀忠（招待講演）： イメージングが拓く T 細胞活性化の時空間的制御機構 –T 細胞
シグナルソームの視点から免疫チェックポイント分子を解釈する− Meet The Specialist
2017 特別講演 （東京） 2017 年 11 月 30 日
185. Nannya Y, Takeda J, Sato S, Shiozawa Y, Shiraishi Y, Kataoka K, Chiba K, Tanaka H, Chiba S,
Asou N, Kiyoi H, Imai K, Hirase C, Dobashi N, Kiguchi T, Nakao S, Ohyashiki K, Miyazaki Y,
Naoe T, Makishima H, Miyano S, Yoshida K, Ogawa S: Molecular signatures that predict-34-

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response to azacitidine treatment for myelodysplastic syndromes. ,59 th ASH annual
meeting and exposition. Dec 9-12, 2017, Atlanta, USA.
186. Tanaka Y, Okabe S, Tauchi T, Ito Y, Ohyashiki K: The therapeutic target of phosphoinositide
3-kinases and histone deacetylases in multiple myeloma: the novel strategies forproteasome resistant myeloma cells. 59th ASH annual meeting and exposition. Dec 9-12,
2017, Atlanta, USA.
187. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Aurora A kinase as a promising therapeutictarget in Philadelphia chromosome positive leukemia cells. 59th ASH annual meeting andexposition. Dec 9-12, 2017, Atlanta, USA.
188. Okabe S, Tauchi T, Tanaka Y, Ohyashiki K: Peroxisome proliferator-activated receptors:
Targets for the treatment of Philadelphia chromosome-positive leukemia cells. 59th ASH
annual meeting and exposition. Dec 9-12, 2017, Atlanta, USA.
189. Tauchi T, Okabe S, Katagiri S, Tanaka Y, Ohyashiki K: Combining effects of the SMO
inhibitor and BCL-2 inhibitor in MDS-derived induced potent stem cells (iPS). 59th ASH
annual meeting and exposition. Dec 9-12, 2017, Atlanta, USA.
190. Yokosuka T, Machiyama H, Hata K, Yanase N, Hashimoto-Tane A, Siato T: Microclusters
as a functional unit for endocytosis of TCRs 第 46 回日本免疫学会総会学術集会 （仙台）2017 年 12 月 12-14 日
191. Sashida G, Kubota S, Tokunaga K, Oshima M, Umezu T, Kanai A, Tan KT, Yang H, Iwanaga E,
Asou N, Maeda T, Iwama A, Ohyashiki K, Osato M: RUNX2 super enhancer promotes thedevelopment of blastic plasmacytoid dendritic cell neoplasm. 21th RUNX meeting. Dec
12-15. Philadelphia, USA.
192. 町山裕亮、横須賀忠： T 細胞シグナルの一分子・超解像イメージング法の開発 新学術
領域「ネオ･セルフ」第 2 回班会議 （淡路） 2018 年 1 月 9 - 10 日
193. 横須賀忠（招待講演）：イメージングが拓く T 細胞活性化の時空間的制御機構 –免疫チェックポイントはなぜ効くのか？− 肝免疫フォーラム 特別講演 （東京） 2018 年 2 月 10 日
194. Yokosuka T: Molecular imaging unveils spatiotemporal regulation of T cell activation byimmune checkpoint receptors. The 1st International Cancer Research Symposium ofTraining Plan for Oncology Professionals 教育講演 （大阪） 2018 年 3 月 10 - 12 日
195. 横須賀忠（招待講演）：シグナルソームによる T 細胞活性化の時空間的制御機構—分子イメージングが明らかにする免疫チェックポイントの分子メカニズム— 第３３回肝疾患研究部勉強会国立国際医療研究センター 特別講演 （国府台） 2018 年 4 月 11 日
196. Umezu T, Imanishi S, Yoshizawa S, Ohyashiki K, Ohyashiki JH: The role of extracellular
vesicle-mediated miR-10a transfer in bone marrow microenvironment of patients withmultiple myeloma. 7th Annual Meeting of ISEV, May 2-6, 2018, Barcelona, Spain. 梶原
直央、前田 純一、垣花 昌俊、前原 幸夫、嶋田 善久、萩原 優、大平 達夫、杉本
昌弘、池田 徳彦．メタボローム解析による肺癌症例の早期診断と予後予測．第 35 回日
本呼吸器外科学会総会・学術集会 （千葉市） 2018 年 5 月 17 日
197. 大平 達夫、濱中和嘉子、牧野洋二郎、松浦 久美、前原 幸夫、嶋田 善久、前田 純
一、萩原 優、垣花 昌俊、岡野 哲也、梶原 直央、池田 徳彦： 肺癌症例を対象とした各種検体を用いた遺伝子変異検査．第 41 回日本呼吸器内視鏡学会学術集会 （東京） 2018 年 5 月 24 日
198. 横須賀忠（招待講演）： イメージングが拓く免疫チェックポイント分子による細胞活性化の-35-

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時空間的制御機構 第２２回日本がん分子標的治療学会学術集会 ランチョンセミナー（東京） 2018 年 5 月 17 日
199. 横須賀忠： チェックポイント分子から学ぶこと 第３５回日本呼吸器外科学会総会 特別
企画３ （幕張） 2018 年 5 月 18 日
200. 横須賀忠（招待講演）： 免疫チェックポイント療法はなぜ効くのか？ 国立がんセンター
東病院免疫セミナー （柏） 2018 年 6 月 12 日
201. 秦喜久美： 胸腺ダブルポジティブ T 細胞における c-Cbl のシグナルソーム形成と胸腺選択との相関 第２８回 Kyoto T Cell Conference （京都） 2018 年 6 月 15 日
202. 大野 慎一郎、老川 桂生、原田裕一郎、黒田 雅彦: miR-34 によるがん抑制遺伝子
BLU の発現誘導 (Up-regulation of BLU tumor suppressor gene by miR-34a) ポスター発表（一般） 第 107 回日本病理学会総会 ロイトン札幌、ニトリ文化ホール、さっぽろ芸文館（北海道） 2018 年 6 月 22 日
203. 梅原龍之介、倉田厚、藤田 浩司、高梨 正勝、黒田 雅彦: ヒト卵巣およびマウス胚性幹細胞由来の奇形腫内神経管を同定するマーカーとしての fascin の有用性（Fascin: amarker for neural tubes in teratomas of human ovary & those derived from murine ES
cells）ポスター発表（学部学生）第 107 回日本病理学会総会 ロイトン札幌、ニトリ文化ホール、さっぽろ芸文館 （北海道） 2018 年 6 月 23 日
204. ＊ ＊ 老 川 桂 生 、 大 野 慎 一 郎 、 黒 田 雅 彦 : DICER1 症 候 群 モ デ ル 細 胞 の 樹 立（Establishment of DICER1 syndrome model cells） ポスター発表（一般） 第 107 回日本病理学会総会 ロイトン札幌、ニトリ文化ホール、さっぽろ芸文館（北海道） 2018 年 6 月 23日
205. 黒 田 雅彦 (招待講演 ): エクソソーム による細胞治療、再生医療の応用（ Clinicalapplication of exosome therapy）第 107 回日本病理学会総会 ロイトン札幌、ニトリ文化ホール、さっぽろ芸文館 （北海道） 2018 年 6 月 22 日
206. Tadashi Yokosuka: Advantages of single molceuls imaging for neo-self. 新学術領域ネオ･
セルフ第１回国際シンポジウム（淡路） 2018 年 7 月 11 日
207. 横須賀忠（招待講演）： 分子イメージングによる免疫チェックポイント療法を介した T 細胞の疲弊解除とその分子メカニズムの解明 第２２回日本がん免疫学会総会 バイオセラ
ピィ学会合同シンポジウム （岡山） 2018 年 8 月 2 日
208. 梅津 知宏、大屋敷 純子、大屋敷 一馬: 新たなソースより得られた骨髄間質細胞 KNTcells の性状解析 ポスター発表（一般） 第 36 回日本ヒト細胞学会学術集会 日本歯科大学 （東京） 2018 年 8 月 4 日
209. 黒田 雅彦: Exosome opens new generation of cell therapy. 第 10 回日本 RNAi 研究会

第 5 回日本細胞外小胞学会（招待講演）グランドプリンスホテル広島 （広島）2018 年 8 月
29 - 31 日
210. ＊＊大野慎一郎、三部 芳裕、老川 桂生、原田裕一郎、黒田 雅彦: miR-34a によるがん抑制遺伝子 BLU の発現制御機構 (Up-regulation og BLU tumor suppressor by
miR-34a). 第 10 回日本 RNAi 研究会 第 5 回日本細胞外小胞学会 グランドプリンスホテル広島 （広島） 2018 年 8 月 29 - 31 日
211. ＊ ＊ 老 川 桂 生 、 大 野 慎 一 郎 、 黒 田 雅 彦 : DICER1 症 候 群 モ デ ル 細 胞 の 樹 立(Establishment of DICER1 syndrome model cells). 第 10 回日本 RNAi 研究会 第 5 回日
-36-

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本細胞外小胞学会 グランドプリンスホテル広島 （広島） 2018 年 8 月 29 - 31 日
212. 黒田 雅彦: AI を用いた病理診断の実際. 第 26 回日本外科学会生涯教育セミナー（教育講演） ヤクルトホール（東京） 2018 年 9 月 15 日
213. Kajiwara N, Maehara S, Shimada Y, Maeda J, Hagiwara M, Okano T, Kakihana M, Ohira T,

Sugimoto M, Ikeda N: Metabolomic analysis in lung cancer for screening and early
detection. IASLC 19th World Conference on Lung Cancer. Sep. 25, 2018, Toronto, Canada.
214. Eriguchi D, Okano T, Kawaguchi Y, Maeda J, Hagiwara M, Kakihana M, Kajiwara N, Ohira T,
Ikeda N: The importance to switch from EGFR-TKI to cytotoxic chemotherapy for EGFR
mutation-positive adenocarcinoma. IASLC 19th World Conference on Lung Cancer. Sep. 26,
2018, Toronto, Canada.
215. Ohira T, Hamanaka W, Shimada Y, Maehara S, Maeda J, Hagiwara M, Kakihana M, Okano

T, Kajiwara N, Ikeda N: The cytology samples and plasma specimens were feasible for the
EGFR molecular testing. IASLC 19th World Conference on Lung Cancer. Sep. 26, 2018,
Toronto, Canada.
216. Shimada Y, Okano T, Ohira T, Ikeda N: UCHL1 has prognostic relevance and is a therapeutictarget in high-grade neuroendocrine lung cancers. 43rd ESMO (European Society for
Medical Oncology) Congress. Oct, 20, 2018, Munich, Germany.
217. 横須賀忠（招待講演）： がん免疫と分子イメージング先端的研究〜免疫チェックポイント
分子とキメラ抗原受容体 CAR の分子メカニズム〜 第７７回日本癌学会学術総会 ランチョンセミナー （大阪） 2018 年 9 月 29 日
218. 横須賀忠（招待講演）：分子イメージングが拓く T 細胞活性化機構の解明とがん免疫応答
三重大学大学院医学系研究科セミナー （津） 2018 年 10 月 9 日
219. 黒田 雅彦: リキッドバイオプシーによる肺がん non-coding RNA の解析（ワークショップ） ： リキッドバイオプシーの up to date 第 59 回日本肺癌学会学術集会 京王プラザ
ホテル （東京） 2018 年 11 月 29 日-12 月 1 日
220. 大平 達夫、濱中和嘉子、牧野洋二郎、松浦 久美、今井健太郎、嶋田 善久、前原 幸
夫、前田 純一、萩原 優、垣花 昌俊、岡野 哲也、梶原 直央、池田 徳彦: リキッド
バイオプシーによる遺伝子変異検査の限界．第 59 回日本肺癌学会学術集会 （東京）2018 年 11 月 29 日
221. 杉本 昌弘、梶原 直央、前田 純一、垣花 昌俊、池田 徳彦: 人工知能とメタボローム
解析を用いたリキッドバイオプシー．第 59 回日本肺癌学会学術集会 （東京） 2018 年 11月 29 日222. 梶原 直央、前田 純一、垣花 昌俊、前原 幸夫、嶋田 善久、萩原優、大平 達夫、
杉本 昌弘、池田 徳彦: Ex-Vivo と血液のメタボローム解析による新規バイオマーカーの創出．第 59 回日本肺癌学会学術集会 （東京） 2018 年 11 月 29 日
223. 嶋田 善久、岡野 哲也、前原 幸夫、前田 純一、萩原 優、垣花 昌俊、梶原 直央、
大平 達夫、黒田 雅彦、池田 徳彦: 臨床的疑問解決のための clinical sequence：扁平上皮癌診断のための網羅的解析，血清 exosome miRNA 解析．第 59 回日本肺癌学会学術集会 （東京） 2018 年 12 月 1 日
224. 松林 純、大平 達夫、垣花 昌俊、嶋田 善久、前原 幸夫、前田 純一、萩原 優、-37-

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岡野 哲也、梶原 直央、長尾 俊孝、池田 徳彦: 病理診断に影響を与える検体採取法．第 59 回日本肺癌学会学術集会 （東京） 2018 年 12 月 1 日
225. ＊＊大野慎一郎、黒田 雅彦: Up-regulation og BLU tumor suppressor by miR-34 family
第 59 回日本肺癌学会学術集会 京王プラザホテル （東京） 2018 年 11 月 29 日-12 月 1日(11/30)226. 横須賀忠： 分子イメージングが明らかにする T 細胞活性化の時空間的制御機構〜免疫チェックポイント分子と CAR〜 第５９回日本肺癌学会学術集会 シンポジウム１２ （大阪）2018 年 11 月 29 日
227. 老川 桂生、大野慎一郎、黒田 雅彦: Establishment of

DICER1 syndrome model cells
第 77 回日本癌学会学術総会 大阪国際会議場 （大阪） 2018 年 9 月 27 - 29 日

228. ＊＊大野慎一郎、老川 桂生、原田裕一郎、黒田 雅彦: Up-regulation og BLU tumor

suppressor by miR-34 family 第 77 回日本癌学会学術総会 大阪国際会議場 （大阪）2018 年 9 月 27 - 29 日
229. ＊＊高梨 正勝、上田しのぶ、須藤カツ子、黒田 雅彦: 樹状細胞由来エクソソームは内包される分子を T 細胞に伝達して免疫機構を活性化する. 第 77 回日本癌学会学術総会
大阪国際会議場 （大阪） 2018 年 9 月 27 - 29 日
230. 梅津 知宏、今西 哲、大屋敷純子、大屋敷一馬: Extracellular vesicle-mediated miRNAtransfer enhances growth and survival of multiple myeloma 第 77 回日本癌学会学術総会
大阪国際会議場 （大阪） 2018 年 9 月 27 - 29 日
231. Yokosuka Tadashi, Wakamatsu Ei, Yanase Noriko, Toyota Hiroko, Furuhata Masae, Hata
Kikumi, Machiyama Hiroaki： Dynamics of the PI3K signaling pathway induced by a T cell
costimulator, ICOS. 第 47 回日本免疫学会総会 ワークショップ （福岡） 2018 年 12 月
10 日
232. Kikumi Hata, Hiroaki Machiyama, Noriko Yanase, Masae Furuhata, Hiroko Toyota, Ei
Wakamatsu, Tadashi Yokosuka： Cooperative regulation of thymic selection by receptor
endocytosis and signal strength through TCR and E3 ubiquitin ligase c-Cbl microcluster
formation. 第 47 回日本免疫学会総会 ワークショップ （福岡） 2018 年 12 月 10 日
233. Yanase Noriko, Machiyama Hiroaki, Wakamatsu Ei, Toyota Hiroko, Furuhata Masae, Hata
Kikumi, Mamonkin Maksim, Brenner Malcolm K, Yokosuka Tadashi：Molecular imaging of
the hCD19 CAR signalosomes, “CAR microclusters”. 第 47 回日本免疫学会総会 ワークシ
ョップ （福岡） 2018 年 12 月 10 日
234. Hiroaki Machiyama, Ei Wakamatsu, Noriko Yanase, Kikumi Hata, Masae Furuhata, Hiroko
Toyota, Tadashi Yokosuka ： Single molecule imaging unveils a distinct difference in
Lck-dynamics between CD4+ and CD8+ T cells. 第 47 回日本免疫学会総会 ワークショッ
プ （福岡） 2018 年 12 月 10 日
235. Tadashi Yokosuka（招待講演）: Molecular imaging unveils a mechanism of T cell activation
regulation by immune checkpoint microclusters. The 23rd JFCR-ISCC New Antitumor Agents
under Develioment in the US, Europa and Japan.（お台場） 2018 年 12 月 13 日
236. 横須賀忠（招待講演）：分子イメージングが拓く T 細胞活性化の解明とがん免疫応答 ―チェックポイント分子のとキメラ抗原受容体 CAR のシグナルソーム形成― State of the
Are Immuno-Oncology Forum in 愛媛 （松山） 2019 年 1 月 25 日
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237. 町山裕亮： Lck キナーゼの構造・局在・ダイナミクスによる T 細胞シグナル制御 理研シンポジウム「細胞システムの動態と理論 XI」 （和光） 2019 年 3 月 28 日
238. Tomohiro Takehara, Ei Wakamatsu Hiroaki Machiyama, Noriko Yanase, Kikumi Hata,
Hiroko Toyoda, Masae Furuhata, Hiroyuki Yasuda, Kenzo Soejima, and Tadashi Yokosuka:
Programmed cell death 2 forms coinhibitory microclusters that directly attenuate T cell
receptor signaling by recruiting phosphatase SHP2. AACR （アトランタ） 2019 年 3 月 30 日4月3日
239. 横須賀忠（招待講演）： 分子イメージングが拓くがん免疫応答と T 細胞活性化メカニズムの解明 第７５回日本口腔科学会学術集会 シンポジウム 3 （松山） 2019 年 4 月 21 日
240. Kuroda M: (招待講演) RNA-based Biomarkers. ANNUAL MEETING ISEV 2019 (Educationday), みやこめっせ (京都) 2019 年 4 月 24 日
241. Takanashi M, Ueda S, Sudo K, Kuroda M: Dendritic cell derived-exosomes activate immune

systems by transferring exosome involved factors to T cell. ANNUAL MEETING ISEV 2019,
みやこめっせ (京都) 2019 年 4 月 27 - 28 日
242. Umezu T, Ohyashiki K, Kuroda M: Extravellular vesicles derived from bone marrow stromalcells promote evasion of multiple myeloma cells from NK cell anti-tumor activity. ANNUAL
MEETING ISEV 2019, みやこめっせ (京都) 2019 年 4 月 27 - 28 日
243. 町山裕亮、若松英、秦喜久美、矢那瀬紀子、古畑昌枝、豊田博子、横須賀忠： 末梢 T 細
胞では Lck と共受容体 CD4/CD8 との協調的クラスター形成よって初期の TCR シグナルが惹起される 第２９回 Kyoto T Cell Conference （京都） 2019 年 6 月 7 日
244. ＊＊老川 桂生: DICER1 症候群モデル細胞の解析（ポスターセッション）第 15 回日本臨床

プロテオゲノミクス研究会 アルカディア市ヶ谷 私学会 （東京） 2019 年 5 月 11 日
245. 倉田 厚、高梨 正勝、藤田 浩司、黒田 雅彦: マウス多能性幹細胞由来奇形腫を用いた chemotherapeutic retroconversion の 検 証 . （ Validation of chemotherapeutic
retroconversion using murine pluripotent stem cell-derived teratomas.） 第 108 回日本病理学会総会 東京国際フォーラム （東京） 2019 年 5 月 11 日
246. ＊＊大野慎一郎、老川 桂生、原田裕一郎、黒田 雅彦: miR-34 family による 3p21.3 領域に存在するがん抑制遺伝子の発現誘導. （Up-regulation of tumor suppressor genes on
3p21.3 locus by miR-34a family.）第 108 回日本病理学会総会 東京国際フォーラム （東京） 2019 年 5 月 9 日
247. 藤田 浩司、倉田 厚、刑部 安弘、黒田 雅彦: 浸潤性乳管癌における癌関連繊維芽細胞の免疫組織学的検索とその意義.（IHC search of cancer-associated fibroblasts ofinvasive breast cancer and its significance.）第 108 回日本病理学会総会 東京国際フォー
ラム （東京） 2019 年 5 月 10 日
248. 渡辺 紀子、原田裕一郎、大野慎一郎、藤田 浩司、急式 政志、柿沼 幹男、巾澤 温
子、黒田 雅彦: 小児悪性固形腫瘍における CD99 発現の網羅的検索―抗 CD99CAR-T 療
法適用の観点から―（A comprehensive detection of CD99 expression in pediatric solid
malignancy for CAR-T cell therapy.） 第 108 回日本病理学会総会 東京国際フォーラム（東京） 2019 年 5 月 10 日
249. 及川 恒輔、佐藤 冬樹、黒田 雅彦、尾崎 敬、村垣 泰光: 粘液型死亡肉腫において-39-

（様式 2）
法人番号プロジェクト番号131054
S1511011

TLS-CHOP は抗腫瘍性サイトカイン IL-24 を多重機構で抑制する.（TLS-CHOP suppresses ananti-tumor cytokine IL-214 by multiple mechanisms in myxoid liposarcoma cells.）第 108 回
日本病理学会総会 東京国際フォーラム （東京） 2019 年 5 月 10 日
250. 大平 達夫、松林純、濱中和嘉子、前田 純一、吉田 浩一、垣花 昌俊、梶原 直
央、筒井 英光、長尾 俊孝、河手 典彦、池田 徳彦: 肺癌に対する Liquid-based
cytology 検体を用いた遺伝子検査．第 60 回日本臨床細胞学会総会（春期大会） （東京）2019 年 6 月 9 日
251. Shigefuku S, Kudo Y, Yunaiyama D, Matsubayashi J, Park J, Nagao T, Shimada Y, Kajiwara N,

Ohira T, Ikeda N: Prognostic factors for surgically resected non-small cell lung cancer. The
65th Annual Congress of International College of surgeons Japan Section. June 22, 2019,
Tokyo.
252. 工藤 勇人、岡野 哲也、垣花 昌俊、濱中和嘉子、田中 裕紀、山田 祐揮、矢崎 裕
紀、松林 純、萩原 優、本多 英俊、梶原 直央、大平 達夫、長尾 俊孝、池田 徳
彦: 当院における EGFR 遺伝子変異検査に対する Liquid Biopsy と Tissue Biopsy の現状．
第 42 回日本呼吸器内視鏡学会学術集会 東京 2019 年 7 月 4 日
253. 黒田 雅彦: 核酸医薬品の臨床応用.（招待講演）第 3 回 バイオ医薬 EXPO、東京ビッグサイト （東京）、2019 年 7 月 4 日
254. 黒田 雅彦: エクソソームとライフサイエンス（招待講演）、御茶ノ水ソラシティカンファレン
スセンター2F（sola city Hall）（東京） 2019 年 7 月 8 日
255. 横須賀忠（招待講演）： 分子イメージングが拓く T 細胞活性化機構の解明とがん免疫療法への応用 〜チェックポイント分子とキメラ抗原受容体 CAR のシグナルソーム形成〜
Urology Conference at Shinanomachi （信濃町） 2019 年 7 月 2 日
256. 黒田 雅彦: The practice of pathological fiagnosis using AI .（招待講演）第 38 回札幌国際がんシンポジウム、ロイトン札幌ホテル（北海道） 2019 年 7 月 12 - 13 日
257. 横須賀忠、若松英、矢那瀬紀子、秦喜久美、竹原朋宏、西航、町山裕亮： CAR マイクロク
ラスターによる腫瘍抗原の認識と CAR-T 細胞活性化の時空間的制御機構 第 23 回日本
がん免疫学会総会 国際シンポジウム （高知） 2019 年 8 月 21 日
258. Ohira T, Kudo Y, Makino Y, Shimada Y, Maehara S, Hagiwara M, Kakihana M, Okano T,

Kajiwara N, Ikeda N: Comparison of molecular testing using various specimens fornon-small cell lung cancers. 2019 World Conference on Lung Cancer. Sep 8, 2019,
Barcelona, Spain.
259. Osawa J, Shimada Y, Maehara S, Hagiwara M, Kakihana M, Kajiwara N, Ohira T, Ikeda N:

Relationship between EGFR mutation and pathological differentiation in patients withclinical stage IA lung adenocarcinoma. 2019 World Conference on Lung Cancer. Sep 9,
2019, Barcelona, Spain.
260. 横須賀忠、若松英、矢那瀬紀子、秦喜久美、竹原朋宏、西航、町山裕亮： 多様な T 細胞
シグナルソームによる腫瘍およびネオ･セルフ抗原認識の分子機構 第９２回日本生化学会大会 シンポジウム１６ （横浜） 2019 年 9 月 18 日
261. 横須賀忠： CAR マイクロクラスターによる腫瘍抗原の認識と CAR-T 細胞活性化の時空間的制御機構．JST さきがけ「慢性炎症」継続領域会議（金沢） 2019 年 9 月 23 日
-40-

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法人番号プロジェクト番号131054
S1511011

262. 大野慎一郎、老川桂生、原田裕一郎、黒田雅彦: がん抑制遺伝子 BLU のプロモーターから発現する lncRNA は miR-34-AGO 複合体を介して BLU の発現を誘導する．第 78 回日本癌学会学術総会 国立京都国際会館 2019 年 9 月 27 日
263. 梅津 知宏、大屋敷一馬、黒田雅彦: がん間質細胞由来エクソソームは NK 細胞の抗腫瘍効果を抑制する．第 78 回日本癌学会学術総会 京都（国立京都国際会館） 2019 年 9月 27 日264. 横須賀忠（招待講演）： 分子イメージングが拓く T 細胞活性化機構の解明とがん免疫療法 第８１回日本血液学会学術集会 モーニングセミナー （東京） 2019 年 10 月 12 日
265. 梅津 知宏、高梨正勝、大野慎一郎、黒田雅彦: アセロラ果汁由来小胞の機能解析とその有用性に関する研究．第 6 回日本細胞外小胞学会学術集会 国立がん研究センター
研究所 （東京） 2019 年 10 月 24 - 25 日
266. 横須賀忠（招待講演）：分子イメージングが拓く T 細胞活性化機構の解明とがん免疫療法〜免疫チェックポイント分子とキメラ抗原受容体のシグナルソーム〜 Scientific Exchange
Meeting in 北九州 2019 （小倉） 2019 年 11 月 26 日
267. 梅津 知宏、高梨 正勝、斎藤 彰、工藤 勇人、嶋田 善久、大平 達夫、池田 徳彦、
黒田 雅彦: 肺がん患者由来エクソソーム miRNA を用いた診断バイオマーカーの探索
第 60 回日本肺癌学会学術集会 大阪（大阪国際会議場） 2019 年 12 月 6 - 8 日
268. Kuroda M: Exosome as new drug delivery tools. (招待講演) APSEV 2019 Inaugural Annual
Meeting ＆ KSEV 2019 Annual Meeting, Jeju, Republic of KOREA （ ICC/International
Convention Center）、Nov 25th 2019269. Yanase N, Machiyama H, Toyota H, Furuhata M, Hata K, Takehara T, Wakamatsu E,

Yokosuka T：Extrinsic and intrinsic inhibition of T cell response by co-inhibitory receptors,
TIGIT and CD96. 第 48 回日本免疫学会総会 ワークショップ （浜松） 2019 年 12 月 12日
270. Takehara T, Wakamatsu E, Machiyama H, Yanase N, Toyota H, Furuhata M, Fukunaga K,

Soejima K, Yokosuka T： Programmed cell death 2 forms coinhibitory microclusters that
directly attenuate T cell receptor signaling by recruiting the phosphatase SHP2. 第 48 回
日本日本免疫学会総会 ワークショップ （浜松） 2019 年 12 月 12 日
271. Wakamatsu E, Machiyama H, Toyota H, Furuhata M, Hata K, Yanase N, Yokosuka T ：Indirect suppression of CD4+ T cell activation by LAG3-mediated trogocytosis of MHC Class
II. 第 48 回日本日本免疫学会総会 ワークショップ （浜松） 2019 年 12 月 12 日
272. Machiyama H, Wakamatsu E, Hata K, Yanase N, Furuhata M, Toyota H, Yokosuka T：

Different requirement of the coreceptors CD4 and CD8 for initiation of T cell activation.
第 48 回日本日本免疫学会総会 ワークショップ （浜松） 2019 年 12 月 12 日

-41-

（様式 2）
法人番号プロジェクト番号131054
S1511011＜研究成果の公開状況＞（上記以外）
シンポジウム・学会等の実施状況、インターネットでの公開状況等＜既に実施しているもの＞
1. 公開シンポジウム：平成 27 年度～平成 31 年度 文部科学省私立大学戦略的研究基盤
形成支援事業『RNA メタボロームによる細胞内小胞体の解明に基づいた革新的がん治療法の開発』中間報告会（平成 30 年 1 月 31 日）【研究機器整備進捗状況】(1) 免疫チェックポイント分子と細胞内微小構造を可視化する超解像顕微鏡システムの確立
免疫学分野 横須賀 忠【研究基盤形成進捗状況】(1)miR-34a によるがん抑制遺伝子 BLU の発現誘導機構の解析 分子病理学分野
大野慎一郎(2)慢性骨髄性白血病における治療中止と extracellular microRNA 血液内科学分野 大屋
敷 一馬(3)Cell free DNA を用いた肺癌に対する Precision Medicine の有用性 呼吸器・甲状腺外科学
分野 大平 達夫
(4)がん微小環境における間質由来分泌小胞の役割 医学総合研究所 梅津 知宏＜これから実施する予定のもの＞
1. 公開シンポジウム：平成 27 年度～令和元年度 文部科学省私立大学戦略的研究基盤形
成支援事業『RNA メタボロームによる細胞内小胞体の解明に基づいた革新的がん治療法の開発』最終報告会（令和 2 年 3 月 11 日予定の報告会は COVID-19 の影響により中止、感染の拡大状況をみながら開催予定）
１４ その他の研究成果等
1. 核酸医薬に関して：ボナック社との共同開発
2. EV の単離に関しての：ニチレイとの共同研究開発
3. 大屋敷純子、梅津知宏：メディア掲載 科学新聞（2017 年 7 月 28 日）「多発性骨髄腫のガン血管新生効果証明」.
4. 横須賀忠：講演 「イメージングが拓く免疫チェックポイント分子によるリンパ球調節機構の解明」 姫路市医師会セミナー 特別講演（2017 年 7 月 29 日、姫路）.
5. 横須賀忠：アウトリーチ活動 「免疫細胞は自分と他人をどう見分けているの？」日本免疫学主催・文部科学省後援 免疫ふしぎ未来 2017 （2017 年 8 月 6 日、東京）.
6. 横須賀忠（実行委員長）：アウトリーチ活動 日本免疫学主催・文部科学省後援 免疫ふしぎ未来 2019 （2019 年 8 月 4 日、東京）.

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（様式 2）
法人番号プロジェクト番号
１５ 「選定時」及び「中間評価時」に付された留意事項及び対応＜「選定時」に付された留意事項＞
特記事項なし＜「選定時」に付された留意事項への対応＞
特記事項なし＜「中間評価時」に付された留意事項＞
特記事項なし＜「中間評価時」に付された留意事項への対応＞
特記事項なし-43-

131054
S1511011

（様式2）
法人番号プロジェクト番号131054
S1511011

１６

（千円）
内
年度・区分 支出額

平 施 設
成
2 装 置
7 設 備
年
度 研究費
平 施 設
成
2 装 置
8 設 備
年
度 研究費
平 施 設
成
2 装 置
9 設 備
年
度 研究費

訳

共同研
受託
究機関
寄付金 その他（
研究等
負担

法 人
負 担

私 学
助 成

18,5706,190

12,38010,036

5,126

4,910

6,210

2,070

4,140

18,34910,357

7,992

13,921

10,91924,832

0

）

0
0

0
0

0
0
0
24,840

平 施 設
成
3 装 置
0 設 備
年
度 研究費

24,832

施 設

0

装 置

0

設 備

0

研究費

24,83824,838

0

施 設

0

0

0

0

0

0

0

総

装 置

0

0

0

0

0

0

0

額

設 備

24,7808,260

16,520

0

0

0

0

研究費

102,895

79,07423,8210000127,675

87,33440,341

0

0

0

0

令
和
元
年
度

総

計

0
0
0

-44-

備 考

（様式2）
法人番号131054

１７《施設》 （私学助成を受けていないものも含め、使用している施設をすべて記載してください。）

施 設 の 名 称

整備年度

研究施設面積小胞体創薬研究センター

H28

73㎡
45㎡
88㎡

免疫学分野研究室
医学総合研究所（分子腫瘍研究部門）

研 究 室 等 数 使用者数

1
1
1

事業経費

補助金額

（千円）
補助主体

8
7
7

※ 私学助成による補助事業として行った新増築により、整備前と比較して増加した面積
0㎡《装置・設備》 （私学助成を受けていないものは、主なもののみを記載してください。）
装置・設備の名称

型

整備年度

番

台

数

稼働時間数

事業経費

（千円）
補助金額

補助主体

（研究装置）ｈｈｈｈｈ（研究設備）
デジタルPCRシステム
全反射蛍光顕微鏡

H28
H27

QS3D-PF
Ti-E-TIRF-TMU

1
1

1580
3224

ｈ
ｈ
ｈ
ｈ
ｈ

6,210
18,5704,140 私学助成
12,380 私学助成

（情報処理関係設備）
ｈｈｈ

１８ 研究費の支出状況
年
度
平成
小 科 目
消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 雑費 ）
計
人件費支出（兼務職員）
教育研究経費支出
計
教育研究用機器備品

図

書
計

（千円）
27

支 出 額
教
3,606

年度
主 な 使 途
育
研
究

40 旅費
2,507 委託料
1,485 課税雑費
7,638
ア
ル
バ
398

イ

金

ト

40 国内学会旅費
2,507 実験補助委託料
1,485 ソフト
7,638
関
係
支
出
398 時給 1,460円，年間時間数 272時間

実人数 1人398
398
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）
1,959
1,959 ノートパソコン、超低温槽
41
41 図書
2,000
2,000
研
究
ス
タ
ッ
フ
関
係
支
出

リサーチ・アシスタント
ポスト・ドクター
研究支援推進経費

計

積 算 内 訳
主 な 内 容額
経費
支
出3,606 試薬、実験器具、文具、消耗図書

0

-45-

（様式2）
法人番号
年

度

小 科 目

28

平成
支 出 額

年度
主 な 使 途育研
究教
16,603

消 耗 品 費光 熱 水 費
通信運搬費
印刷製本費
旅費交通費
報酬・委託料（ 諸会費 ）
（ 雑務費 ）
（ 修理費 ）
計金

積 算 内 訳額主 な 内 容経費
支出16,603 試薬、実験器具、文具、消耗図書

6 旅費

6 国内学会旅費

30 課税諸会費
166 雑務費
473 修理費
17,278
ア
ル
バ
1,071

人件費支出
（兼務職員）
教育研究経費支出
計131054イ

30 学会参加費
166 解析代
473 機器備品修理費
17,278関係
支
出
1,071 時給 1,460円，年間時間数 732時間

ト

実人数 2人1,071
1,071
設 備 関 係 支 出（１個又は１組の価格が５００万円未満のもの）

教育研究用機器備品

図

書
0

計
研

究

ス

タ

ッ

フ

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プロジェクトの成果-48-

Gene Therapy (2015) 22, 127–137© 2015 Macmillan Publishers Limited All rights reserved 0969-7128/15
www.nature.com/gt

ORIGINAL ARTICLE

Inhibition of choroidal ﬁbrovascular membrane formation
by new class of RNA interference therapeutic agent targeting
periostin
T Nakama1, S Yoshida1, K Ishikawa1, Y Kobayashi1, Y Zhou1, S Nakao1, Y Sassa1,2, Y Oshima1, K Takao3, A Shimahara3, K Yoshikawa3,
T Hamasaki4, T Ohgi4, H Hayashi4, A Matsuda5, A Kudo6, M Nozaki7, Y Ogura7, M Kuroda8 and T Ishibashi1
Age-related macular degeneration (AMD) is a vision-threatening disease characterized by choroidal ﬁbrovascular membrane (FVM)
formation, choroidal neovascularization (CNV) and choroidal ﬁbrosis. No safe and effective therapeutic method has been developed
for the choroidal ﬁbrosis, although anti-vascular endothelial growth factor therapy can partially shrink the CNV. We recently
reported that periostin (POSTN), which is produced by retinal pigment epithelial cells, has an important role in the formation of
preretinal FVMs, but its role in choroidal FVMs has not been determined. In this study, we used Postn knockout mice to investigate
the role played by POSTN in choroidal FVM formation. In addition, we used a new class of RNA interference (RNAi) agent (NK0144)
that targets POSTN and determined its effect on choroidal FVM development. Genetic ablation of Postn had an inhibitory effect not
only on CNV formation but also on choroidal ﬁbrosis in a mouse CNV model. NK0144 also had a greater inhibitory effect on both
the CNV and choroidal ﬁbrosis than control RNAi with no apparent adverse effects. These ﬁndings suggest a causal relationshipbetween POSTN and choroidal FVM formation, and also a potential therapeutic role of intravitreal NK0144 for AMD.
Gene Therapy (2015) 22, 127–137; doi:10.1038/gt.2014.112; published online 11 December 2014

INTRODUCTION
Age-related macular degeneration (AMD) is a leading cause of asevere reduction of vision in the elderly population of developed
countries.1–3 It is estimated that the prevalence of AMD will
increase, which would then increase both the social and medical
burdens of the countries. At the advanced stage of AMD, choroidal
ﬁbrovascular membranes (FVMs), which consist of choroidal
neovascularization (CNV) and choroidal ﬁbrosis form, and they
can lead to severe vision reduction.4–6 In this process, there is
proliferation, adhesion and migration of various types of cells, forexample, vascular endothelial cells, retinal pigment epithelial (RPE)
cells, glial cells, ﬁbroblasts and macrophages/monocytes. There isalso deposition of extracelluar matrices.7–10 Several growth factors,
such as vascular endothelial growth factors (VEGFs), placental
growth factor, connective tissue growth factor and transforming
growth factor βs (TGF-βs) and their receptors, are involved in thisprocess.6,11–13 Among these, the VEGFs have been well characterized and are known to have a critical role in CNV formation. AntiVEGF therapies have been proven to inhibit the progression of
CNVs to some degree, but the different types of therapies havenot been able to inhibit choroidal ﬁbrosis.14 In addition, it wasrecently reported that the intravitreal injection of anti-VEGF
antibodies can cause retinal damage and promote choroidal
ﬁbrosis.8,14–16 Therefore, choroidal FVM formation, especiallychoroidal ﬁbrosis, remains a severe vision-threatening pathological condition.

We have recently demonstrated that there was an increase in
the expression of periostin (POSTN) in the vitreous and FVMs ofpatients with proliferative diabetic retinopathy and proliferative
vitreoretinopathy.17–20 POSTN, a 90-kDa secreted matricellular
protein, is a member of the fasciclin (fas) family. It is composed ofan amino-terminal EMI domain, tandem repeat of four fas I
domains and a carboxyl-terminal domain including a heparinbinding site at its C-terminal end.21 POSTN interacts directly withthe extracelluar matrices, including ﬁbronectin, tenascin c andcollagen type I, through its EMI and fas I domains. Furthermore,
POSTN also serves as a ligand for integrins, such as αvβ3 and αvβ5.
Through these interactions, POSTN is associated with thedevelopment and remodeling of tissues.22 Recent studies haveshown that POSTN promotes a regeneration of heart tissue after
myocardial infarction, cutaneous wound healing and chronic
allergic inﬂammation.23–28 These ﬁndings indicate that POSTN has
fundamental roles in tissue repair in several organs, and thus
raises the possibility that POSTN may also be involved in thedevelopment of choroidal FVMs.
RNA interference (RNAi) is a natural mechanism of posttranscriptional silencing of gene expression that has been recently
considered to be a new type of therapeutic system.29 As a result of
their high selectivity and potency, RNAi-based therapy has several
advantages over conventional therapeutic approaches such as
antibody, aptamer and antisense therapy. In addition, RNAi agentscan be easily synthesized and the steps that are necessary1
Department of Ophthalmology, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan; 2Department of Ophthalmology, Fukuoka University Chikushi Hospital,
Fukuoka, Japan; 3AQUA Therapeutics Co., Ltd., Kobe, Japan; 4Strategic Headquarters for Research and Development, BONAC Corporation, BIO Factory, Fukuoka, Japan;
5
Department of Ophthalmology, Juntendo University School of Medicine, Tokyo, Japan; 6Department of Biological Information, Tokyo Institute of Technology, Yokohama, Japan;
7
Department of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan and 8Department of Molecular Pathology, Tokyo
Medical University, Tokyo, Japan. Correspondence: Dr S Yoshida, Department of Ophthalmology, Kyushu University Graduate School of Medical Sciences, Fukuoka, 812-8582,
Japan.
E-mail: yosida@eye.med.kyushu-u.ac.jp
Received 13 May 2014; revised 23 September 2014; accepted 24 October 2014; published online 11 December 2014-49-

New RNAi therapy targeting POSTN for AMD
T Nakama et al128
to identify and optimize them are rapid. However, previous
investigations using canonical double-stranded small interfering
RNAs (siRNAs) showed several obstacles including the lack of asafe drug delivery system (DDS), adverse off-target effects throughToll-like receptor 3 (TLR3) activation, and the lack of stability.30–34
In this study, we used a new class of single-stranded RNAitherapeutic agent, which can overcome those obstacles.35 In
addition, it is expected that the cost of large-scale production of
single-stranded RNAi can be reduced because of the simple
synthesizing method.
Based on these reports including ours, we hypothesized that
POSTN is involved in choroidal FVM formation and can be used asa therapeutic target. To test this, we determined the expression
and function of POSTN in human FVM specimens and in a mouse
CNV model. We also studied the inhibitory effect of a new class of
single-stranded RNAi agent targeting POSTN on choroidal FVM
formation.
RESULTS
Expression of POSTN in RPE cells of choroidal FVMs in CNV mousemodel and AMD patients
To determine whether POSTN is involved in the development ofchoroidal FVMs, real-time reverse transcription-PCR (RT-PCR) was
performed and the expression of the mRNA of Postn in the RPEchoroid complexes after laser injury were compared with thatin normal RPE-choroid complexes. The expression of Postn mRNAin a mouse CNV model group was signiﬁcantly upregulatedcompared with the control group and reached a peak on day 14(P o 0.05, n = 4; Figure 1a).
To conﬁrm the result of real-time RT-PCR, we performed
enzyme-linked immunosorbent assay (ELISA) to determine theconcentration of the protein of POSTN. ELISA showed that thelevel of POSTN protein in the RPE-choroid complexes of CNV
model mice was signiﬁcantly higher from day 3 (75.41 ± 10.95 pg
POSTN per mg total protein, Po 0.05; n = 4) than that in thecontrol group (31.97 ± 0.65 pg POSTN per mg total protein; n = 4).
The concentration reached a peak on day 14 (125.18 ± 16.76 pg
POSTN per mg total protein, P o0.001; n = 4; Figure 1b).
Next, sections were stained with antibodies to determine thelocalization of the POSTN in the choroidal FVMs. Immunohistochemical analyses showed POSTN-positive staining in RPE65positive RPE cells after the laser injury (Figure 2a). In the humanchoroidal FVMs, POSTN stained the cytokeratin-positive RPE cells(Figure 2b). These ﬁndings indicated that the expression of the
POSTN was increased in the RPE cells in both mouse CNV modeland in the FVMs of AMD patients.

Choroidal FVM formation is reduced in Postn KO mouse
To investigate whether POSTN enhances the development ofchoroidal FVMs, we quantiﬁed the volume of the CNVs at day 7
and ﬁbrous volumes at day 21 in both wild-type (WT) and Postn
knockout (KO) mice. The average CNV volumes stained by isolectin
B4 was 63 400 ± 2891 μm3 in the Postn KO mice group (n = 6),
which was signiﬁcantly smaller by 34% than that of WT micegroup at 96 222 ± 311 μm3 (P o 0.01, n = 6; Figures 3a and b). There
was a 59% reduction in average ﬁbrosis volume (stained bycollagen type I antibody) in Postn KO mice group at 21
166 ± 2886 μm3 (n = 6) than in the WT mice at 51 332 ± 5665 μm3(P o 0.05, n = 6; Figures 3a and b).
NK0144-mediated inhibition of POSTN mRNA in cultured human
RPE cells
Four types of RNAi agents including controls were synthesized as
described in the Materials and methods section. A summary ofRNAi agents were shown in Table 1, and the structure of RNAi
agents were shown in Figure 4 (Figures 4a and b). The synthesizedtargeted sequence of RNAi agents for POSTN is present in not only
human POSTN but also in mouse, rat, rabbit and rhesus macaque
POSTN. This indicates that the direct animal testing of NK0144
would also be suitable for human clinical trials.
To determine if transfection with NK0144 inhibited theexpression of the mRNA of POSTN, cell culture studies of human
RPE cells, which are the cellular source of POSTN and important
cells for ocular ﬁbrosis, were carried out. Real-time RT-PCR showedthat the levels of POSTN mRNA were signiﬁcantly decreased
following exposure of ARPE19 cells to 0.01, 0.1 and 1 nM of NK0144
in a dose-dependent manner, and almost completely blocked theexpression of the mRNA of POSTN following exposure to 1 nM
NK0144 (Po 0.0001, n = 4; Figure 4c). In contrast, the expression
was not inhibited after exposure to 0.01, 0.1 and 1 nM of NI0000 as
the negative control siRNA.
To determine whether transfection by NK0144 was cytotoxic,
we performed cell viability assays. No signiﬁcant difference in cell
viability was observed at 24, 48 and 72 h after transfection
between human primary RPE cells with 10 nM NK0144 transfection
and those with control siRNA transfection (n = 4; Figure 4d).
Inhibitory effect of NK0144 on proliferation, adhesion andmigration of human RPE cells in vitro
Next, we examined the effect of NK0144 on the proliferation,adhesion and migration of human RPE cells because thesebiological processes are involved in FVM formation, and they are
facilitated by POSTN.20 The proliferation of RPE cells was
signiﬁcantly inhibited by 10 nM NK0144 transfection (P o 0.01,
n = 4; Figure 5a) as was cell adhesion (P o0.001, n = 4; Figure 5b).

Figure 1. Expression of the mRNA of POSTN determined by real-time RT-PCR (a) and the concentration of POSTN protein measured by ELISA(b) in mouse eyes with laser-induced CNV. Bruch’s membrane was ruptured at 20 locations in each eye. On days 3, 7, 14, 21 and 28 after thelaser photocoagulations, mice were killed, eyes were removed, and both the total RNA and protein were extracted from the RPE-choroidal
complexes. The POSTN expression was signiﬁcantly increased and reached a peak at 14 days after the laser photocoagulation (n = 4 pergroup). *Po 0.05, **P o0.001 vs control.
Gene Therapy (2015) 127 – 137© 2015 Macmillan Publishers Limited-50-

New RNAi therapy targeting POSTN for AMD
T Nakama et al

129

Figure 2. Localization of POSTN in laser-induced CNV in a mouse model (a) and human choroidal FVMs (b). Parafﬁn-embedded sections ofeyes from the CNV mouse model and human choroidal FVMs resected during vitrectomy were immunohistochemically stained for POSTN,
RPE65 and cytokeratin. Nuclei were stained with DAPI. These sections were examined by ﬂuorescence microscopy. (a) POSTN (red) costainedwith RPE65 (green) in the choroidal FVM of CNV mice both at day 7 (top row) and day 14 (bottom row) when the expression of POSTN in RPEchoroid complexes is maximal. Scale bars = 50 μm. (b) Double staining for POSTN and cytokeratin in the human choroidal FVM. POSTN (green)staining in cytokeratin-positive RPE cells can be seen. A POSTN-stained section in the top panel is shown at higher magniﬁcation at thebottom. Scale bars = 200 μm.

In situ distribution of ﬂuorescein isothiocyanate (FITC)-labeled
NK0144 in eyes after intravitreal injection
To determine the distribution and kinetics of NK0144 after
intravitreal injection in mouse eyes, we used FITC-labeled
NK0144. Twenty-four hours after the intravitreal injection of 1 μlof 100 μM FITC-labeled NK0144, there was staining for FITC in the
RPE cells adjacent to the CNV site (Figure 6a). The degree ofstaining was increased at 72 h and then decreased at 120 halthough it was still detectable (Figure 6a). These results showedthat NK0144 rapidly diffused to the RPE cells around the CNV sites
after the intravitreal injection and remained in that area for several
days after the injection.

intravitreally in the CNV mouse model. Laser-capture microdissection was used to extract pure mRNA from the CNV sites(Figure 6b). On day 14 at the peak of Postn expression, the levelof Postn mRNA in CNV site was signiﬁcantly decreased after the
intravitreal injection of 10 μM of NK0144, whereas there was no
signiﬁcant difference between NK0000 injection group andphosphate-buffered saline (PBS) injection group (Figure 6c).
At 7 days after rupturing Bruch’s membrane, eyes that received
an intravitreal injection of 1 μl of 10 μM NK0000 as a negative
control developed larger areas of CNV than eyes after 1 μl of 10 μM
NK0144 injections (Figure 7a). Compared with the control NK0000treated eyes, the volume of the CNV in eyes treated with NK0144
was reduced by 75% (P o0.05, n = 8; Figure 7b).
We further investigated the effect of NK0144 on choroidal ﬁbrosis.
At day 21, the volume of the choroidal ﬁbrous tissues in eyes treatedwith 1 μl of 10 μM NK0144 was 30% smaller than that treated with
NK0000 as a negative control (Po0.05, n = 8; Figures 7a and b).
These results are consistent with those of Postn KO mice (Figure 3).

In vivo inhibitory effect of NK0144 on expression of Postn mRNAand progression of choroidal FVM formation
To determine whether NK0144 also causes a speciﬁc knock-downof Postn in vivo and has therapeutic potential, it was injected

No off-target effect on TLR3 activation by NK0144 in CNV
development
As earlier reports showed that naked canonical double-stranded
siRNA can suppress CNV formation not by RNAi but by target

In addition, the inhibitory effect of NK0144 on cell adhesion wasby approximately 40%, which was signiﬁcantly greater than after
NI0079 transfection (P o 0.01, n = 4; Figure 5b). Finally, themigration of RPE cells was signiﬁcantly impaired by NK0144
transfection (P o0.01, n = 4; Figure 5c).

© 2015 Macmillan Publishers Limited

Gene Therapy (2015) 127 – 137-51-

New RNAi therapy targeting POSTN for AMD
T Nakama et al

130

Figure 3. The mean volume of the CNV at day 7 and choroidal ﬁbrosis at day 21 in laser-induced CNV mouse model. WT and Postn KO mice
had laser photocoagulation (4–6 shots per eye) on day 0. The choroidal ﬂat mounts were stained with isolectin B4 and collagen type I andwere examined by laser scanning confocal microscopy. The volumes of the CNV on day 7 and choroidal ﬁbrous tissue on day 21 at the lasersites were measured. (a) Representive immunohistochemical studies showing that the areas of CNV and choroidal ﬁbrosis are smaller in Postn
KO mice than in WT mice. (b) Both CNV and choroidal ﬁbrosis volumes in Postn KO mice were signiﬁcantly smaller than those in WT mice (n = 6
per group). *Po 0.05.

Table 1.

Sequence of RNAi agents directed against POSTN mRNAName

RNA classSequence

NI0079Canonical double-stranded target siRNA

NI0000Canonical double-stranded scrambled siRNANK0144

New class single-stranded target RNAi agent

NK0000New class single-stranded scrambled RNAi agent

5′-GCACCAAAAAGAAAUACUUTT-3′(sense)/5′-AAGUAUUUCUUUUUGGUGCTT-3′ (antisense)
5′-UACUAUUCGACACGCGAAGTT-3′(sense)/5′-CUUCGCGUGUCGAAUAGUATT-3′ (antisense)
5′-AGCACCAAAAAGAAAUACUUUUCCCCACACCGGAAAAGUAUUUCUUUUUGGUGCUUCUUCGG-3′5′-AUACUAUUCGACACGCGAAGUUCCCCACACCGGAACUUCGCGUGUCGAAUAGUAUUCUUCGG-3′

Abbreviations: POSTN, periostin; RNAi, RNA interference; siRNA, small interfering RNA.

sequence-independent activation of cell surface TLR3,31,33 we
examined the effect of a new class of RNAi agents, NK0000 and
NK0144, on TLR3 phosphorylation in RPE cells in vitro by westernblot. TLR3 phosphorylation was not increased 30 min after
transfection with NK0000 or NK0144, whereas transfection withcanonical siRNAs, NI0000 and NI0079, promoted TLR3 phosphorylation (Figure 8a). To further conﬁrm the absence of off-target
effects by the new class of RNAi, we performed ELISA ofinterleukin-12 (IL-12), which is a downstream signaling marker of
TLR3 activation.31 There was no signiﬁcant difference between thelevel of IL-12 protein in the supernatant from RPE cells transfectedwith NK0000 or NK0144 (NK0000; 168.42 ± 26.08 pg IL-12 per mg
total protein, NK0144; 140.86 ± 11.36 pg IL-12 per mg total
protein; n = 4) and that from RPE cells without transfection(129.50 ± 23.02 pg IL-12 per mg total protein; n = 4). In contrast,
the IL-12 level from RPE cells transfected with NI0000 or NI0079
were signiﬁcantly higher (NI0000; 243.25 ± 38.06 pg IL-12 per mg
total protein, NI0079; 300.48 ± 71.24 pg IL-12 per mg total protein,
Po 0.05; n = 4) compared with the normal control (Figure 8b).
We also performed ELISA of interferon gamma, which is anotherdownstream signaling marker for TLR3 activation.31 The level ofinterferon gamma protein in the supernatant from RPE cells
transfected with 10 nM each NI0000, NI0079, NK0000 or NK0144
Gene Therapy (2015) 127 – 137

and that from RPE cells without transfection was below the levelof detection (data not shown).
We next compared the effect of canonical double-stranded
siRNAs and a new class of RNAi agents on CNV formation in vivo.
The average CNV volumes at day 7 after the injection of 1 μl of
10 μM of each canonical double-stranded siRNAs (NI0000 orNI0079) tended to be lower than that in the PBS injection group,
although the difference between PBS injection group and NI0000
or NI0079 injection group was not signiﬁcant (n = 8; Figure 8c). In
contrast, the volume of the CNV in eyes treated with 1 μl of 10 μM
NK0000 was not reduced compared with the PBS-treated eyes. An
intravitreal injection of 1 μl of 10 μM NK0144 led to signiﬁcantly
smaller volumes of CNV than eyes after 1 μl of 10 μM NK0000
injections (Figures 7a and 8c).
These results indicated that the new class of RNAi agents had
no off-target effects and the observed inhibitory effect of CNV by
NK0144 was the result of a speciﬁc knock-down of POSTN.
No obvious adverse effects by NK0144 on retina
To evaluate retinal function after intravitreal injections of NK0144,
we recorded electroretinographies (ERGs) from mice at 21 days
after 1 μl of 10 μM NK0144 injection on days 0 and 7. The mean
amplitude of the a- and b-waves in the mice eyes treated with© 2015 Macmillan Publishers Limited-52-

New RNAi therapy targeting POSTN for AMD
T Nakama et al

131

Figure 4. Structure of canonical double-stranded siRNA (a) and new class single-stranded RNAi agent (b), and the NK0144-mediated effects on
POSTN mRNA and cell survival in cultured human RPE cells. Cultured RPE cells were transfected or not transfected with negative control siRNA,or NI0079, or NK0144. Total RNA was extracted at 24 h after transfection and was analyzed by real-time RT-PCR (c). Cell viability at 24, 48 and
72 h after transfection was assessed by MTT assay (d). (a) Canonical double-stranded siRNA was prepared as double-stranded RNA oligomers.
(b) New class of RNAi agent was prepared as single-stranded RNA oligomers that then self-anneal as shown. Nucleotides in red indicate sense
strand of the target (POSTN), nucleotides in yellow are the antisense strand, and nucleotides in blue are the scaffold. (c) Expression of themRNA of POSTN in ARPE19 cells exposed to 0.01, 0.1 and 1 nM of NK0144 was signiﬁcantly reduced in a dose-dependent manner comparedwith those with negative control siRNA treatment (n = 4 per group). *Po 0.05, **P o0.0001. (d) No signiﬁcant difference in cell viability wasobserved up to 72 h between RPE cells with NK0144 treatment and those with control siRNA treatment (n = 4 per group).

NK0144 (scotopic a-wave = 386.1 ± 13.5 μV, scotopic b-wave =744.8 ± 59.1 μV) were not signiﬁcantly different from that of theuntreated control eyes (scotopic a-wave = 370.3 ± 10.5 μV, scotopic
b-wave = 703.1 ± 55.0 μV; n = 4; Figure 9a).
The histological structure observed by hematoxylin and eosin
staining of the mouse retina at 21 days after NK0144 injection
appeared normal and did not differ from that of the untreatedeyes (Figure 9b). The retina of the CNV mouse model showedterminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)-positive apoptotic cells in the outer nuclear layer.
In contrast, the eyes injected with NK0144 showed no
TUNEL-positive staining in the retina (Figure 9c). These results
showed that NK0144 had no adverse effects on the function andmorphology of the retina.
DISCUSSION
Anti-VEGF therapy has been used to treat the CNV in eyes with
AMD, and this treatment has led to signiﬁcant advances in theunderstanding of the pathogenesis of AMD.36–38 However, theCNV activity does not respond to multiple treatments as isrequired in many patients. In addition, there is currently no
therapy to treat choroidal ﬁbrosis. Therefore, ﬁnding additional
therapeutic methods is still a goal.
To the best of our knowledge, this is the ﬁrst study providing
evidence that POSTN is expressed in choroidal FVMs in both a CNV
mouse model and AMD patients. Furthermore, our results showed
the usefulness of a new class of single-stranded RNAi moleculethat targeted POSTN and can be considered for the treatment ofchoroidal FVMs. Intravitreal injections of NK0144 signiﬁcantly
inhibited the volume of the induced CNVs and subsequentﬁbrosis, which supports our ﬁndings that POSTN is a positive
regulator of choroidal FVMs formation.

Our data demonstrated that POSTN is expressed by the RPE cells
associated with choroidal FVMs in both a CNV mouse model andAMD patients. Moreover, genetic ablation of Postn gene can inhibit
both CNV and choroidal ﬁbrosis. Thus, an elevated expression of
POSTN by RPE cells may promote the development of choroidal
FVMs. These ﬁndings are in line with our earlier reports showingthat the POSTN produced by RPE cells in eyes with proliferative
vitreoretinopathy promotes preretinal ﬁbrous proliferation of RPE
cells through integrin αv-mediated focal adhesion kinase in an
autocrine manner.17,18,20 In addition, because integrin αvβ3 isinvolved in the development of CNVs in eyes with AMD,39,40 the
POSTN expressed by RPE cells may also promote CNV development
through integrin αvβ3 in a paracrine manner.
It was recently reported that anti-VEGF therapy may have arole in the development of geographic atrophy in AMD patientscompared with that following other treatments.16 This is partly
because VEGF has an important role in the normal function of
the retina and the maintenance of the choriocapillaris.41 Thus,
the therapies that block VEGF to inhibit pathologic neovascularization could result in unexpected disturbances of the normal
homeostasis in the retina and should be used cautiously. In
contrast to VEGF, we have reported that the mRNA and proteinof POSTN were barely detectable in the normal retina.20 Together
with the fact that anti-POSTN therapy in this study showed noapparent adverse effects, these results raise the possibility that
POSTN may be an attractive therapeutic target to regulate‘disease-speciﬁc’ pathways involved in the development ofchoroidal FVMs, while minimizing the unfavorable side effects
to the normal retina.
Although canonical double-stranded siRNAs have attractive andpromising aspects for new therapy, some obstacles still remain to
be overcome before their clinical application.30 These hurdles
include the lack of a safe DDS, adverse off-target effects through
TLR3 activation and the lack of stability. In this study, we tried to© 2015 Macmillan Publishers Limited

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Figure 5. NK0144-mediated effects on human RPE cell proliferation, adhesion and migration in vitro. BrdU-ELISA was used to assess thetransfected cell proliferation, and MTT assay was used to assess cell migration. The migration of RPE cells was examined on ﬁbronectin-coatedwell after 48-h incubation by staining with calcein AM and quantiﬁed with a ﬂuorescence microplate reader. (a) NK0144 signiﬁcantly inhibitedthe proliferation of human RPE compared with negative control siRNA (n = 4 per group). *P o0.01. (b) Human RPE cells adhesion was reduced
more by NK0144 transfection than by both negative control siRNA and NI0079 (n = 4 per group). *P o0.001, **P o0.01. (c) NK0144 signiﬁcantly
reduced the degree of migration of human RPE cells more than that with negative control siRNA (n = 4 per group). *Po0.01.

overcome these obstacles by using a new class of single-stranded
RNAi agents that self-anneals into a unique structure containing acanonical double-stranded RNA. Previous studies showed that the
new single-stranded RNAi is not associated with off-target effectand is more stable against nuclease than canonical doublestranded siRNA.35,42 Our results showed that naked NK0144 cansigniﬁcantly inhibit the expression of POSTN, proliferation,adhesion and migration of RPE cells without target sequenceindependent effect through TLR3 activation and inﬂuence on cell
viability. Furthermore, we showed that labeled single-stranded
nkRNA without any DDS was detected in the RPE-choroid for atleast 5 days following an intravitreal injection. This suggests that it
was taken up and retained within cells at the CNV site for a
substantial period of time. Unlike canonical double-stranded
siRNAs, we also demonstrated that naked NK0144 signiﬁcantly
inhibited choroidal FVM formation and had POSTN sequencedependent CNV suppression without serious toxicity. These results
strongly indicate that intravitreal injections of naked NK0144 may
be a safer and more efﬁcient therapeutic method to inhibit
choroidal CNV. This is because the naked single-stranded RNAitherapy may be safer than canonical siRNA because of its stability,
no DDS need, and much less proinﬂammatory or toxicologicalresponses induced by siRNA itself or by accompanying DDS.43
Unlike previous reports, target sequence-independent CNV
suppression by canonical siRNAs was not signiﬁcant in this study.
This is probably due to the smaller injection amounts of canonical
Gene Therapy (2015) 127 – 137

double-stranded siRNAs (0.1 μg per eye). In the previous reports,
the inhibitory effect was only observed with the 10 times larger
amount (1 μg per eye).31
In conclusion, our results show a causal link between POSTN
and choroidal FVM formation, and the effects of a naked,
unmodiﬁed new class of RNAi agent targeting POSTN. Additional
preclinical studies concerning the toxicity, stability and effect of
duration are underway for establishing the new POSTN-targeting
RNAi agent for combating choroidal FVM formation.
MATERIALS AND METHODS
Animals
All animal experiments were performed following the guidelines of the
Association for Research in Vision and Ophthalmology (ARVO) Statementon the Use of Animals in Ophthalmic and Vision Research. The
experimental protocols were approved by the Institutional Animal Careand Use Committee of Kyushu University.
C57BL/6J WT mice (CLEA, Tokyo, Japan) and Postn KO mice between 6
and 8 weeks were used for the animal experiments. PCR was used todetermine the genotype of the experimental mice. Primer sequences forgenotyping were as follows:
5′-GTTCTTACAGAAAGCAGAAGGATAC-3′ and5′-TTAAATCACTCCACAGCAGAACACG-3′
were used to detect a 436-bp product for the WT allele; and5′-CATGATAGCTTCTCTCCCAGTTCTC-3′ and5′-CTTGCAATAAGTAAAACAGCTCCCC-3′
were used to detect a 372-bp product for the Postn KO allele.
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Figure 6. Distribution of FITC-labeled NK0144 in the mouse retina at different time points after intravitreal injection (a) and inhibitory effect ofNK0144 on the expression of Postn mRNA in vivo (b, c). FITC-labeled NK0144 was given by intravitreal injection at the same time as laser
treatment. The mice were killed at 24, 72, 120 h after the injection. The eyes were enucleated and cryosections were cut, and the cell nuclei
were stained with DAPI (blue) followed by examination by ﬂuorescence microscopy. The mRNAs at CNV sites were extracted using lasercapture microdissection at day 14 and analyzed by real-time RT-PCR. (a) NK0144 (green) can be clearly seen at the CNV site from 24 h after
intravitreal injection. The staining for NK0144 at the laser site increased at 72 h, and decreased at 120 h although it was still detectable.
(b) Immunostaining before (left) and after (right) laser microdissection. (c) The level of Postn mRNA in CNV site was signiﬁcantly decreased byintravitreal injection of 10 μM NK0144 compared with NK0000 injection group and PBS injection group (n = 4 per group). *Po 0.05.

Figure 7. Effect of NK0144 on progression of CNV at day 7 and choroidal ﬁbrosis at day 21 in laser-induced CNV mouse model. On days 0 or 7
after CNV induction, mice were injected intravitreally with 1 μl of 10 μM negative control siRNA in one eye and 1 μl of 10 μM of NK0144 in the
fellow eye. The mice were killed 7 days after the injection for CNV and 21 days after injection for choroidal ﬁbrosis assessments. The eyes wereenucleated and choroidal ﬂat mounts were stained with isolectin B4 and collagen type I and examined by laser scanning confocal microscopy.
(a) Representative immunohistochemical analysis shows that the areas of CNV and choroidal ﬁbrosis in NK0144 treatment group were smaller
than those in negative control siRNA treatment group. (b) Both CNV and choroidal ﬁbrosis volumes in eyes treated with NK0144 weresigniﬁcantly smaller than that in the negative control siRNA group (NK0000; n = 8 per group). *P o0.05.

Laser-induced CNV model
CNVs were generated as described in detail.44 Brieﬂy, mice wereanesthetized with intraperitoneal injections of 100 mg kg–1 ketamineand 10 mg kg–1 xylazine, and the pupils were dilated with a mixture of
0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin P;
Santen, Osaka, Japan). Laser photocoagulation (532 nm, 100 mW,
100 ms, 75 μm; Verdi; Coherent, Santa Clara, CA, USA) was applied toeach fundus using a coverslip as a contact lens on day 0 (4–6 shots pereye for CNV volume studies: 20 shots per eye for mRNA and protein
analyses). The formation of a bubble at the time of laser application
indicating a rupture of Bruch’s membrane was an important signthat sufﬁcient injury had been done to induce a CNV. Thus, only burns
with bubble were included in the study. We excluded burns withbleeding.

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Quantitative real-time RT-PCR
Total RNA was extracted from the homogenized RPE-choroid complexes atthe selected time points and the human RPE cells treated with RNAi
agents. The MagDEA RNA kit (Precision System Science, Pleasanton, CA,
USA) was used according to the manufacturer’s protocol.44,45 After
quantiﬁcation of the RNA concentration, complementary DNAs weresynthesized with a First Strand cDNA Synthesis Kit (Roche, Mannheim,
Germany). Quantitative RT-PCR was performed and analyzed using a SYBR
Premix Ex Taq (Takara, Shiga, Japan) and a LightCycler 96 PCR system(Roche). A volume of 20 μl containing 0.2 μM of primers was loaded into
each LightCycler well. The primer sequences were as follows:
for mouse Postn,
5′-CTTTCGAGAAACTGCCACGAG-3′ and5′-CCTTCCATGGTCTCAAACACG-3′;
for mouse β-actin,
5′-GATGACCCAGATCATGTTTGA-3′ and
5′-GGAGAGCATAGCCCTCGTAG-3′;
for human POSTN,
5′-TGCCCAGCAGTTTTGCCCAT-3′ and5′-CGTTGCTCTCCAAACCTCTA-3′;
and for human β-actin,
5′-ATAGCACAGCCTGGATAGCAACGTAC-3′ and 5′-CACCTTCTACAATGAG
CTGCGTGTG-3′.
The quality and speciﬁcity of the PCR reaction were determined bymelting curves. A standard curve was used to quantify the expression levels.

Enzyme-linked immunosorbent assay
Total protein was isolated from the sonicated RPE-choroid complexes
using Tissue Protein Extraction Reagent with protease inhibitor (T-PER;
Thermo, Waltham, MA, USA). The concentrations of POSTN in the mouse
RPE-choroid complexes, and IL-12 and interferon gamma in the supernatant from the cultured human RPE cells were measured with a mouse
POSTN immunoassay kit (R&D Systems, Minneapolis, MN, USA), human
IL-12 ELISA kit (Thermo) and human interferon gamma immunoassay kit(R&D Systems), respectively, according to the manufacturer’s instructions.

Immunohistochemistry

Figure 8. RNAi agent-mediated effects on TLR3 phosphorylation andIL-12 levels in cultured human RPE cells in vitro, and target sequenceindependent CNV suppression in vivo. In vitro, cultured RPE cells weretransfected or not transfected with NI0000, NI0079, NK0000 or
NK0144. Total protein was extracted at 30 min after transfection forwestern blot (a) or 24 h after transfection for ELISA (b). In vivo, mice
were injected intravitreally with 1 μl of 10 μM each RNAi agents on day
0 after CNV induction. CNV volumes 7 days after the injection wereanalyzed using choroidal ﬂat mounts stained with isolectin B4 (c).
(a) TLR3 phosphorylation was detected in RPE cells after exposure to
NI0000 or NI0079, whereas NK0000 and NK0144 had no effect on TLR3
phosphorylation. β-Actin served as loading control. (b) IL-12 levels in
RPE cells exposed to NI0000 or NI0079 were signiﬁcant higher,
whereas no signiﬁcant difference was observed between RPE cells
without treatment and those transfected with NK0000 or NK0144(n = 4 per group). *Po0.05. (c) Target sequence-independent CNV
suppression was observed in the double-stranded scrambled siRNA(NI0000) injection group, whereas there was no decrease of CNV in thecorresponding single-stranded scrambled RNAi (NK0000) injectiongroups compared with PBS injection group (n = 8 per group).
Intravitreal injection of NK0144 showed signiﬁcantly smaller areas of
CNV than eyes after NK0000 injections. *Po0.05.

Human specimens
Choroidal FVM was isolated from the sensory retina and RPE using 33G
subretinal canula and incised from the subretinal space of patients with
AMD who were undergoing pars plana vitrectomy.
Gene Therapy (2015) 127 – 137

Eyes enucleated from a CNV mouse model and human choroidal FVMs
resected during vitrectomy were ﬁxed in 4% paraformaldehyde, thenembedded in parafﬁn and cut at 3 μm thickness. After deparafﬁnization,
rehydration and blocking, the sections were incubated with the primary
antibodies overnight at 4 °C, and the second antibodies were added for 1 h
at room temperature. Nuclei were counterstained with 4,6-diamidino-2phenylindole (DAPI; Hoechst 33342; Molecular Probes, Eugene, OR, USA).
After washing with PBS, the cover slides were mounted with an aqueousmounting medium (Thermo). A ﬂuorescent microscope (BZ-9000; KEYENCE, Osaka, Japan) was used to examine and analyze the specimens.46–48
The primary antibodies were POSTN (MAB 3548: 5 μg ml–1; R&D
Systems), RPE65 (MAB 5428: 5 μg ml–1; Millipore, Temecula, CA, USA) andpan-cytokeratin (sc-8018: 1:100 dilution; Santa Cruz, Dallas, TX, USA). The
second antibodies were Alexa Fluor 488 and 647 (1:100 dilution; Molecular
Probes).

CNV and choroidal ﬁbrosis volume measurement
The volumes of the CNVs on day 7 and that of the choroidal ﬁbrous tissue
on day 21 after laser photocoagulations were measured in choroidal ﬂat
mounts. Choroidal ﬂat mounts were prepared as described in detail.44
Brieﬂy after ﬁxation in 4% paraformaldehyde, the cornea, lens andneurosensory retina of the enucleated eyes were removed from the eye.
The remaining eyecups were washed with PBS and placed in 50 and 100%
methanol for 10 min each at room temperature. The samples were thenplaced in PBS containing 1% bovine serum albumin and 0.5% TritonX-100
as a blocking buffer for 1 h at room temperature. This was followed byincubation with the primary antibodies overnight at 4 °C and second
antibodies for 1 h at room temperature. Several radial cuts were madefrom the edge to the equator and the eyecups were ﬂat mounted in
mounting medium. Choroidal ﬂat mounts were observed with a laser
scanning confocal microscope (Nikon A1; Nikon, Tokyo, Japan), and thevolumes at each burn were measured using a software (NIS-Elements
Software; Nikon).
The primary antibodies were ﬂuorescein-labeled isolectin B4 (FL1201;
1:200 dilution; Vector Laboratories, Burlingame, CA, USA) for CNV and© 2015 Macmillan Publishers Limited-56-

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Figure 9. Absence of apparent adverse effects of NK0144 intravitreal injection on function and morphology in mouse retinas. Intravitreal
injection of NK0144 was performed on days 0 and 7. (a) Scotopic ERGs recorded on day 21 in eyes without any treatment and eyes with
NK0144 treatment. The differences in the amplitudes of the a-wave and b-wave between untreated group and NK0144 treatment group were
not signiﬁcant (n = 4). (b) Hematoxylin and eosin (H&E) staining of retinas obtained on day 21 from mice with NK0144 treatment appeared
normal as well as those from untreated mice. (c) TUNEL staining of retinas from CNV model mice (left) was used as a TUNEL-positive control.
No TUNEL-positive cells were observed in retinas of eyes at 21 days after NK0144 treatment (right). Scale bars = 50 μm.
collagen type I (600-401-103; 1:100 dilution; ROCKLAND, Gilbertsville, PA,USA) for choroidal ﬁbrous tissue. The second antibody for collagen type I
was Alexa Fluor 647 (1:100 dilution; Molecular Probes).

Preparation of new class of RNAi agent targeting POSTN
New RNAi agents were synthesized on solid phase as single-stranded RNAs
as described in detail.35 NI0079 was a canonical double-stranded siRNAdesigned to target the mRNA of human and mouse Postn (Figure 4a), andNI0000 was a scrambled double-stranded siRNA. A long single-stranded
oligonucleotides, denoted as NK0144, was constructed by incorporating
the sense and antisense nucleotides of NI0079 into the scaffold of a unique
RNAi platform named nkRNA (Figure 4b).35 The nkRNAs are new class ofRNAi agents that spontaneously anneal to form a helical structure
containing a double-stranded central stem and two loops within amolecule.35 NK0000 was also a new class of RNAi agent, but the sequenceswere scrambled. The sequences of the RNAi agents were described indetail in Table 1.

Cell culture and transfection
Cells from the ARPE19 cell line and human primary RPE cells isolated fromfetal human eyes (Lonza, Walkersville, MD, USA) were used for the in vitro
studies. Cells were cultured in Dulbecco’s modiﬁed Eagle’s medium(DMEM) containing 100 U ml–1 penicillin, 100 μg ml–1 streptomycin and
10% heat-inactivated fetal bovine serum at 37 °C in 5% CO2.

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When the cultured cells in 24-well plates were 50–70% conﬂuent, they
were transfected with canonical double-stranded siRNA (NI0079), or the
new single-stranded RNAi agent (NK0144) for POSTN, or a negative control
RNAi. The incubation time for the transfection was 24 h using the RNAi
MAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The ﬁnal concentrations of RNAi agents were 0.01, 0.1 and1 nM to determine the knock-down effect of NK0144 transfection on theexpression of POSTN in ARPE19 cells. To assay the primary RPE cells, a ﬁnal
concentration of 10 nM RNAi agents was chosen. The reagent mixtures fortransfection were replaced by DMEM containing 3% fetal bovine serumwith recombinant TGF-β2 at a ﬁnal concentration of 3 ng ml–1 as theinducer of POSTN, followed by each assay.

Cell viability and adhesion assay (MTT assay)
Cell viability was assessed at 24, 48 and 72 h after transfection by staining
with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide(MTT: 5 mg ml–1; Sigma-Aldrich, St Louis, MO, USA) as described indetail.49 In brief, the reagent mixture for transfection was removed andfresh medium with 5 mg ml–1 MTT was added for 5 h to detect live cells.
The MTT assay was also used to measure cell adhesion as described.20
Brieﬂy, transfected RPE cells were placed in DMEM containing 3% fetal
bovine serum and recombinant TGF-β2 for 24 h. The cells were thentrypsinized and 1 × 104 cells were injected on ﬁbronectin-coated wells andallowed to attach for 60 min. After washing gently with PBS twice, fresh
medium with 5 mg ml–1 MTT was added for 5 h.
Each MTT assay was quantiﬁed by measuring the level of formazan
precipitates using 100% DMSO (Sigma-Aldrich). The absorbance at 590 nm
was measured using an Immuno Mini NJ-2300 plate reader (NJ InterMed,
Tokyo, Japan).

Cell proliferation assay
For cell proliferation assays, 1 × 104 transfected RPE cells were seeded ineach well of 96-well plates and incubated with TGF-β2. After 24 h,
bromodeoxyuridine (BrdU)-ELISA (Roche) was used to assess the cellproliferation according to the manufacturer’s instructions.

Laser-capture microdissection
Laser-capture microdissection was used to purify the CNV sites from whole
retinas. Eyes from mice injected with each RNAi agents were enucleated atday 14 and embedded in OCT compound (4583; Sakura Finetech) and kept
at –80 °C until sectioning. Then, 10 μm thick sections were cut with acryostat and placed on glass slides designed for laser microdissection
microscopy (LMD6500; Leica Microsystems, Wetzlar, Germany). Sections
were stained with toluidine blue and only CNV sites were collected by laser
microdissection.

Western blot analysis
RPE cells were seeded in collagen-coated six-well plates. After starvation
with serum-free DMEM for 24 h, the cells were transfected with each RNAi
agents. After 30-min incubation, the total cell lysates were extracted using
lysis buffer with protease inhibitor and phosphatase inhibitor (Thermo).
The extracted cell lysates were subjected to 4–12% sodium dodecyl
sulfate-NuPAGE, and the blots were incubated with an Ab against
phosphorylated TLR3 (Tyr759, NBP2-24904: 0.1 μg ml–1; Novus Biologicals,
Littleton, CO, USA). Visualization was performed using a SuperSignal West
Femto Maximum Sensitivity Substrate (Thermo) detection system. Laneloading differences were determined by blotting the membranes with an
Ab against β-actin (4970: 1:1000; Cell Signaling Technology, Danvers,
MA, USA).

Electroretinography
ERGs were recorded as described in detail.50 Brieﬂy, mice were darkadapted overnight and were anesthesized under dim red light. The pupils
were dilated and recordings were made using the PuREC system (MAYO,
Aichi, Japan). ERGs were recorded on day 21 after the intravitreal injectionsof 1 μl PBS containing 100 pmol NK0144 on days 0 and 7. The ERGs of theuntreated eyes were used as controls. The scotopic ERGs were elicited by astimulus intensity of 10 000 cd m–2, and the responses were differentially
ampliﬁed and ﬁltered between 0.3 and 500 Hz.

Histology and TUNEL
Cell migration assay
The Oris 96-well cell migration assay kit (Platypus Technologies, Madison,
WI, USA) was used to assess the degree of cell migration according to the
manufacturer’s instructions. Brieﬂy, 5 × 104 RPE cells were seeded in eachﬁbronectin-coated well and transfected with each RNAi agents. After 1 h ofpretreatment in DMEM with 3% fetal bovine serum containing recombinant TGF-β2 and 5 μg ml–1 aphidicolin (Sigma-Aldrich) to inhibit cell
division, the stoppers were removed to start the migration of cells into thedetection zone. After 48 h from the initiating of migration, the cells were
stained with DMEM containing calcein AM (2 μg ml–1, Molecular Probes) for
1 h. Then, the signal intensity of the stained cells that migrated into thedetection zone was measured with a Spectra Max M5 microplate reader inthe ﬂuorescence mode (Molecular Devices, Sunnydale, CA, USA). The
ﬂuorescence ﬁlter set (excitation 485 nm and emission 528 nm) with an
Oris detection mask attached to the bottom of the plate was used.

POSTN RNAi treatment in vivo
Immediately after the laser photocoagulation, mice were given an
intravitreal injection of 1 μl PBS containing 10 μM of control siRNA in one
eye and NK0144 in the fellow eye. An additional injection was given on day
7 for the choroidal ﬁbrosis volume assessments. The intravitreal injections
were performed 0.5 mm away from the limbus using a 1 ml Hamilton
syringe (Hamilton, Reno, NV, USA) and a 33-gauge needle under a surgicalmicroscope.

Hematoxylin and eosin and TUNEL staining of parafﬁn-embedded sections
were performed. An ApopTag Fluorescein In Situ Apoptosis Detection Kit(Millipore) was used for the TUNEL staining, and the procedures wereaccording to the manufacturer’s protocol. The sections were costainedwith DAPI (Molecular Probes) to observe the cell nuclei, and the stained
sections were examined under a ﬂuorescence microscope. A retina fromthe mouse CNV model was used as a TUNEL-positive control.

Statistical analyses
All results are expressed as the means ± s.e.m. All statistical analyses of thedifferences between groups were made using the Dunnett's test or
Wilcoxon rank sum test. Differences were considered signiﬁcant for
Po0.05. Statistical analyses were performed using JMP 9.0.2 (SAS Institute,Cary, NC, USA).

CONFLICT OF INTEREST
The patent on periostin (WPO Patent WO/2013/147140) became public, and in thesepatents, the names of TN, SY, KI, TH, TO and TI are included. This work was supported
in part by A-STEP (Adaptable & Seamless Technology Transfer Program throughTarget-driven R&D) governed by Japan Science and Technology Agency. SY is
supported by JSPS 26293374. KT, AS and KY are employees of AQUA Therapeutics
and hold equity. TH, TO and HH are employees of BONAC Corporation and holdequity. The remaining authors declare no conﬂict of interest.

Assessment of distribution of FITC-labeled NK0144 in retina after
intravitreal injections

ACKNOWLEDGEMENTS

Mice that received an intravitreal injection of FITC-labeled NK0144 were
killed at 24, 72 and 120 h after the injection, and the enucleated eyes werefrozen in optimal cutting temperature embedding compound (OCTcompound; Sakura Finetek, Tokyo, Japan). Then, 10 μm thick frozen
sections were cut with a cryostat and ﬁxed in acetone. After blocking with
3% skim milk, the nuclei were stained with DAPI and mounted withaqueous mounting medium. The sections were examined with aﬂuorescent microscope.

We thank the staff of the Research Support Center (Graduate School of Medical
Sciences, Kyushu University) for technical support and Masayo Eto, Kinuko Sasadaand Hiroko Miura for their excellent technical assistance. This work was supported inpart by JSPS KAKENHI grant numbers 24249083, 23592574, 26293374, 26670757,
Takeda Science Foundation, and Adaptable and Seamless Technology Transfer
Program through Target-driven R&D from the Japan Science and Technology Agency.
T Nakama is supported by a fellowship from the Japan Society for the Promotion of
Science for Young Scientists.

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expression of periostin in vitreous and ﬁbrovascular membranes obtained frompatients with proliferative diabetic retinopathy. Invest Ophthalmol Vis Sci 2011; 52:
5670–5678.
19 Asato R, Yoshida S, Ogura A, Nakama T, Ishikawa K, Nakao S et al. Comparison ofgene expression proﬁle of epiretinal membranes obtained from eyes with proliferative vitreoretinopathy to that of secondary epiretinal membranes. PLoS One
2013; 8: e54191.
20 Ishikawa K, Yoshida S, Nakao S, Nakama T, Kita T, Asato R et al. Periostin promotes
the generation of ﬁbrous membranes in proliferative vitreoretinopathy. FASEB J
2014; 28: 131–142.
21 Kudo A. Periostin in ﬁbrillogenesis for tissue regeneration: periostin actions insideand outside the cell. Cell Mol Life Sci 2011; 68: 3201–3207.
22 Conway SJ, Izuhara K, Kudo Y, Litvin J, Markwald R, Ouyang G et al. The role ofperiostin in tissue remodeling across health and disease. Cell Mol Life Sci 2014; 71:
1279–1288.
23 Rios H, Koushik SV, Wang H, Wang J, Zhou HM, Lindsley A et al. periostin null mice
exhibit dwarﬁsm, incisor enamel defects, and an early-onset periodontal diseaselike phenotype. Mol Cell Biol 2005; 25: 11131–11144.
24 Snider P, Hinton RB, Moreno-Rodriguez RA, Wang J, Rogers R, Lindsley A et al.
Periostin is required for maturation and extracellular matrix stabilization of
noncardiomyocyte lineages of the heart. Circ Res 2008; 102: 752–760.

25 Malanchi I, Santamaria-Martinez A, Susanto E, Peng H, Lehr HA, Delaloye JF et al.
Interactions between cancer stem cells and their niche govern metastatic colonization. Nature 2012; 481: 85–89.
26 Shimazaki M, Nakamura K, Kii I, Kashima T, Amizuka N, Li M et al. Periostin isessential for cardiac healing after acute myocardial infarction. J Exp Med 2008;
205: 295–303.
27 Ontsuka K, Kotobuki Y, Shiraishi H, Serada S, Ohta S, Tanemura A et al. Periostin,a matricellular protein, accelerates cutaneous wound repair by activating dermalﬁbroblasts. Exp Dermatol 2012; 21: 331–336.
28 Masuoka M, Shiraishi H, Ohta S, Suzuki S, Arima K, Aoki S et al. Periostin promotes
chronic allergic inﬂammation in response to Th2 cytokines. J Clin Invest 2012; 122:
2590–2600.
29 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of
21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
Nature 2001; 411: 494–498.
30 Pecot CV, Calin GA, Coleman RL, Lopez-Berestein G, Sood AK. RNA interference inthe clinic: challenges and future directions. Nat Rev Cancer 2011; 11: 59–67.
31 Kleinman ME, Yamada K, Takeda A, Chandrasekaran V, Nozaki M, Bafﬁ JZ et al.
Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.
Nature 2008; 452: 591–597.
32 Yang Z, Stratton C, Francis PJ, Kleinman ME, Tan PL, Gibbs D et al. Toll-likereceptor 3 and geographic atrophy in age-related macular degeneration. N Engl J
Med 2008; 359: 1456–1463.
33 Cho WG, Albuquerque RJ, Kleinman ME, Tarallo V, Greco A, Nozaki M et al. Smallinterfering RNA-induced TLR3 activation inhibits blood and lymphatic
vessel growth. Proc Natl Acad Sci USA 2009; 106: 7137–7142.
34 Kleinman ME, Kaneko H, Cho WG, Dridi S, Fowler BJ, Blandford AD et al. Shortinterfering RNAs induce retinal degeneration via TLR3 and IRF3. Mol Ther 2012; 20:
101–108.
35 Hamasaki T, Suzuki H, Shirohzu H, Matsumoto T, D'Alessandro-Gabazza CN, GilBernabe P et al. Efﬁcacy of a novel class of RNA interference therapeutic agents.
PLoS One 2012; 7: e42655.
36 Rosenfeld PJ, Brown DM, Heier JS, Boyer DS, Kaiser PK, Chung CY et al. Ranibizumab
for neovascular age-related macular degeneration. N Engl J Med 2006; 355: 1419–1431.
37 Gragoudas ES, Adamis AP, Cunningham Jr ET, Feinsod M, Guyer DRGroupVISiONCT. Pegaptanib for neovascular age-related macular degeneration. N Engl J
Med 2004; 351: 2805–2816.
38 Stewart MW, Rosenfeld PJ. Predicted biological activity of intravitreal VEGF Trap.
Br J Ophthalmol 2008; 92: 667–668.
39 Mousa SA, Lorelli W, Campochiaro PA. Role of hypoxia and extracellular matrixintegrin binding in the modulation of angiogenic growth factors secretion by
retinal pigmented epithelial cells. J Cell Biochem 1999; 74: 135–143.
40 Friedlander M, Theesfeld CL, Sugita M, Fruttiger M, Thomas MA, Chang S et al.
Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovasculardiseases. Proc Natl Acad Sci USA 1996; 93: 9764–9769.
41 Saint-Geniez M, Kurihara T, Sekiyama E, Maldonado AE, D'Amore PA. An essential
role for RPE-derived soluble VEGF in the maintenance of the choriocapillaris. Proc
Natl Acad Sci USA 2009; 106: 18751–18756.
42 Fujita Y, Takeshita F, Mizutani T, Ohgi T, Kuwano K, Ochiya T. A novel platform toenable inhaled naked RNAi medicine for lung cancer. Sci Rep 2013; 3: 3325.
43 Kariko K, Bhuyan P, Capodici J, Weissman D. Small interfering RNAs mediate
sequence-independent gene suppression and induce immune activation by signaling through Toll-like receptor 3. J Immunol 2004; 172: 6545–6549.
44 Lambert V, Lecomte J, Hansen S, Blacher S, Gonzalez ML, Struman I et al. Laserinduced choroidal neovascularization model to study age-related maculardegeneration in mice. Nat Protoc 2013; 8: 2197–2211.
45 Ishikawa K, Yoshida S, Kadota K, Nakamura T, Niiro H, Arakawa S et al.
Gene expression proﬁle of hyperoxic and hypoxic retinas in a mouse model ofoxygen-induced retinopathy. Invest Ophthalmol Vis Sci 2010; 51: 4307–4319.
46 Ishikawa K, Yoshida S, Nakao S, Sassa Y, Asato R, Kohno R et al. Bone marrow-derived
monocyte lineage cells recruited by MIP-1beta promote physiological revascularization in mouse model of oxygen-induced retinopathy. Lab Invest 2012; 92: 91–101.
47 Arima M, Yoshida S, Nakama T, Ishikawa K, Nakao S, Yoshimura T et al. Involvement of periostin in regression of hyaloidvascular system during ocular development. Invest Ophthalmol Vis Sci 2012; 53: 6495–6503.
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© 2015 Macmillan Publishers Limited

Gene Therapy (2015) 127 – 137-59-

Citation: Molecular Therapy—Nucleic Acids (2015) 4, e258; doi:10.1038/mtna.2015.34
Official journal of the American Society of Gene & Cell Therapy All rights reserved 2162-2531/15
www.nature.com/mtna

Novel Types of Small RNA Exhibit Sequence- andTarget-dependent Angiogenesis Suppression Without
Activation of Toll-like Receptor 3 in an Age-related
Macular Degeneration (AMD) Mouse Model
Masakatsu Takanashi1, Katsuko Sudo2, Shinobu Ueda1, Shin-Ichiro Ohno1, Yuko Yamada1, Yasuhiro Osakabe1, Hiroshi Goto3,
Yoshimichi Matsunaga3, Akio Ishikawa1, Yoshihiko Usui3 and Masahiko Kuroda1

RNA interference (RNAi) has become a powerful tool for suppressing gene expression in vitro and in vivo. A great deal ofevidence has demonstrated the potential for the use of synthetic small interfering RNAs (siRNAs) as therapeutic agents.
However, the application of siRNA to clinical medicine is still limited, mainly due to sequence-independent suppression ofangiogenesis mediated by Toll-like receptor 3 (TLR3). Here, we describe novel types of synthetic RNA, named nkRNA and
PnkRNA, that exhibit sequence-speciﬁc gene silencing through RNAi without activating TLRs or RIG-I–like receptor signaling. In
addition, we conﬁrmed the therapeutic effect for the novel types of RNA in an animal model of age-related macular degeneration(AMD) without retinal degeneration. These data indicate that nkRNA and PnkRNA are of great potential utility as therapies
against blinding choroidal neovascularization due to AMD.
Molecular Therapy—Nucleic Acids (2015) 4, e258; doi:10.1038/mtna.2015.34; published online 20 October 2015
Subject Category: Nucleic acid chemistries; Therapeutic proof-of-concept
Introduction
Age-related macular degeneration (AMD) is a complex disorder that primarily affects the macula, including the retinal pigment epithelium but also to a certain extent the photoreceptor
layer and retinal neurons.1,2 The most common cause ofvision loss from AMD is pathologic choroidal neovascularization (CNV), vascular leakage, and subsequent ﬂuid accumulation in the neurosensory retina. Therefore, pharmacologic
inhibition of vascular endothelial growth factor-A (VEGF-A)
via targeted monoclonal antibody therapy has become thefront line treatment for this blinding disease; this approach
has high efﬁcacy against AMD.3,4 Several other modalities of
VEGF-A–targeted therapies have been investigated including posttranscriptional gene silencing via small interfering
RNA (siRNA), an approach that showed great promise in
preclinical studies. However, the clinical trials studying theﬁrst generation of siRNA-based drugs targeting VEGF-A for
treatment of neovascular AMD were abruptly halted because
they failed to reach the primary endpoints.4
Toll-like receptors (TLRs) belong to a family of innate
immune receptors that detect and clear invading microbial
pathogens. Speciﬁcally, intracellular TLRs such as TLR3,
TLR7/8, and TLR9 recognize microbe-derived nucleic acids
such as double-stranded RNA, single-stranded RNA, andCpG DNA, respectively.5–8 Many siRNAs with a wide rangeof targets, as well as siRNAs that are not competent for RNA
interference (RNAi), suppress CNV in mice.9,10 Moreover,nontargeted (i.e., not targeted against mammalian gene)and targeted siRNAs (against Vegfa or Vegfr1) suppressed

CNV via TLR3, its adaptor TRIF, and induction of interferon-γ(IFNγ) and interleukin-12.10
To address some of these issues, we developed two novel
types of synthetic RNA named nkRNA and PnkRNA.11 Bothof these types of synthetic RNAs are single stranded and notchemically modiﬁed. Notably, nkRNA and PnkRNA exhibitedsequence-speciﬁc gene silencing through RNAi. In addition,
we transfected nkRNA and PnkRNA into murine endothelial
cells and conﬁrmed that they did not activate TLR3 signaling. Furthermore, we found that nkRNA and PnkRNA against
VEGF suppressed CNV in laser-induced mouse models.
These ﬁndings suggested that nkRNA and PnkRNA represent a suitable technology platform for molecularly targeted
therapy.
Results
Novel types of RNAs induced downregulation of VEGF
mRNA expression as effectively as siRNA
To determine whether our new synthetic RNAs, nkRNA and
PnkRNA, could induce knockdown of VEGF gene expression,
we transfected these RNAs and conventional siRNA (NI) into
the mouse endothelial cell line UVfemale2 and analyzed theexpression of VEGF in the culture medium by ELISA. VEGF
expression in the culture medium was reduced in a dosedependent manner in cells transfected with all three types ofRNAs. On the other hands, we observed no apparent knockdown of VEGF expression in cells transfected with scrambled
RNAs (Figure 1b). These data indicate that nkRNA and
PnkRNA can silence gene expression as efﬁciently as siRNA.

This work was done in Shinjuku, Tokyo, Japan.
1
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 2Animal Research Center, Tokyo Medical University, Tokyo, Japan; 3Department ofOphthalmology, Tokyo Medical University, Tokyo, Japan. Correspondence: Masahiko Kuroda, Department of Molecular Pathology, Tokyo Medical University, 6-1-1
Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. E-mail: kuroda@tokyo-med.ac.jp
Keywords: innate immune; molecular target therapy; RNAi; TLR
Received 21 May 2015; accepted 15 August 2015; published online 20 October 2015. doi:10.1038/mtna.2015.34-60-

Novel Types of Small RNA Do Not Activate the Innate Immune System
Takanashi et al.

2asiRNA(NI)nkRNA(n)PPbanalyzed type I IFN expression in nkRNA- and PnkRNAtransfected cells using the IFN ELISA kit. Although IFNα andIFNβ expression increased in a dose-dependent manner,
there was no apparent difference between cells transfectedwith nkRNA, PnkRNA, and NIRNA (Figure 3). Expression of
IFNβ was higher in cells transfected with NIRNA than in cells
transfected with nkRNA or PnkRNA (Figure 3). According tothese data, nkRNA and PnkRNA did not induce IFNβ expression. Therefore, we concluded that nkRNA and PnkRNA do
not activate an innate immune response in vitro.

PnkRNA(Pnk)

8

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Figure 1 Novel types of RNAs induced downregulation of VEGF
mRNA expression as effectively as siRNA. (a) Structure of novel
RNA interference agents. Both nkRNA and PnkRNA were preparedas single-stranded RNA oligomers that underwent self-annealing, asshown. P indicates a proline derivative. (b) 10, 50, 100, and 200 μg
of NIRNA, nkRNA, and PnkRNA against VEGF were transfected into
mouse endothelial cells. Twenty-four hours after transfection, VEGF
expression in the culture medium was measured using a VEGF
ELISA kit. siRNA, small interfering RNA; VEGF, vascular endothelial
growth factor.

nkRNA and PnkRNA induced TLR3 phosphorylation at
low levels
Double-stranded RNA, such as siRNA, is recognized by TLR3
and activates innate immunity via phosphorylation of TLR3 attyrosine 759.12,13 To determine whether nkRNA and PnkRNA
induce TLR3 phosphorylation, we transfected nkRNA,
PnkRNA, NIRNA, or p(I: C) to mouse endothelial cells. Western blot analysis of lysates from cells 1 hour after transfection
revealed that TLR3 was phosphorylated in cells that receivedp(I: C) or NIRNA. On the other hand, cells transfected with
nkRNA or PnkRNA exhibited lower levels of TLR3 phosphorylation (Figure 2a), indicating that these novel RNAs do not
activate TLR3. Phosphorylation of TLR3 leads to the activation of the NF-κB transcription factor.14–16 Activation of TLR3 by
stimuli, such as viral dsRNA, leads to elevated expression of
inﬂammatory cytokines, mediated by the NF- κB transcription
factor. Therefore, to determine whether nkRNA and PnkRNA
can induce NF-κB activation, we performed an NF-κB reporter
assay using a vector encoding secreted alkaline phosphatase(SEAP) cDNA under the control of NF-κB response element.
SEAP expression was lower in nkRNA- or PnkRNA-transfected
cells than in NIRNA-transfected cells, and this reduction wasdose dependent (Figure 2b). From these data, we concluded
that nkRNA and PnkRNA were barely recognized by TLR3.
nkRNA and PnkRNA did not induce an innate immune
response, as demonstrated by expression of type I IFNs
The type I IFN pathway is typically associated with the innate
immune response to the adaptors of TLR3.17–19 Hence, weMolecular Therapy—Nucleic Acids-61-

nkRNA and PnkRNA did not induce expression of IFNβ
in murine eyes
Next, we asked whether administration of nkRNAs and
PnkRNA into murine eyes would induce expression of type
I IFNs. To address this question, we injected 1 μg of nkRNA,
PnkRNA, or NIRNA into murine eyes. Twenty-four hours after
injection, we excised the eyes, extracted RNA from them, andanalyzed expression of IFNα and IFNβ by real-time quantitative PCR. IFNα was expressed at comparably low levels inall samples, whereas, IFNβ was expressed at lower levels
in transfected with nkRNA and PnkRNA than in cells transfected with NIRNA (Figure 4). Thus, nkRNA and PnkRNAbarely induced innate immunity in murine eyes.
nkRNA and PnkRNA inhibited laser-induced CNV in amouse model
To determine whether the novel RNAs, which do not extensively activate innate immunity via TLR3, were able to reduce
CNV in mice, we evaluated angiogenesis, as determined byleakage of blood, from neovascularization in response tolaser coagulation (three shots per eye). The blood leakage
scores in CNV mice were scored on a scale from 0 to 3; weestimated score for leakage of blood from vessels in eye. The
scores were higher in mice that received NIRNA, nontreated,and scramble RNAs than in mice that received nkRNA or
PnkRNA (Figure 5a).
We next examined the ability of nkRNA and PnkRNA to
reduce angiogenesis by administering novel RNAs into thevitreous cavity of mice and comparing the CNV volume following laser coagulation 7 days after injection of RNAs.
CNV volume was signiﬁcantly reduced in eyes that received
nkRNA and PnkRNA than in eyes that received NIRNA orscramble RNAs (Figure 5b). These data show that nkRNAand PnkRNA suppressed angiogenesis, indicating that thenovel RNAs could be useful as therapies against CNV due
to AMD.
Discussion
siRNAs have attracted a great deal of attention as a new
therapeutic platform for achieving target-speciﬁc gene silencing via double-stranded RNA (dsRNA)-mediated RNAi. However, the immune side effects due to TLRs activation are still
problematic issues in the context of therapeutic applications.
Recent work showed that siRNA activates TLR3, a long
double-stranded viral RNA sensor, and activated TLR3 mediates target-independent angiogenesis suppression of CNV inmouse models.10

Novel Types of Small RNA Do Not Activate the Innate Immune System
Takanashi et al.

b

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control

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pTLR/TLR3 expression ratio

2.0

Figure 2 nkRNA and PnkRNA induced TLR3 phosphorylation at low levels. (a) Phosphorylated TLR3 was analyzed by western blotting.
Ten micrograms of protein was loaded in each lane. The ratio of phosphorylated TLR3 to total TLR3 is shown in the graph. (b) NF-κB activity
was measured using an NF-κB reporter in which SEAP cDNA was placed under the control of the κB response element. The numbers on
x-axis show RNA concentrations (nM). SEAP, secreted alkaline phosphatase; siRNA, small interfering RNA; TLR, Toll-like receptor.

baIFN-β expression (pg/ml)

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250

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Figure 3 nkRNA and PnkRNA did not induce an innate immune response, as demonstrated by expression of type I IFNs. (a,b) Levelsof IFNα (a) and IFNβ (b) from mouse endothelial cells in culture medium 24 hours after transfection of nkRNA, PnkRNA, NIRNA, or TLR
ligands were measured by specific ELISA. The numbers in x-axis showed concentrations (nM) of RNAs. TLR3, TLR7, or TLR9 revealed theligand against each receptor. IFN, interferon; TLR, Toll-like receptor.

Recently, we developed a novel class of RNAi agents.11
Each of these agents is prepared as a single-stranded RNA
that self-anneals into a unique structure containing a double-stranded RNA with an unpaired site, bound at the rightand left ends by an oligonucleotide loop or a non-nucleotide
molecule (proline derivative). Here, we showed that thesenovel RNAi therapeutic agents suppress expression of target genes without inducing type I IFN responses. In general, double-stranded RNAs are recognized by members ofthe membrane TLR and RIG-I–like receptor families (RIG-I,
MDA5, and LGP2).20,21 When double-stranded RNA enters
cells, both types of receptors activate NF-κB and/or IRF3/
IRF7.20,22–25 In this study, we conﬁrmed that nkRNA and

PnkRNA induced low levels of TLR3 phosphorylation and
NF-κB activation. In addition, we examined the type I IFN
response. Together, these data demonstrated that nkRNAand PnkRNA can achieve sequence-speciﬁc gene silencing
without activating the TLRs and RIG-I–like receptor.
How do nkRNA and PnkRNA evade the TLRs and RIGI–like receptors? TLR3 recognizes double-stranded RNA,and recognition of ligands by TLR3 triggers signaling pathways that lead to the activation of transcription factors
such as NF-κB, AP-1, and IRFs, which regulate the production of proinﬂammatory cytokines and type I IFNs.23,26–29
Because NIRNA is a double-stranded RNA, it is recognized
by TLR3 and triggers the innate immune system. On the
www.moleculartherapy.org/mtna-62-

Novel Types of Small RNA Do Not Activate the Innate Immune System
Takanashi et al.

4

b

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Relative expression of IFNβ mRNA

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3.5

nk-sc

a

Figure 4 nkRNA and PnkRNA did not induce expression of IFNβ in murine eyes. To evaluate of activation for innate immune in vivo by
nkRNA and PnkRNA, eyes of C57BL/6J mice were injected 1 μg of nkRNA, PnkRNA, or NIRNA. (a) IFNα and (b) IFNβ mRNAs from murine
eyes 24 hours after administration mRNA were quantitated by real-time PCR. IFN, interferon.

ab*(P < 0.05)*(P < 0.05)*(P < 0.05)

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Nontreat

Figure 5 nkRNA and PnkRNA inhibited laser-induced CNV in a mouse model. (a) Fluorescein angiography. Neovascularization was
evaluated based on leakage of blood fluid from neovasculature by fluorescein angiography. (b) Confocal micrograph of laser-induced CNV inmice. The retinas of eyes on RNA-treated mice after laser-induced coagulation were used to generate choroidal flatmounts, which were thenexamined with a scanning laser confocal microscope to visualize vessels. Vessels in the laser-treated area and superficial to this reference
plane were judged as CNV. The area of CNV-related fluorescence was measured. CNV, choroidal neovascularization.

other hand, nkRNA and PnkRNA are prepared as singlestranded RNAs, but form double-stranded RNAs via selfannealing11 (Figure 1a). These double-stranded forms may
enable the novel RNAs to escape recognition by TLR3. In
addition, the ligand that activates TLR3 is double-stranded
RNA or oligonucleotides of at least 40–50 base pairs inlength.30,31 Although in this study we used oligonucleotides
41 (NIRNA), 62 (nkRNA), and 53 (PnkRNA) base pairs inlength, only NIRNA activated TLR3. The structural conformation of the ligand of TLR3 plays an important role in recognition of TLR3.6,32–37 Furthermore, mutated virus with analtered conformation can escape the innate immune system, because such viruses are not recognized by TLRs.38,39
Molecular Therapy—Nucleic Acids-63-

Because nkRNA and PnkRNA have loops at both ends, inbetween the sense and antisense nucleotide sequences,
we concluded that the conformations of these unique RNA
structures might be altered; therefore, these recognition ofthese RNAs by TLR3 was signiﬁcantly impaired. PnkRNAs
have prolines in both loops of the nucleotide sequence, andwe predicted that the resultant alteration in structural conformation affected recognition of ligand by TLRs. The resultsof TLR3 phosphorylation indicated at low levels of thephosphorylation in the cell gene transferred with PnkRNA
than that in the cell transferred with nkRNA. In addition, thedata regarding NF-κB activation and IFNβ expression wereconsistent with the results described above.

Novel Types of Small RNA Do Not Activate the Innate Immune System
Takanashi et al.

5

The application of RNAi to clinical medicine is still limited, mainly due to activation of the innate immune system via TLRs. The VEGF silencing reagent causes side
effects and exhibits sequence-independent suppression ofangiogenesis.10
We conﬁrmed the therapeutic effect of the novel RNAs ina mouse model of laser-induced CNV. nkRNA and PnkRNA
were not associated with off-target expression of inﬂammatory cytokines in mice. In vivo data revealed that the novelRNAs, nkRNA and PnkRNA, repressed angiogenesis due
to laser-induced coagulation. Thus, these novel RNAs may
be effective as therapeutic reagents against CNV. Notably,
escape from TLR3 recognition and VEGF mRNA silencing
were better for PnkRNA than for nkRNA, indicating that is thesuperior therapeutic reagent. Accordingly, we concluded thatnkRNA and PnkRNA may have great clinical utility as therapies against blinding CNV due to AMD. In particular, PnkRNA
may become useful as a therapeutic agent against CNV.
Materials and methods
Cells. The mouse endothelial cell line, UVfemale2,40,41 waspurchased from the RIKEN cell bank and cultured in Dulbecco’s Modiﬁed Eagle’s medium (Life Technology, Carlsbad,CA) containing 10% fetal calf serum (FCS) penicillin andstreptomycin. siRNA (NIRNA) or novel RNA oligonucleotides(nkRNA and PnkRNA) targeting mouse VEGF were transfected into endothelial cells using HiPerFect (Qiagen, Valencia,
CA). One hour after transfection, cells were collected, andproteins were extracted for subsequent investigations.
RNA interference. siRNA (NI) and novel RNAs used in thisstudy were as follows. Mouse VEGF (NI): sense 5′-ACCUCACCAAAGCCAGCACT-3′, antisense 5′- GUGCUGGCUUUGGUGAGGUTT-3′; mouse NI scramble: sense (NIsc) 5′- GCACAAC
ACCCGCUCACAATT-3′, antisense 5′- UUGUGAGCGGGUGUUGUGCTT-3′; mouse VEGF nkRNA (nk): 5′- AACCUCACCAAAGCCAGCACUUCCCCACACCGGAAGUGCUGGCUUUGGUGAGGUUUCUUCGG-3′; mouse nk scramble RNA(nksc): 5′- AGCACAACACCCGCUCACAAUUCCCCACACCGGAAUUGUGAGCGGGUGUUGUGCUUCUUCGG-3′;
mouse VEGF PnkRNA (Pnk): 5′-AACCUCACCAAAGCC
AGCACUUCC-P5-GGAAGUGCUGGCUUUGGUGAGG
UUUC-P5-G-3′; mouse Pnk scramble RNA (Pnksc):
5′- AGCACAACACCCGCUCACAAUUCC-P5-GGAAUUGUG
AGCGGGUGUUGUGCUUC-P5-G-3′.
Enzyme-linked immunosorbent assay. IFN and VEGF protein
levels were measured in culture medium of cells transfectedwith nkRNA, PnkRNA, or NIRNA, 24 hours after transfection,using cytokine-speciﬁc ELISA kits as follows: IFNα, VeriKine
Interferon ELISA kit (PBL Biotechnology, Norwich, UK); IFNβ,
EN424001 (Thermo Fisher Scientiﬁc, Waltham, MA); IFNγ,
EM1011 (Thermo Fisher Scientiﬁc); and VEGF, Quantikine
Mouse VEGF kit (R&D Systems, Minneapolis, MN).
SEAPorter assay. NF-κB activation by nkRNA, PnkRNA, or
NIRNA transfection in cells was assayed using the SEAPorter
assay kit (IMK-515; IMGENEX, San Diego, CA). Twenty-fourhours after transfection, culture media from the oligo-transfected endothelial cells were analyzed.
Real-time quantitative PCR. RNAs were isolated from mouseeyes 24 hours after inoculation of 1 μg nkRNA, PnkRNA, or
NIRNA per body using the Isogen reagent (Nippon Gene,
Tokyo, Japan). Complementary DNA was synthesized using
SuperScript II and Random Hexamers (Life Technology).
Quantitative PCR (FastStart Universal SYBR Green Master;Roche, Basel, Switzerland) reactions were run on a Stratagene
MX3000P thermocycler and analyzed using MxPro (Stratagene; Agilent Technologies, Santa Clara, CA). Glyceraldehyde
3-phosphate dehydrogenase mRNA was used as an internal control. IFN primers used in this study were as follows:
IFNα (forward) 5′-TCTGATGCAGCAGGTGGG-3′, (reverse)
5′-AGGGCTCTCCAGACTTCTCGC-3′; IFNβ (forward)
5′-AGCACTGGGTGGAATGAGAC-3′, (reverse) 5′-TCCCACG
TCAATCTTTCCTC-3′; IFNγ (forward) 5′- CACGGCACAGTCATTGAAAG-3′, (reverse) 5′-TTTTGCCAGTTCCTCCAGAT-3′;
glyceraldehyde 3-phosphate dehydrogenase (forward) 5′-AA
AATGGTGAAGGTCGGTGTG-3′, (reverse) 5′-TGGCAACAATCTCCACTTTG-3′.
Western blot method. Protein samples were suspended in
sodium dodecyl sulfate loading buffer. After boiling, equalamounts (10 μg) of the proteins were run on 7.5% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis gels andthen transferred to Immobilon membranes (Millipore, Bedford, MA) by semidry blotting. The membranes were probedwith anti-TLR3 (Sc-86912; Santa Cruz Biotechnology, Dallas,
TX) or anti-phospho-TLR3 (IMG-5348A; Imgenex/Novus Biologicals, Littleton, CO) antibodies using standard techniques.
The signals were visualized using the ECL Plus Western
Blotting Detection System (GE Healthcare, Piscataway, NJ)
and detected on a LAS-3000 mini (Fujiﬁlm, Tokyo, Japan).
Animals. All animal experiments were in accordance with theguide lines of Tokyo Medical University Institutional Animal
Care and Use Committee. Female C57BL/6J mice (Japan
SLC, Hamamatsu, Japan) between the age of 6 and 8 weeks
were used to minimize variability. For all procedures, anesthesia was achieved by injection of under ketamine–xylazine
anesthesia (120 and 6 mg/kg, respectively, i.p.).
Induction of CNV. Laser photocoagulation (532 nm, 200 mW,
100 ms, 75 μm, GYC-2000 Green/Dye; Nidek, Gamagori,
Japan) was performed in three spots on one eye of eachmouse. The laser spots were evaluated for the presence of
CNV on day 7 by ﬂuorescence angiography.
Injection of siRNA or novel RNAs into mice. On day 7 afterlaser photocoagulation, mice were anesthetized, and weadministrated 1 μg of NIRNA, nkRNA, or PnkRNA into thevitreous cavity using a 32-gauge needle attached to a Hamilton syringe (Hamilton, Reno, NV).
CNV volume. Laser-induced CNV volume was measured
by ﬂuorescence angiography of Phoenix Micron III Retinal
Imaging Microscope (Phoenix Research Lab, Pleasanton,CA) following i.p. injection of 0.1 ml of 1% FLUORESCITE
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Novel Types of Small RNA Do Not Activate the Innate Immune System
Takanashi et al.

6(Alcon, Tokyo, Japan) into each mouse. CNV of retina wasevaluated with ﬂuorescence leakage of three categories (bygrading score: I–III). Mice were anesthetized (ketamine/xylazine) and perfused through the heart with 1 ml of phosphatebuffered saline containing 50 μg ﬂuorescein-labeled dextran(FITC-Dextran; Sigma-Aldrich, St. Louis, MO). The eyes were
removed and ﬁxed with 10% buffered formalin. The cornea
and the lens were removed and the neurosensory retina wascarefully dissected from the eyecup. Four relaxing radial incisions were made, and the remaining retinal pigment epithelium–choroid–sclera complex was ﬂatmounted, Flatmounts
were examined with a scanning laser confocal microscope(LMS710; Carl Zeiss, Oberkochen, Germany). Vessels werevisualized by laser excitation and capture and capturing.
About 1-μm step of horizontal optical sections were obtained
from the surface of the retinal pigment epithelium–choroid–sclera complex. The area of CNV-related ﬂuorescence wasmeasured by computerized image analysis using the microscope software (ZEN; Carl Zeiss). The sum of the all areaof CNV-related ﬂuorescence in each horizontal section wasused as an index of the CNV volume.
Statistical analysis. Statistical evaluation was performed on
the basis of Student’s t-test or Welch’s t-test. P values lessthan 0.05 were considered signiﬁcant.
Acknowledgments. This research was supported in part by
Grants-in-Aid for scientiﬁc research (B) and Grant-in-Aid for
Exploratory Research from Japan Society for the Promotionof Science (JSPS) and supported in part by Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Scienceand Technology, Japan (MEXT).
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RESEARCH ARTICLE

Angiopoietin-Like 7 Is an Anti-Angiogenic
Protein Required to Prevent Vascularizationof the Cornea
Tetsuya Toyono1, Tomohiko Usui1*, Seiichi Yokoo1, Yukako Taketani1,
Suguru Nakagawa1, Masahiko Kuroda2, Satoru Yamagami1, Shiro Amano1
1 Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan,
2 Department of Pathology, Tokyo Medical University, Tokyo, Japan* tomohiko-tky@umin.ac.jp

Abstract
PurposeWe sought to identify the anti-angiogenic molecule expressed in corneal keratocytes that is
responsible for maintaining the avascularity of the cornea.
OPEN ACCESS
Citation: Toyono T, Usui T, Yokoo S, Taketani Y,
Nakagawa S, Kuroda M, et al. (2015) AngiopoietinLike 7 Is an Anti-Angiogenic Protein Required toPrevent Vascularization of the Cornea. PLoS ONE
10(1): e0116838. doi:10.1371/journal.pone.0116838
Academic Editor: Atsushi Asakura, University of
Minnesota Medical School, UNITED STATES
Received: March 26, 2014

Methods
Human umbilical vein endothelial cells (HUVECs) were cultured with either human dermal
fibroblasts or with human corneal keratocytes under serum-free conditions. The areas thatexhibited blood vessel formation were estimated by immunostaining the cultures with anantitibody against CD31, a blood vessel marker. We also performed microarray geneexpression analysis and selected one molecule, angiopoietin-like 7 (ANGPTL7) for further
functional studies conducted with the keratocytes and in vivo in mice.

Accepted: December 15, 2014

Results

Published: January 26, 2015

Areas showing blood vessel formation in normal serum-free medium were conditions weremarkedly smaller when HUVECs were co-cultured with corneal keratocytes than when they
were co-cultured with the dermal fibroblasts under the same conditions. Microarray analysis
revealed that ANGPTL7 expression was higher in keratocytes than in dermal fibroblasts. In
vitro, inhibiting ANGPTL7 expression by using a specific siRNA led to greater tube formation
than did the transfection of cells with a control siRNA, and this increase in tube formation
was abolished when recombinant ANGPTL7 protein was added to the cultures. In vivo,
intrastromal injections of an ANGPTL7 PshRNA into the avascular corneal stroma of mice
resulted in the growth of blood vessels.

Copyright: © 2015 Toyono et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source arecredited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.
Funding: A grant-in-aid for Scientific Research fromthe Japanese Ministry of Education, Culture, Sports,
Science and Technology. The funders had no role instudy design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declaredthat no competing interests exist.

Conclusions
ANGPTL7, which is abundantly expressed in keratocytes, plays a major role in maintaining
corneal avascularity and transparency.

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Angiopoietin-Like 7 Is a Corneal Anti-Angiogenic Protein

Introduction
The cornea is present on most of the surface of the eyeball, and this transparent tissue is avascular. Corneal avascularity is the result of an active process involving the production of antiangiogenic factors that counterbalance pro-angiogenic and pro-inflammatory factors. For example, corneal epithelial cells express soluble vascular endothelial growth factor receptor-1(sVEGFR1) to neutralize VEGF activity, even in normal corneas [1]. Keratocytes are the predominant cellular components of the corneal stroma and are similar to fibroblasts. When keratocytes are activated by pathological conditions such as trauma and inflammation, these cells
often transform into myofibroblasts, which produce an extracellular matrix, collagen degrading
enzymes, and cytokines [2]. These factors orchestrate the cellular behavior that is associatedwith the development of corneal neovascularization, which is one of the major causes of theloss of corneal transparency [2, 3]. Conversely, in the normal cornea, the keratocytes are quiescent [3] and likely maintain the corneal transparency and avascularity. However, the contributions of keratocytes to the preservation of corneal avascularity have not yet been
fully elucidated.
Angiogenesis is a complex biological process involving cell-to-cell interactions between thevessel-forming endothelial cells and the host microenvironment. Previously, an in vitro systemfor co-culturing human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) was previously established to mimic the anigiogenic microenvironment, and it
was used as a model system to explore the growth factors and regulatory networks that control
angiogenesis [4]. This system relies on fibroblasts secreting the necessary matrix componentsthat act as a scaffold for tube formation [4]. In this study, we first attempted to establish the coculture system by using HUVECs and cultivated human corneal keratocytes (HCKs) in order
to examine the properties of keratocytes that contributes to blood vessel tube formation. Next,
we performed microarray analysis to compare the gene-expression profiles of the HCKs withthat of the HDFs to identify potential anti-angiogenic genes in the HCKs. Lastly, the functionof one molecule identified from the microarray analysis, angiopoietin-like 7 (ANGPTL7), wasexamined both in vitro and in vivo.

Materials and Methods
Cell culture
Primary HDFs and HUVECs were purchased from Lonza (Allendale, NJ, USA). HDFs wereseeded in culture dishes with Dulbecco’s modified Eagle medium (DMEM)/F12 containing 2%
fetal bovine serum (FBS). The medium was changed every two days until the cells reached confluence. Primary HCKs were isolated from a corneoscleral button retrieved from an eye bank
(SightLife, Seattle, WA, USA) as previously described [5, 6]. Briefly, the corneal epithelium wasremoved from the stroma by scraping with a razor blade, and the Descemet’s membrane andendothelial cells were peeled away separately as a sheet by using fine forceps. A central corneal
button was cut using an 8.0 mm trephine (Biopsy Punch, Kai Medical, Gifu, Japan), and subsequently incubated overnight at 37°C in a basal medium DMEM/ F12 medium supplemented
with B27 (Invitrogen, Carlsbad, CA, USA) containing 0.02% collagenase (Sigma-Aldrich,
St. Louis, MO, USA). Next, the digested tissue and cells were dispersed by pipetting, and thencentrifuged at 800 x g for 5 min. After removing the supernatant, the keratocytes were resuspended in 1.0 mL of the basal culture medium and seeded into culture dishes. The medium waschanged every two days until the cells reached confluence. Second-passage cells were used in
the co-culture assays.

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Angiopoietin-Like 7 Is a Corneal Anti-Angiogenic Protein

Co-culture tube formation assay
HDFs and HCKs were seeded in 24-well plates (4 × 104 cells/well), and cultured in DMEM/F12
containing 2% FBS for five days. The medium was then changed to DMEM/F12 supplemented
with serum free B27 supplementation for two days. On day seven, HUVECs were seeded onto
confluent HDFs or HCKs (2 × 104 cells/well), and these co-cultures were grown in the DMEM/
F12 supplemented with serum-free B27 for an additional 11 days. The formation of blood vesseltubes by the HUVECs was quantified by immunohistochemistry with the blood vessel marker,
CD31. On day seven, the number of cells in several wells was determined by staining them with
40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) and performing a nonradioactive colorimetric assay with water-soluble Tetrazolimum salt (WST-1); Takara Bio, Inc., Shiga, Japan).

In vitro immunohistochemistry in vitro
Tube formation was quantified by using a tubule-staining kit (Kurabo, Okayama, Japan) as per
the manufacturer’s protocol. Briefly, on day 18, co-cultured cells were fixed in 70% ice-coldethanol for 30 min at room temperature. Next, the cells were washed thrice in blocking solution(phosphate buffered saline (PBS) with 1% bovine serum albumin) and then incubated with amonoclonal anti-human CD31 antibody (1:4000 dilution; Kurabo) and incubation at 37°C for
60 min. The cells were again washed with blocking solution and then incubated with a secondary antibody (1:500, goat anti-mouse IgG conjugated to alkaline phosphatase; Kurabo) at 37°C
for 60 min. The immunoreactivity was detected by incubating cells for 5 min with 5-bromo-4chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT, Kurabo).
Quantitative image analysis of the CD31 immunoreactivity was conducted using NIH ImageJ software (http://rsb.info.nih.gov/ij/). Twelve fields (40x magnification) of each group werechosen in a randomly for the analysis. All compared images were captured using the same exposure and all photography settings were used at under-saturation levels. The images of theimmunostained cells (CD31-positive cells) were transformed to gray-scale images and an optical density plot of the selected areas was generated using the Histogram tool. The immunostaining area (arbitrary unit, AU) was calculated as the difference between membrane orcytoplasm immunostaining and the background. Lastly, the area was normalized using the
pixel area. All of the data were analyzed using the statistical software JMP Pro version 10.0.2(SAS Institute Inc., Cary, NC, USA), and the Mann-Whitney U test was used for comparing
the differences.

WST-1 assay
To confirm the cellular viabilities of the HDFs and HCKs, a WST-1 assay (Takara Bio) was performed as described previously. Briefly, the WST-1 reagent was applied to the confluent HDFs
and HCKs in DMEM/F12 medium, and after incubation at 37°C for 1 h, the dye intensity at
450 nm was measured using a plate reader (Victor 3V Multilabel Counter Model 1420; Perkin
Elmer, Waltham, MA, USA). The results were expressed as the mean percentage of viable
cells ± standard deviation (SD).

Microarray analysis
HDFs and HCKs were seeded at 4 × 104 cells/well in 24-well tissue culture plates and cultured
in DMEM/F12 supplemented with serum-free B27 for 48h. Total RNA was extracted from thecells by using Isogen (Nippon gene, Toyama, Japan) as per the manufacturer’s instructions.
Quantification and quality assessment of the isolated RNA samples were performed using a
NanoDrop2000 (Thermo Scientific, Waltham, MA, USA) in accordance with the

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Angiopoietin-Like 7 Is a Corneal Anti-Angiogenic Protein

manufacturer’s instructions. A260/A230 and A260/A280 ratios between 1.8 and 2.1 were confirmed in all RNA samples. RNA was amplified into cRNA and labeled according to the Agilent
One-Color Microarray Based Gene Expression Analysis protocol (Agilent Technologies, Palo
Alto, CA, USA). Global gene-expression analysis was performed using a SurePrint G3 humangene expression 8×60 microarray kit (G4851A, Agilent Technologies) which contained 27,958
Entrez Gene RNAs and 7419 large intergenic noncoding RNAs (lincRNAs). To confirm the results of microarray analysis, we performed quantitative RT-PCR (qRT-PCR) and western
blot analysis.

qRT-PCR
Total RNA was extracted from the cells or corneas by using Isogen, and cDNA was synthesizedusing reverse transcriptase (PrimerScript RT Master Mix, Takara Bio). qRT-PCR was performed using SYBR green master mix reagents (SYBR Premix Ex Taq (Tli RNase H Plus),
Takara Bio) with the following temperature profile; 95°C for 5 sec, followed by 40 cycles of
95°C for 5 sec and 60°C for 30 sec. Total reaction volume was 25 mL including SYBR green
master mix (Takara Bio) with cDNA transcribed from 100 ng of RNA and 0.4 mM forward
and reverse primers (Table 1). The comparative Ct (ΔΔCT) method was used to compare theTable 1. Primer sets for qPCR analysis.
Target gene

Sequence

Human ANGPTL1

forward, 50 - GGATTCTATGATGTGGCATAATGGT −30
reverse, 50 - AATTCCATCTTGGTGCTTGCT −30

Human ANGPTL2

forward, 50 - GGAGGTTGGACTGTCATCCAGAG −30
reverse, 50 - GCCTTGGTTCGTCAGCCAGTA −30

Human ANGPTL3

forward, 50 - ACAGTTCCACGTTGCTTGAA −30

Human ANGPTL4

forward, 50 - GGACAAGAACTGCGCCAAGAG −30

reverse, 50 - CCCAACTGAAGGAGGCCATT −30
reverse, 50 - AGGTTGGAATGGCTGCAGGT −30
Human ANGPTL5

forward, 50 - TTAAGGATACCATTGGCTCTGTCAC −30
reverse, 50 - CCTCTGGAAATCAATTATCCCATCA −30

Human ANGPTL6

forward, 50 - CAGTACCATGGTGATGCTGGAGA −30
reverse, 50 - AGTGGGCACAGGCATGGTA −30

Human ANGPTL7

forward, 50 - CGGCTGCGTGTAGAGATGGA −30

Human AKR1B10

forward, 50 - GGCAACCATACTCAGCTTCAACAG −30

reverse, 50 - CCTTGGTGCTGAAGGCTGTGT −30
reverse, 50 - GGGACATGAGTGGAGGTAGTCACA −30
Human GRP

forward, 50 - TTTGCTGGGTCTCATAGAAGCAAAG −30
reverse, 50 - TCACGTTGAGAACCTGGAGCA −30

Human PIP

forward, 50 - GCTTGCTCCAGCTCCTGTT −30
reverse, 50 - TGGACGTACTGACTTGGGAATG −30

Human COL1A1

forward, 50 - GCTTGCTCCAGCTCCTGTT −30

Human GAPDH

forward, 50 - TTGATTTTGGAGGGATCTCG −30

reverse, 50 - TGGACGTACTGACTTGGGAATG −30
reverse, 50 - GAGTCAACGGATTTGGTCGT −30
Mouse ANGPTL7

forward, 50 - AATGAACATATCCACCGGCTCAC −30
reverse, 50 - AGTTCATTGCCCAACGCAAAG −30

Mouse GAPDH

forward, 50 -CACATTGGGGGTAGGAACAC−30
reverse, 50 -AACTTTGGCATTGTGGAAGG−30

doi:10.1371/journal.pone.0116838.t001

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Angiopoietin-Like 7 Is a Corneal Anti-Angiogenic ProteinmRNA expression levels of the genes of interest. The gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chosen as an internal control.

Western blot analysis
Samples of the culture media were also collected to determine the ANGPTL7 production. An
equal volume of the SDS sample buffer (Lane Marker Reducing Sample Buffer; Thermo Scientific,
Waltham, MA, USA) was added to the collected medium, mixed and boiled for 5min. The samples (50 mg/lane) were electrophoresed on 12% acrylamide gel (Bio-Rad, Hercules, CA, USA) andsubsequently transferred to poly-vinylidene difluoride membranes (Bio Rad) for western blot
analysis. Membranes were incubated with an anti-angptl7 polyclonal antibody (1:100; Abcam,
Cambridge, UK) for 60 min at room temperature and immunostaining was detected using the alkaline phosphatase technique performed using a BCIP/NBT immunoblotting kit (Bio Rad).

Suppression of Angptl7 expression in vitro
HCKs were seeded in 24-well tissue culture plates and seeded at 4 × 104 cells/well in DMEM/
F12 supplemented with 2%FBS and grown for 3 days. The ANGPTL7 short interfering RNA(siRNA) (50 nM, sc-88201, Santa Cruz Biotechnology, Dallas, TX, USA) and Control siRNA(50 nM, sc-37007, Santa Cruz Biotechnology) were transfected into the cells by using Lipofectamine RNAiMAX (Invitrogen), and the cells were incubated for an additional 48 h. The medium was then replaced with DMEM/F12 supplemented with serum-free B27 for another 48 or
96 hours. Semiquantitative RT-PCR was performed using specific primers to monitor the
ANGPTL7 gene-expression knockdown.
We also transfected the ANGPTL7 siRNA and Control siRNA into co-cultures of HCKs and
HUVECs and then examined tube formation. The HUVECs were seeded onto confluent HCKs
(20,000 cells/well) and were cultured in serum-free medium with ANGPTL7 siRNA and Control siRNA (serum free) for 6 days. To several of the wells, we also added human recombinant
ANGPTL7 (500 ng/mL, 914-AN-025/CF, R&D systems, MN, USA). Tube formation by the
HUVECs was then quantified by means of CD31 immunohistochemistry. Furthermore, we
also measured the mRNA expression levels of other ANGPTL family members (1–6) after
transfecting cells with ANGPTL7 siRNA for 48 h.

In vivo suppression of Angptl7 expression in mouse cornea
Under direct microscopic observation, a custom made 33-gauge needle (30° bevel) attached toa 5-mL syringe (ITO, Shizuoka, Japan) was manually introduced into the corneal stroma andadvanced to the center, where 5 ml of 1 mM ANGPTL7 PshRNA or Control PshRNA (Bonac,
Fukuoka, Japan) was forcibly injected into the stroma. PshRNAs were synthesized in solid
phase as single-stranded RNAs that, following synthesis, self-anneal into a unique helical structure containing a single stem loop (Fig. 1). This stem loop contained proline derivatives similar

Figure 1. PshRNA platform. PshRNA was synthesized in solid phase as single-stranded RNAs that,
following synthesis, self-anneal into a unique helical structure containing a single stem loop.
doi:10.1371/journal.pone.0116838.g001

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to PnkRNA as previously described [7]. Seven days after the injection, the corneas were harvested to quantify the corneal neovascularization.

Quantification of hemangiogenesis in vivo
Hemangiogenesis was quantified by performing corneal whole-mount CD31 staining as previously described [8]. Briefly, following neovascularization, we obtained a corneal button thatwas excised 0.5mm posterior from the limbal vessel and rinsed in PBS, and then fixed in acetone for 10 min at-20°C. Subsequently, the corneas were washed thrice in PBS, blocked with
3% BSA in PBS for 48 h at room temperature, stained with a rat anti-mouse CD31 antibody(BD Biosciences, Franklin Lakes, NJ) and incubated at 4°C overnight. The corneas were again
washed with PBS and stained with a secondary antibody (Alexa Fluor 594 donkey anti-rat IgG;
Invitrogen) for 3 h at room temperature. The stained whole mounts of corneas were placed on
glass slides and examined under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).
ImageJ was used for image analysis. Neovascularization was quantified by setting a thresholdlevel of CD31-positive fluorescence, above which only vessels were depicted. Neovascularization was quantified in a masked manner. The vascularization area was outlined by using the innermost vessel of the limbal arcade as the border, and the surface area of corneal
neovascularization (vascularized area/total corneal area) was quantified.

Ethics statement
This study adhered to the Association for Research in Vision and Ophthalmology Statement
for the Use of Animals in Ophthalmic and Vision Research and was approved by the University of Tokyo Hospital Animal Care Committee. The animals were allowed free access to foodand water. A 12-h light-dark cycle was maintained. All surgical procedures were performed
under general anesthesia by injecting the mice (8-week-old males C57BL/6) with xylazine hydrochloride (5mg/kg of body weight) and ketamine hydrochloride (35mg/kg of body weight).
The animals were euthanized by an intraperitoneally injecting them with Nembutal (60 mg/kg
of body weight) (Sigma) before harvesting the eyes. All efforts were made tominimize suffering.

Statistical analysis
All of the data were analyzed using the statistical software (JMP Pro version 10.0.2), and valuesof p < 0.05 were considered statistically significant.

Results
Tube formation was weaker on corneal cells than on dermal fibroblasts
The HCKs and HDFs were cultured for 7 days and then HUVECs were co-cultured with them.
Before the adding HUVECs to HCKs and HDFs, the numbers of the HCKs and HDFs at confluence were not significantly different, as shown by DAPI staining (cell counts/field; HCKs
158.2±15.2, HDFs 149.8±18.7, p = 0.12). Moreover, results of the WST-1 assay revealed thatthe viability the HCKs and HDFs at confluence did not differ significantly (relative viabilities,
HCKs:HDFs = 1:0.94; p = 0.43).
We assessed tube formation in both co-culture models by immunostaining the cultures withan anti-CD31 monoclonal antibody. The areas of tube formation by HUVECs under the normal serum-free medium conditions were 6.81 ± 1.07% in co-culture with HCKs and 10.37 ±
2.52% in the co-culture with HDFs (p < 0.0001, Fig. 2). This result suggested that HCKs provided a more anti-angiogenic environment for the HUVECs than did the HDFs.

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Figure 2. Tube formation by HUVECs co-cultured with corneal cells or dermal fibroblasts underserum-free conditions. The areas of tube formation by HUVECs under the normal serum-free medium
conditions were 6.81 ± 1.07% in co-culture with the human corneal keratocytes (HCKs, A) and 10.37 ± 2.52%
in co-culture with the human dermal fibroblasts (HDFs, p < 0.0001, B and C). N = 12 per condition.
doi:10.1371/journal.pone.0116838.g002

Microarray gene expression analysis
To investigate the difference between the tube-formation responses in the two co-culture models, we performed microarray analysis. Table 2 shows the genes that were most upregulated in
HCKs relative to those in HDFs; the Supplemental Data section lists 100 upregulated anddownregulated genes in HCKs. We performed qRT-PCR to confirm the differential expression
Table 2. Upregulated genes in HCKs relative to those in the HDFs.
Ofﬁcial Full Name

Gene Symbol

Fold change

Location

Unigene ID

aldo-keto reductase family 1, member B10

AKR1B10

13.38

hsj7q33

Hs.116724

gastrin-releasing peptide

GRP

12.78

hsj18q21.32

Hs.153444

angiopoietin-like 7

ANGPTL7

12.34

hsj1p36.22

Hs.146559

aldo-keto reductase family 1, member B15

AKR1B15

12.22

hsj7q33

Hs.116724
Hs.99949

prolactin-induced protein

PIP

10.06

hsj7q34

cancer/testis antigen 1A (CTAG1A)

CTAG1A9.77

hsjXq28

Hs.534310

carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 6

CHST6

9.46

hsj16q23.1

Hs.655622

transmembrane 4 L six family member 1 (TM4SF1)

TM4SF1

9.40

hsj3q25.1

Hs.351316

secretogranin II

SCG2

9.36

hsj2q36.1

Hs.516726

cytokine-like 1

CYTL1

9.34

hsj4p16.2

Hs.13872

ATPase, aminophospholipid transporter (APLT), class I, type 8A, member 1

ATP8A1

9.08

hsj4p13

Hs.435052

solute carrier family 4, sodium bicarbonate cotransporter, member 4

SLC4A49.01

hsj4q13.3

Hs.5462transmembrane protein 155 (TMEM155)

TMEM155

8.53

hsj4q27

Hs.27524

Glutathione S-transferase theta 1

GSTT1

8.30

hsj22q11.23

Hs.268573

sushi domain containing 2

SUSD2

8.01

hsj22q11.23

Hs.131819

aldehyde dehydrogenase 3 family, member A1

ALDH3A1

7.78

hsj17p11.2

Hs.531682

nuclear receptor subfamily 0, group B, member 1

NR0B1

7.78

hsjXp21.2

Hs.268490

transcription factor AP-2 beta (activating enhancer binding protein 2 beta)

TFAP2B7.67

hsj6p12.3

Hs.33102hydroxysteroid (11-beta) dehydrogenate 1

HSD11B1

7.51

hsj1q32.2

Hs.195040

sclerostin

SOST

7.07

hsj17q21.31

Hs.349204

doi:10.1371/journal.pone.0116838.t002

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Figure 3. Relative expression of ANGPTL7 in HCKs and HDFs. AKR1B10, GRP, ANGPTL7, and PIPlevels were elevated in the cDNA derived from HCKs as compared to with that from HDFs (A). In western blot
analysis, ANGPTL7 was detected in culture media collected from HCKs but not i HDFs (B). N = 3
per condition.
doi:10.1371/journal.pone.0116838.g003

of several genes identified by means of microarray analysis. The expression of AKR1B10, GRP,ANGPTL7, and PIP was higher in HCKs than in HDFs (Fig. 3A). Furthermore, in western blot
analysis, ANGPTL7 was detected in the culture medium collected from the HCKs but not the
HDFs (Fig. 3B).

Inhibition of ANGPTL7 increased tube formation in the HCK-HUVEC coculture model
To elucidate the function of ANGPTL7 in angiogenesis, we analyzed the tube formation by the
HUVECs co-cultured with HCKs that had been transfected with an ANGPTL7 siRNA.
ANGPTL7 mRNA expression was drastically inhibited following the administration of the
ANGPTL7 siRNA, even at 240 h after transfection (Fig. 4A). The areas of tube formation innormal serum-free medium and after control siRNA treatment were 0.13 ± 0.15% and 0.31 ±
0.09%, respectively (Fig. 4B), but when the cells were transfected with the ANGPTL7 siRNA,the areas of tube formation were significantly increased when compared with the areas in thecontrol siRNA group (1.41 ± 0.54%, p < 0.001, Fig. 4B). However, this effect of the ANGPTL7
siRNA was significantly suppressed when human recombinant ANGPTL7 was added to thecells (0.69 ± 0.30%, p < 0.001, compared with the ANGPTL7 siRNA group; Fig. 4B).
We also analyzed whether the mRNA expression of other ANGPTL family members was altered after the suppression of ANGPTL7 expression. The expression levels of ANGPTL1 andANGPTL4 were decreased to < 60% relative to the control level, whereas the expression of
ANGPTL2 was increased by nearly 120% compared with control (Fig. 4C).

Blood vessel growth on the avascular corneas of mice after suppressionof gene expression by using an ANGPTL7 PshRNA
Next, to confirm the results derived from the tube formation assay in vitro, the hemangiogenic
responses in mice treated with ANGPTL7 PshRNA were investigated. The ANGPTL7 mRNA
expression was dramatically inhibited (almost 80% inhibition) by the intrastomal injection of
the ANGPTL7 PshRNA on 48 h after injection, but had been restored by 150 h (Fig. 5A). By
contrast, the corneas of mice injected with the control PshRNA did not exhibit any alteration(Fig. 5). Notably, the intrastromal injection of the ANGPTL7 PshRNA resulted in the spontaneous development of blood vessel growth from the limbal areas in the avascular mouse corneas
by day 4 after the injection (Fig. 5).

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Angiopoietin-Like 7 Is a Corneal Anti-Angiogenic Protein

Figure 4. ANGPTL7 siRNA tranfection increased tube formation in HCK-HUVEC co-culture. ANGPTL7 mRNA expression was drastically inhibitedfollowing the administration of ANGPTL7 siRNA for up to 240 h after transfection (A). The tube-formation areas under the normal serum-free medium(normal) and control siRNA treatment were 0.13 ± 0.15% and 0.31 ± 0.09%, respectively (B). In the cells transfected with the ANGPTL7 siRNA the tube
formation areas increased significantly (1.41 ± 0.54%, p < 0.001, B). This siRNA effect was potently suppressed when human recombinant ANGPTL7 (A7siRNA+rhANGPTL7) was added to the cells (0.69 ± 0.30%, p < 0.001, B). N = 12 per condition. In the cells in which ANGPTL7 expression was suppressed,
the mRNA expression levels of ANGPTL1 and ANGPTL4 were also decreased (by 48.4% and 56.1%, respectively), but the expression of ANGPTL2 wasincreased (119%). N = 2 per each condition.
doi:10.1371/journal.pone.0116838.g004

Discussion
In this study, we demonstrated that blood vessel formation by HUVECs in co-cultured together with corneal cells under serum-free conditions was considerably weaker than that by
HUVECs co-cultured with dermal fibroblasts. These results suggested that HCKs potentiallyprovide an anti-angiogenic environment for the HUVECs. Microarray analysis revealed that
ANGPTL7 was abundantly expressed in HCKs in comparison to HDFs. The suppression of
ANGPTL7 expression resulted in an angiogenic response both in vitro and in vivo. These results suggested that ANGPTL7, which is abundantly expressed in corneal cells, might be responsible for maintaining the corneal avascularity.
The ANGPTLs comprise a family of proteins structurally related to the angiopoietins [9].
ANGPTLs contain an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain,but, ANGPTLs do not bind to the angiopoietin receptor Tie1 or Tie2 [9]. ANGPTL7 was originally named corneal-derived transcript 6 (CDT6) [10], and it is still considered an orphan ligand. Because stable transfection of human melanoma cells with ANGPTL7/CDT6 led to a
reduction of tumor growth in a mouse xenograft model relative to the growth observed in controls, ANGPTL7 was speculated to function as a negative regulator of angiogenesis and to possibly contribute to corneal avascularity [10]. However, subsequent studies by Bouis et al

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Figure 5. Blood vessel growth on the avascular corneas of mice after suppression of gene expression
by using an ANGPTL7 PshRNA. ANGPTL7 mRNA expression was inhibited by almost 80% inhibition at 48
h after intrastomal injection of the ANGPTL7 PshRNA, but normal expression was restored by 150 h (A, n = 3
per condition). The corneas of mice injected intrastromally with the control PshRNA did not exhibit any
expression change (B, left panel). Notably, intrastromal injection of the ANGPTL7 PshRNA resulted in the
spontaneous development of blood vessel growth from the limbal areas in the avascular mouse corneas byday 4 after the injection (B, right panel). Areas of vessels in the mouse corneas were significantly different
following the injection of control PshRNA injection and ANGPTL7 PshRNA injection (p < 0.001, C, n = 4 percondition)
doi:10.1371/journal.pone.0116838.g005

demonstrated that ANGPTL7/CDT6 had no effect on tumor growth [11, 12], casting doubt on
the angiogenic properties of this gene. Our results have shown that siRNA-mediated suppression of ANGPTL7 expression led to blood vessel formation on HCKs and this was coupled witha change in the expression of other ANGPTLs (Fig. 4); When we suppressed ANGPTL7 expression in vitro using the siRNA, the expressions of ANGPTL1 and ANGPTL 4 were also decreased
but that of ANGPTL2 was increased (Fig. 4C). ANGPTL1 and ANGPTL4 have been reported
to function as anti-angiogenic factors under certain conditions [13]. Guo et al suggested that
ANGPTL4 exerts anti-angiogenic effects under physiological conditions [14]. By contrast,
ANGPTL2 has been considered to function as a pro-angiogenicfactor [8]. Therefore, this findings might partially explain why ANGPTL7 suppression could accelerate tube formation invitro (Fig. 4). Furthermore, although tube formation was decreased following the addition of

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Angiopoietin-Like 7 Is a Corneal Anti-Angiogenic Protein

recombinant ANGPTL7 (Fig. 4), this reversal effect of ANGPTL7 was not observed in the
HDF-HUVEC co-culture model (data not shown). The expression of ANGPTL7 was observed
to be elevated in the corneal tissue, but not in other tissues such as brain, liver, and heart [15],further suggesting that the corneal environment might be critical for normal ANGPTL activity.
This variation in ANGPTL7 activity could result from the the lack of expression of its receptor
in tissues other than the corneal tissue.
The expression of ANGPTL7 has also been observed in the trabecular meshwork, which islocated adjacent to the cornea and is the drainage site for aqueous humor [16, 17]. This tissue
is also avascular and the vessel invasion to the trabecular meshwork in hypoxic retinal diseases
such as diabetic retinopathy is observed in the form of severe glaucoma (neovascular glaucoma). Therefore, the expression of ANGPTL7 in the trabecular meshwork might also play a crucial role in maintaining its avascularity. Furthermore, ANGPTL7 expression was upregulated
together with that of type I collagen following the addition of dexamethasone to cultured
human trabecular meshwork cells [16–20]. Type I collagen is widely recognized one of themain components of the corneal stroma and the application of topical corticosteroids inhibits
corneal neovascularization [21, 22]. The mechanisms by which ANGPTL7 exerts antiangiogenic effects have thus far remained unknown. Because type I collagen retards tube formation in vitro [23], the assembly of dense type I collagen fibers in the corneal stroma might
prevent the entry of vessel sprouts into avascular corneas. Thus, we speculate that the synthesisof type I collagen stimulated by ANGPTL7 might serve as one possible mechanism by which
corneal avascularity is maintained. In light of our results, it is possible that application of a topical corticosteroid inhibits corneal neovascularization through the upregulation of ANGPTL7
and type I collagen expression.
In this study, we intrastromally injected a PshRNA in order to suppress the expression of
the ANGPTL7 in mice. Although siRNAs are widely used in typical RNA interference experiments, siRNAs are double stranded and might act as ligands for toll-like receptor 3, which
could induce inflammatory reactions. The PshRNA used in our in vivo studies, is a singlestranded RNA. This short hairpin single-stranded RNA does not induce IFN-gamma inflammatory reactions (data not shown). Thus, our treatment procedure might be capable of inducing corneal neovascularization without stimulating an inflammatory response that is observed
when other reagents are used. Furthermore, this drug delivery system might facilitate the development of a novel in vivo RNA interference-based therapeutic strategy.
A key feature of our in vitro study is that we could use it to observe tube formation under
non-serum conditions. HUVECs typically cannot grow by themselves under serum-free conditions. In the original co-culture system developed using fibroblasts and HUVECs, allogenic
serum such as FBS was used [4]. Because FBS contains various growth factors and hormones,
variations among FBS lots could present a problem. Another potential limitation of the systemis that observing the pure effects of the various factors on tube formation might be challenging.
Thus, a non-serum co-culture system would be an ideal tool for investigating gene-expression
profiles and screening for drugs in the case of angiogenesis, and this could lead to the development of an angiogenesis assay.
The results of our co-culture assays suggested that HCKs express fewer pro-angiogenic factors
and/or more anti-angiogenic factors than do HDFs. Although the anti-angiogenic properties ofkeratocytes remain to be fully elucidated, keratocytes have been reported to potentially express
anti-angiogenic molecules [24, 25]. For example, although keratocytes expressed thrombospondin, which is widely recognized as an anti-angiogenic molecule, the expression of this molecule
was attenuated following the infection of cultured keratocytes with herpes simplex virus-1 [24].
Based on using in vitro assays, Azar et al also reported that keratocyte-derived membrane-type

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Angiopoietin-Like 7 Is a Corneal Anti-Angiogenic Protein

matrix metalloproteinase exerts potent anti-angiogenic effects [25]. Taken together with our results, this suggests that keratocytes likely protect against corneal neovascularization.
In conclusion, we determined that ANGPTL7, which is abundantly expressed in keratocytes,
plays a major role in maintaining corneal avascularity, and that the corneal environment might
be crucial for its activity. Because the receptor of ANGPTL7 has not yet been identified, in future studies, it will be critical to explore ANGPTL7 signaling pathways and the molecularmechanisms of its action in corneal cells.

Supporting Information
S1 Table. Relative up-regulated genes in HCKs.
(PDF)
S2 Table. Relative down-regulated genes in HCKs.
(PDF)

Author Contributions
Conceived and designed the experiments: TT TU S. Yokoo. Performed the experiments: TT
YT. Analyzed the data: TT TU S. Yamagami YT SN. Contributed reagents/materials/analysis
tools: TU MK SA. Wrote the paper: TT TU MK S. Yamagami SA.

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33: 954–962. doi: 10.1080/02713680802461106 PMID: 19085378

PLOS ONE | DOI:10.1371/journal.pone.0116838 January 26, 201513 / 13-79-

Am J Physiol Lung Cell Mol Physiol 310: L1143–L1154, 2016.
First published April 29, 2016; doi:10.1152/ajplung.00357.2015.

CALL FOR PAPERS

Translational Physiology

Biomarkers in Lung Diseases: from Pathogenesis to Prediction

to New Therapies

Cord blood-derived endothelial colony-forming cell function is disrupted incongenital diaphragmatic hernia
Hideshi Fujinaga,1,2,3 Hiroko Fujinaga,2 Nobuyuki Watanabe,4 Tomoko Kato,5 Moe Tamano,5Miho Terao,5 Shuji Takada,5 Yushi Ito,3 Akihiro Umezawa,2 and Masahiko Kuroda1
1

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 2Department of Reproductive Biology,
National Institute for Child Health and Development, Tokyo, Japan; 3Division of Neonatology, Center for Maternal-Fetal andNeonatal Medicine, National Center for Child Health and Development, Tokyo, Japan; 4Department of Human Genetics,
National Institute for Child Health and Development, Tokyo, Japan; and 5Department of Systems BioMedicine, National
Institute for Child Health and Development, Tokyo, Japan
Submitted 13 October 2015; accepted in final form 22 April 2016

Fujinaga H, Fujinaga H, Watanabe N, Kato T, Tamano M,
Terao M, Takada S, Ito Y, Umezawa A, Kuroda M. Cord
blood-derived endothelial colony-forming cell function is disrupted in congenital diaphragmatic hernia. Am J Physiol Lung Cell
Mol Physiol 310: L1143–L1154, 2016. First published April 29,
2016; doi:10.1152/ajplung.00357.2015.—Vascular growth is necessary for normal lung development. Although endothelial progenitor
cells (EPCs) play an important role in vascularization, little is known
about EPC function in congenital diaphragmatic hernia (CDH), a
severe neonatal condition that is associated with pulmonary hypoplasia. We hypothesized that the function of endothelial colony-forming
cells (ECFCs), a type of EPC, is impaired in CDH. Cord blood (CB)was collected from full-term CDH patients and healthy controls. We
assessed CB progenitor cell populations as well as plasma vascularendothelial growth factor (VEGF) and stromal cell-derived factor 1
(SDF1) levels. CB ECFC clonogenicity; growth kinetics; migration;
production of VEGF, SDF1, and nitric oxide (NO); vasculogenic
capacity; and mRNA expression of VEGF-A, fms-related tyrosine kinase
1 (FLT1), kinase insert domain receptor (KDR), nitric oxide synthase(NOS) 1–3, SDF1, and chemokine (C-X-C motif) receptor 4 (CXCR4)
were also assessed. Compared with controls, CB ECFCs were decreasedin CDH. CDH ECFCs had reduced potential for self-renewal, clonogenicity, proliferation, and migration. Their capacity for NO production wasenhanced but their response to VEGF was blunted in CDH ECFCs. In
vivo potential for de novo vasculogenesis was reduced in CDH ECFCs.
There was no difference in CB plasma VEGF and SDF1 concentrations,
VEGF and SDF1 production by ECFCs, and ECFC mRNA expressionof VEGF-A, FLT1, KDR, NOS1-3, SDF1, and CXCR4 between CDH
and control subjects. In conclusion, CB ECFC function is disrupted in
CDH, but these changes may be caused by mechanisms other thanalteration of VEGF-NO and SDF1-CXCR4 signaling.
endothelial progenitor cells; endothelial colony-forming cells; congenital diaphragmatic hernia; cord blood
WHILE THE SURVIVAL RATE OF congenital diaphragmatic hernia(CDH) has improved over the decades due to advances inperinatal management, the mortality rate remains over 30%

Address for reprint requests and other correspondence: H. Fujinaga, Division of
Neonatology, Center for Maternal-Fetal and Neonatal Medicine, National Centerfor Child Health and Development, Tokyo, Japan 2-10-1, Okura, Setagaya-ku,
Tokyo 157-8535, Japan (e-mail: fujinaga-h@ncchd.go.jp).
http://www.ajplung.org(21, 40, 41). Furthermore, survivors often suffer from multisystem morbidity in long-term outcome (18).
In CDH, due to the defect in the diaphragm, the abdominal
viscera invade the thoracic cavity, impair lung development,
and cause lung hypoplasia (25). Lung pathology demonstrated
low lung volume, decreased airway generation and alveolar
count, air-blood barrier thickness, decreased vascular bed, andincreased vascular smooth muscles in precapillary vessels (50).
The exact mechanisms of how CDH leads to pulmonary
hypoplasia have not been fully elucidated.
As a developmental lung disease, bronchopulmonary dysplasia (BPD) has been intensively studied. The pathophysiology of BPD is considered to be impaired lung vascular andalveolar growth (37). In clinical settings and animal BPD
models, disruption of vascular endothelial growth factor(VEGF)-nitric oxide (NO) signaling in the lungs (12, 13, 36,
42, 43) and its adverse effect on endothelial progenitor cells(EPCs), especially endothelial colony-forming cells (ECFCs),
have been demonstrated (10, 12, 24). Therefore, a “vascularhypothesis,” suggesting the contribution of injured lung vascular growth during lung development to the etiology of lung
hypoplasia (1), was proposed. Recently, the existence of lungresident ECFCs and their dysfunction in BPD were suggested(3, 4). Some studies showed the relationship between decreased circulating ECFCs and the occurrence of BPD (8, 15),
while others did not (52, 54). Additionally, one paper demonstrated the possibility of cord blood (CB)-derived ECFCs as atherapeutic option for BPD via paracrine effects (3), butanother suggested the limitation of CB ECFC-conditioned
media for that purpose (11). Therefore, the depletion anddysfunction of circulating and/or lung-resident ECFCs might
be one of the possible mechanisms underlying arrested lungvascular growth in BPD (3, 14).
Lung vascular growth occurs via angiogenesis and vasculogenesis (20, 29) with the involvement of EPCs (38). Several distinct
populations of cells have been reported as EPCs (5, 28, 45). By
flow cytometry, circulating progenitor cells can be classified as
ECFCs (CD45CD34AC133CD31), angiogenic circulatingprogenitor cells (CPCs) (CD45dimCD34AC133CD31), andnonangiogenic CPCs (CD45dimCD34AC133CD31) (22). In

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cell culture, EPCs are distinguished into two types, early outgrowth EPCs [known as colony-forming unit endothelial cells(CFU-ECs)] and late-outgrowth EPCs (known as ECFCs) (30,53). CFU-ECs have the characteristics of both angiogenic macrophages and hematopoietic cells and are not highly proliferative(65). ECFCs have a cobblestone-like appearance, express endothelial cell surface markers, can be highly proliferative, have thecapacity for self-renewal and to develop a vascular network invivo, and do not ingest bacteria (30, 31, 53).
VEGF and stromal cell-derived factor 1 (SDF1) are among
the factors involved in EPC mobilization, recruitment, homing,and differentiation in neovascularization (5, 27, 35, 47, 59, 63,
66). In the lungs of CDH patients, VEGF-A mRNA expression
was decreased at the alveolar stage (60). In rat CDH models
with nitrofen, lung VEGF-A protein expression was reduced(26, 55, 57). On the other hand, increased plasma VEGF wasassociated with the clinical severity and adverse outcome in
CDH (51). Surgically induced CDH in fetal sheep markedly
reduced highly proliferative pulmonary artery endothelial cells(PAECs) and caused PAEC dysfunction (2). Additionally,antenatal treatment with simvastatin, sildenafil, or BY 41–
2272, which were thought to affect VEGF-NO signaling or
EPC function, improved pulmonary hypertension or pathological features of lung hypoplasia in animal CDH models (48, 49,
61). These studies suggest that disrupted vascularization was
involved in the pathophysiology of pulmonary hypoplasia in
CDH. However, few studies have investigated the role of EPCs
and SDF1 signaling in pulmonary hypoplasia in CDH (9, 17).
We hypothesized that the function of EPCs, especially
ECFCs, in CDH patients is impaired due to disrupted
VEGF-NO or SDF1-chemokine (C-X-C motif) receptor 4(CXCR4) signaling. Therefore, the objectives of this study
were to compare ECFC functions and VEGF-NO and SDF1CXCR signaling in CDH patients and in healthy newborns.
MATERIALS AND METHODS

Subject Enrollment and Data Collection
This study was approved by the ethics committee of the National
Center for Child Health and Development (NCCHD), Tokyo, Japan.
Written informed consent was obtained from the parents whose
unborn child was diagnosed with CDH (CDH group) or had noabnormal findings in their screening tests (control group). Exclusion
criteria included preterm or postterm birth infants, known congenital
infection and chromosomal abnormalities, as well as infants treatedprenatally with fetoscopic tracheal occlusion (FETO). CB samples
were obtained at birth at the NCCHD. Clinical data of infants and theirmothers were collected from electronic medical records.
CB Samples and Endothelial Colony-Forming Cell Culture for
Isolation
CB was collected directly from the umbilical vein using a heparinized syringe immediately after birth. All CB samples were processed within 4 h after collection. CB was diluted with PBS (Invitrogen, Carlsbad, CA), underlaid with Ficoll-Paque PLUS (GE Healthcare Bio-Sciences, Uppsala, Sweden), and centrifuged at 740 g for 30
min. Then, the mononuclear cell (MNC) buffy coat was retrieved.
MNCs were plated on collagen-coated culture plates (Corning, Corning, NY) at a density of up to 5  106 cells/cm2, incubated for 14 daysin EGM-2 (Lonza, Walkersville, MD) with 10% FBS (Life Technologies, Carlsbad, CA), and expanded by standard techniques.
Isolated ECFCs were characterized by light microscopy, flow
cytometry, single cell colony formation assay, in vitro tube formation

assay and in vivo vasculogeneisis assay as previously described (10,24, 45). Low passage cells (p3 or p4) were used for each experiment.
Flow Cytometry Analysis
Measurement of circulating progenitor cell populations in CB. We
measured circulating progenitor cell populations in CB using polychromatic flow cytometry (PFC). MNCs (0.5–1.0  106) from CB processed
as described in the previous section were resuspended in PBS with 2%
FBS. Cells were stained with the following antibodies: CD14-PE-Cy5.5(abcam, Cambridge, UK), CD31-FITC (BD Pharmingen, San Jose,CA), CD34-PE (BD Pharmingen), CD45-Pacific Orange (Invitrogen),CD133/1 (AC133) pure (Miltenyi Biotec, Auburn, CA) conjugated byZenon Pacific Green Mouse IgG1 Labeling Kit (Molecular Probes,
Eugene, OR), glycophorin-A (R&D Systems, Minneapolis, MN) andCD41 (R&D Systems) conjugated by Pacific Blue Monoclonal Antibody
Labeling Kit (Molecular Probes), and the LIVE/DEAD Fixable Violet
Dead Cell Stain Kit (Invitrogen). We used AbC Anti-Mouse Bead Kit(Molecular Probes) and ArC Amine Reactive Compensation Bead Kit(Molecular Probes) as single stained controls for appropriate multicolor
compensation. Gating was established using an unstained control andfluorescence-minus-one control samples with biexponential gating (22).
Cell populations were quantified as a percentage of the gated CD14negative MNCs after erythrocytes, platelets, and dead cell exclusion as
described before (8). ECFCs (CD45CD34AC133CD31), angiogenic CPCs (CD45dimCD34AC133CD31), and nonangiogenic
CPCs (CD45dimCD34AC133CD31) were measured. The CPC/nonCPC ratio was calculated by dividing angiogenic CPCs by non-angiogenic CPCs. The observers who evaluated the results were blinded to theidentity of the samples.
Characterization of Isolated ECFCs
Cells (0.5–1.0  106) were resuspended in 80 l of DNase Staining
Buffer [PBS with 2% FBS, 80 units/ml Dornase alfa (Sigma-Aldrich,
St. Louis, MO)] with 20 l of mouse serum (Sigma-Aldrich). Cells
were stained with one of following: AC133-PE (Miltenyi Biotec),
CD45-FITC, CD31-FITC, CD34-PE, CD38-FITC, CD90-FITC,
CD105-FITC, CD146-FITC, and the appropriate FITC-mouse IgG1
and PE-mouse IgG1 isotype controls. All antibodies except for
AC133-PE were purchased from BD Pharmingen. Propidium iodide
was applied to the samples to identify dead cells immediately before
analysis.
Cells were analyzed using the Attune Acoustic Focusing Cytometerand the Attune Cytometric Software v1.2.5 (Applied Biosystems,
Carlsbad, CA).
ECFC Colony Assay
In cell culture for ECFC isolation from CB, colonies of ECFCs
were identified as well-circumscribed monolayers of cells with cobblestone appearance and counted by visual inspection with an inverted
microscope, Nikon Eclipse TS100 (Nikon, Tokyo, Japan) on day 14.
The numbers of ECFC colonies were expressed as the frequency ofcolonies per 107 plated MNCs or per milliliter of CB. The observers
who evaluated the results were blinded to the identity of the samples.
Single Cell Colony Formation Assay
To assess self-renewal potential of ECFCs, a single ECFC wasplaced into each well of a 96-well collagen-coated culture plate using
a FACSAria III (Becton Dickinson, San Jose, CA) and cultured in 200
l of EGM-2 with 2% FBS. Media were changed every 4 days. On
day 14, each well was examined for colony growth. To enumerate thenumber of cells per well, we counted cells by visual inspection withan inverted microscope or trypsinized the cells and counted them witha hemocytometer. Additionally, to test the ability of the clonal
progeny of a single ECFC to form secondary colonies, cells inindividual wells containing more than 50 cells were subcultured to a

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24-well collagen-coated culture dish containing 500 l of EGM-2
with 2% FBS. After 7 days, wells were examined for colony growthor cell confluence by visual inspection with an inverted microscope.
The observers who evaluated the results were blinded to the identityof the samples.

differences in mean fluorescence intensity between ECFCs treatedeither with or without DAF-FM DA (24). All experiments were donein triplicate.

ECFC Growth Assay

ECFCs were seeded at 20,000 cells/well in 96-well plates coatedwith 50 l of Matrigel (Corning, 10 mg/ml) or growth-factor reduced
Matrigel (Corning, 6 mg/ml) in EGM-2 with 2, 5, or 10% FBS. Six
and 12 h after plating, images of capillary-like structures werecollected using an inverted microscope. The total length of capillarylike structures per well was quantitated using the ImageJ software.
Each experiment was done in triplicate (44). The observers who
evaluated the results were blinded to the identity of the samples.

ECFCs were plated on six-well collagen-coated culture plates at
2.5  104 cells/well in EGM-2 with 10% FBS and incubated overnight. ECFCs were cultured in EGM-2 with 2% FBS for 6 days.
Media were changed once every 2 days. Cell counts were conducted
on day 0, 3, and 6 with the Vi-CELL XR (Beckmann Coulter;
Pasadena, CA) and the results in triplicate were averaged for eachtime point.

Tube Formation Assay

Migration Assay with Modified Boyden Chamber Method

In Vivo Vasculogenesis Assay

Cell chemotaxis was assessed using a “modified Boyden chamber
assay” with Cultrex In Vitro Angiogenesis Assay Endothelial Cell
Invasion Kit (Trevigen, Gaithersburg, MD) as described by the
manufacturer. Briefly, ECFCs were seeded at 20,000 cells/well in 50
l of EBM-2 to the top chambers coated with coating buffer. To
create a positive chemotactic gradient, 150 l of EGM-2 with 2 or 5%
FBS were added to the bottom chambers. After incubation for 48 h,
150 l of Cell Dissociation Solution/Calcein-AM were added to thebottom chambers and incubated for 1 h. The bottom chambers wereread at 485-nm excitation and 520-nm emission to obtain relative
fluorescence units using the Wallac 1420 ARVO SX multilabel
counter (Perkin Elmer, Waltham, MA). The numbers of cells that
migrated from the top chambers to the bottom ones were calculated
according to the standard curve. Data were represented as percent (%)
migration [i.e., the percentage of these cell numbers to those in the top
chambers at the start of the experiments (20,000 cells/well)]. All
experiments were done in triplicate wells.

Transplantation of ECFCs. Cellularized gel implants were transplanted into mice as previously described (31, 32, 56, 65). ECFCs
were suspended in a solution composed of EGM-2, 10% FBS, 1.5
mg/ml rat tail collagen I (BD Biosciences), 100 ng/ml human fibronectin (Millipore, Billerica, MA), 1.5 mg/ml sodium bicarbonate(Sigma-Ardrich), and 25 mM HEPES (Lonza) at 2  106 cells/ml.
Then, 1 ml of the cell suspension was transferred into the 24-wellculture plate, incubated at 37°C for 30 min for polymerization, andthen covered with EGM-2 with 2% FBS and incubated overnight. The
following day, gels with ECFCs from a newborn were implanted into
the flank of three 8-wk-old NOD/SCID mice (CLEA, Tokyo, Japan).
The right flank of each mouse was implanted with ECFCs fromcontrols, and the left flank with CDH ECFCs. Fourteen days after
transplantation, grafts were harvested and analyzed for vessel formation by immunohistochemistry. All experiments were approved by theInstitutional Animal Care and Use Committee of the National Research Institute for Child Health and Development.

Scratch Wound Healing Assay

Immunohistochemistry

Cell chemokinesis was assessed using a “scratch wound healing
assay” (7). ECFCs were plated on 24-well collagen-coated culture
plates in EGM-2 with 10% FBS at 80,000 cells/well. When cells
reached 70 – 80% confluence, media were changed to EBM-2 with 2%
FBS. After 24 h, cell monolayers were scratched with a sterile cell
scraper to make wounds of equivalent sizes and media were replacedwith EGM-2 with 2, 5, or 10% FBS to remove nonadherent cells.
Images of the wound area were collected using Olympus CKX41
inverted microscope (Olympus; Tokyo, Japan) and were captured
using Olympus DP21. Nonpopulated scratch areas were measured
with ImageJ version 1.48 software (NCBI), and the percent (%)
closure (percentage of the original wound area occupied at 6, 12, 18,
and 24 h by cells that had migrated into the wound area) was
calculated. All experiments were done in quadruplicate wells. The
observers who evaluated the results were blinded to the identity of thesamples.

Paraffin-embedded tissue sections of 7-m thickness were deparaffinized and immersed in the Target Retrieval Solution (Dako,
Carpenteria, CA) for 15 min at 121°C at 15 psi above atmosphericpressure. Slides were incubated at room temperature with anti-humanCD31 (clone JC70A; Dako) for 30 min followed by 30-min incubation with EnVision  Dual Link, Single Regents (Dako) and thendeveloped with DAB solution (Dako) for 5 min. Isotype-matched
nonbinding antibody (Dako) was used to control for nonspecificreactivity. Tissue sections were counterstained with hematoxylin andeosin. Slides were dehydrated and mounted using Mount-Quick(Daido Sangyo, Tokyo, Japan). Enumeration of vessels perfused andstained positively for anti-human CD31 was performed by visualinspection using Olympus BX51 upright microscope and Olympus
DP71. The observers who evaluated the results were blinded to theidentity of the samples.

NO Production Assay

ELISA

ECFCs were plated at 2.5  105 cells/well into the six-wellcollagen-coated culture plates containing EGM-2 with 10% FBS andincubated overnight. After pretreatment in EGM-2 with 2% FBS
without VEGF supplementation for 24 h, cells were incubated in
HBSS (IBCO, Carlsbad, CA) either with or without 0.5 mol/l of
3-amino,4-aminomethyl-2=,7=-difluorofluorescein diacetate (DAF-FM
DA; Santa Cruz Biotechnology, Dallas, TX) for 30 min for loading
and then incubated in EBM-2 either with or without 25 ng/ml ofrecombinant human VEGF (rhVEGF; R&D Systems) for 30 min andharvested enzymatically with 0.25% trypsin/0.02% EDTA. Fluorescence intensity was quantified by flow cytometry in the system for
FITC. Intracellular NO concentration of ECFCs was expressed by

VEGF and SDF1 concentrations in CB plasma were measured
with the Quantikine ELISA Kits (R&D Systems) as described by the
manufacturer.
In addition, we compared VEGF and SDF1 protein production by
ECFCs between both study groups. Briefly, ECFCs were plated in a
six-well collagen-coated culture plate at 0.6  106 cells/well in
EGM-2 with 10% FBS. On the next day, media were changed toEBM-2 with 2% FBS to exclude the effects of exogenous VEGF orother growth factors and minimize the effects of FBS. After 24-h
incubation, cell culture supernatants were collected and VEGF and
SDF1 protein concentrations were measured with the Quantikine
ELISA Kits.

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Fig. 1. Flowchart of the number of screened and included newborns, as well as the cord blood and endothelial colony-forming cells (ECFC) samples available
for analysis.

RT-PCR and Quantitative Real-Time PCR
Total RNA extraction from ECFCs was performed using RNesay
Plus Mini (Qiagen, Venio, the Netherlands). The cDNA was synthesized by RT-PCR with Superscript III First-Strand Synthesis System
for RT-PCR (Invitrogen). Quantitative PCR was performed on the
Applied Biosystems 7900HT Fast Real-Time PCR System (Applied
Biosystems). Reaction products of VEGF-A, fms-related tyrosine
kinase 1 (FLT1), kinase insert domain receptor (KDR), NO synthase(NOS) 1, NOS2, NOS3, SDF1, CXCR4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were quantified by Thunderbird
SYBR qPCR Mix (Toyobo Life Science, Osaka, Japan) with 0.3
mol/l of gene specific primers as described by the manufacturer.
Each experiment was done in triplicate. Results were normalized to
GAPDH, and relative mRNA expression to those of ECFCs from aparticular participant in the control group was analyzed with the
2Ct method.
The gene specific primer sequences were as follows: VEGFA: forward 5=-AGGGCAGAATCATCACGAAGT-3=, reverse
5=-AGGGTCTCGATTGGATGGCA-3=; FLT1: forward 5=GAAAACGCATAATCTGGGACAGT-3=, reverse 5=-GCGTGGTGTGCTTATTTGGA-3=; KDR: forward 5=-GTGATCGGAAATGACACTGGAG-3=, reverse 5=-CATGTTGGTCACTAACAGAAGCA3=; NOS1: forward 5=-TTCCCTCTCGCCAAAGAGTTT-3=, reverse
5=-AAGTGCTAGTGGTGTCGATCT-3=; NOS2: forward 5=TTCAGTATCACAACCTCAGCAAG-3=, reverse 5=-TGGACCTGCAAGTTAAAATCCC-3=; NOS3: forward 5=-TGATGGCGAAGCGAGTGAAG-3=, reverse 5=-ACTCATCCATACACAGGACCC-3=;
SDF1: forward 5=-ATTCTCAACACTCCAAACTGTGC-3=, reverse
5=-ACTTTAGCTTCGGGTCAATGC-3=; CXCR4: forward 5=ACTACACCGAGGAAATGGGCT-3=, reverse 5=-CCCACAATGCCAGTTAAGAAGA-3=; and GAPDH: forward 5=-ACAACTTTGG-TATCGTGGAAGG-3=, reverse 5=-GCCATCACGCCACAGTTTC-3=.
Statistics
Statistical analysis was performed with the Prism software package
version 4 (GraphPad, La Jolla, CA). For analyses of participants’clinical characteristics, data were presented as count (relative frequency, %), mean  SD, or median (range). Fisher’s exact test,
unpaired t-test, or Mann-Whitney’s U-test were conducted. For analyses of experiments with CB and ECFC function assays, data arepresented as mean  SE, and paired or unpaired t-tests were conducted. All statistical tests were two-sided. P  0.05 was consideredsignificant.
RESULTSCB Samples
Figure 1 shows the number of newborns who were screened
and included in this study, as well as the number of CB
samples and ECFCs available for each analysis.
Briefly, we obtained CB samples from 10 newborns in eachgroup and analyzed the data of 5–10 samples from each groupin each experiment.
Clinical characteristics of the study newborns
Among the 20 newborns enrolled, there was no significant
difference in maternal, prenatal, and perinatal factors (maternal age, complications and smoking, chorioamnionitis,
premature rupture of membrane, prenatal corticosteroids,

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Fig. 2. Characterization of ECFCs. A: images of typical ECFCs. ECFCs had a cobblestone-like appearance. Scale bars  500 and 200 m, respectively.
B: flow cytometry of ECFCs. ECFCs express endothelial cell surface markers. Each single-stain result (black) was compared with the corresponding
isotype control (gray). ECFCs were strongly positive for CD31, CD105, and CD146, weakly positive for CD34, but negative for CD133, CD38, CD45,
and CD90. C: representative image of in vitro tube formation assay. ECFCs formed capillary-like structures. Scale bars  500 m. D: representative imageof in vivo vasculogenesis assay. ECFCs formed chimeric vessels stained positively for anti-human CD31 (brown) and perfused with mouse erythrocytes.
Scale bars  50 m.

delivery mode, meconium staining, gestational age, birthweight, small for gestational age, sex, and so on) between
both groups, except for Apgar score. In each experiment, thecomparison of clinical characteristics of the participants
between both groups yielded the same results as describedabove. The CDH group had a lower Apgar score at 1 and 5
min than that of the control group [4 (1– 8) vs. 8 (7–9); P 
0.001 and 4 (1–9) vs. 9 (8 –9); P  0.001, respectively],
because in our hospital CDH patients were intubated andmechanically ventilated with muscle relaxant immediately
after birth. However, umbilical artery pH that correlatedwith severity of perinatal asphyxia was not significantly
different. All CDH were left-sided.

Characterization of ECFCs
Isolated ECFCs developed a cobblestone-like morphology(Fig. 2A). On flow cytometry, these cells were positive for
CD31, CD105, and CD146 (endothelial cell markers), weakly
positive for CD34, but negative for CD133 (markers for cell
immaturity), CD38 and CD45 (hematopoietic markers), as wellas CD90 (a fibroblast marker) (Fig. 2B). In both in vitro tube
formation assay and in vivo vasculogenesis assay, ECFCs
formed capillary-like structures (Fig. 2, C and D). Additionally, in single cell colony formation assays, ECFCs demonstrated self-renewal capacity and some ECFCs from eachnewborn showed highly proliferative potential.

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ECFCs Are Reduced in CB of CDH Patients
In PFC of CB, the ECFC population in CB from CDH
patients was 80% lower than that from controls (0.0020 
0.0005 vs. 0.0098  0.0020%; P  0.008; Table 1). There wasno significant difference in other CPC populations and the
CPC/non-CPC ratio.
In addition, the numbers of ECFC colonies yielded on day
14 in ECFC culture for isolation from CB were enumerated.
There was no significant difference in the number of MNCs in
CB [3.8  0.8 vs. 4.1  0.7 ( 106 MNCs/ml of CB); P 
0.824]. The colony counts of CDH ECFCs per 107 MNCs andthat per ml of CB were 72 and 76% lower than those ofcontrols respectively (5.0  2.5 vs. 17.8  4.0 colonies/107
MNCs, P  0.015; Fig. 3A; 1.6  0.7 vs. 6.7  1.4 colonies/mlof CB, P  0.005; Fig. 3B).
ECFC Self-Renewal, Clonogenic, and Proliferative
Potentials Are Impaired in CDH Patients
In a single cell colony formation assay, ECFCs are classified into three categories according to their proliferative
potential; high proliferative potential-endothelial colonyforming cell (HPP-ECFC), which forms a colony that contains 2,000 cells, low proliferative potential-endothelialcolony-forming cell (LPP-ECFC), which forms a colonythat has 51 to 2,000 cells, and endothelial cell (EC) cluster
which forms a colony composed of 2 to 50 cells (31, 33, 34).
Although the percentage of single cells undergoing at least
1 cell division was not significantly different between bothgroups (55.7  9.1 vs. 41.7  6.5%; P  0.260), thepercentages of HPP-ECFCs and EC clusters among thesecells were 92% lower and 320% higher in CDH, respectively, compared with those of control (HPP-ECFC: 2.8 
2.8 vs. 35.9  9.8%, P  0.032; EC cluster: 52.1  9.2 vs.
12.4  2.3%, P  0.009; Fig. 4A). The average number ofcell progeny derived from a single cell was 85% smaller in
CDH than in control (293  155 vs. 1,943  344 cells/colony or cluster, P  0.001; Fig. 4B). Additionally, thepercentage of single cell progeny forming a secondarycolony was also 77% smaller in CDH than in control(16.5  6.8 vs.70.4  9.8%, P  0.001; Fig. 4C).
Moreover, on day 0 of the ECFC growth assay, there was no
significant difference in ECFC counts between both groups[0.023  0.005 vs. 0.018  0.001 (106 cells/well), P 
0.412; Fig. 5]. On day 6, the number of CDH ECFCs was 61%
smaller than that of control [0.200  0.072 vs. 0.517  0.084(106 cells/well), P  0.018; Fig. 5].
Table 1. Circulating progenitor cell populations in cord
blood
Cell Populations

Control (n  7)CDH (n  6)P Value

ECFCs,a %bAngiogenic CPCs,c %bNonangiogenic CPCs,d %b
CPC/non-CPC ratioe

0.0098  0.0020
0.0018  0.0005
0.0322  0.0113
0.1057  0.0434

0.0020  0.0005
0.0012  0.0004
0.0130  0.0085
0.4930  0.1942

0.008
0.325
0.215
0.109

Data are presented as mean  SE. ECFCs, endothelial colony-forming cells;
CPCs, circulating progenitor cells; CDH, congenital diaphragmatic hernia.
aCD45CD34AC133CD31 cells; bpercentage of the gated CD14-negative MNCs after erythrocytes, platelets, and dead cell exclusion;c
CD45dimCD34AC133CD31 cell; dCD45dimCD34AC133CD31 cells;eangiogenic CPCs/nonangiogenic CPCs.

Fig. 3. ECFCs are decreased in cord blood of congenital diaphragmatic hernia(CDH) patients. The number of ECFC colonies yielded on day 14 in ECFC
culture for isolation from cord blood (CB) was enumerated and expressed ascolony numbers per 107 mononuclear cells (MNCs) (A) or per ml of CB (B).
The number of CB ECFC colonies of CDH patients was significantly lower
than that of controls (P  0.015, P  0.005). Bars represent mean  SE.
Unpaired t-test was used to analyze statistical significance (n  7; control
group, n  8; CDH group).

ECFC Migration Is Impaired in CDH Patients
As for ECFC chemotaxis assessed by modified Boydenchamber assay, ECFC count that migrated toward EGM-2 with
2% FBS from EBM-2 in 48 h was 89% smaller in the CDH
group than in the control group (%migration; 5.4  2.6 vs.
50.6  18.0%, P  0.023; Fig. 6A). The result of migration
toward EGM-2 with 5% FBS was the same (data not shown).
As for ECFC chemokinesis assessed by scratch woundhealing assay, the area refilled by CDH ECFCs was 38%
smaller than that by controls 18 h after wounding in EGM-2
with 2% FBS (%closure; 24.6  4.4 vs. 39.7  1.5%, P 
0.017; Fig. 6, B and C). The results at other time points and inother concentrations of FBS were the same (data not shown).
Capacity of ECFCs for NO Production Is Enhanced but
Response to VEGF Stimulation Is Blunted in CDH PatientsTo assess ECFC capacity for NO production, intracellular
NO concentrations of ECFCs in basal and VEGF-stimulated
conditions were measured with DAF-FM DA. NO concentrations of ECFCs from all controls, as well as those from all
CDH patients but one, were increased by VEGF stimulation.
Although the increase by VEGF stimulation in NO concentration of ECFCs from controls was statistically significant, thatfrom CDH patients was not. (VEGF-stimulated condition vs.
basal condition; CDH; P  0.114 and control, P  0.010,

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Fig. 4. ECFC self-renewal and clonogenic capacity is impaired in CDH patients. A: the percentage of high proliferative potential (HPP)-ECFCs was significantly
lower and that of EC clusters was significantly higher in CDH than control in a single cell colony formation assay (P  0.032, P  0.009). B: average
number of cell progeny derived from a single cell was significantly smaller in CDH than control (P  0.001). C: percentage of single cell progeny forminga secondary colony was also significantly smaller in CDH than control (P  0.001). Bars represent mean  SE. Unpaired t-test was used to analyze statisticalsignificance (n  5; control group, n  6; CDH group).

respectively; Fig. 7). Also, the increase in NO concentration in
CDH ECFCs was not statistically significant when the result of
ECFCs from one patient without an increase in NO concentration by VEGF was excluded from statistical analysis. On theother hand, NO concentrations of CDH ECFCs in basal and
VEGF-stimulated conditions were 146 and 129% higher, respectively, compared with those of the control (basal condition: P  0.047; VEGF-stimulated condition: P  0.048,
respectively; Fig. 7).
Capacity of ECFCs to Form Capillary-Like Structures in In
Vitro Tube Formation Assay Is Not Significantly Different
Between CDH Patients and Controls
In the ECFC tube formation assay with the growth-factor
reduced Matrigel, there was no significant difference between
both groups in the total length of capillary-like structures (datanot shown).
The Potential for In Vivo Vasculogenesis Is Reduced in
ECFCs from CDH Patients
Since the capacity for de novo vasculogenesis in vivo is oneof the most important criteria and functional parameters for
ECFCs (30, 44, 53), we compared the in vivo ability of ECFCs
from both groups to form vascular structures in xenotransplan-tation to mice, although there was no difference in an in vitro
tube formation assay. The number of chimeric vessels perfusedwith mouse erythrocytes in the transplanted graft of the CDH
group was 85% smaller than that of the control group (16.9 
3.5 vs. 113.2  18.4 vessels/mm2; P  0.007; Fig. 8, A and B).
Plasma VEGF and SDF1 Concentrations of CB Are Not
Significantly Different Between CDH Patients and Controls
To investigate the difference in ECFC exposure to VEGF
and SDF1 in fetal circulation, CB plasma VEGF and SDF1
concentrations were measured by ELISA. VEGF concentrationof CDH patients tended to be higher than that of controls, but
the difference was not statistically significant (60.9  25.7 vs.
27.2  12.7 pg/ml, P  0.261). There was no difference
between groups in SDF1 concentration (572.4  17.8 vs.
536.8  22.8 pg/ml, P  0.235).
Gene Expression of VEGF-A, FLT1, KDR, NOS1-3, SDF1,
and CXCR4 Is Not Significantly Different Between ECFCs of
CDH Patients and Controls
To elucidate the relationship of VEGF-NO signaling and
SDF1-CXCR4 signaling with these differences in ECFC functions between CDH patients and controls, mRNA expression of
ECFCs for several angiogenic genes was compared by quantitative real-time PCR. The difference in relative mRNA expression of all of these genes between both groups was notstatistically significant (VEGF-A: 5.04  2.41 vs. 1.57  0.49,
P  0.218; FLT1: 1.99  0.54 vs. 1.64  0.26, P  0.599;
KDR: 0.87  0.37 vs. 0.59  0.12, P  0.502; NOS1: 2.61 
1.53 vs. 0.69  0.19, P  0.267; NOS2: 1.29  0.65 vs.
0.82  0.18, P  0.518; NOS3: 1.08  0.51 vs. 0.92  0.18,
P  0.791; SDF1: 2.65  2.19 vs. 0.86  0.28, P  0.457;
CXCR4: 0.70  0.33 vs. 0.88  0.47, P  0.747).
VEGF and SDF1 Production In Vitro Is Not Different in
ECFCs from CDH Patients and Controls

Fig. 5. ECFC growth is impaired in CDH patients. On day 0 of the growth
assay, there was no difference in ECFC counts between both groups. On day
6, the number of CDH ECFCs was significantly smaller than that of controls(P  0.018). Graph represents mean  SE. Unpaired t-test was used toanalyze statistical significance (n  5; control group, n  6; CDH group).

Because there was a trend for higher VEGF-A and SDF1mRNA expression in CDH ECFCs, we measured VEGF-A andSDF1 protein concentrations in culture media after 24-h
incubation of ECFCs from both groups. VEGF concentrations
in culture supernatants of all ECFCs were lower than theminimum detectable limit (5.0 pg/ml according to the man-

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Fig. 7. Capacity of nitric oxide production of ECFCs is enhanced but response
to VEGF stimulation is blunted in CDH patients. Intracellular NO concentrations of ECFCs in basal and VEGF-stimulated (rhVEGF; 25 ng/ml) conditions
were measured with DAF-FM DA. NO concentration of ECFCs from controls
was significantly increased by VEGF stimulation (P  0.010), and that from
CDH patients was also increased but not significant (NS: not significant; P 
0.114). However, NO concentrations of CDH ECFCs were higher than thoseof controls in both basal and VEGF-stimulated conditions (#P  0.047 and}P  0.048, respectively). Bars represent mean  SE. Paired or unpaired t-test
was used to analyze statistical significance (n  5; control group, n  6; CDH
group).

SDF1-CXCR4 signaling. This study had three main findings.
First, ECFCs were decreased in the CB of CDH patients.
Second, ECFCs derived from the CB of CDH patients had
reduced in vitro potential for self-renewal, clonogenicity, proliferation, and migration and decreased in vivo capacity for denovo vasculogenesis. Their capacity for NO production wasFig. 6. ECFC migration is impaired in CDH patients. A: ECFC chemotaxis wasassessed by modified Boyden chamber assay. Compared with control, thenumber of ECFCs, which migrated in 48 h was significantly lower in CDH(P  0.023). ECFC chemokinesis was assessed by a scratch wound healing
assay. B: representative images of the assay. Black lines indicate the boundaries of the wound area. Scale bars  500 m. C: 18 h after wounding, the area
refilled by CDH ECFCs was significantly less than that by controls (P 
0.017). Bars represent mean  SE. Unpaired t-test was used to analyze
statistical significance (n  5; control group, n  6; CDH group).

ufacturer). Additionally, there was no difference in SDF1
protein concentration between both groups (292.4  0.2 vs.
292.5  0.2 pg/ml, P  0.714).
DISCUSSION

In the present study, we demonstrated that CB-derived
ECFC function was disrupted in CDH. The disruption might becaused by mechanisms other than alteration of VEGF-NO and

Fig. 8. The potential for in vivo vasculogenesis is reduced in ECFCs from
CDH patients. A: representative images of xenograft implants into NOD/SCID
mice on day 14. Grafts were stained with anti-human CD31 (brown) to identify
human blood vessels perfused with murine red blood cells (indicated by blue
arrows). Scale bars  200 m. B: number of chimeric vessels perfused withmouse erythrocytes of the CDH group was smaller than that of the control
group (P  0.007). Bars represent mean  SE. Unpaired t-test was used
to analyze statistical significance (n  5; control group, n  6; CDH group).

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enhanced but response to VEGF was blunted. Third, alteredexposure to VEGF and SDF1, together with gene expressionof VEGF-A, FLT1, KDR, NOS1-3, SDF1, and CXCR4, was
not involved in these ECFC functional changes.
Notably, this is the first report that demonstrates detailed
functions of ECFCs in CDH. Our findings suggest that pulmonary hypoplasia in CDH may potentially be related to impaired
ECFC functions and ECFC may be a new therapeutic target for
CDH. As noted in studies of EPCs in adult vascular diseases (6,16, 23, 58, 62), CB ECFCs may potentially serve as a biomarker to predict disease severity or as a therapeutic option in
the management of CDH.
In this study, ECFC colonies from CB were decreased and
ECFC growth was impaired in CDH compared with controls.
However, in an earlier study, Baker et al. (9) showed contrary
results. Although we cannot precisely explain why the resultsof the two studies differ, it could be attributed to the differences in the study populations and methods between the twostudies. First, CDH patients in the study of Baker et al. (n  6)were of lower gestational age (GA; 37.1  2.6 wk in the Baker
study vs. 38.4  0.9 wk in ours) and included two prematureinfants (GA: 33 and 34 wk; personal communication with Dr.
C. D. Baker). The control group (n  33) was larger than the
one in our study and, on average, had fewer CB ECFCs. Baker
et al. (10) also reported in a separate paper that preterm infants
have increased CB ECFCs with increased proliferation compared with term infants. Therefore, GA and sample size may
explain some of the differences between the two studies. Haraet al. (26) found that the developmental pattern of lungVEGF-A mRNA expression was inhibited in a rat CDH modelwith nitrofen but was recovered by tracheal occlusion. Cloutier
et al. (19) demonstrated that tracheal occlusion stimulated
distal capillary growth as well as expression of specific angiogenic genes in fetal mouse lung. Both our study and that ofBaker et al. excluded patients who underwent FETO (personalcommunication with Dr. C. D. Baker) as they hypothesizedthat FETO may modify lung vascular growth and EPC function
in CDH. Additionally, the impaired proliferative and selfrenewal potential of ECFCs in CDH was demonstrated in ourstudy not only by growth assay but also by single cell colony
formation assay, which was not done in the study by Baker et
al. even though it is the most rigorous test for the proliferativeand clonogenic potential of progenitor cells (30, 44, 53). There
were also several minor methodological differences, including
the duration between the acquisition of CB and ECFC isolation(within 24 h in the Baker study vs. within 4 h at our center).
The studies were completed at two different geographicallocations (Denver, CO, altitude 1,600 m vs. Tokyo, Japan, sealevel). The concentration of FBS differed between the growth
assays (2.5 and 2% serum, respectively). However, we cannot
explain how these methodological differences would cause
opposing results. Finally, all CDH patients in the Baker study
were of white or Hispanic origin (personal communication
with Dr. C. D. Baker), while those in the present study were
Asian. However, the effect of race or ethnicity on ECFC
number and function has not been described. Therefore, further
studies in similar conditions with larger sample sizes areneeded to confirm the results.
We showed that the capacity of ECFCs for NO production
was enhanced in CDH patients overall but the response to
VEGF stimulation was blunted. Additionally, the result of invitro tube formation assay of CDH ECFCs was not different
when compared with that of controls. These were unexpected.
Especially, we expected impaired ECFC capacity for in vitro
tube formation, which is perhaps the most relevant to the invitro capacity of angiogenesis, in CDH.
Although growth factors contained in medium or growthfactor reduced Matrigel might obscure the difference in ECFC
capacity for in vitro tube formation between both groups, thesefindings might suggest that some ECFC functions (mobilization, proliferation, and migration, etc.) are impaired whileothers (such as NO production) are enhanced (partially) tocompensate for the impairment in CDH. As a result, when thesame numbers of ECFCs from both study groups participate in
an in vitro tube formation assay, there might be no difference
in developing capillary-like structures. However, comparedwith control, the capacity of CDH ECFCs for vascular formation was reduced in in vivo vasculogenesis assay. Because thecapacity of de novo vasculogenesis in vivo is one of the mostimportant criteria and functional parameters for ECFCs, it
might be reasonable to consider that the in vivo potential of CB
ECFCs to form vascular network is impaired in CDH. The
reason why there were different results between in vitro and invivo tests to evaluate vascular formation could be attributed tothe differences in methodology, such as the duration of experiments, which was much longer in the latter experiment (14days) compared with that of the former (6 to 12 h). Hence, thelatter experiment might have better reflected the differences inthe effect of the proliferative potential of ECFCs on vascularformation in both groups. In summary, because of inhibitedmobilization, migration, and proliferation, fewer ECFCs might
be involved in vascular growth, resulting in fewer vessels
formed in the developing lung of CDH patients.
VEGF and SDF1 are involved in the roles of EPCs, including ECFCs, in neovascularization (5, 27, 35, 47, 59, 63, 66).
VEGF plays a role via the activation of endothelial NOS(eNOS: a synonym of NOS3) (24, 47, 63). Therefore, we
focused on VEGF-NO signaling and SDF1-CXCR4 signaling
to reveal the mechanisms of ECFC functional changes in CDH.
There were no significant differences in CB plasma VEGF and
SDF1 concentration, in vitro VEGF and SDF1 protein
production by ECFCs, and gene expression of ECFCs for
VEGF-A, FLT1, KDR, NOS3, SDF1, and CXCR4 between
CDH patients and controls. These results suggest that ECFC
functional changes in CDH might be caused by mechanisms
other than altered exposure to VEGF and SDF1 and alterationof gene expression of VEGF-NO and SDF1-CXCR4 signaling.
It would be necessary to investigate not only the other aspectsof VEGF-NO or SDF1-CXCR4 signaling but also other signal
pathways in CDH ECFCs to elucidate the mechanisms of
ECFC functional changes in CDH.
With regard to ECFC capacity for NO production, since
NOS3 mRNA expression was unaltered but NO production
was increased in CDH ECFCs, we also measured mRNA for
NOS1 and NOS2 in ECFCs. There were no significant differences in these gene expressions between ECFCs from bothgroups. Therefore, the possible mechanisms could be an increase in enzyme activity of NOS, substrate, or cofactor levels
in CDH ECFCs. As for the blunted increase of NO production
in CDH ECFCs in response to VEGF-stimulation, there might
be two possibilities: 1) the reactivity to VEGF with increasing
NO production was impaired in CDH ECFCs, and 2) there was

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no room to increase NO production because NO production
had already reached a near-maximum level by such possible
mechanisms as described above.
ECFCs are thought to reside throughout the vascular endothelium contributing to vascular integrity and be mobilized intoa circulating pool of EPCs and involved in neovasculogenesis(33, 64). A recent study suggested the existence of lungresident ECFCs and their impaired functions in BPD (3, 4).
The depletion and dysfunction of circulating and/or lungresident ECFCs might be one of the possible mechanisms
underlying arrested lung vascular growth in BPD (3, 8, 14, 15),and several study have investigated the possibility of CB
ECFCs as a therapeutic option for BPD (3, 11). Also, in adultchronic obstructive pulmonary disease, the number and function of ECFCs from peripheral blood were negatively correlated with the severity of pulmonary hypertension (46). Similarly, in our study, circulating ECFCs were decreased and theircell functions were disrupted in CDH. Although we evaluatedonly circulating ECFCs but not lung ECFCs, similar underlying mechanisms for decreased lung vascular growth might beinvolved in lung hypoplasia in CDH. Further studies areneeded to establish a link between the depletion and dysfunction of circulating and/or lung-resident ECFCs and the developmental lung disease like BPD in preterm infants and lung
hypoplasia in CDH.
Our study has several limitations. In this study, only circulating ECFC functions were examined. Hence, the results may
not reflect lung ECFC functions in CDH. Our study excluded
CDH patients who underwent FETO. In our hospital, CDH
patients who are expected to be the most severe cases, that isclassified as liver-up with more than half of the stomach
herniated into the right chest in their fetal diagnosis (39), will
be candidates for FETO. No patients involved in this study met
these criteria. Therefore, some of the results may not apply tothe most severe cases of CDH. Furthermore, except for mRNA
expression of ECFCs for several angiogenic genes and their
exposure to VEGF or SDF1, the more precise mechanisms
for altered ECFC functions in CDH were not investigated.
Basically, we used EGM-2 with FBS in ECFC functional
assays. As EGM-2 with FBS contains various growth factors(EGF, FGFb, IGF1, VEGF, hydrocortisone, and so on), experiments that include a period of serum starvation and growth
factor omission might be helpful to reveal the important signaling pathways involved in the mechanisms of altered ECFC
functions in CDH. Finally, the sample size was small. Therefore, important differences might be missed, for example,
altered VEGF exposure as one of the possible mechanisms for
ECFC functional change in CDH. However, this is an inherent
limitation, as CDH is not a common disease and estimated tooccur in 1 in every 3,500 live births (40). Therefore, further
study is necessary to gain a deeper understanding of the rolesof ECFCs in lung development in CDH and their potential asa new biomarker and/or therapeutic target for CDH.
In conclusion, our results showed that circulating ECFCs
were reduced in CDH and CDH ECFCs had impaired in vitro
potential for self-renewal, clonogenicity, proliferation and migration and in vivo capacity for de novo vasculogenesis.
Additionally, the capacity of ECFCs for NO production wasenhanced while response to VEGF was blunted, indicating thatcirculating ECFC functions are disrupted in CDH. However,these changes might be caused by mechanisms other thanaltered exposure to circulating VEGF and SDF1 and alterationof gene expression of VEGF-NO and SDF1-CXCR4 signaling.
ACKNOWLEDGMENTS
We gratefully acknowledge the staff of the Center for Maternal-Fetal andNeonatal Medicine, NCCHD, for collecting CB samples and clinical data. We
also thank Dr. Julian Tang and Dr. Eisuke Inoue of the Department of
Education for Clinical Research, NCCHD, for proofreading and editing thisarticle and statistical analysis, respectively. Also, we thank Dr. Christopher D.
Baker, Pediatric Heart Lung Center, University of Colorado School of Medicine, for devoted discussion with us.
GRANTSWe are grateful for support from the Japan Society for the Promotion of
Science (JSPS KAKENHI Grant 25670493).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Hideshi Fujinaga and A.U. conception and design of research; Hideshi
Fujinaga, Hiroko Fujinaga, N.W., T.K., M. Tamano, and M. Terao performed experiments; Hideshi Fujinaga, Hiroko Fujinaga, and A.U. analyzed data; Hideshi Fujinaga, Y.I., A.U., and M.K. interpreted results ofexperiments; Hideshi Fujinaga prepared figures; Hideshi Fujinaga drafted
manuscript; Hideshi Fujinaga, Hiroko Fujinaga, N.W., T.K., S.T., Y.I.,
A.U., and M.K. edited and revised manuscript; Hideshi Fujinaga, Hiroko
Fujinaga, N.W., T.K., M. Tamano, M. Terao, S.T., Y.I., A.U., and M.K.
approved final version of manuscript.
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doi:10.1111/jog.12995

J. Obstet. Gynaecol. Res. Vol. 42, No. 6: 612–617, June 2016

MicroRNA and gynecologic cancers
Kohsuke Kanekura1, Hirotaka Nishi2, Keiichi Isaka2 and Masahiko Kuroda1
Departments of 1Molecular Pathology and 2Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan

Abstract
Aim: Gynecologic malignancies are serious problems in female health. Here we aim to discuss the involvement of
microRNA (miRNA) in the pathogenesis of gynecologic cancers and use of miRNA proﬁles for diagnosis of
diseases.
Methods: In order to obtain information needed for this review, we searched the PubMed database with thefollowing keywords: miRNA and ovarian cancer; miRNA and cervical cancer; and miRNA and endometrial
cancer.
Results: Recent explosive investigations in the ﬁeld have dramatically expanded our knowledge of the roles ofmiRNA in the pathology of gynecologic malignancies. In ovarian cancer, miRNA participates in the developmentof drug resistance. In cervical cancer and endometrial cancer, miRNA play essential roles in important oncogenic
processes, including cell proliferation, migration and metastasis. miRNA also have high potentials to be used as
biomarkers in these diseases.
Conclusion: Further validation of the studies and improvement of the methods will result in the broader use ofmiRNA in the diagnosis of diseases as well as in understanding of the pathomechanisms of gynecologic cancers.
Key words: cervical cancer, endometrial cancer, miRNA, ovarian cancer.

Introduction
MicroRNA (miRNA) are a class of small (approximately
18–25 nt in length) single-stranded non-coding RNA
molecules that mediate protein translational repression
in up to 30% of human entire genes by decapping 5′
Cap structure, deadenylating the poly-A tail anddegrading the mRNA.1 miRNA are encoded on the
genome as a cluster of precursors (pri-microRNA),2,3
and once transcribed, miRNA precursors are processed
through multistep enzymatic reactions mediated byendoribonucleases, including Dicer, Drosha and
Argonaute, to form mature miRNA, and function byinterfering with their target mRNA. Since the discoveryof miRNA, an explosively emerging understanding of
their roles in post-transcriptional gene regulation has
provided new insight into the control of cell proliferation,
cell death, metabolism, stem cell maintenance anddifferentiation and development of diseases. Recent
investigations into gynecologic disorders have clariﬁed

the involvement of miRNA in the pathogenesis ofovarian cancer, cervical cancer and endometrial cancer.
In this review, we discuss the progress of miRNA researchin the ﬁeld and the possibility of miRNA-mediateddiagnosis of diseases.

miRNA in gynecologic cancers
During the oncogenesis, tumor cells acquire hallmarks ofcancer, such as resistance against apoptosis, increased
cell proliferation, enhanced migration and invasion.
The miRNA clusters called oncomiR and anti-oncomiRare newly emerged players in carcinogenesis. Hyperactivation of oncomiR and dysfunction of anti-oncomiR
confer cells the characteristics of cancer. The oncogenic
oncomiR are known to target apoptotic molecules.
The most characterized oncomiR cluster, miR-17-92,
suppresses the expression of apoptosis executor Bim4
and transcription factor E2F1, which affect apoptosis

Received: December 28 2015.
Accepted: February 18 2016.
Correspondence: Dr Kohsuke Kanekura, Department of Molecular Pathology, Tokyo Medical University, 6-1-1, Shinjuku, Tokyo 160-8402, Japan.
Email: kanekura@tokyo-med.ac.jp612© 2016 Japan Society of Obstetrics and Gynecology-92-

MiRNA and gynecologic cancers

throughARF-p53signaling.5 EnhancedexpressionofmiR17-92 is conﬁrmed in the tissues from patients with several
malignant tumors, including B cell lymphoma6 and lungcancer.7 ThesedatastronglysupportthatmiR-17-92playsa
pivotalroleintumordevelopment.Interestingly,however,
the miR17–92 cluster is deleted in 16.5% of ovariancancer and 21.9% of breast cancer, implying some tumorsuppressing effect of this oncomiR in gynecologic
malignancies.8 Introduction of miR-17 reduced the rate ofproliferation in breast cancer cell lines via inhibition of theampliﬁed in breast cancer (AIB) 1 gene, which encodes atranscriptional coactivator of the estrogen receptor andE2F1,9 suggesting that estrogen-dependent gynecologic
tissues might have unique responsive mechanisms tomiRNA-mediatedoncogenesis.

which is an enhancer of invasiveness and a metastatic
promoter in EOC as well as a poor prognostic factor.16,17
Using different cell lines, Kumar et al. found that 11 more
miRNA signatures were differentially expressed in thecisplatin-sensitive cell line (A2780) compared with thecisplatin-resistant cell line (A2780/CP70). These miRNA
could potentially target many pathways important forcancer development, including MAPK, transforming
growth factor (TGF)-β signaling, cytoskeleton, Wnt
signaling, Notch signaling and apoptosis.18 From 443
advanced EOC specimens, Parikh et al. found thathigh expression of miR-181a was associated with shorterrecurrence time (P = 0.013).19 miR-181a promotes
TGF-β-mediated epithelial mesenchymal transition(EMT) via repression of its functional target, Smad7,
suggesting that miR-181a might play a central role in
the oncogenesis of EOC.

miRNA in ovarian cancer
Ovarian cancer is the third most common cause of deathfrom women’s cancers.10 Epithelial ovarian cancer(EOC), the most dominant form of ovarian malignant
tumors accounting for 90% of all ovarian malignant
tumors, has the highest mortality rate among all
gynecologic malignancies.10 One reason for this highmortality rate is the lack of appropriate biomarkers forthe early stages of EOC and over 70% of women with
EOC are diagnosed at advanced stages.11 The root causeof EOC remains to be elucidated, but multiple risk
factors, including inherited gene mutations in breast
cancer gene (BRCA) 1 and BRCA2, long-term treatment
with estrogen hormone replacement therapy and lackof pregnancy history have been reported.12–14 Involvement of miRNA in EOC was ﬁrst described by Zhang
et al.8 and since then a number of studies have shown
the proﬁles of miRNA differentially expressed in EOC
tissues compared with those of normal control. During
the clinical course, most patients have surgery followedby chemotherapy with carboplatin and paclitaxel.
Although a signiﬁcant proportion of patients have ahigh initial response rate to the chemotherapy, survival
rates remain poor because chemotherapy resistancedevelops in most (70%) patients with advanced
EOC.14,15 Sorrentino et al. performed high-throughput
analysis of the miRNA in different groups of chemoresistant EOC cells (platinum and paclitaxel resistant)and found that miR-30c, miR-130a and miR-335 wereconsistently downregulated in both chemoresistant
EOC compared with wild-type EOC, suggesting thatthese miRNA are involved in the development ofchemoresistance. A possible miR-130a target is M-CSF,

miRNA in cervical cancer
Cervical cancer is the most common cause of deathfrom women’s cancers in developing countries.20 The
pathogenesis of cervical cancer is highly associated with
the infection of human papilloma virus (HPV), especiallytype 16 and type 18.21 HPV has a circular 8-kb doublestranded DNA genome, encoding eight genes, E1, E2,
E4, E5, E6, E7, L1 and L2.21,22 Among these genes, E5,
E6 and E7 oncoproteins are supposed to be responsible
for the oncogenic activity of HPV. At the protein level,
HPV16-E5 enhances epidermal growth factor (EGF)EGF receptor signaling and promotes cell proliferationin vitro.23 E5 can also suppress apoptotic cascades
mediated by tumor necrosis factor ligand (FasL) andtumor necrosis factor-related apoptosis-inducing ligand(TRAIL) by downregulation of FAS receptor andinhibition of the formation of death-inducing signaling
complex activated by TRAIL.24 The E6 oncoprotein
consists of 158 amino acids and contains two zinc-ﬁnger
binding motifs.25 The most established target of E6 is thetumor suppressor gene, p53, and E6 enhances proteasomal degradation of p53, resulting in uncontrolled cell
cycle progression.26 Although the inhibition of p53 is
one of the main pathomechanisms by HPV-E6, it has
been accepted that E6 has oncogenic activities that are
independent of p53. For instance, E6 interacts with
pro-apoptotic Bcl2 family protein, Bax and suppresses
apoptosis in vitro.27 Furthermore, E6 associates with
c-Myc and induces the expression of human telomerase reverse transcriptase (hTERT) to contribute toimmortalization of the cervical tumor cells.28 The E7© 2016 Japan Society of Obstetrics and Gynecology

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K. Kanekura et al.

proteins, especially encoded by high-risk HPV, such astype 16 and 18, have a strong afﬁnity to Rb protein andblock the anti-tumor activity of Rb.29 Rb-related
cell-cycle regulating proteins, such as p107 and p130,
are also targeted by E7 via the surface pocket
structure.30–32 One of the major functions of Rb is tobind and negatively regulate E2F-family transcription
factors whose functions are essential for controlling
G1/S transition.33 By binding to Rb, HPV-E7 inactivates Rb and enables the E2F to trigger transcriptionsof cyclins, cyclin-dependent kinases and checkpoint
regulators to stimulate cell cycles. Because E6 and E7
are involved in the activation of transcription factors,
it is little wonder that E6 and E7 cause dysregulationof miRNA, resulting in the enhancement of malignancy seen in cervical cancer. To date, more than 20
miRNA have been identiﬁed as dysregulated miRNAin human cervical cancer samples.21 Wang et al.
demonstrated that the miRNA-25-92a cluster was
highly induced by overexpression of HPV-E7 in vitro
and also highly expressed in the samples of humancervical cancer compared with normal control andcervical intraepithelial neoplasia (CIN).34 In contrast,
the expression of miR-22-29a cluster was not affectedin the presence or absence of HPV infection, suggesting that the ratio of miR-25-92a over miR-22-29a
might be a useful tool to distinguish normal cervix
from CIN and from CIN to cervical cancer. Tang et al.
screened miRNA from cervical cancer cell lines
and identiﬁed that miR-182 is highly expressed in
cervical cancer tissue compared with normal
tissue.35 Antagonistic anti-miR-182 signiﬁcantly
suppressed the growth of tumor cells in vivo,
suggesting that miR-182 plays an onco-miRNA rolein human cervical cancer. Luo et al. found that theexpression level of miR-497 negatively correlateswith the prognosis of cervical cancer.36 The
possible target of miR-497 is insulin-like growth
factor 1 receptor, thus miR-497 possibly functions
as a tumor suppressor.
Qian et al. reported that the HPV genome itself
encodes viral microRNA, which can target cell cycle
proteins CYP26B1 and ﬁbroblast growth factor 7(FGF7), also known as keratinocyte growth factor(KGF).37 KGF is a potent epithelial growth factor
involved in wound-healing and can modulate thegrowth of HPV16 immortalized cervical epithelial cells.
The TEAD1 gene, encoding a transcriptional enhancer
factor TEF1, which is another possible target of HPV16miRNA, regulates cell proliferation, migration andinduction of EMT.38

614

miRNA in endometrial cancer
Endometrial cancer (EC) is the most common
gynecologic cancer, especially in Western countries.39
Risk factors of EC include obesity, excessive estrogen
exposure, high blood pressure and diabetes.40 It has
been demonstrated that continuous high levels of
estrogen stimulate the proliferation of the endometrium.
Genetic change that causes endometrial malignant
transformation occurs during this prolonged endometrial proliferation. Upon estradiol binding, estrogen
receptors mediate transcription by direct binding to
speciﬁc estrogen response elements in the promoter/enhancer region of the target genes, such as atranscription factor SP1.40,41 Estrogen receptor α (ERα),but not ERβ, is reported to interact with and suppress
Drosha activity, which is essential for the processing ofpro-miRNA to pre-miRNA.42 The interaction between
ERα and Drosha is enhanced by estradiol, and a numberof miRNA are downregulated by treatment with
estradiol in vitro.43 One of the miRNA downregulatedby the estrogen signal is the tumor suppressor miR-30c,
whose target is metastasis-associated protein (MTA)-1.
Kong et al. reported that estrogen downregulatesmiR-30c and increases the expression of MTA-1,
resulting in enhanced cellular proliferation of EC cells.44
Another example of miRNA downregulated by estrogenis miR-194. miR-194 suppresses the expression ofpolycomb complex protein B cell-speciﬁc Moloneymurine leukemia virus integration site (BMI)-1, which
regulates EMT.45 BMI-1 promotes EMT by repressing
expression of both E-cadherin and p16INK4a.46 In EC,
a low expression level of miR-194 causes poor prognosis
compared with EC with a high expression level of miR194, showing the importance of miRNA regulation in
the metastasis of EC.
As well as repressing the expression of miRNAby interfering Drosha, estrogen-ERα complex also
upregulates multiple miRNA by distinct mechanisms.
Estrogen-ERα has versatile targets to exert its biological
functions and one of the targets is c-Myc.47 Once
estrogen binds to ERα, the estrogen-ERα complex
immediately induces the expression of c-Myc.47,48
Induced c-Myc triggers expression of a miRNA cluster,miR-17-92.47 Overexpression of miR-17-92, conﬁrmed
in human EC samples,49 suppresses the proapoptotic
Bcl2 family protein Bim and thus promotes tumor
progression.50,51 Furthermore, Karaayvaz et al. reported
that the expression levels of miR-200c and miR-205 weresigniﬁcantly increased in EC compared with normal
tissue, and especially, high levels of miR-205 were

© 2016 Japan Society of Obstetrics and Gynecology-94-

MiRNA and gynecologic cancers

associated with poor prognosis.52 The possible target ofmiR-205 was a tumor suppressor PTEN and theexpression of PTEN was suppressed in EC.

Circulating miRNA as diagnostic tools forgynecologic malignancies
The change of proﬁles of circulating miRNA in humanblood has been reported for a variety of diseases.53,54
Because of difﬁculty in diagnosis of gynecologic cancers
at early stages, many patients are diagnosed at advanced
stages. In attempts to facilitate early and accurate
diagnosis of gynecologic malignancies, several studies
have identiﬁed possible biomarkers for gynecologic
cancers.55 Taylor et al. reported that the levels of eightmiRNA (miR-21, -141, -200a, -200c, -200b, -203, -205
and -214) were differentially expressed in the serum ofpatients with ovarian cancer compared with normal
controls.56 Resnick et al. also reported that miR-21, -29a,-92, -93 and -126 were upregulated whereas miR-127, 155 and -99b were downregulated in the serum ofpatients with ovarian cancer compared with normal
controls.57 The proﬁles of circulating miRNA in theserum of patients with endometrial cancer have been lesscharacterized compared with those of ovarian cancer,
but recently, miR-194 was found as a prognostic marker
in EC and patients with higher miR-194 levels had better
prognosis than those with lower miR-194 levels (P =0.0067).8 In the case of cervical cancer, levels of miR-218
were signiﬁcantly reduced in the sera of patients withcervical cancer, and levels of miR-219 were also usefulfor disease staging due to lesser levels of miR-219
associated with advanced stages and lymph nodemetastasis.58 Zhao et al. found that the levels of miR20a were signiﬁcantly higher and the levels of miR-203
were signiﬁcantly lower in the sera of patients withcervical cancer compared with those of normal
controls.59 Furthermore, higher levels of miR-20a werecorrelated with lymph node metastasis, tumor diameterand histological grade whereas downregulation of miR203 was only correlated with lymph node metastasis.

human samples and immature techniques of quantiﬁcation of miRNA. In addition, for clinical use, sensitivity
and speciﬁcity of miRNA in each disease need to befurther investigated. Improvement of techniques forliquid biopsy and quantiﬁcation of miRNA will open a
new horizon for expanding our knowledge in gynecologic cancer research. The beneﬁt of miRNA proﬁling
will help us to obtain more accurate diagnoses and evenbetter prognoses.

Acknowledgments
Our research was supported by Grants-in-Aid for
Scientiﬁc Research (26293088), Grants-in-Aid for
Exploratory Research from the Japan Society for thepromotion of Science (15 K15100) and supported inpart by Strategic Research Foundation Grant-aided
Project for Private Universities from the Ministry of
Education, Culture, Sports, Science and Technology(S1511011), Japan.

Disclosure
The authors have no conﬂicts of interest related to thisarticle.

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Conclusions
We have proposed the possibility of miRNA as
diagnostic tools for gynecologic malignancies. There is
no doubt that proﬁles of circulating miRNA have great
potential as novel biomarkers for these diseases;
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International Journal of

Molecular Sciences
Review

Focus on Extracellular Vesicles: Development ofExtracellular Vesicle-Based Therapeutic Systems
Shin-ichiro Ohno 1 , Gregor P. C. Drummen 2, *,† and Masahiko Kuroda 1, *,†
1
2

*

†

Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402,
Japan; s-ohno@tokyo-med.ac.jp
Cellular Stress and Ageing Program, Bionanoscience and Bio-Imaging Program, Bio & Nano-Solutions,D-33647 Bielefeld, Germany
Correspondence: gpcdrummen@bionano-solutions.de (G.P.C.D.); kuroda@tokyo-med.ac.jp (M.K.);
Tel.: +49-521-3988-2959 (G.P.C.D.); +81-333-516-141 (ext. 234) (M.K.); Fax: +49-3222-240-7500 (G.P.C.D.);+81-333-526-335 (M.K.)
These authors contributed equally to this work.

Academic Editor: William Chi-shing Cho
Received: 22 August 2015; Accepted: 29 January 2016; Published: 6 February 2016

Abstract: Many types of cells release phospholipid membrane vesicles thought to play key roles
in cell-cell communication, antigen presentation, and the spread of infectious agents. Extracellular
vesicles (EVs) carry various proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs), likea “message in a bottle” to cells in remote locations. The encapsulated molecules are protectedfrom multiple types of degradative enzymes in body fluids, making EVs ideal for deliveringdrugs. This review presents an overview of the potential roles of EVs as natural drugs and novel
drug-delivery systems.
Keywords: exosome; microvesicle; extracellular vesicle; exosome; dexosome; outer membrane vesicle;
stem cell; drug delivery system; vaccine; clinical trial

1. Introduction
Membrane vesicles secreted from various types of cells are categorized as exosomes, microvesicles(MVs), and apoptotic vesicles according to their size, pathway of origin, and constituent molecules [1,2].
Exosomes, which are 30–100 nm sized vesicles derived from the late endosome, can be isolated fromthe conditioned medium of various cells or from body fluids using a sucrose gradient (1.13–1.19 g/mL)or ultracentrifugation (100,000ˆ g for 70 min) [3]. Exosomes are enriched in heat shock proteins(HSP70, HSP90), tetraspanin family molecules (CD9, CD63, CD81), and components of the ESCRT(endosomal sorting complex required for transport) machinery (e.g., Alix and TSG101) [1,2]. MVs, also
called shedding vesicles, ectosomes or microparticles, are between 100 and 1000 nm in diameter andare enriched in phosphatidylserine, integrins, selectins, and CD40 ligand. Unlike exosomes, MVs areformed through outward budding of the plasma membrane [4,5]. Apoptotic vesicles are derived from
apoptotic cells and are distinctly different from exosomes because they abundantly contain histones
associated with membranes that float at high sucrose densities (1.24–1.28 g/mL) and because they arevery heterogeneous in size and morphology when observed by EM [6].
Because the methods used to isolate and purify membrane vesicles differ significantly between
studies, we do not strictly distinguish these categories in this review, but instead collectively refer tosuch vesicular structures as “extracellular vesicles” (EVs) and explicitly identify the subtype whennecessary. EV proteins that are expressed on lipid bilayer membranes stimulate receptors on thesurfaces of physically separated cells, and the encapsulated materials play functional roles in the cells
that take up the EVs. These characteristics raise the possibility that EVs might be used therapeutically.
Int. J. Mol. Sci. 2016, 17, 172; doi:10.3390/ijms17020172

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Research
aimed atapplyingaimed
EVs inata applying
clinical setting
candividedinto cantwo bebroad
categories(Figure1):
therapeutically.
Research
EVs ina beclinicalsetting
dividedinto two
broad
(1)
EVs
as
biological
medicines,i.e.,therapeutics
that
take
advantageofthepositive
effectexertedbycategories (Figure 1): (1) EVs as biological medicines, i.e., therapeutics that take advantage of themolecules
contained
themolecules
EVs secreted
by particular
types
of cells.by particular types of cells.
positive effectexertedinby
contained
in the EVs
secreted

1. Research aimed at developing
developing extracellular
extracellular vesicles (EVs) forfor clinicalclinical applications.
applications. APC:
Figure 1.
presenting cell;
cell; DCs:
DCs: Dendritic cells; iDCs: Immature dendritic cells; DDS: Drug delivery
Antigen presenting
Graft-versus-host disease;disease; MSCs:
MSCs: Mesenchymal
Mesenchymal stem
stem cells.
cells.
system; GVHD: Graft-versus-host

The majority
majorityof oftheseefforts
are focusedon immune-regulatory(i.e.,
immuneThe
theseefforts
are focusedon immune-regulatory
effects (i.e.,effects
immunesuppression
suppression
or immunecontext
of vaccines(Figure1, upper).
In have
addition,haveor
immune activation)in activation)
the contextin
ofthe
vaccines(Figure1, upper).
In addition,
EVs
beenEVs
reportedbeenreportedtomediateeffectsontissuerepairandangiogenesis(Figure1,
lowerleft);(2)EVs
asto mediate effects on tissue repair and angiogenesis (Figure 1, lower left); (2) EVs as carriers for
carriersfordrug-delivery
systems(DDSs),
takingadvantageofEVs’
naturalcharacteristics
todeliverdrug-delivery systems (DDSs), taking advantage of EVs’ natural characteristics to deliver molecules
molecules
to target
cells(Figure1, lowerright).
In thiswe summarize
the developmentoftotarget cells(Figure1, lowerright).
In thisreview,
wereview,
summarize
the developmentof EV-based
EV-baseddrugs
anddrug-delivery
systems,anddiscuss
futurepossibilitiesforclinicalapplicationsofdrugs and drug-delivery systems, and discuss future possibilities for clinical applications of EVs.
EVs.
A
comprehensive
introduction
to
EVs
is
provided
in
this
focus
editioninthearticleby
A comprehensive introduction to EVs is provided in this focus edition in the article by Kalra et al. [2].
Kalra et al. [2].
2. EVs as Therapeutic Vehicles
2. EVs as Therapeutic Vehicles
2.1. Liposomes vs. EVs
2.1. Liposomes
vs. EVs
Historically,
artificial vesicular carriers, e.g., liposomes (Figure 2A), have been used to carrytherapeutics
to target
tissuesvesicularand cells.carriers,
These liposomes
generally
have various
sizeswithused
eitherHistorically,
artificial
e.g., liposomes(Figure2A), havebeen
to single
carryormultiplelipidbilayers,
e.g.,
Small
or
Large
Unilamellar(SUVs;LUVs)
vs.
Multilamellar
Vesicles.
therapeutics to target tissues and cells. These liposomes generally have various sizes with either
The
lipid
compositions
liposomeson(SUVs;
their intended
application,butsingleor multiple
lipid ofbilayers,
e.g.,generally
Small or vary,
Largedepending
Unilamellar
LUVs) vs.
Multilamellar
mostcommonlyphosphatidylcholine(lecithin)andphosphatidylethanolamineorderivatives
areusedVesicles. The lipid compositions of liposomes generally vary, depending on their intended
incombination
control membrane fluidity
andprevent
physical instability andapplication,
butwithmostcholesterolcommonlytophosphatidylcholine(lecithin)andphosphatidylethanolamineorprematurecontent
leakage(highpermeability).
Dependingonthepolarityoftherapeutic
agent,
derivatives are used in combination with cholesterol to control membrane thefluidityand prevent
eitherencapsulationin theliposomal
lumenleakage
or incorporation
into the lipid
bilayer occurs.
At least
physicalinstability andprematurecontent(high permeability).
Dependingon the polarity
19
drugs
with
liposomal
DDSs
have
been
approved
by
the
FDA
and
EMA
[7,8],
whilstnearlyof the therapeutic agent, either encapsulation in the liposomal lumen or incorporation into 450
the
phase
I-III open
interventional
studiescurrently
being
conducted[9]. Despite
severaladvantages,
lipid bilayer
occurs.
At least 19
drugsarewithliposomal
DDSs
have been
approved
by the
FDA andliposomes
drawbacks
that
includeproblems related
to stability,
fatty acyl
moietyoxidation,
EMA [7,8],suffer
whilstfrom
nearly450 phaseI‒III
open interventional
studiesare currently
being
conducted[9].

Despite several advantages, liposomes suffer from drawbacks that include problems related tostability, fatty acyl moiety oxidation, drug loading, liposome targeting, drug leakage and release,-99-

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drug loading, liposome targeting, drug leakage and release, and others. Most importantly, intravenous
and others. Most importantly, intravenous injection of liposomal drugs has been known to induce
injectionof liposomal drugs has been known to induce complement activation-related pseudoallergy
complement activation-related pseudoallergy (CARPA) in some cases; an acute and severe(CARPA)in some cases;
an acute
and severe hypersensitivity reaction [10,11].
hypersensitivity
reaction[10,11].

Figure 2. Comparison of liposomes and extracellular vesicles (EVs) as drug-delivery-systems (DDS).
Figure2. Comparison of liposomes and extracellular vesicles (EVs) as drug-delivery-systems (DDS).
(A) Schematic depiction of lipid-based vesicular carriers (not to scale). Note that in comparison to(A) Schematic depiction of lipid-based vesicular carriers (not to scale). Note that in comparison toliposomes, EV membranes not only consist of a diverse number of lipid classes, but also containliposomes, EV membranes not only consist of a diverse number of lipid classes, but also containtrans-membrane and membrane-associated proteins, receptors, adhesion molecules, as well as a naturaltrans-membrane
membrane-associatedreceptors,
adhesionmolecules,
as wellcorona. Such andfeaturesmust be engineered proteins,
into liposomes
to obtaina stableand suitable
DDS;as anaturalcorona.
Suchfeaturesmust
beengineered
intoliposomes
toobtainastableandsuitable
DDS;(B) Accumulation of exosomes is profoundly higher (B2) than accumulation of artificial nano-carriers(B) Accumulationofexosomesisprofoundly
higher(B)
thanaccumulationofartificial
nano-carriers
such as liposomes (B1; white arrows) in PC12 neuronalcells after 24 h incubation [12]. Red (Dil,
2
97ClN2O4): (Bexosomes
nano-carriers;
Green(rabbitcells
anti-PGP9.5):
microfilaments;
Blue(Dil,such C
as59Hliposomesin PC12neuronal
after 24 hactin
incubation[12]. Red
1 ; whiteorarrows)(DAPI):
nuclei.
C59 H97 ClN2 O4 ): exosomes or nano-carriers; Green (rabbit anti-PGP9.5): actin microfilaments; Blue(DAPI): nuclei.
Unlike liposomes, EVs have the distinct advantage that their membranes are structurallycomparable to other membranous structures found in cells. This means that both their lipid
Unlikeliposomes,
EVsthehave
the distinct
advantage
thatpotential
their membranes
are docking
structurallycomposition,
fluidity,presenceof membraneproteins,
targeting andcomparable
to otherstructures
foundin cells.EVsThis
meansthat theboththeir lipid
modalities
are all membranouspreserved in EVs(Figure 2A).
Furthermore,are likely
to carryrequiredfusogenic
properties
or uptakeofmachinery
theirpotential
corpora. targeting
In comparison,
liposomes
arecomposition,
fluidity,
the presence
membranewithinproteins,
and docking
modalities
vesicularsome measurecontrolregardingbeare all primitive
preserved
in EVsstructures,(Figure over2A).whichFurthermore,
EVsofarelikelyto carrytheir
theproperties
requiredcan
fusogenic
obtainedthrough
control
of
their
size,
lipid
composition,
corona,andadditionofpotential
targetingproperties or uptake machinery within their corpora. In comparison, liposomes are primitive
peptides or other surface modifications. However, the difficulty of obtaining suitable coronas thatvesicular structures, over which some measure of control regarding their properties can be obtained
ensure sufficiently decreased clearance by the mononuclear phagocyte system (long circulation
through
controlof theirsize, lipid composition,
and additionof potentialfibronectintargeting andpeptidestimes),
prevent
opsonization(fouling with corona,complement,
immunoglobulins,
or othersurface modifications.
However,the difficulty
obtainingthat ofensureapolipoproteins)and non-specificinteractions
with cellsofillustrates
thesuitable
relative coronasprimitivenesssufficiently
decreasedclearance
bywiththe mononuclear
phagocyte
systemcirculation
times),
liposomes
as DDSs
comparedEVs. For instance,
a commonway to(longreducesurface foulingis preventbycoating with
polyethylene
glycol (PEG). However,corona design
requires significant
engineering, as andopsonization(foulingwith complement,
immunoglobulins,
fibronectin
and apolipoproteins)recentresearch
intonano-sized
DDS
shows
that
for
long
circulation
times
and
anti-foulingboth
non-specific interactions with cells illustrates the relative primitiveness of liposomes as DDSs compared
mixed
lengthPEG
chainswithparticularconformationsandasufficiently
high
PEG
chain
densitywith EVs. For instance, a common way to reduce surface fouling is by coating with polyethylene glycol(PEG). However, corona design requires significant engineering, as recent research into nano-sized
DDS shows that for long circulation times and anti-fouling both mixed length PEG chains withparticular conformations and a sufficiently high PEG chain density are required [13,14]. Although
PEGylation might reduce the aforementioned limitations, it also potentially reduces interaction of the-100-

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DDS with target cells, negatively affecting the DDS’s biodistribution. Furthermore, Armstrong andco-worker report that preexisting PEG antibodies are present in 22%–25% of healthy blood donors
as a consequence of exposure to PEG in cosmetics and foodstuffs [15,16], which might negatively
influence the effectiveness of therapeutic interventions [17]. Nonetheless, PEGylation is the only
coating strategy for DDSs that is regarded as safe for use in humans and carries the GRAS classification(Generally Recognized As Safe) issued by the FDA [18]. Overall and because of the nature andstructural composition of EVs, these should be considered superior as DDSs compared with liposomes
and consequently open up new ways for drug delivery.
2.2. Cellular Uptake and Drug-Loading
Cellular uptake of EVs surpasses that of more traditional carriers such as liposomes or nanoparticles(compare Figure 2B1,2). Batrakova’s group not only demonstrated unparalleled accumulation of
drug-loaded exosomes compared with 100 nm poly(lactic-co-glycolic acid) or liposomes in PC12 cells,
but also that ex vivo catalase-loaded macrophage-derived exosomes significantly accumulated in mousebrain neurons and microglial cells upon intranasal administration [12], circumventing the limitationsof various DDSs regarding mucosal and blood brain barrier traversal.
Fuhrman and collaborators recently compared various passive and active drug-loading
methods, i.e., electroporation, saponin treatment, extrusion and dialysis, and used porphyrins
of various hydrophobicities as model drugs [19]. They determined that porphyrin-loaded EVs
from MDA-MB231 breast cancer, human umbilical vein endothelial and human mesenchymal
stem cells all showed superior cellular uptake and light-induced cellular killing than drug-loaded
phosphatidyl-choline/cholesterol liposomes or free porphyrins. Furthermore, passive loading ofhydrophobic compounds into EVs was significantly higher than in liposomes, which might be a resultof the presence of particular domains within EV membranes that are absent in the artificial membranesof liposomes. Although it has long been known that, through the right combination of phospholipids
and cholesterol in liposomal formulations, the properties of liposomal membranes can be steered, such
artificial membranes are unlikely to be able to compete with the more natural structural properties of
EV membranes (Figure 2A). Finally, Fuhrman et al. determined that of all nondestructive active loading
methods, particularly the saponin-assisted method allowed high loading efficacies. Similar results wereobtained in the aforementioned catalase-loaded exosomes; albeit that saponin treatment, sonication andextrusion all showed high loading efficiencies and no significant degradation of catalase [12]. Despite
the reported high loading capacities, there are limits to the maximal attainable loading, in particularin exosomes, due to size restrictions and the native presence of numerous proteins and nucleic acids.
For small RNAs, electroporation is generally used to load EVs, as established by Alvarez-Erviti andco-workers [20]. We also successfully used electroporation to load miRNA into exosomes targeted tothe epidermal growth factor receptor (EGFR) of breast cancer cells [21]. However, electroporation has
been known to induce aggregation in particular when loading siRNA and as a result the true siRNAcontent is often overestimated [22]. Beside the aforementioned loading methods, genetic manipulationof the parental cell has been used to incorporate particular proteins into EVs. This strategy can berealized by, for instance, introducing genetic material, e.g., cDNA, encoding the protein of interest
and a protein that localizes in EVs such as C1C2 to the parental cell [23]. Alternatively, plasmid-based
introduction has been successfully used to modify macrophages for reproduction, packaging, andtargeted therapeutics delivery to treat neurodegenerative disorders [24]. Finally, Maguire et al. took
it one step further and proposed exploiting viral packaging systems (from non-enveloped viruses)
to improve gene delivery by creating hybrid vesicles called “vexosomes” (vector-exosomes) [25].
Although they achieved significantly better transfection and reduced immunogenicity compared withthe free viral vector, such hybrid systems would require additional scrutiny.
Overall, the abovementioned research results show that drug-loaded EVs are superiors as DDS
both in terms of loading, circulation times, and cellular uptake and payload delivery. Furthermore,-101-Int. J. Mol. Sci. 2016, 17, 172

5 of 19due to the nature of EVs, therapeutic options built into EVs are significantly better protected from theharsh outside environment than other lipid-based DDS.
3. Medicinal Use of Native EVs
3.1. EVs from Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are self-renewing precursor cells (multipotent stem cells) that areable to differentiate into a variety of cell types, including bone, cartilage, muscle, marrow, ligament,
adipose and connective tissues [26,27]. Consequently, MSCs have predominantly been investigatedin the context of clinical applications aimed at repairing damaged tissue. These studies revealed that
MSCs exert tissue-repair functions in blood vessels, lung, kidney, bone, and cartilage tissue in various
animal disease models [28]. In addition, MSCs have potent immunosuppressive activities that caninhibit both innate and adaptive immune responses. Research and clinical trials have been conducted
to investigate the inhibitory effects of MSC transplantation on graft-versus-host disease (GVHD) after
allogeneic tissue transplantation, rheumatism, uveitis, diabetes, and inflammatory bowel disease [29].
Furthermore, via their neuroprotective effects, MSCs can prevent amyotrophic lateral sclerosis, multiple
sclerosis, Parkinson’s disease, and glaucoma [30–34]. As noted above, MSCs have multiple functions
that include tissue repair, immunosuppression, and neuroprotection, but the mechanisms underlying
these functions remain largely unknown. Recent work showed that administration of the exosome
fraction isolated from MSCs has an effect similar to that of MSC transplantation [35]. Treatment
of GVHD with MSC-derived EVs decreased the pro-inflammatory cytokine response of peripheral
blood mononuclear cells (PBMCs) and diarrhea volume. In lung disease, EVs derived from bonemarrow MSCs inhibit pulmonary arterial hypertension in rats [36] and acute lung injury inducedby E. coli endotoxin in mice [37,38]. Tissue-repair functions are not limited to MSCs, but have also
been reported in embryonic stem cell (ESC). Although transplantation of ESCs is considered very
effective in vascular disorders, serious side effects such as teratomas may occur [39,40]. Nonetheless,
Khan et al. successfully treated myocardial infarction (MI) in a MI mouse model without side-effects
when administrating ESC-derived EVs [41]. EVs derived from MSCs also have useful functions beyond
activation or suppression of immune responses. For example, EVs derived from MSCs protect against
acute kidney injury and may activate a proliferative program in surviving tubular cells after injury via
horizontal transfer of mRNA [42,43]. These results indicate that the identities and functions of MSCs
are reflected in their EVs. Consequently, the EVs of MSCs have attracted attention as a novel cell-free
approach for stem cell therapy of various diseases [44].
3.2. EVs from Antigen Presenting and Other Immune Cells
Antigen-presenting cells (APCs) include B-cells, macrophages, and dendritic cells (DCs) that
display foreign antigens on their surfaces in association with major histocompatibility complex (MHC)
proteins. These antigen-MHC complexes activate T-cells via T-cell receptors (TCRs). Recent work
showed that EVs secreted from APCs also express MHCs and costimulatory receptors on theirsurfaces [45–47]. Moreover, these EVs can stimulate T-cells and activate the adaptive immune system.
For example, EVs from peripheral blood of BALB/c mice infected with the reticulocyte-prone non-lethal
Plasmodium yoelii strain 17X contain parasite proteins. Moreover, immunization of mice with purified
EVs elicits IgG antibodies capable of recognizing P. yoelii-infected red blood cells and protects against
lethal challenge with the normocyte-prone lethal P. yoelii strain 17XL [48]. Furthermore, peptide- orprotein-loaded EVs derived from DCs (dexosomes) can inhibit tumor development in a CD4+ T- andB-cell-dependent manner [49,50]. Damo and co-workers recently showed that dexosomes producedwith the Toll-like receptor 3 (TLR-3) agonist polyinosinic-polycytidylic acid (poly(I:C)) in conjunction
with ovalbumin (OVA) stimulate OVA-specific CD8+ and CD4+ T-cell proliferation and acquisition ofeffector functions [51]. This effect was significantly higher compared with using lipopolysaccharide(LPS) for TLR-4 or CpG-B for TLR-9. Furthermore, when DCs were loaded with a B16F10 melanoma-102-

Int. J. Mol. Sci. 2016, 17, 172

6 of 19cell lysate to produce dexosomes containing tumor antigens, the resulting vaccine strongly induced
activation of melanoma-specific CD8+ T-cells and recruitment of cytotoxic CD8+ T-cells, NK and
NK-T-cells to subcutaneously grafted B16-F10 melanoma tumors in C57BL/6 mice. As a consequence,tumor growth was significantly more reduced and survival prolonged compared with comparable
vaccines that lack the aforementioned pretreatments.
In the presence of IL-4 and IL-10, cultured bone marrow DCs develop into immunosuppressive
DCs. Surprisingly, EVs derived from immunosuppressive DCs inhibit delayed-type hypersensitivity(DTH) and rheumatism in animal models [52]. This mechanism is still not well understood, but
probably depends on MHC Class II and the CD95-CD95L signaling pathway. In addition to theimmunosuppressive EVs generated by cultured cells in vitro, as described above, immunosuppressive
EVs also exist in vivo. EVs purified from the serum or bronchoalveolar lavage fluid (BALF) ofantigen-sensitized mice have immunosuppressive activity [53]. EVs from genetically modified bonemarrow-derived DCs expressing IL-4 or FasL suppress inflammation of DTH in a mouse model ofcollagen-induced arthritis [54,55]. In addition, the proportion of Foxp3+ CD4+ CD25+ regulatory T-cells
is elevated and production of IL-2 and IFN-γ is inhibited in human PBMCs incubated with EVs frombreast milk [56].
Beside induction of immune responses by bioactive membrane constituents, microRNA (miRNA)luminal cargos have been assumed to mediate biological functions via gene regulation of the target
cell. Although EV transfer of small RNAs have now been observed in various systems involving
immune cells, the exact role of EVs in this transfer remains unclear. For instance, endogenous miR-142
and miR-223 from human macrophages were shown to be transferred to hepatocellular carcinoma
cells (HCC), which resulted in decreased expression of stathmin-1 and insulin-like growth factor-1
receptor and inhibition of proliferation [57]. However, this transfer only occurred to a limited extent
via exosomes, whereas the majority of miRNA transfer required intercellular contact and involved
gap junctions. Recent results from investigations of exosome-mediated miRNA transfer between
Dicer-proficient macrophages to Dicer-deficient endothelial cells equally only showed modest exosomaltransfer [58]. Consequently, it is questionable that exosomal miRNA transfer alone could exert theobserved biological effects in the aforementioned investigations. However, these results do neither
exclude the potential for therapeutic effects from treatment with previously isolated and purified
macrophage-derived exosomes nor the involvement of EVs in signal transduction of other cellular
and tissue systems. For instance, the potentially deranged metabolism in cancerous cells might affectboth packing of small RNA into EVs and the degree of their release. One recent exciting developmentin using EVs to modulate immune responses is based on helminth exosomes. Given the ability ofhelminths to exploit host immunity, the small RNAs and proteins in secreted helminth exosomes havebeen shown to suppresses the Th2 innate responses and eosinophilia against particular allergens, or
suppress immune functions by inducing apoptosis and tolerogenic properties in DCs [59–61].
Overall the aforementioned reports all suggest that EVs play important roles in regulating theimmune system in vivo and therefore have therapeutic potential against immune system-relateddiseases [62].
4. EVs as Drug-Delivery System Carriers
4.1. EVs as Carriers for Small Molecular Drugs
Different methods can be employed for loading small molecules into EVs. These include loading
EVs after isolation from parental cells, loading parental cells with the drug, followed by isolationof released EVs, and transfecting parental cells with DNA encoding for the drug and subsequentisolation of the EVs. For instance, EVs loaded with doxorubicin have effectively been used to inhibit
growth of breast and colon cancers [63,64]. These studies suggest that EVs can effectively deliver
chemotherapeutics to treat malignant tumors.
Guo et al. showed that therapy with EVs purified from L1210 lymphocytic leukemia cell
antigen/lipopolysaccharide-pulsed DCs and loaded with cyclophosphamide and poly(I:C) were-103-Int. J. Mol. Sci. 2016, 17, 172

7 of 19able to induce DC maturation and most effectively suppress tumor growth and prolong survival timein tumor-bearing DBA2 mice [65]. These findings suggest that the combination of a tumor vaccine, aconventional anti-cancer agent, and a promoter of DC maturation might represent a generally usefulstrategy for anti-cancer therapy. MSCs have been proposed as delivery vehicles for anticancer agents
because of their ability to migrate to tumor microenvironments. Pascucci et al. reported a new approachto drug delivery using EVs from MSCs. Following priming with paclitaxel (PTX), MSCs were able tostrongly inhibit pancreatic tumors via their capacity to release the drug from PTX-loaded MSC EVs
that were taken up by the cancer cells [66]. Tian and co-workers used a different approach to deliver
doxorubicin (Dox) to tumors in BALB/c nude mice [63]. These authors loaded exosomes purified
from immature dendritic cells (iDCs) with Dox via electroporation. Tumor targeting was enhanced bypromoting expression of the exosomal membrane protein Lamp2b fused to αv integrin-specific iRGD
peptide (CRGDKGPDC) in iDCs, whereas iDCs were used to reduce immunogenicity and toxicity.
Interestingly, Federici et al. recently showed that the opposite to drug loading of EVs
occurs in resistance to cytotoxic anti-cancer drugs, i.e., “drug unloading” of cisplatin [67]. Not
only does extracellular acidosis impair cisplatin uptake by cancer cells per se, but also induces
exosome-mediated elimination of cisplatin from cancer cells; both could be inhibited by additionof a proton-pump-inhibitor.
4.2. EV-Encapsulated Curcumin
Curcumin, a natural polyphenol found in the rhizomes of Curcuma longa (turmeric), has beenshown to exert various biological activities, including anti-inflammatory, anticancer, and antioxidant
effects. However, due to its hydrophobicity, curcumin has poor aqueous solubility and a poor
absorbability and overall bioavailability. To overcome these limitations, various delivery strategies havebeen used, including encapsulation in lecithin liposomes (small unilamellar vesicles) [68], micelles [69],cyclodextrins [70], and chitosan [71]. In addition, absorption enhancers, such as piperine have beenused in conjunction with encapsulation to improve bioavailability [72].
Sun et al. on the other hand used EVs as a delivery vehicle for curcumin and showed that curcumin
preferentially interacts with EVs in lipid membranes [73]. Additionally, curcumin delivered by EVs ismore stable and more concentrated in blood, which indicates a significantly superior absorption andbioavailability. In a LPS-induced mouse model of septic shock, EV-encapsulated curcumin inhibited
LPS-induced inflammation significantly more effectively than free curcumin [73]. This study provided
evidence for the potential of EVs as drug-delivery carriers. Zhuang et al. improved the methodof administration of EV-encapsulated curcumin: intranasal administration of EVs curcumin led to
rapid delivery of curcumin to the brain and significantly inhibited brain tumor growth, LPS-induced
brain inflammation, and experimental autoimmune encephalitis (EAE) in mouse models [74]. This
strategy may provide a noninvasive and novel therapeutic approach for treating inflammatory brain
diseases. In cancer therapy, Phase I and II clinical trials have yielded promising results on the use of
curcumin as a component of pancreatic cancer therapeutic strategies [75]. In association with these data,
EV-encapsulated curcumin also decreases the viability of pancreatic adenocarcinoma cell lines [76].
4.3. Nucleic Acid-Based Drugs and EVs
EVs carry multiple types of molecules including proteins and nucleic acids, and these cargoes
are more stable than they would be if exposed to body fluids. In particular, mRNA and miRNAencapsulated in exosomes are called esRNA (exosome shuttle RNA). esRNA in body fluids such asserum, urine, and saliva is a biomarker for early diagnosis and surveillance of various disorders [77].
The fact that EVs can carry nucleic acids in stable form has attracted the attention of researchers who
are developing nucleic acid-based drugs.
Alvarez-Erviti and co-workers developed a DDS for the central nervous system using modified
EVs [20]. To reduce immunogenicity, they used autologous (self-derived) DCs for EV production.
Targeting of nerve cells was achieved using a neuron-specific RVG peptide and loading of nucleic-104-

Int. J. Mol. Sci. 2016, 17, 172

8 of 19acid drugs was achieved by electroporation. siRNA delivered by intravenously injected RVG-targeted
EVs can specifically inhibit target genes in the brain. The therapeutic potential of EV-mediatedsiRNA delivery was demonstrated by knockdown of BACE1; a therapeutic target in Alzheimer’s
disease [20]. These results suggested that EVs can cross the blood-brain barrier and can be targetedby surface modification and loaded with nucleic acid drugs. We recently developed cancer-targeting
EVs that inhibit progression of breast cancer in an in vivo mouse model [21]. Targeting was achievedby engineering the donor cells to express the transmembrane domain of platelet-derived growth
factor receptor fused to the GE11 peptide; an artificial ligand of EGFR. The GE11-EVs delivered
let-7a tumor suppressor microRNA and inhibited development of xenograft breast cancer cells in
RAG2KO mice. Our results suggest that EVs can be used therapeutically to target cancer tissues with
nucleic-acid drugs [21]. Additionally, MSC EVs-mediated transfer of miR-133b has been shown topromote neurite growth in neural cells [78]. Likewise, MSC-derived EVs loaded with miR-146b inhibit
glioma growth [79]. Because MSCs are efficient producers of exosomes, they are well suited for mass
production of exosomes, which are ideal for drug delivery [80]. Immune cells in particular are suitabletarget cells for EV-based therapeutic interventions. Bryniarski et al., recently demonstrated effective
T-cell inhibition via systemic transit of exosome-like nanovesicles delivering the inhibitory miRNA
miR-150 to target effector T-cells in an antigen-specific manner via surface coating with antibody lightchains [81].
5. EVs in Clinical Trials
Because of the unique properties of EVs and overall encouraging preclinical results, it is
not surprising that development of these vesicles as therapeutic agents is vigorously pressed on.
Consequently, a number of clinical trials have been completed or are currently underway to determine
their efficacy and safety as therapeutics or DDSs in vivo (Table 1). Phase I clinical trials have been
conducted using tumor peptide/drug–loaded exosomes in patients with metastatic melanoma,
advanced non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and malignant glioma [82–85].
Advanced stage III/IV melanoma patients with tumors expressing the MAGE-3 antigen wereenrolled and treated with MAGE-3 peptides (HLA-A1/B35) loaded dexosomes from autologous DCs
in conjunction with MAGE-3 class II peptides (DP04) or tetanus toxin class II epitopes for helper
effects [82]. Five out of the 15 enrolled patients experienced some measure of clinical benefit withrespect to the primary tumor or lymph node lesions. Thirteen enrolled patients with pre-treated Stage
IIIb and IV NSCLC expressing MAGE-A3 or A4 were treated with MAGE-A3, -A4, -A10, and DP04
peptides loaded dexosomes from autologous DCs [83]. Of the 13 enrolled patients, 9 completed thetherapy and some patients benefited from long term disease stabilization and moderate immune
effector activation, but overall no significant induction of MAGE peptide-specific T-cell responses
could be monitored in patients. Consequently the authors engineered a new process for the isolation of
second generation dexosomes with adjusted peptide formulation and improved immune stimulatory
capacities [86]. A phase II follow-up with this improved vaccine is currently underway aimed atdetermining progression-free survival in 41 enrolled non-operable NSCLC patients after vaccination(Table 1). Forty patients with HLA-A*0201+ CEA+ stage III or IV CRC were either treated withascites-derived exosomes alone or in conjunction with granulocyte-macrophage colony-stimulating
factor (GM-CSF) [84]. Thirty-seven patients completed the vaccinations and the results showed that
only in patients receiving GM-CSF a beneficial anti-tumor cytotoxic T-cell response against colon cancer
peptide CAP-1 could be observed. The fact that no significant effects were observed in patients who
were treated with exosomes only shows that further preclinical research might be required to determine
if sufficient immune inductive components are present in tumor-derived exosomes in general or if thiswas an exceptional case. A recent innovative pilot study used a vaccine based on autologous glioma
tumor cells (isolated during surgical craniotomy), treated with an antisense oligodeoxynucleotide(AS-ODN) targeting insulin-like growth factor receptor-1 (IGF-1R), encapsulated in small diffusion
chambers and re-implanted into the patient’s abdomen 24 h post-surgery for 24 h [85].

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Table 1. Completed and running clinical trials of extracellular vesicle (EV)–based therapies.
Vesicle Type

Admin.1 Patients

DiseaseDrug

EV Source

Autologous
dexosomes

Metastaticmelanoma

Melanoma
peptideantigens

DCs

i.d./s.c.

15Autologous
dexosomes

Non-smallcell lungcancer

MAGE peptides

DCsi.d./s.c.

9Autologous
exosomes

Colorectal
cancerEVs, EVs +GM-CSF

Ascites fluid

s.c.

37 4

Autologous
exosomes

MalignantGliomaEVs + AS-ODN

GliomaAllogeneic
exosomes/MVs

Type I
diabetes
mellitus

EVs

Umbilical
cord-blood
derived MSC

i.v.

Allogeneic
exosomes

GVHD

EVs

MSC

OMVs

Meningitis

Vaccine

OMVs

Meningitis

Plexosomes 5
Plexosomes 5

Colon cancer
Mucositis

Therapeutic Results 2

Side-Effects 2

Status 3

Ref.

C

[82]

C

[83]

C

[84]

Phase I clinical trials
MART1–HLA-A2 T-cell
response and tumor
shrinkage (1); minor
response (1); mixed
response (1); stabilization (2)MAGE-specific T cell
responses (3); NK cell
lysis (2)EVs+GM-CSF: cytotoxic T
cell resp. to CAP-1 (76.9%);
stabilization (1); minor
response (1)No major toxicity; moderate
pain (1), swelling at injection
site (8); mild fever (1)No major toxicity; moderate
pain, swelling, pruritus at
injection site (37); fever (1),fatigue (3) and nausea (1)

N.D.

N.D.

C

[85]

20

N.D

N.D

E

[87]

i.v.

1

GVHD symptoms improved;
stabilization for several
months. Patient died ofpneumonia 7 months post
exosome application

C

[35]

B:4:P1.7-2,4;
B:15:P1.7,16
strainsi.m.

91

MenBVac/MeNZB vaccines:
high efficacy

C

[88]

VaccineFetA
modifiedstrain 44/76

i.m.

52

MenPF-1 vaccine:highadditive efficacy

C

[89]

CurcuminCurcumin

Fruit
Fruit

oral
oral

35
60

N.D.
N.D.

N.D.
N.D

R
R

[90]
[91]

DCs

i.d./s.c.

41

N.D

N.D

E

[86]

i.m.

147

rMenB+OMV: high additive
efficacy

C[92]

Implanted
biodiffusion 12
chamber

No major toxicity; minor
inflammation; grade 1
fever (5)No major side-effects

No major toxicity; moderate
pain, swelling, induration at
the injection site; mild fever,malaise/headacheNo major toxicity; moderate
pain, swelling at injection
site; mild fever, malaise,nausea/headachePhase II clinical trials
Autologous
dexosomes

Non-smallcell lungcancerIFN-γ, MAGE
peptidesOMVs

Meningitis

rMenB vaccine,
B:4:P1.7-2,4
NadA/fHBP/NHBA
strains

Moderate pain, swelling,induration at the injection
site; mild fever; serious
adverse events (18)

N.D.: No data; 1 Administration: i.d.: intradermal; s.c.: subcutaneous; i.m.: intramuscular; i.v.: intravenous;
2 Number of patients between brackets; 3 C: completed; E: evaluating; R: recruiting; 4 13 patients also
received GM-CSF; 5 Plant-derived exosomes; AS-ODN: antisense oligodeoxynucleotide targeting insulin-likegrowth factor receptor-1 (IGF-1R); fHBP: factor H binding protein; GM-CSF: granulocyte-macrophagecolony-stimulating factor; GVHD: Graft-versus host disease; IFN-γ: interferon gamma; MSC: Mesenchymal
stem cell; MVs: microvesicles; NadA: Neisserial adhesin A; NHBA: Neisserial heparin binding antigen;
MenPF-1: FetAon PorAon .

The rationale is that AS-ODN loaded exosomes slowly diffuse out of the chamber and induce
T-cell activation and immune memory. Although the study is listed as completed, no results have
yet been published. However, the proposed methodology is currently being further developed ina pre-clinical setting [93,94] and the results of the clinical trial are forthcoming shortly (personalcommunication [95]). Treatment of a single patient suffering from GVHD as a consequence of
allogeneic stem cell transplantation with peripheral blood stem cells for secondary acute myeloid
leukemia with MSC-derived exosomes showed marked improvement of GVHD and stabilization of
disease [35]. The authors isolated exosomes with anti-inflammatory profiles containing high quantities
of IL-10, transforming growth factor beta and human leukocyte antigen-G. The only trail that does
not target cancerous tissue is currently in phase I and aims to reduce inflammation and improve
islets β-cells mass and glycemic control in patients with type I diabetes mellitus (T1DM) [87]. To
this end, 20 enrolled patients are treated via intravenous infusion of cell free umbilical cord-blood
derived MSC exosomes/MVs without additional modification with therapeutic agents. To conclude,
the development of an ovarian cancer vaccine based on exosomes from the ascites fluid of ovariancancer patients and the TLR3 agonist poly[I]:poly[C12 U] (Ampligen® ) is currently being prepared forphase I clinical testing [96].
Overall, the aforementioned phase I trials highlight the feasibility of large-scale EV production
and show that administration of autologous EVs in particular is generally devoid of serious adverse
side-effects, with one patient tolerating treatment as long as 21 months [82]. Reported side-effects
included inflammatory responses at the site of injection and low-grade fever. None of the phase I-106-

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trials reported serious toxicity or adverse immune responses. However, these trials were limited
to vaccination attempts to induce or boost endogenous immunogenic responses to cancer antigens(Table 1) and at an early stage of evaluation, which makes drawing a definite conclusion regarding
long-term safety premature.
In recent times, strategies were developed based on EVs from non-mammalian cells and were
therefore not discussed above, but because this approach entered phase I clinical testing will briefly be
included here, i.e., bacterial outer membrane vesicles (OMVs) and plant-based exosomes (plexosomes)
as therapeutic carriers. OMVs have long been investigated in the context of anti-bacterial vaccines
since OMVs are naturally released by Gram-negative bacteria and are part of their virulence [88,97].
A 2005 phase I trial showed that OMV-based vaccines from B:4:P1.7-2,4 and B:15:P1.7,16 NZ 98/254
meningococcal strains were 93%´97% effective against Neisseria meningitidis serogroup B and achieved
significantly higher serum bactericidal titers than vaccines based on capsular polysaccharides [88,92].
A phase II follow-up trial was recently concluded in which 147 infants from the United Kingdom were
immunized with either a recombinant vaccine (rMenB) containing Neisserial adhesin A (NadA), factor
H binding protein (fHBP) and Neisserial heparin binding antigen (NHBA) alone or in combination
with OMV from B:4:P1.7-2,4 (NZ 98/254) [92]. Both rMenB and rMenB+OMV were immunogenic
against strains expressing homologous or related NadA and fHBP, but significantly less effective
against heterologous fHBP variants. Overall, rMenB+OMV demonstrated greater immunogenicity
and was also immunogenic against strains expressing homologous PorA. In addition, both vaccines
stimulated immune memory (anamnestic) responses after the fourth dose and were overall welltolerated. Another approach to broaden the protective effect of OMV vaccines against N. meningitidis
was tested by Marsay et al. based on ferric enterobactin receptor (FetA) and PorA expression (MenPF-1)
in 52 adults [89]. The MenPF-1 vaccine was generated through genetic modification of N. meningitides
strain 44/76 by replacing the promoter region of fetA with a 17 bp spacer region derived from thepromoter region of porA, which resulted in constitutively expressed FetA and wild-type PorA. Theirresults show that FetA-specific reactions could be induced per se and that MenPF-1 (FetAon PorAon )showed broad and additive responses with titers ě1:4 in 96%´100% of subjects after the final dose.
In the case of these OMV-based vaccines, immunogenicity is wanted. Gujrati and collaborators
further developed OMVs as more general therapeutic vehicles by loading OMVs shed with a humanepidermal growth factor receptor 2 (HER2)-specific affibody for active targeting from a mutant
E. coli strain with siRNA targeting kinesin spindle protein (KSP) [98]. KSP silencing prevents theformation of bipolar mitotic spindles in rapidly dividing cells, which results in cell-cycle arrest andapoptosis [99,100]. In order to reduce endotoxicity and immunogenicity, the authors used a msbB
mutant E. Coli strain that produces under-acylated (lacks the myristic acid moiety of lipid A) andshorter length LPS [101,102].
Results from the N. meningitides phase I clinical trials demonstrate that OMV-based vaccines
induced no serious vaccine-related adverse events. Nonetheless, most patients displayed local
reactions, including moderate pain, redness, swelling and induration at the injection site, whereas
approximately 40%´50% of patients reported systemic reactions such as mild fever, malaiseand headache [88]. Although the aforementioned modified OMV-based DDS displayed low
immunogenicity and reduced toxicity as a consequence of the msbB mutant, safety concerns may
still arise because of the bacterial origin of OMV-based therapeutics. Consequently, further clinicalevaluation might be necessary when utilizing OMVs as vaccines, whereas OMVs that are used as DDSs
require surface modification strategies and additional preclinical evaluation to meet particular safety
criteria as therapeutics or DDSs.
Plexosomes have recently attracted attention because of their potentially low toxicity andimmunogenicity-plexosomes are consumed by humans on a daily basis-and because they represent anunlimited source of DDS. Two phase I clinical trials have recently been initiated that predominantly
evaluate if oral plexosome-based curcumin delivery to normal and cancerous colon tissue is more
effective than with conventional DDSs [90] and if oral mucositis associated with chemoradiation-107-

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treatment of head and neck cancer can be prevented [91]. Plant-derived EVs may solve isolation andpurification restrictions, since plant-based EVs represent a potentially unlimited source of EVs for
drug delivery.
6. EVs Caveats and Challenges
The application of EVs as therapeutics or DDSs has become an active area of research. However,many obstacles must still be overcome in order to successfully and safely use EVs as therapeutics
in a routine clinical setting. The clinical trials that have been conducted at the phase I stage seem toindicate that no serious acute side effects occur. Nonetheless, those trials were limited to a particularbiological action, i.e., immune-modulation, and long-term experience is lacking. In the light of themany uncertainties regarding EVs, clinical trials with EVs, albeit attractive and highly desirable,
should be performed with care. As deliberated in the introduction by Karla et al., [2] these uncertainties
include a lack of unequivocal characterization and nomenclature based on cellular origin, size, physicalproperties, and unique markers, and a lack of suitable and standardized isolation methods. As aconsequence of the aforementioned caveats, it is impossible to firmly determine EV classes, theirbiogenesis, physiological function, transport, how they reach their target cell, what their role in various
disease processes is, whether EV networks should be targeted in therapeutics development and if
therapeutics show additive, antagonistic, or synergistic effects, to name but a few. Furthermore, EVsfrom aberrant cells have been shown to carry the cell of origin’s divergent phenotype and therefore
might carry tumorigenic potential [103–106]. Equally, pathogens have been shown to utilize EVs
to spread infection and avoid the host’s immune system [107–109]. This clearly suggests that EVs
may have an inherent disease-bearing potential, which justifies caution when isolating, manipulatingor engineering EVs for therapeutic purposes. These are all safety concerns for which substantial
precedence and experience exist with other types of biological therapeutics.
Given concerns about their immunogenicity, safety, and efficiency, EVs should be purified fromself-derived and rapidly proliferating cells such as immune or induced pluripotent stem cells (iPS) for
autologous treatment. Hu et al. successfully purified EVs from iPS cells-derived MSCs and showedthat pretreatment with these EVs resulted in protection of limbs from ischemic injury in a mousemodel [110]. Currently, because it would be very challenging to identify and understand all constituent
elements of EVs, it is extremely difficult to realize clinical use of EVs. However, EVs are easier toapply clinically than iPS cells because they are not intact cellular material and consequently have alower potential for malignant alterations. Furthermore, transplantation of self-derived MSCs and DCs
has extensively been investigated in many clinical trials. Therefore, the safety issue regarding severe
adverse immunogenic responses as a result of the administration of EVs purified from patient-derived
MSCs and DCs has essentially already been resolved.
To manufacture EVs as medicines, it will be necessary to establish methods for stable production
and bulk preparation. Jang et al. developed bioinspired exosome-mimetic nanovesicles from monocytes
or macrophages by serial extrusion through filters with diminishing pore sizes (10, 5 and 1 µm) [64].
Such a method represents an effective way to ensure sufficiently high quantities of exosomes forclinical use. If self-derived EVs are to be used for therapy, it will be necessary to develop a system forpersonalized medicine. The most common method for EV purification involves ultracentrifugation,
but this method is not well suited for bulk preparation. Consequently, novel isolation, separation andrefinement technologies capable of sufficiently high yields are urgently required.
The function and the treatment effect of the purified EVs are mainly summarized in this review.
On the one hand, the physiological function of EVs in several diseases may be therapeutic targets.
Because some pathways regulate the secretion machinery of EVs such as ceramide and the ESCRT
machinery [111–113], cell and mouse models completely lacking EVs have not been created. Therefore,
the physiological significance in vivo and involvement of EVs in various diseases remains unclear.
Nonetheless, recent research is increasingly implicating EVs in particular disorders. For instance, theaccumulation of pathological tau protein, a major hallmark of Alzheimer’s disease, was shown to-108-

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be promoted via EVs derived from microglia [114]. In addition, inhibition of the ESCRT machinery
decreased infectious prion release [115]. Exosomes from HCV-infected cells were shown to be capableof transmitting infection to naive human hepatoma cells and able to establish a productive infection
by exosome-mediated transmission of HCV RNA [116]. These results suggest that the inhibition of
EV secretion pathways and/or the depletion of EVs from blood from the blood of patients throughapheresis may represent a novel strategy to modulate or interrupt disease progression.
7. Conclusions
In recent years, the number of EV-related studies has increased rapidly, and specialized journals
and conferences have been established to disseminate these findings. The knowledge about EVs
obtained from research of multiple life science disciplines shows great potential for various applications,particularly in regenerative medicine and to treat diseases refractory to conventional approaches. EVs
may be considered to be the smaller “alter egos” of secretory cells and, therefore, may function
equivalent to implantation of secretory cells; at least partially. However, compared to living celltransplantation, administration of EVs will decrease the burden on both physicians and patients withregard to production, quality management and therapy cost. Thus, one important characteristic of
EVs in the context of therapeutic applications is that they might replace cell transplantation in thelong run. On the other hand, the DDS field has focused on other characteristics of EVs. To date, thedevelopment of DDS has been hampered by biological toxicity and antigenicity of carriers. The abilityof EVs to deliver molecules to distant cells via body fluids without significant toxicity and antigenicity
demonstrates their utility as DDS. Nonetheless, further studies are needed to establish EVs as thenext-generation DDSs or novel biological drugs. Additional information about various aspects ofextracellular vesicles is provided in the other reviews in this focus edition [2,117–120].
Acknowledgments: We would like to thank Elena Batrakova (Centre for Nanotechnology and Drug Delivery,
Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA) for providing the
micrographs in Figure 2B1,2.
Author Contributions: Shin-ichiro Ohno wrote the first draft of the manuscript; Masahiko Kuroda and
Gregor P. C. Drummen revised the manuscript and Gregor P. C. Drummen made the figures and tables. All
authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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original article© The American Society of Gene & Cell Therapy

MiR-29a Assists in Preventing the Activation of
Human Stellate Cells and Promotes Recovery From
Liver Fibrosis in Mice
Yoshinari Matsumoto1,2,3, Saori Itami1, Masahiko Kuroda4, Katsutoshi Yoshizato5, Norifumi Kawada1 andYoshiki Murakami1
1
Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan; 2Department of Medical Nutrition, Graduate School of
Human Life Science, Osaka City University, Osaka, Japan; 3Current address: Department of Nutrition Management, Osaka University Medical Hospital,
Osaka, Japan; 4Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 5Phoenixbio Co. Ltd, Hiroshima, JapanThe microRNA-29 (miR-29) family is known to suppress the activation of hepatic stellate cells (HSCs) andreversibly control liver fibrosis; however, the mechanismof how miR-29a controls liver fibrosis remains largely
unknown. This study was conducted to clarify the mechanism of anti-fibrotic effect of miR-29a and to explore
if miR-29a is a promising candidate for nucleic acidmedicine against liver fibrosis. Two liver fibrosis murine
models (carbon tetrachloride or thioacetamide) wereused. MiR-29a mixed with atelocollagen was systemically administered. Hepatic fibrosis was evaluated byhistological analysis and the expression levels of fibrosisrelated genes. We observed that miR-29a treatment dramatically accelerated the reversion of liver fibrosis in vivo.
Additionally, miR-29a regulated the mRNA expression ofcollagen type I alpha 1 (COL1A1) and platelet-derived
growth factor C (PDGFC). We also noted that miR-29a
significantly suppressed COL1A1 mRNA expression andcell viability and significantly increased caspase-9 activity(P < 0.05) in LX-2 cells. Pretreatment of miR-29a inhibited activation of LX-2 cell by transforming growth factor beta treatment. MiR-29a exhibited anti-fibrotic effectwithout cell toxicity in vivo and directly suppressed theexpression of PDGF-related genes as well as COL1A1 andinduced apoptosis of LX-2 cells. MiR-29a is a promising
nucleic acid inhibitor to target liver fibrosis.
Received 22 January 2016; accepted 2 June 2016; advance online
publication 2 August 2016. doi:10.1038/mt.2016.127

INTRODUCTION

Fibrosis and tissue remodeling in the liver lead to cirrhosis. Liverfibrosis is characterized by excessive production and depositionof extracellular matrix (ECM) materials, leading to the destruction of the normal hepatic parenchyma and disruption of theliver structure.1,2 The development of hepatic fibrosis is triggered by the activation and proliferation of resident hepatic stellate cells (HSCs).3 Activation of HSCs is induced by paracrine
peptide and lipid mediators derived from damaged hepatocytes,

activated Kupffer cells, and endothelial cells. The autocrine loopof transforming growth factor β1 (TGFβ1) also contributes toliver fibrogenesis.4
Clinically, cirrhosis is observed in patients with alcoholinduced liver injury, chronic hepatitis B and C, nonalcoholicsteatohepatitis, and other hepatitis.5 In spite of the cause, therisk of hepatocellular carcinoma increases by the stage of liver
fibrosis, therefore, controlling liver fibrosis is key to preventing
hepatocarcinogenesis.
There is increasing evidence that fibrosis is a dynamic andreversible process and removing the primary cause is the mosteffective approach for fibrosis regression. In chronic hepatitis
C virus infection, treatment with antiviral drugs is usually recommended for hepatitis C virus eradication. Other approaches
to improving liver fibrosis involve controlling fibrosis-related
molecules include: (i) receptor–ligands, for example, peroxisome proliferator-activated receptor alpha agonist or adiponectin, (ii) inhibitors of TGFβ1, and (iii) inducers of the apoptosisof activated HSCs or the matrix degradation (reviewed in ref. 6).
However, since side effects, such as liver toxicity, make it difficult
to apply these methods clinically, it is critical to develop directacting and safe agents to control liver fibrosis.
MicroRNAs (miRNAs) are endogenous small noncoding
RNAs that control gene expression by degrading or suppressing
the translation of target mRNAs.7 Aberrant expression of miRNAis associated with the progression of liver fibrosis.8–10 For example,
the expression of miR-132 and the miR-29 family (miR-29a, miR29b, and miR-29c) is significantly underexpressed in fibrotic livers, as demonstrated in human liver cirrhosis and in liver injury
models induced by bile duct ligation and carbon tetrachloride(CCl4) in mice. Downregulation of these miRNAs can modify
the activity of HSCs.11–13 Serum miR-29a is significantly lower
in patients with advanced liver cirrhosis than in healthy controls
or patients with early fibrosis.12 Further it has been shown thatoverexpression of miR-29 in murine HSC resulted in the downregulation of collagen expression12,14 through a mechanism that
directly targets the mRNA expression of ECM genes. These findings suggest that miR-29 may be an important therapeutic target
in chronic liver disease.

The first two authors contributed equally to this work.
Correspondence: Yoshiki Murakami, Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka 545–8585, Japan. E-mail:yoshimurak@med.osaka-cu.ac.jp

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MiR-29a Assists Recovery From Liver Fibrosis

Recently, various forms of nucleic acid have emerged as potential treatments in the medical field. For instance, the use of nucleic
acid against miR-122 for chronic hepatitis C genotype 1 infection
resulted in prolonged dose-dependent reductions in hepatitis C
virus levels without viral resistance.15 Besides miR-122, several
alternative nucleic acid therapies are likely to be tested in future
clinical trials.16
Based on these applications of miRNAs, it is expected thatmiR-29a can control liver fibrosis regardless of the primary cause.
If liver fibrosis can be treated directly, the development of cirrhosis and carcinogenesis could be repressed. Thus, the aim of ourstudy is to establish methods of nucleic acid therapy for liver fibrosis using miR-29a and to elucidate the mechanism of how miR29a represses liver fibrosis.

RESULTS
Liver fibrosis models

Liver fibrosis was induced in mice by administering CCl4 or
thioacetamide (TAA) for 5 weeks. Liver fibrosis level and spontaneous resolution after ceasing fibrosis induction were examined (Figure 1a,g). CCl4 and TAA livers demonstrated bridging
fibrosis, but this gradually reversed after CCl4 (Figure 1b) or TAA(Figure 1h) treatment was ceased. During the 5-week administration period, sirius red (SR)-positive area was significantly
larger in both CCl4 and TAA than in both groups of control livers (corn oil or saline) (P < 0.05) and time-dependently decreasedin the 1- or 2-week observation groups (CCl4 + 1 week, CCl4 +2 weeks; TAA + 1 week, TAA + 2 weeks observation) (P < 0.05)(Figure 1c,i). Hydroxyproline level in the liver was higher in CCl4
and TAA models than in the control groups; in particular, there
was a significant difference between control livers and TAA at
5 weeks (P < 0.05) (Figure 1d,j). To evaluate the activation of
HSC, the expression of four representative genes was examined.
The expression level of collagen type I alpha 1 (COL1A1) in CCl4
or TAA was significantly higher than in the control and 1- or
2-week observation groups (Figure 1e,k). The expression levelof alpha-smooth muscle actin (α-SMA) in CCl4 was significantly
higher than the other groups. In CCl4 and TAA, the expression
level of matrix metallopeptidase (MMP) 2 was significantly higher
than the other groups (P < 0.05). In addition, tissue inhibitor of
metalloproteinase (TIMP) 2 in TAA was significantly higher thanin the others (P < 0.05). The expression level of mmu-miR-29a waslower in CCl4- or TAA-treated liver than in the controls but was
similar among all the groups (Figure 1f,l).

Controlling liver fibrosis by administering miR-29a
Previous reports have suggested that miR-29 can control HSC
activity.9,10 The sequence between mmu-miR-29a and hsa-miR29a was conserved across species (http://www.mirbase.org). We
evaluated the ability of miR-29a to improve liver fibrosis using
CCl4 and TAA models established in Figure 1. MiRNA wasadministered for 1 or 2 weeks after inducing fibrosis for 5 weeks(Figure 2a).
In CCl4 model, histological examination confirmed thatmiR-29a administration for 1 or 2 weeks improved liver fibrosis (Figure 2b,c). Moreover, based on the SR-positive area, we
determined that miR-29a accelerated the resolution of liver

fibrosis significantly (P < 0.05) compared to the negative control(NC) miRNA groups (Figure 2d). Hydroxyproline level was significantly lower in 1-week/2-week miR-29a groups than in NC
miRNA groups (P < 0.05) (Figure 2e). The expression level of
COL1A1 mRNA was significantly lower in the miR-29a-treatedgroups than in the CCl4 + 1-week observation groups (P < 0.05)(Figure 2f).
In the TAA model, histological examination showed that miR29a improved liver inflammation and fibrosis (Figure 3a,b). The
SR staining area in the groups administered miR-29a had significantly decreased compared to NC miRNA or both vehicle groups(P < 0.05) (Figure 3c). Hydroxyproline levels in miR-29a-treatedliver was significantly lower than in NC miRNA or both vehicle
livers (P < 0.05) (Figure 3d). The expression level of COL1A1 in
the miR-29a group was lower than in the 1-week vehicle group(Figure 3e). Elastica van Gieson staining of liver samples showedthat elastic fibers decreased in the 1 week/2 weeks miR-29a groupcompared with NC miRNA group (Supplementary Figure S1).

Gene expression profile in miR-29a-treated livertissueTo clarify the mechanism of the anti-fibrotic effect of miR-29a,
we performed a comprehensive gene expression analysis using
microarray. We chose genes with expression levels that had a 1.5
times difference between miR-29a-treated group and NC miRNAtreated group. CCl4 or TAA livers that underwent 1 and 2 weeks
of miR-29a treatment (1- and 2-week miR-29a) had 126 and 152
upregulated genes and 276 and 128 downregulated genes, respectively, compared to those treated with NC miRNA (Figure 4a andSupplementary Tables S2 and S3). In CCl4 and TAA groups thatreceived 1 or 2 weeks of miR-29a treatment, nine and seven genes
were commonly upregulated and downregulated, respectively(Figure 4a and Supplementary Table S4).
Target genes for miR-29a were predicted from the 276 and128 downregulated genes using the online search algorithmsof Targetscan, mirtarbase, and microRNA.org. In addition,
genes that were found in previous studies to be associated withfibrogenesis were selected from the predicted target genes.
Fibrinogen-like protein 217 and platelet-derived growth factor C (PDGFC)18,19 were chosen and the expression level ofthese genes in 1-week miR-29a was lower than in NC miRNA.
Mitogen-activated protein kinase kinase kinase kinase 4 (ref. 20)
and PDGFC were also chosen and genes with expression levels
that were lower in 2-week miR-29a livers than in the NC miRNA(Figure 4b,c). Applying these constraints, we validated theexpression of three genes by real-time polymerase chain reaction (PCR) analysis (Figure 4d). In addition, the expression of
PDGFC was commonly lower in 1-week/2-week miR-29a livers
than in the NC miRNA groups. COL1A1, a representative ECM
gene, expressed lower in miR-29a-treated CCl4 mouse than in
NC miRNA-treated CCl4 mouse. We also identified COL1A1 asa miR-29a target gene.
MiRNA disposition kinetics of miR-29a
We examined miRNA disposition kinetics of administered miR29a in mice. Mice in which liver fibrosis was not induced were
killed at 3, 6, 24, and 48 hours after being administered miR-29a.
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MiR-29a Assists Recovery From Liver Fibrosisa0 week5 weeks

6 weeksc

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Figure 1 Preparation of two chronic liver injury models. (a,g) Treatment schedules for carbon tetrachloride (CCl4) and thioacetamide (TAA).
(b,h) Hematoxylin and eosin (upper rows) and sirius red (SR) staining (lower rows) of liver. Scale bar = 200 μm. (c,i) SR staining positive area ofliver. (d,j) Liver hydroxyproline levels. (e,k) The expression pattern of four fibrosis-related genes (COL1A1, α-SMA, MMP2, and TIMP2) in liver.
(f,l) The expression pattern of miR-29a in liver. *P < 0.05 in Tukey HSD. Horizontal axis in (c–f) and (i–l) shows whether CCl4 treatment was doneor not (CCl4) and the timing of sacrifice (observation).

In these mice, elevated miR-29a expression was observed in theearly phase after miR-29a was administered (Figure 5a). LivermiR-29a expression was similar among the CCl4 and TAA groups
that underwent 1-week and 2-week miR-29a treatment (Figure5b). They were killed at 72 hours after miR-29a was administered.
In addition, to confirming that miR-29a is associated with fibrosisregulated genes physiologically and functionally, we performed
an argonaute2-coimmunoprecipitation (Ago2-IP) analysis to1850

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MiR-29a Assists Recovery From Liver Fibrosisa

5 weeks

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Figure 2 Therapeutic effect of miR-29a in carbon tetrachloride (CCl4)-treated mouse liver for 1 and 2 weeks. (a) Summary of schedule for
miRNA treatment. (b,c) Hematoxylin and eosin (upper row) and sirius red (SR) (lower row) staining in livers. Scale bar = 200 μm. (d) SR staining positive area of livers. (e) Liver hydroxyproline levels. (f) The expression pattern of COL1A1 in livers. *P < 0.05 in Tukey HSD, **P < 0.05 in student’s t-test.

observe whether RNA-induced silencing complex (RISC) could
retain fibrosis-regulated genes when miR-29a was overproducedin CCl4 + 1-week NC miRNA and miR-29a liver (Figure 5c).
The miR-29a disposition kinetics in other organs is shownin Supplementary Figure S2A and the expression of COL1A1
appears in Supplementary Figure S2B.

To determine if there was any toxicity associated with miR29a treatment, the ration of body weight (BW) (Supplementary
Figure S3) and the ratio of liver to BW (Supplementary Figure S4)
were measured. There was not different among each miRNAtreated groups. Alanine aminotransferase and aspartate aminotransferase in serum increased with CCl4 treatment, but returned

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MiR-29a Assists Recovery From Liver Fibrosis

1 week NC miRNA

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Figure 3 Therapeutic effect of miR-29a in thioacetamide (TAA)-treated mouse liver for 1 and 2 weeks. (a,b) Hematoxylin and eosin (upper row)and sirius red (SR) (lower row) staining in livers. Scale bar = 200 μm. (c) SR staining positive area of livers. (d) Liver hydroxyproline levels. (e) The
expression pattern of COL1A1 in livers. *P < 0.05 in Tukey HSD, **P < 0.05 in student’s t-test.

to normal when CCl4 treatment was ceased. MiR-29a injection
did not increase these markers (Supplementary Figure S5).

Functional analysis of miR-29a in vitro
To evaluate the effect of miR-29a on HSC activation, we used
an in vitro system. LX-2 is a human HSC cell line that can be
activated by TGFβ1 (Figure 6a). TGFβ1 treatment suppressed
the expression level of miR-29a in LX-2 cells (Figure 6b) andincreased the expression levels of COL1A1, PDGFC, and MMP2
(Figure 6c). MiR-29a suppressed expression of these genes in

HSCs in the presence or absence of TGFβ1 (Figure 6c). An
immunoblot showed that COL1A1 expression was decreased bymiR-29a treatment in presence of TGFβ1 (Figure 6d).
XTT assay revealed that overexpression of miR-29a suppressed cell viability regardless of TGFβ1 treatment (Figure 6e).
In LX-2 cells with overexpressed miR-29a, caspase 9 activity was higher than in NC miRNA-transfected LX-2 cells(Figure 6f).
The effect of miR-29a in mouse-primary HSC was also examined. In this culture system, HSC was activated and COL1A1

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miR-29a>NC miRNA: 1 week

miR-29a>NC miRNA: 2 weeks

miR-29a>NC miRNA: 1 and 2 weeks

miR-29a>NC miRNA: 1 week

miR-29a>NC miRNA: 2 weeks

miR-29a>NC miRNA: 1 and 2 weeks

b
′

′

′
′

′
′
′

′
′

′

′
′

′

′
′

′

′
′
′

′
′

c

d

Micro array##

Figure 4 Microarray analysis of carbon tetrachloride (CCl4) and thioacetamide (TAA) liver fibrotic models and miR-29a target genes.
(a) Venn diagram of the fibrosis-related genes selection criteria in six patterns. Number depicted in common genes in two or four groups. Black circle
shows CCl4 1 week, dotted circle shows TAA 1 week, bold black circle shows CCl4 2 weeks, and bold dotted circle shows TAA 2 weeks. (b) MiR-29atarget site sequences in 3ʹ UTR of PDGFC, FGL2, MAP4K4, and COL1A1. Numbers left of 5ʹ-UTR sequences show nucleotide from 5ʹ end. MAP4K4and COL1A1 have pleural recognition site of miR-29a. (c) The expression pattern of three target genes from microarray data. (d) The expression
pattern of three target genes from real-time PCR data. PDGFC shows four sample’s data (CCl4 1 week/2 weeks and TAA 1 week/2 weeks). Fgl2 shows
two sample’s data (CCl4/TAA 1 week). Map4k4 shows two sample’s data (CCl4/TAA 2 weeks). *P < 0.05 in student’s t-test.

expression increased over time. MiR-29a was transfected at
24 hours after isolation (day 1). The cells were harvested at day 3
and 4 (Supplementary Figure S6A). The expression of COL1A1
in miR-29a-treated HSC was inhibited compared to NC miRNAtreated HSC (Supplementary Figure S6B).

Pretreatment of miR-29a can inhibit HSC activation
MiR-29a was transfected with LX-2 cells and the culture medium
was changed 6 hours later to remove any remaining miR-29a;
TGFβ1 was then added at a concentration of 2.5 ng/ml. The cells
were harvested at 24 and 48 hours after transfection (Figure 7a).

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MiR-29a Assists Recovery From Liver Fibrosis

0

0

DISCUSSION

24 hours 48 hoursc

mmu-miR-29a

1

1 week 2 weeks

2

1

0

1 week 2 weeks

2

#

N

C

m
iR

m
iR

0

mmu-miR-29a

TAA
Relative expression

2

CCI4

9a

3 hours 6 hours

NC miRNAmiR-29a

-2

0

1

A

1

2

N

Relative expression

2b
Relative expression

1

The expression of COL1A1 mRNA was higher in LX-2 cells treatedwith TGFβ1 than those without TGFβ1 treatment. However, pretreatment with miR-29a significantly suppressed COL1A1 gene
expression especially in the TGFβ1-treated groups. Moreover,on day 2, in LX-2 cells transfected with miR-29a, COL1A1 gene
expression did not differ between TGFβ1-treated cells and nontreated cells (Figure 7b). The expression level of PDGFC in LX-2
cells with overexpressed miR-29a was lower than in NC miRNAtreated cells on day 1 in the TGFβ1 nontreated group (Figure 7b).
In TGFβ1-treated LX-2 cells, Western blot analyses showed thatthe expression level of COL1A1 was lower in miR-29a overexpressed cells than in NC miRNA-treated cells in presence of
TGFβ1 (Figure 7c).

mmu-miR-29a

Relative expression

Relative expressionaFigure 5 Pharmacodynamics of miR-29a in mice. (a) The expression patterns of miR-29a in liver 3, 6, 24, and 48 hours after injecting miR-29a without treatment with any fibrotic inducing reagents.
(b) The expression pattern of miR-29a in 1 week/2 weeks therapeutic carbon tetrachloride (CCl4) models. (c) The expression pattern of
miR-29a in 1 week/2 weeks therapeutic thioacetamide (TAA) models. (d) The expression pattern of miR-29a in co-immunoprecipitatedmiRNA with Ago2 in 1-week therapeutic CCl4 model liver. White bars
show NC miRNA-treated liver and black bars show miR-29a-treatedliver. *P < 0.0001 in student’s t-test.

a−Day 1

Day 0

SpreadmiRNA transfection

In this study, we demonstrated two ways of controlling liver fibrosis with miR-29a: one is through prevention and the other is byrepair.

Controlling liver fibrosis through prevention
In the course of preventing liver fibrosis, ECM production must
be repressed by controlling mRNA expression of ECM, inhibiting inflammation, and by inducing apoptosis or quiescence of
ECM-producing cells. MiR-29a can directly control COL1A1 as
a target gene and repress the production of ECM (Figure 8a).

Day 1

Day 2

b
#

Harvest

TGFβ1

c
#

#

#

#

#

d

e
COL1A1

f
#

#

#

#

βactinFigure 6 In vitro analysis of miR-29a function. (a) Time course of experiment for LX-2 cells. (b) The expression level of miR-29a in LX-2 treated with
TGFβ1. (c) The expression level of fibrosis-related genes in LX-2. (d) Western blotting of COL1A1 and β-actin in LX-2 cells. (e) Cell viability test in
LX-2 measured by XTT assay. (f) Apoptosis test in LX-2 verified by Caspase 9 activity. (c,e,f) White bars show NC miRNA transfected LX-2 and blackbars show miR-29a transfected LX-2. *P < 0.05 in student’s t-test.

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Day 0

SpreadmiRNA transfection

6 hours

Day 1

Day 2

HarvestHarvest

TGFβ1

b

PDGFC

COL1A1

Relative expression

*

*
*

*

MMP2

*

*

#

c
TGFβ(−)*

TGFβ(+)

TGFβ1(−)

TGFβ1(+)
Day2

miR-29aDay1βactinNC miRNADay2

TGFβ1(+)

TGFβ1(−)

TGFβ1(+)

0

miR-29aDay1

TGFβ1(−)

TGFβ1(+)

TGFβ1(−)

TGFβ1(+)
Day2

0

NC miRNADay1

TGFβ1(−)

TGFβ1(+)

0

TGFβ1(−)

COL1A1Figure 7 Inhibitory effect of TGFβ activation in LX-2 cells. (a) Time course of experiment. MiR-29a was transfected to LX-2 cells; the medium waschanged after 6 hours and TGFβ1 was added. The cells were isolated 24 and 48 hours after transfection. (b) The expression of fibrosis-related genes
in LX-2 cells. White bars show NC miRNA transfected LX-2 and black bars show miR-29a transfected LX-2. (c) Western blotting of COL1A1 and β-actin
in LX-2 cells on 2 days. *P < 0.05 in Tukey HSD, **P < 0.05 in student’s t-test.

MiR-29a treatment downregulated the COL1A1 expression in
activated HSC (Figure 6c,d; Supplementary Figure S6), and pretreatment with miR-29a inhibited the upregulation of COL1A1
resulting from TGFβ1 treatment (Figure 7b,c). The expression of
COL1A1 and α-SMA mRNA increased after CCl4 or TAA treatment and decreased after these reagents were ceased. COL1A1
mRNA in TAA model and α-SMA mRNA in both models had
already declined to a nonstimulated state by week 1 of the spontaneous recovery period (Figure 1e,k), and no miR-29a effect wasobserved. However, the inhibition of COL1A1 mRNA expression
in miR-29a-treated mice was observed in CCl4 model (Figure 2f).
In regards to the target genes for miR-29a introduced inthis study, cell viability was downregulated (Figure 6e) and caspase9 activity was upregulated (Figure 6f) in miR-29a-treatedLX-2 cells. The PDGF pathway is well known to be associatedwith fibrosis and cell proliferation,19 and miR-29a directly recognized PDGFC (Figure 4b). PDGF has also been implicatedin ras homolog family member A mediating actin reorganization, hyperplasia of stress fiber, and cell adhesion.21,22 In addition, PDGF accelerated cell proliferation via mitogen-activatedprotein kinase 8.23 Previous research has shown a connection
between v-raf-leukemia viral oncogene 1 and serum response factor in the PDGF pathway, and these genes regulate cell proliferation.24,25 Microarray analysis showed that the expression level ofthese PDGF-related genes were lower in miR-29a liver than in NC
miRNA liver (Figure 8a). Thus, we believe miR-29a may directly
regulate PDGFC expression as target genes, and then suppress cell
migration and cell proliferation. Prior studies have shown that adecrease in fibrinogen-like protein 2 expression in T cells inhibited HSC activation.26 Our findings concur as microarray confirmed that miR-29a treatment decreased fibrinogen-like protein
2 (Figure 4b–d and Figure 8a). Additional reports have suggested
that mitogen-activated protein kinase kinase kinase kinase 4 plays

a role in hepatocellular carcinoma proliferation and epithelial–mesenchymal transition and metastasis.26,27 We therefore believethat miR-29a may inhibit cell activation and proliferation by targeting these genes (Figure 4b–d and Figure 8a).
In our microarray analysis, we observed genes related toliver fibrosis progression that were downregulated in miR29a-treated liver, however they were not target genes for miR29a (Figure 8b). Interleukin 1 beta regulates inflammation
signal pathway28 and accelerates the expression level of MMP
and TIMP.29 It was reported that N-myc downstream regulated 1
regulates angiogenesis in hepatocellular carcinoma,30 and B-cellleukemia/lymphoma 2 protein promotes apoptosis. MiR-29a
indirectly regulated these fibrosis-related genes resulting in the
suppression of liver fibrosis in this study. This has led us to conclude that miR-29a can prevent and improve liver fibrosis directlyor synergistically.

Controlling liver fibrosis through repair
In healthy liver, ECM is regulated by several gene expressions, forexample, MMP and the TIMP family. In damaged liver, interleukin 1 beta can promote the expression of MMPs. In addition, theexpression of TIMPs is also upregulated to inhibit MMP activity
and lead to ECM accumulation29 (Figure 1e,k and Figure 8b). It
was reported that MMP2 expression increased and remained elevated during ECM accumulation, however the upregulated TIMP2
expression decreased after ceasing fibrosis induction in CCl4
model mice.29 The declined TIMP expression promoted MMP
activation and then led fibrolysis to the spontaneous recovery
process. In both CCl4 and TAA liver fibrosis models, hydroxyproline and SR staining area declined more rapidly in the miR-29atreated group than in the NC miRNA-treated group (Figure 2b–e
and Figure 3a–d). To clarify how miR-29a contribute to theliver fibrolysis process, we used quantitative PCR to compare the1855

Molecular Therapy vol. 24 no. 10 oct. 2016-124-© The American Society of Gene & Cell Therapy

MiR-29a Assists Recovery From Liver FibrosisamiR-29a

Cell migration
Cell proliferationb

miR-29a00

Inflammation

0

Angiogenesis

Apoptosis

ECM resolution

Figure 8 Schema of miR-29a for liver fibrosis. The summary of hypothetical effect of miR-29a on liver fibrosis in this study. The graphs show microarray data. White bars show NC miRNA-treated liver and black bars show miR-29a-treated liver. (a) MiR-29a can control COL1A1, PDGFC, FGL2, and
MAP4K4 as target genes. The expression of four PDGFC pathway genes, RhoA, MAPK8, RAF1, and SRF, were downregulated in the miR-29a-treatedgroup. (b) IL-1B, NDRG1, and BCL2 were indirectly controlled by miR-29a and expression pattern of these genes was observed in microarray.

expression ratio of MMP2 and TIMP2 between miR-29a- and NC
miRNA-treated mice liver were analyzed (Supplementary FigureS7). CCl4 or TAA treatment upregulated MMP2/TIMP2 expression ratio, however it became downregulated during the spontaneous recovery process. In the group administered miR-29a, theratio of MMP2/TIMP2 remained higher than in the NC miRNAgroup, and this may have led to the more rapid progression offibrolysis.
MiR-29a treatment improved liver fibrosis through a repair
process and could control fibrolysis-related genes indirectly
because MMP2 and TIMP2 were not target gene for miR-29a.
In the spontaneous recovery period, liver fibrosis in TAA model
improved more rapidly than in CCl4 model. The effects of miR29a in both models were not completely replicated because of
the difference of their spontaneous recovery time. However, thepotential of miR-29a to improve liver fibrosis was common inboth models. This suggests that liver fibrosis resulting from different causes may be cured using only one miRNA.

Pharmacodynamics of miR-29a
Atelocollagen, a pepsin-treated type I collagen lacking telopeptides at the N and C terminals that confer antigenicity, has beendeveloped as a highly biocompatible material.31,32 Surface charge
is important for cellular uptake of nanoparticles, and positively
charged nanoparticles show higher cell uptake than negatively
charged particles.33 We confirmed the presence of pharmacodynamics in miR-29a in mouse liver model using atelocollagen. We
surmised that miR-29a reached the liver in the early phase of treatment (Figure 5a). Elevated miR-29a expression was not observedin fibrotic mice (Figure 5b) that were killed 72 hours after the
final injection of miR-29a. It would be fair to assume therefore
that although miR-29a reached the liver in the very early phase

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MiR-29a Assists Recovery From Liver Fibrosisof treatment it did not stay for a long time. In addition, Ago2-IP
analysis showed that miR-29a expression was concentrated in
RISC in CCl4 + 1-week miR-29a-treated liver (Figure 5c).
Administering miR-29 has effects on anti-fibrosis in CCl4 andTAA chronic liver injury models. Generally, miRNA has lower
gene-silencing ability than siRNA and miRNA expression vector; however, miRNA controls more genes than both. To clinically
apply nucleic acid-based medicine, miRNA expression by viral
vector is a highly efficient gene transporter; however, the drawback is it results in high antigenicity. In our study, hematoxylin andeosin staining showed that administering miR-29a did not resultin cell toxicity or inflammation (Figure 2b,c and Figure 3a,b),
body and liver weight (Supplementary Figures S3 and S4), andserum alanine aminotransferase and aspartate aminotransferase(Supplementary Figure S5).
We have presented a method for reversing fibrosis without
hepatotoxicity and have shed light on how miR-29a controls liver
fibrosis. The results presented here can make an impact on theclinical application and nucleic acid drug technology. Additional
research that clarifies the anti-fibrotic effect of miR-29a would be
instrumental not only for understanding liver fibrosis but also
other fibrotic diseases.

MATERIALS AND METHODS

Mouse model of liver fibrosis. C57BL/6J 6-week-old male mice (Japan SLC,
Shizuoka, Japan) were acclimated for 1 week, and lighting conditions were
controlled in a 12 hours light and 12 hours dark cycle. The animal protocol
was approved by the Institutional Animal Care and Use Committee of the
Osaka City University (Permission Number: 13043).
Two chronic liver injury mouse models were created to induce
fibrosis by using: (i) CCl4 and (ii) TAA. CCl4 was mixed with corn oil
and intraperitoneally injected at a dose of 0.5 µl/g BW every 3 days over
5 weeks for a total of 12 injections (Figure 1a).34 TAA was resolved insaline and given to mice at escalated doses from 100 µg/g BW (1stadministration), 200 µg/g BW (from 2nd to 5th administration), 300µg/g BW (from 6th to 9th administration), to 400 µg/g BW (from 10th
to 12th administration) every 3 days (Figure 1g).35 Control mice for CCl4
and TAA were administered corn oil only or saline only, respectively, andkilled at week 5.
Development of liver fibrosis and its spontaneous resolution wereconfirmed in both CCl4 and TAA models. Mice were observed for 1 or
2 weeks without any treatment after ceasing 5 weeks of intoxication.
Each mouse model was divided into three groups depending on theobservation period: 0 week (5 week CCl4 or TAA), 1 week (5 week CCl4 or
TAA + 1 week observation), or 2 weeks (5 week CCl4 or TAA + 2 weeks
observation) (Figure 1a,g). At least three mice were prepared for eachgroup.
Administration of miR-29a in vivo. To evaluate the effect of miR-29a on

the resolution of liver fibrosis, mice with developed liver fibrosis by CCl4
or TAA were administered miR-29a for 1 or 2 weeks immediately after
the fibrosis induction period. Fifty micrograms of double-strand mature
mmu-miR-29a (miR-29a) or nonspecific oligonucleotide (negative control for miR-29a: NC miRNA) (Bonac, Fukuoka, Japan) was mixed with
200 µl of atelocollagen (Atelo gene; KOKEN, Tokyo, Japan) and injected
via the tail vein every 3 days.36 MiRNA was administrated two times inmice treated for 1 week and four times in mice treated for 2 weeks. Thus,
mice for each fibrotic model were now spread over eight groups: observed
without any treatment (1 week observation/2 weeks observation), administrated only atelocollagen, a vehicle for nucleotides (1 week vehicle/2

weeks vehicle), administrated NC miRNA (1 week NC miRNA/2 weeks
NC miRNA), or administrated miR-29a (1 week miR-29a/2 weeks miR29a) (Figure 2a).
Histological analysis. Livers were fixed with 10% formalin and two serial

paraffin sections were made at 5 μm. One section was used for hematoxylin
and eosin. The other section was stained in a picrosirius red (Sigma-Aldrich,
St. Louis, MO) solution and counter stained with Fast Green (SigmaAldrich).37 The SR-positive area was quantified by MICROANALYZER(Nihon Poladigital, Tokyo, Japan). To validate the positive area of SR, fiveimages containing at least one portal vein were chosen.
Hydroxyproline assay. Liver hydroxyproline was measured using
Hydroxyproline Assay kit (BioVision, Milpitas, CA) following the manufacturer’s instruction. The detailed procedures are as previously described.37
Cell culture and miRNA transfection. Human HSC cell line LX-2, donatedby Scott L. Friedman (Division of Liver Diseases, Mount Sinai Hospital),
was cultured with Dulbecco’s modified medium (WAKO, Osaka, Japan)
and supplemented with 10% fetal bovine serum, 100 U/ml penicillin,
100 μg/ml streptomycin (Thermo Fisher Scientific, Waltham, MA), andnonessential amino acid (Thermo Fisher Scientific). 1 × 105 of LX-2 cells
were seeded to six-well plate (Thermo Fisher Scientific).38 Forty micromolar of miR-29a or NC miRNA was transfected to cells by lipofectamine
RNAiMAX reagent (Thermo Fisher Scientific).
In case of TGFβ1 treatment (Figure 5a), cells were seeded at -day
1 supplemented with 2.5 ng/ml TGFβ1 (Sigma-Aldrich). In the next day(day 0), miRNAs were transfected for 48 hours and cells were harvested
at day 2.
In the study that analyzing the effect of miR-29a to prevent HSCs
activation (Figure 6a), the cells were seeded at -day1. At day 0, miRNAs
were transfected to cells and 2.5 ng/ml TGFβ1 was added to medium
6 hours after transfection. The cells were harvested at day 1 or day 2,
respectively.
RNA extraction and mRNA and miRNA expression analysis. Total RNA
was extracted by miRNAeasy mini kit (QIAGEN, Venlo, Netherlands)
according to the manufacturer’s instruction. Complementary DNA wassynthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche,
Basel, Switzerland). For real-time PCR analysis, FastStart Universal SYBRGreen Master mix (Roche) was used. The sequences of primers are listed in
Supplementary Table S1. The reaction was performed in triplicate.
TaqMan miRNA assay (Thermo Fisher Scientific) was used toquantify the relative expression level of hsa-miR-29a (assay ID 002447)
and U18 (assay ID 001204) as an internal control. Complementary
DNA was synthesized using the TaqMan miRNA RT Kit (Thermo Fisher
Scientific). For real-time PCR analysis, Fast Start Universal probe master(Roche) was used. StepOnePlus Real-Time PCR System (Thermo Fisher
Scientific) was used to detect miRNA and mRNA. The reaction was
performed in triplicate.
Cell viability analysis. 1 × 104 LX-2 cells were seeded to 96-well plate. Cell

Proliferation Kit II (XTT) (Roche) was used according to the manufacturer’s instruction. Briefly, 48 hours after miRNA transfection, 50 μl of XTTreagent was added to cells and incubated for 6 hours at 37 °C. The absorbance at 450 nm was measured.

Caspase 9 activity assay. 1 × 104 LX-2 cells were seeded to 96-well plate.

Caspase-Glo 9 Assay kit (Promega, Madison, WI) was used according to
the manufacturer’s instruction.

Immunoprecipitation of Ago2. CCl4-treated 1-week miR-29a and NC
miRNA mice livers were used. MicroRNA Isolation Kit, Mouse Ago2
(WAKO) was used according to the manufacture’s instruction. The expression of miR-29a conjugated with Ago2 was confirmed by real-time PCR.

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MiR-29a Assists Recovery From Liver Fibrosis

Microarray analysis. Complementary RNA from 100 ng total RNA waslabeled with cyanine-3 using the Low Input Quick Amp Labeling Kit, onecolor (Agilent Technologies, Santa Clara, CA) according to the manufacturer’s protocol. Fragmented RNA samples were hybridized to SurePrint
G3 Mouse Gene Expression 8 × 60 K Microarray Kit (G4852A) (Agilent).
After hybridization, plates were scanned (Scanner G2539A) and analyzed
by Feature Extraction (v10.10.1.1). Data were analyzed with GeneSpring
GX (v13.0.0) and registered in NCBI Gene Expression Omnibus repository (GenBank accession no. GSE67423).
Immunoblot. Whole cell lysate was homogenized in 50 mmol/l Tris-HCl(pH 8.0), 150 mmol/l NaCl, 0.1% sodium dodecyl sulfate, 0.5% deoxycholic acid, and 1% NP-40. The quantified supernatants (20 µg/well) wereboiled in equal volumes of 20% glycerol containing 125 mmol/l Tris-HCl(pH 6.8), 4% sodium dodecyl sulfate, 10% 2-mercaptoethanol, and 0.01%
bromophenol blue. PAGEL (5–20%) (ATTO, Tokyo, Japan) was used for
sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA)
by submarine type blotting. Membranes were incubated with 5% skim
milk/Tris-buffered saline with Tween 20 for blocking. Primary antibodies
against COL1A1 (sc-8784; Santa Cruz, Dallas, TX) or β-actin (sc-69879;
Santa Cruz), and horseradish peroxidase-conjugated goat anti-rabbit oranti-mouse secondary antibodies (Dako, Glostrup, Denmark) were used.
Immunoreactivity was visualized by chemiluminescence (GE Healthcare,
Buckinghamshire, England).
Statistical analysis. For statistical analysis, analysis of variance and Tukey hon-

estly significant difference (HSD) post hoc test were used for multiple groupdata, and student’s t-test was used for independent two group data (SPSS; IBM,
Chicago, IL). P values <0.05 were considered statistically significant.

SUPPLEMENTARY MATERIAL
Figure S1. EVG staining of mice liver.
Figure S2. The expression of miR-29a and Col1a1 in tissues after
miR-29a administration.
Figure S3. The changes of body weight in 2 weeks treated mice.
Figure S4. The rate of liver weight/body weight.
Figure S5. ALT and AST in serum.
Figure S6. The effect of miR-29a on COL1A1 expression in primary
mice HSC.
Figure S7. The balance of MMP2 and TIMP2 expression by real-time
PCR analysis in time course.
Table S1. Primer list for real-time PCR.
Table S2. Genes with changing expression level in liver treated with
miR-29a for 1 week.
Table S3. Genes with changing expression level in liver treated bymiR-29a for 2 weeks.
Table S4. Genes with changing expression level in liver treated with
miR-29a for 1 and 2 weeks.

ACKNOWLEDGMENTS
Study concept and design: Y.M. and Y.M. Acquisition of data: Y.M. and
S.I. Analysis and interpretation of data: Y.M., S.I., and Y.M. Drafting
the manuscript: Y.M., S.I., and Y.M. Critical revision of the manuscriptfor important intellectual content: M.K., K.Y., N.K., and Y.M. Statisticalanalysis: Y.M. All authors read and approved the final manuscript. We
thank Takao Kenkou, Keisuke Inoue, and Emi Donoue for technical support performing histological analysis (Central Laboratory of Osaka City
University Medical School).. The authors have no conflict of interest to
declare. N.K. and Y. Murakami received financial support from KOSEIKAKEN (H25-kanen-ippan-012).

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Citation: Molecular Therapy—Nucleic Acids (2016) 5, e292; doi:10.1038/mtna.2016.1
Official journal of the American Society of Gene & Cell Therapy All rights reserved 2162-2531/16
www.nature.com/mtna

Topical Use of Angiopoietin-like Protein 2
RNAi-loaded Lipid Nanoparticles Suppresses
Corneal Neovascularization
Yukako Taketani1, Tomohiko Usui1, Tetsuya Toyono1, Nobuyuki Shima1, Seiichi Yokoo1, Mikiko Kimakura1, Satoru Yamagami1,
Shinichiro Ohno2, Risako Onodera3, Kohei Tahara3, Hirofumi Takeuchi3 and Masahiko Kuroda2

Corneal neovascularization (CNV) is a sight-threatening condition that is encountered in various inflammatory settings including
chemical injury. We recently confirmed that angiopoietin-like protein 2 (ANGPTL2) is a potent angiogenic and proinflammatory
factor in the cornea, and we have produced a single-stranded proline-modified short hairpin anti-ANGPTL2 RNA interference
molecule that is carried in a lipid nanoparticle (ANGPTL2 Li-pshRNA) for topical application. In this study, we have further
examined the topical delivery and anti-ANGPTL2 activity of this molecule and have found that fluorescence-labeled ANGPTL2
Li-pshRNA eye drops can penetrate all layers of the cornea and that ANGPTL2 mRNA expression was dramatically inhibitedin both epithelium and stroma at 12 and 24 hours after administration. We also examined the inhibitory effect of ANGPTL2
Li-pshRNA on CNV in a mouse chemical injury model and found that the area of angiogenesis was significantly decreased incorneas treated with ANGPTL2 Li-pshRNA eye drops compared to controls. Together, these findings indicate that this modified
RNA interference agent is clinically viable in a topical formulation for use against CNV.
Molecular Therapy—Nucleic Acids (2016) 5, e292; doi:10.1038/mtna.2016.1; published online 8 March 2016
Subject Category: nanoparticles

Introduction
Corneal neovascularization (CNV) is a sight-threatening
condition that is encountered in various inflammatory settings including chemical injury. CNV can lead to corneal
scarring, edema, and lipid deposition that may compromise
visual function and decrease the success rate of subsequent
penetrating keratoplasty.1 Topical corticosteroids have been
the mainstay for suppressing CNV.2 However, the inhibitory
effects of corticosteroids are limited and may be accompanied by adverse effects including delayed wound healing,
infection, posterior subcapsular cataracts, and elevationof intraocular pressure. As an alternative, antibody-based
anti-vascular endothelial growth factor molecular targetingtherapy has recently become available for the treatment of
CNV.3,4 However, this treatment also has several drawbacks,
including high costs and questionable efficacy in terms ofvessel regression and repair of epithelial defects.5
RNA interference (RNAi) occurs when small interfering
RNA (siRNA) molecules, in association with a nucleolytic
cytoplasmic protein complex, mediate sequence-specificdegeneration of target RNA molecules. Theoretically, thismechanism can silence the expression of virtually any
gene in a sequence-directed manner with high specificity.
The potential clinical applications of this phenomenon are
now being explored.
Angiopoietin-like protein 2 (ANGPTL2) is a member of theangiopoietin-like protein family and is essential for various
physiologic activities, including lipid metabolism, angiogenesis, and inflammation.6,7 Kanda et al. have reported that

ANGPTL2 in the ocular tissue was related to acute inflammation in the retina in an endotoxin-induced uveitis model via
activation of an NF-κB signaling pathway, and they indicatedthat ANGPTL2 could be a therapeutic target in acute inflammatory disease.8 In addition, we have previously found that
ANGPTL2 is a potent proangiogenic factor in mouse corneal
inflammation and that subconjunctival injection of ANGPTL2siRNA in mice dramatically suppressed local expression of
ANGPTL2 mRNA and reduced the extent of CNV.9 This finding represents a new approach to the treatment of angiogenesis-associated corneal diseases. However, it is not ideal toperform repeated subconjunctival injections in the clinicalsetting, and a common problem with RNAi agents has been
the lack of an effective in vivo drug delivery system. Moreover, because siRNAs are double stranded, they might act
as ligands for Toll-like receptor 3,10 which may induce innate
immune reactions.
To address these problems, we generated a singlestranded RNAi that can be delivered in a lipid nanoparticle
for use as a topical anti-ANGPTL2 agent in the treatment ofocular diseases. In this study, we have examined its efficacy
in terms of drug delivery and inhibition of CNV in a mousealkali-burn model.
Results
Safety of proline-modified short hairpin RNA-loaded
lipid nanoparticles in vitro
Cell viability was examined using simian virus 40-immortalized human corneal epithelial cells. There was no significant

1
Department of Ophthalmology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Department of Molecular Pathology, Tokyo Medical University,
Tokyo, Japan; 3Laboratory of Pharmaceutical Engineering, Gifu Pharmaceutical University, Gifu, Japan. Correspondence: Tomohiko Usui, Department of Ophthalmology,
Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: tomohiko-tky@umin.ac.jp
Keywords: angiogenesis; cornea; lipid nanoparticles; topical RNAi eye drops
Received 25 September 2015; accepted 20 December 2015; published online 8 March 2016. doi:10.1038/mtna.2016.1-129-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

2**

Cell viability (saline = 100%)

1,000%

100%

10%

1%
SalineANGPTL2
pshRNAANGPTL2
Li-pshRNA

Negative
pshRNA

Negative
Li-pshRNA

Amphotericin

Figure 1 WST-1 assay of Li-pshRNA. WST-1 assay was performed to confirm cell viability after treatment with Li-pshRNA. There was no
significant difference in cell viability among the treatment groups (n = 4, **P < 0.01). ANGPTL2, angiopoietin-like protein 2; Li-pshRNA, prolinemodified short hairpin RNA wrapped with liposome.

a
Epi
St

Epi

Endo

St
Endo

b

Time posteye drops

Control

Li-pshRNA

1 hour200 µm200 µm200 µ m200 µm200 µm200 µm3 hours

6 hours

Figure 2 Delivery of fluorescence-labeled pshRNA eye drops. (a) Left panel: bare siRNA with fluorescent label; right panel: fluorescencelabeled pshRNA enclosed in the lipid nanoparticle. The pshRNA was distributed only in the superficial layer of the corneal epithelium, while theLi-pshRNA was observed in almost all layers. (b) Delivery of fluorescence-labeled Li-pshRNA drops to mouse eyes. Samples were extracted at
1, 3, and 6 hours after instillation of the eye drops. Left panel: saline as control. Right panel: fluorescence-labeled Li-pshRNA. The Li-pshRNA
was distributed to all layers as time advanced. Epi, epithelium; Endo, endothelium of cornea; St, stroma. Li-pshRNA, proline-modified short
hairpin RNA wrapped with liposome.
Molecular Therapy—Nucleic Acids-130-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

3difference in posttreatment viability among five groups (control group, negative proline-modified short hairpin RNA(pshRNA) group, ANGPTL2 pshRNA group, negative
pshRNA wrapped with liposome (Li-pshRNA), and ANGPTL2 Li-pshRNA group), and cell viability in all five groups
was greater than that of cells treated with amphotericin
B (P < 0.01 for comparison, Figure 1).
Drug delivery and efficacy in normal mouse eyes
Fluorescence-labeled siRNA was distributed only in the
superficial layer of the corneal epithelium, while fluorescence-labeled Li-pshRNA showed almost full-thickness distribution (Figure 2a) and was detected in the stroma andthe endothelium at 3 and 6 hours after instillation of the eye
drops (Figure 2b).
ANGPTL2 mRNA expression was strongly inhibited in bothepithelial and stromal cells at 12 and 24 hours (P = 0.049 and<0.001, respectively) after instillation of eye drops in mice,but normal expression of ANGPTL2 mRNA was restored by
72 hours (Figure 3, n = 7 for all experiments). In contrast,
there was no change in ANGPTL2 mRNA expression in thecorneas of control mice (Figure 3).

Relative mRNA expression of ANGPTL2

a
*
1e+01

**

1e−01

1e−03

1e−05
Control

Negative 12 hours
Li-pshRNA

24 hours

48 hours

72 hours

48 hours

72 hours

Relative mRNA expression of ANGPTL2

b
50.00

*

*

5.00

0.50

0.05
0.01
Control

Negative 12 hours
Li-pshRNA

24 hours

Figure 3 Reduction of ANGPTL2 mRNA expression in normal
cornea after topical administration of ANGPTL2 Li-pshRNA.
Reverse transcriptase PCR showed that ANGPTL2 mRNA
expression was significantly reduced in the corneal epithelium (a)at 12 and 24 hours (P = 0.049 and <0.001, respectively; n = 7) andin stromal cells (b) at 12 and 24 hours (P = 0.0030 and 0.0021,
respectively, n = 7). Expression returned to baseline levels by 72
hours. *P < 0.05, **P < 0.01. ANGPTL2, angiopoietin-like protein
2; Li-pshRNA, proline-modified short hairpin RNA wrapped withliposome.

CNV in alkali-burn models
The effect of Angptl2 Li-pshRNA on CNV after alkali injury
was investigated. Drug delivery in the alkali model was also
evaluated by fluorescence assay, which confirmed that ANGPTL2 Li-pshRNA was distributed in all layers of the injured
cornea, while ANGPTL2 pshRNA alone was not (Figure 4a).
Expression of ANGPTL2 mRNA was inhibited at 36 hours
after alkali injury by topical treatment with ANGPTL2 LipshRNA, but not by topical administration of negative LipshRNA or by pshRNA alone (P = 0.043, n = 6; Figure 4b).
However, there was no significant difference in the levelsof expression of ANGPTL2 mRNA expression between the
ANGPTL2 Li-pshRNA group and the control group at 48
hours after alkali injury (Figure 4c). After 10 days of oncedaily treatment, the area of CNV was markedly reduced in
medicated eyes compared to the controls (P < 0.01, n = 7;
Figure 5).
Discussion
Several researchers have evaluated the therapeutic potential of gene silencing by RNAi for CNV in animal models.
Kim et al., using a corneal micro-pocket assay and herpetic
stromal keratitis models in mice, reported that the subconjunctival injections and systemic administration of vascularendothelial growth factor pathway-specific siRNAs inhibited
CNV,11 and Qazi et al. reported that intrastromal injection ofpoly nanoparticles loaded with plasmid-expressing anti-vascular endothelial growth factor siRNA also inhibited CNV ina more sustained and robust manner than the naked plasmid alone.12 Thus, the concept of using RNAi agents against
CNV seems promising. However, the invasiveness of subconjunctival or intrastromal injections could pose a hurdle torepeated treatments. A topical formulation would be a moredesirable and convenient approach. In this study, we haveshown that effective topical delivery of a modified RNAi agent(ANGPTL2 Li-pshRNA) can be accomplished using a lipid
nanoparticle, and that this agent reduced the expression of
ANGPTL2 mRNA and inhibited CNV in a mouse alkali-burnmodel.
It is crucial to confirm the safety of any therapy before clinical application. RNAi is generally thought to have minimal
cellular toxicity because of its endogenous nature, and in aprevious study, the intravitreal injection of RNAi did not cause
any obvious adverse effects in mouse retina.13 In the present
study, Li-pshRNA did not affect cell viability (Figure 1), and webelieve that the highly biocompatible nature of RNAi-loaded
lipid nanoparticles could be ideal for clinical application.
We used single-stranded RNAi (pshRNA) because double-stranded siRNAs have been associated with adverse offtarget effects due to activation of innate immunity, specifically
due to Toll-like receptor 3 activation.10 Further, annealing is
mandatory for generation of double-stranded siRNA, but notfor pshRNA, which is a single-stranded RNAi in which thelinker region has been replaced by amino acid derivatives.
This substitution and the short hairpin construct exhibit highstability in vivo and preliminary data (not shown) suggest that
pshRNA is less susceptible to degeneration due to RNase
activity. Because annealing is not necessary to generatewww.moleculartherapy.org/mtna-131-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

4a1 hour3 hours
200 µ m200 µ m

b

c
6

*

5

Relative mRNA expression of ANGPTL2

Relative mRNA expression of ANGPTL2

6

*
4

3

2

1

0

5

4

3

2

1

0
Control

Negative
Li-pshRNA

ANGPTL2
Li-pshRNA

Control

Negative
Li-pshRNA

ANGPTL2
Li-pshRNA

Figure 4 Inhibition of ANGPTL2 mRNA by ANGPTL2 Li-pshRNA eye drops after chemical injury. (a) Fluorescence-labeled topical
ANGPTL2 Li-pshRNA permeated the entire thickness of the injured cornea at 1 and 3 hours after injury. (b) ANGPTL2 mRNA expression was
reduced after topical administration of ANGPTL2 Li-pshRNA eye drops 36 hours after injury (P = 0.017 and 0.032 for saline as control andnegative Li-pshRNA, respectively; n = 6). ANGPTL2 mRNA expression was not inhibited by negative Li-pshRNA. *P < 0.05. (c) At 48 hours
after injury, levels of ANGPTL2 expression were the same among all three groups. ANGPTL2, angiopoietin-like protein 2; Li-pshRNA, prolinemodified short hairpin RNA wrapped with liposome.

the pshRNA, the cost can be also reduced. Moreover, previous research showed that pshRNA did not have off-target
effects and it was more stable against nuclease than doublestranded RNAi constructs.14 Thus, pshRNA overcomes many
disadvantages hitherto encountered with double-stranded
siRNAs.
The corneal epithelium contains tight junctions that forma barrier to paracellular drug permeation of the inner layerof the cornea in vivo. The corneal epithelium is lipoidal innature, which poses considerable resistance to permeation
by topically administered hydrophilic agents,15 as demonstrated by the failure of unmodified pshRNA, which is hydrophilic, to penetrate the epithelial surface in our experiments(Figure 2). The charge of a drug also affects permeation.
Positively charged molecules exhibit poor permeation, presumably because they bind to the negatively charged corneal and scleral proteoglycan matrix.16 To overcome this, thepshRNA was loaded into lipid nanoparticles (Li-pshRNA) thatwere specifically designed to facilitate drug delivery to theretina.17 In this study, we showed that ANGPTL2 Li-pshRNA
could reduce in vivo corneal ANGPTL2 mRNA expression(Figures 3 and 4). This result and others, for example, that

Molecular Therapy—Nucleic Acids-132-

the topical administration of edaravone-loaded lipid nanoparticles can protect against light-induced retinal dysfunction,18
support the potential usefulness of lipid nanoparticle-loaded
topical agents in the treatment of corneal diseases.
We found that the marked reduction in the level of ANGPTL2 mRNA was not sustained beyond 48 hours after alkali
injury. Even in the control group, ANGPTL2 expression
dropped to the same level as that of GAPDH, suggesting that
ANGPTL2 is produced in the very early stage of inflammation. As alkali damage progresses, the cells are not able toproduce as much ANGPTL2, and it becomes more difficult to
detect the effects of ANGPTL2 RNAi.
In conclusion, extensive corneal injuries such as chemicalburns cause conjunctivalization of the corneal surface andmassive neovascularization, leading to corneal opacity andsevere reduction in and visual acuity. We have successfully
generated a topical RNAi agent (Li-pshRNA) that inhibits
corneal inflammation and CNV by inhibiting expression of
ANGPTL2. Li-pshRNA eye drops show promise for preventing CNV and subsequent visual impairment without the side
effects commonly experienced with steroids or immunosuppressive agents.

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

5

a

d

b

Control

Negative Li-pshRNAe Negative Li-pshRNA

Control

2.0 mmg

ANGPTL2 Li-pshRNAf ANGPTL2 Li-pshRNA

2.0 mm

2.0 mm**45%
Percent are of angiogenesis in cornea

c

**

40%
35%
30%
25%
20%
15%
10%
5%
0%
Control

Negative
Li-pshRNA

ANGPTL2
Li-pshRNA

Figure 5 Inhibition of corneal neovascularization by ANGPTL2
Li-psh RNA eye drops. Corneal neovascularization after alkali injury wasmarkedly reduced after 10 days of once-daily treatment with ANGPTL2
Li-pshRNA eye drops (c) compared to eyes treated with control (a) or
negative Li-pshRNA (b). Corneas treated with ANGPTL2 Li-pshRNA
showed smaller areas of neovascularization (f) compared to control (d)and negative Li-pshRNA (e) corneas. The samples were stained with
CD31 to visualize the neovascularization. (g) Corneal neovascularization(CNV) was quantified by area (vascularized area/total cornea area) using
image J software. The extent of CNV after ANGPTL2 treatment wassignificantly smaller compared to the other groups (P < 0.01, n = 9). **P <0.01. ANGPTL2, angiopoietin-like protein 2; Li-pshRNA, proline-modified
short hairpin RNA wrapped with liposome.

Figure 6 ANGPTL2 pshRNA platform. PshRNA was synthesized
in solid phase as single-stranded RNAs that self-anneal after
synthesis into a helical structure containing a single stem loop. Red
characters indicated guide sequence, and green characters showed
nucleotide cassette sequence of pshRNA. ANGPTL2, angiopoietinlike protein 2; pshRNA, proline-modified short hairpin RNA.
A, adenine; C, cytosine; G, guanine; U, uracil; P, proline derivative.

Materials and methods
Animals. All animal experiments were approved by the University of Tokyo Hospital Animal care committee and conformed
to the Association for Research in Vision and Ophthalmology

Statement for the Use of Animals in Ophthalmic and Vision
Research. All surgical procedures were performed underketamine hydrochloride (35 mg/kg) and xylazine chloride(5 mg/kg) anesthesia, and all efforts were made to minimize
suffering. C57BL/6 mice aged 8–12 weeks were used in all
experiments.
Reagents. The ANGPTL2-specific single-stranded RNAisegment was contained in a short hairpin structure (ANGPTL2 pshRNA) with a stem loop, which was synthesized in
the solid phase.14 The stem loop contained proline derivatives similar to PnkRNA, as previously described.19 The basesequence of the ANGPTL2 pshRNA was 5′-CCA GAA AGC
GAG UAC UAU AUU CC proline modified-3′, 5′-GGA AUAUAF UAC UCG CUU UCU GGU U-3′ (structure shown in
Figure 6), and the negative control was 5′-UAC UAU UCGACA CGC GAA GUU CC proline modified-3′, 5′-GGA ACUUCG CGU GUC GAA UAG UAU U-3′. The reagents were
supplied by BONAC (Fukuoka, Japan). The linker region of
the pshRNA was replaced by a fluorescent pigment for drugdelivery experiments.
The lipid nanoparticle was prepared as described previously.17
1,2-Distearoyl-sn-glycero-3-phosphocholine,
cholesterol,and stearylamine (7:3:1 molar ratio, lipid concentrations: 20
mmol/l) were dissolved in a small amount of chloroform anddried in a rotary evaporator under reduced pressure at 40°C to form a thin lipid film. The film was dried in a vacuum
oven overnight to ensure complete removal of the solvent.
After hydration of the film with a suitable amount of Tris–ethylenediaminetetraacetic acid buffer containing RNAi, theresultant lipid suspension was incubated in a water bath at
70 °C. The multilamellar vesicle suspension was incubated atroom temperature for 15 minutes. Submicron-sized liposomes
were prepared using an extruder (LipoFast-Pneumatic, Avestin, Ottawa, Ontario, Canada) equipped with a polycarbonate membrane (0.1-µm membrane filter pore size; Whatman
Japan KK, Tokyo, Japan). Extrusion was performed 41 times
with nitrogen pressure (200 psi). The final phospholipid andRNAi concentrations in liposomal suspensions were 20
mmol/l and 0.5 or 1 mol/l, respectively.
Cell toxicity of Li-pshRNA in vitro. Cellular viability wasevaluated using a nonradioactive colorimetric assay (WST1; TAKARA Bio, Shiga, Japan) according to the manufacturer’s protocol. Simian virus 40-immortalized human corneal
epithelial cells were cultured in Dulbecco’s modified Eagle
medium (Wako Pure Chemical Industry, Osaka, Japan)/F12
containing 10% fetal bovine serum supplemented with cholera toxin (100 ng/ml), human recombinant epidermal growth
factor (10 µg/ml), and insulin (5 µg/ml). After the culture
reached confluence, ANGPTL2 Li-pshRNA, negative control
Li-pshRNA, Amphotericin B (0.1% Fungizone, Bristol-Myers,
Tokyo, Japan), or saline was applied for 30 minutes followed
by WST-1 reagent in phenol red-free Dulbecco’s modified
Eagle medium/F12 medium for 1 hour at 37 °C, and the dye
was measured at 450 nm by a Victor 3V Multilabel Countermodel 1420 plate reader (Perkin Elmer, Waltham, MA). The
results were expressed as the mean ± SD of the percentage of cell viability compared with that of the control-treatedcontrols.
www.moleculartherapy.org/mtna-133-

Topical RNAi Suppresses Corneal Neovascularization
Taketani et al.

6
Table 1 Quantitative reverse transcriptase PCR primersGenesymbolForward 5′–3′Reverse 5′–3′

MusAACCATGGAGGACTGGTCTG
ANGPTL2TGAGGTTGGAGTGAGCACAG

MusGAPDH

AACTTTGGCATTGTGGAAGGCACATTGGGGGTAGGAACAC

Drug delivery to the cornea. Drops of Li-pshRNA labeledwith fluorescent pigment (5 µl) were instilled on the corneal
surface of anesthetized mice, after which the eyes wereenucleated. Specimens were embedded in optimal cutting
temperature compound (Sakura Finetechnical, Tokyo, Japan)
after 1, 3, and 6 hours. The samples were immediately frozen
in liquid nitrogen and cut into 10-µm sections. Corneal distribution of the labeled pshRNA was examined by fluorescencemicroscopy (BZ-9000; Keyence, Osaka, Japan). The sameprocedure was performed in the alkali-burn models withdrops instilled 1, 3, and 6 hours after alkali burn.
To examine changes in mRNA expression after administration of the Li-pshRNA RNAi agent, 5 µl of ANGPTL2 LipshRNA was instilled on the corneal surface in anesthetized
mice. Eyes were enucleated at 12, 24, 48, and 72 hours afteradministration, and the corneal epithelium was separated
from the corneal stroma using trypsin-ethylenediaminetetraacetic acid. The total RNA of each sample was extracted
and ANGPTL2 expression was quantified by quantitative
real-time reverse transcriptase PCR.
Real-time reverse transcriptase PCR. cDNA was synthesizedusing PrimerScript RT Master Mix (TAKARA Bio). The PCR
amplifications were performed in an instrument using Takara
PCR Thermal Cycler Dice (TAKARA Bio) as described previously.9 The primer sets are shown in Table 1.
Mouse model of alkali-induced inflammatory CNV. The
mouse alkali-burn models were produced as previously
described20–22 with modifications to suit our experiment.
Chemical injury was induced by placing 5 µl of 0.5 N sodiumhydroxide (NaOH) on the cornea of anesthetized mice for
30 seconds. The eyes were then washed with 20 ml saline(day 0). On day 1, the injured eyes were treated with 5 µl of
ANGPTL2 Li-pshRNA or negative Li-pshRNA for 5 minutes,
after which reagents were absorbed (KimWipe: Nikkei Products, Osaka, Japan) and eyes were subsequently treatedwith ofloxacin ophthalmic ointment (Talibit, Santen, Osaka,
Japan). The RNAi reagent and antibiotic treatments wererepeated daily until day 10, then the mice were euthanized
and the cornea with limbus was excised.
CNV was quantified using CD31 staining as previously
described.9 Briefly, corneas were fixed in acetone for 10 minutes at −20 °C, washed three times in phosphate-bufferedsaline, stained with rat anti-mouse CD31 antibody (BD Biosciences, Franklin Lakes, NJ), and incubated at 4 °C overnight.
The samples were washed again with phosphate-bufferedsaline and stained with the secondary antibody (Alexa Fluor
594 donkey anti-rat IgG, Invitrogen, San Diego, CA) for 5
hours at room temperature. The stained samples were evaluated by fluorescence microscopy (BZ-9000, Keyence), andMolecular Therapy—Nucleic Acids-134-

the CD31-positive blood vessels were quantified by area
using Image J software (http://rsb.info.nih. gov/ij/). The results
are expressed as vessel area/total field.
To examine changes in mRNA expression in our model,
the corneas were extracted 36 and 48 hours after injury, andquantitative real-time reverse transcriptase PCR of ANGPTL2 was performed for each group (control, negative LipshRNA, and ANGPTL2 Li-pshRNA) using the conditions
described above.
Statistical analysis. All of the data were analyzed by Mann–
Whitney U-test using statistical software R (Ver. 3.1.1), andvalues of P < 0.05 were considered statistically significant
after Holm’s correction.
Acknowledgments This work has been supported by agrant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
We would like to specially thank Azusa Kamikawa, assistantresearcher, for her assistance in conducting our experiments.
This work was done in Tokyo, Japan.
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6. Kadomatsu, T, Endo, M, Miyata, K and Oike, Y (2014). Diverse roles of ANGPTL2 inphysiology and pathophysiology. Trends Endocrinol Metab 25: 245–254.
7. Thorin-Trescases, N and Thorin, E (2014). Angiopoietin-like-2: a multifaceted protein withphysiological and pathophysiological properties. Expert Rev Mol Med 16: e17.
8. Kanda, A, Noda, K, Oike, Y and Ishida, S (2012). Angiopoietin-like protein 2 mediates
endotoxin-induced acute inflammation in the eye. Lab Invest 92: 1553–1563.
9. Toyono, T, Usui, T, Yokoo, S, Kimakura, M, Nakagawa, S, Yamagami, S et al. (2013).
Angiopoietin-like protein 2 is a potent hemangiogenic and lymphangiogenic factor incorneal inflammation. Invest Ophthalmol Vis Sci 54: 4278–4285.
10. Kleinman, ME, Yamada, K, Takeda, A, Chandrasekaran, V, Nozaki, M, Baffi, JZ et al.
(2008). Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.
Nature 452: 591–597.
11. Kim, B, Tang, Q, Biswas, PS, Xu, J, Schiffelers, RM, Xie, FY et al. (2004). Inhibition ofocular angiogenesis by siRNA targeting vascular endothelial growth factor pathway genes:
therapeutic strategy for herpetic stromal keratitis. Am J Pathol 165: 2177–2185.
12. Qazi, Y, Stagg, B, Singh, N, Singh, S, Zhang, X, Luo, L et al. (2012). Nanoparticlemediated delivery of shRNA.VEGF-a plasmids regresses corneal neovascularization.
Invest Ophthalmol Vis Sci 53: 2837–2844.
13. Nakama, T, Yoshida, S, Ishikawa, K, Kobayashi, Y, Zhou, Y, Nakao, S et al. (2015).
Inhibition of choroidal fibrovascular membrane formation by new class of RNA interference
therapeutic agent targeting periostin. Gene Ther 22: 127–137.
14. Takanashi, M, Sudo, K, Ueda, S, Ohno, S, Yamada, Y, Osakabe, Y et al. (2015). Noveltypes of small RNA exhibit sequence- and target-dependent angiogenesis suppression
without activation of Toll-like receptor 3 in an age-related macular degeneration (AMD)mouse model. Mol Ther Nucleic Acids 4: e258.
15. Gaudana, R, Ananthula, HK, Parenky, A and Mitra, AK (2010). Ocular drug delivery. AAPS
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17. Murata, M, Yonamine, T, Tanaka, S, Tahara, K, Tozuka, Y and Takeuchi, H (2013). Surface
modification of liposomes using polymer-wheat germ agglutinin conjugates to improve theabsorption of peptide drugs by pulmonary administration. J Pharm Sci 102: 1281–1289.
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Topical RNAi Suppresses Corneal Neovascularization
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original article© The American Society of Gene & Cell Therapy

Development of Novel Small Hairpin RNAs That do
not Require Processing by Dicer or AGO2
Shin-ichiro Ohno1, Karen Itano1, Yuichirou Harada1, Koutaro Asada1, Keiki Oikawa1, Mikie Kashiwazako1,
Hikaru Okuyama1, Katsuyoshi Kumagai2, Masakatsu Takanashi1, Katsuko Sudo2, Norihiko Ikeda3 andMasahiko Kuroda1
1
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 2Department of Animal Research Center, Tokyo Medical University, Tokyo,
Japan; 3Department of Respiratory Surgery, Tokyo Medical University, Tokyo, Japan

The innate cytokine response to nucleic acid is the mostchallenging problem confronting the practical use of nucleic
acid medicine. The degree of stimulation of the innate cytokine response strongly depends on the length of the nucleic
acid. In this study, we developed a 30-nucleotide singlestrand RNA, termed “guide hairpin RNA (ghRNA, ghR)”,
that has a physiological function similar to that of miRNAand siRNA. The ghR caused no innate cytokine response
either in vitro or in vivo. In addition, its structure does notcontain a passenger strand seed sequence, reducing theunwanted gene repression relative to existing short RNA
reagents. Systemic and local injection of ghR-form miR-34a(ghR-34a) suppressed tumor growth in a mouse model of
RAS-induced lung cancer. Furthermore, Dicer and AGO2
are not required for ghR-34a function. This novel RNA
interference (RNAi) technology may provide a novel, safe,and effective nucleic acid drug platform that will increase
the clinical usefulness of nucleic acid therapy.
Received 2 October 2015; accepted 4 April 2016; advance online
publication 24 May 2016. doi:10.1038/mt.2016.81

INTRODUCTION

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene
expression and fundamental cell processes such as development, differentiation, proliferation, and apoptosis. miRNAs are generated ina series of steps from a primary transcript (pri-miRNA) with a cap
structure and a poly-A tail.1 In the nucleus, the hairpin structure ofpri-miRNA is cleaved by Drosha, an RNase III enzyme, to yield premiRNA. The pre-miRNA is transported to the cytoplasm, where it isprocessed into shorter double-stranded RNA (dsRNA) by the RNase
III enzyme Dicer, ultimately yielding mature miRNA. One notable
exception to this is miR-451, which does not depend on Dicer and isinstead processed by Argonaute (Ago) 2 (refs. 2,3). The short dsRNA
generated by Dicer is taken in by AGO family protein (AGO1-4) of
the RNA-induced silencing complex (RISC). One of the two strands
is discarded, and the RISC binds to the remaining single-stranded
mature miRNA and an mRNA with a complementary sequence.
In particular, the 2–8-base pair sequence on the 5’ side the mature
miRNA, called the seed sequence, is important for target recognition; this sequence binds to target mRNAs with complementary

sequences in their 3’ UTRs.4 miRNA can repress expression of its
target mRNA in different ways. The recruitment of RISC to targetedmRNA can induce deadenylation of the poly-A tail and inducedmRNA degradation.5,6 The RISC can also affect the formation offunctional ribosomes at the 5’-end of the mRNA by competing withtranslation initiation factors and/or abrogating ribosome assembly.7,8In addition, the RISC can inhibit protein production by targeting thedegradation of the growing polypeptides.9
Reduced or elevated expression of miRNAs has been implicatedin the development of various human diseases, including cancer.10
Members of the miR-34 family of tumor-suppressive miRNAs,
comprising miR-34a, miR-34b, and miR-34c, are direct transcriptional targets of p53; their upregulation induces apoptosis, cell-cycle
arrest, and inhibition of invasion/migration by targeting AXL, MET,
and CDK6.11–13 Recent studies showed that exogenously supplied
miR-34a induces apoptosis and cell-cycle arrest in lung cancer celllines, and is effective against RAS-induced lung cancer in mousemodels.14–16 Therefore, miRNA replacement therapy represents apromising approach for the treatment of lung cancer.17 However,
the immunological response to nucleic acids mediated by Toll-likereceptors (TLRs), RIG-I-like receptors, and dsRNA-activated protein kinase (PKR) restricts the practical use of nucleic acid drugs
in clinical settings.18–24 The first clinical trial of a small RNA-based
therapeutic, bevasiranib (formerly known as Cand5), utilized 21-nt
dsRNA against vascular endothelial growth factor for the treatmentof neovascular age-related macular degeneration; however, the trial
was cancelled during phase 3. Kleinman et al.25 reported that 21-nt
dsRNA suppresses neovascularization in mice not via RNAi, butinstead via sequence-independent activation of cell surface TLR-3.
In other words, 21-nt dsRNA exerts its cellular activity as a pathogen-associated molecular pattern. In order to avoid recognition by
TLR-3, dsRNA must be shorter than 19 nucleotides. The development of minimal size of interfering RNA have been well studied
at short hairpin RNAs (shRNAs)-based.26,27 Short shRNA, duplex
lengths does not exceed 19 bp, are too short for Dicer1 substrate andprocessed by AGO2-mediated slicer activity.26 Such shRNAs are oneof the effective shortest interfering RNA. However, the total lengthof short shRNA is still about 40 nucleotides that may cause immune
responses.25,28 Therefore, we attempted to develop shorter nucleic
acid drugs that can escape immune surveillance.

This work was done in Shinjyu-ku, Tokyo, Japan.
Correspondence: Masahiko Kuroda, Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160–8402,
Japan. E-mail: kuroda@tokyo-med.ac.jp

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RESULTS
Consideration of shortening of the mimic miRNA

Development of Novel Short Hairpin RNA

Before loading onto the RISC complex, miRNAs form a doublestranded structure consisting of a guide strand and a passenger strand, each of which is 20–23 nt in length. However, otherclasses of miRNAs exist. For example, miR-451 is a single-stranded
miRNA identified in AGO2-knockout zebra fish and DICER1
knockout MEFs.2,3 The 3’ side of the guide chain of pre-miR-451
assumes a loop structure and is connected to a passenger chain.
Because it does not require a complementary sequence in the loop,
miR-451 is shorter than normal double-stranded miRNA. Initially,
we designed a mimic miRNA with a structure similar to that ofpre-miR-451 (Figure 1a). For the mimic sequence, we selected
miR-34a, the most potent tumor-suppressive miRNA. miR-34a
expression levels are correlated with relapse and overall survival
in surgically resected non–small-cell lung cancer patients.29 We
constructed a miR-451-like form of miR-34a (miR-451-form miR34a), in which the sequence of mature miR-34a assumes the samestructure as pre-miR-451 (Figure 1a). miR-34a is a 44 nt dsRNA(two strands of 22 nt each), whereas miR-451-form miR-34a isslightly shorter (40 nt). It has been reported that the mimicking
Pre-miR-451 structure retains the inhibitory activity of the original miRNA.2,3,30 We confirmed whether miR-451-form miR-34a
maintained inhibitory activity in the non–small-cell lung cancer lung cancer cell line NCI-H1299 (H1299). In order to evaluate the effect of miR-34, we focused on three genes (AXL, MET,
CDK6) targeted by this miRNA for mRNA degradation.11–13 Wetransfected the miR-451-form miR-34a mimic into H1299 andconfirmed the downregulation of AXL and MET (Figure 1b). We
then attempted further shortening based on the structure of miR451-form miR-34a. Based on the assumption that the nucleotide on
the 5’ side of the passenger chain was unnecessary, we synthesized
a nucleic acid in which the passenger sequence was shortened by
2 nt from the 3’ end of the sequence (Supplementary Figure S1a).
When we evaluated the activity of these shortened miRNAs compared with full-length miR-451-form miR-34a, we found thatknockdown activity decreased proportion to the length of deleted
nucleotide and activity was completely lost when the passenger
sequence was reduced to 8 nt (Supplementary Figure S1b).
The stability of the secondary structure is important for the
inhibitory activity of both duplex siRNA and miRNA.31,32 The freeenergy balance of G22/P10 (guide: 22 nt, passenger: 10 nt) is 0
kcal/mol, as determined using the secondary structure prediction
algorithm CentroidFold (http://www.ncrna.org/centroidfold/).33
Therefore, in order to maintain the hairpin structure of the miR451-form miRNA, we deleted 1 nt in both directions from theconnection point of the guide sequence and passenger sequences,and evaluated the resultant RNAs (Figure 1c). Surprisingly, inhibitory activity equivalent to that of miR-34a was preserved evenwhen the sequence was shortened to 30 nt (G17/P13) (Figure 1d,
Supplementary Figure S2). Moreover, miR-34a with a 4 nt hairpin loop structure (G19/P15, G17/P13) retained the inhibitory
activity, whereas miR-34a with 6 nt in the hairpin loop structure(G20/P16, G18/P14, G16/P12) lost the activity (Figure 1d). We
next synthesized miR-34a with four nt in the hairpin loop andreduced the length below 30 nt, and then evaluated the inhibitory activity of the resultant RNAs (Supplementary Figure S3a).

If the loop contained 4 nt, it retained a strong suppressive effecteven when shortened to G15/P11 (Supplementary Figure S3b).
However, because the inhibitory activity of the 26-nt variant of
miR-34a against AXL was slightly reduced, we continued the study
with the 30-nt variant of miR-34a, which we named “guide hairpin
RNA (ghR-) 34a” and characterized its properties in detail.

Analysis of the inhibitory activity and characterizationof ghR-34a
Chemical modification and shortening of nucleic acids often
diminishes their original RNAi activity. Therefore, we next examined whether the physiological function and activity of miR-34a
was preserved in the ghR structure. To this end, we analyzed
the introduction density and the sustained time of the suppressant effect; in these experiments, we could not detect a difference
between miR-34a and ghR-34a (Figure 2a,b; Supplementary
Figure S4). Furthermore, we confirmed by immunoblotting analysis that ghR-34a reduced target protein expression (Figure 2c).
Furthermore, because expression of miR-34a suppresses lung cancer cell growth,16,34 we examined the ability of ghR-34a to exert thiseffect. We confirmed that ghR-34a regulated proliferation of thelung cancer cell lines H1299 and Calu-3 as effectively as miR-34a(Figure 2d; Supplementary Figure S5).
ghR-34a was designed based on the structure of miR-451, a
DICER1-independent miRNA generated by the Slicer activity of
AGO2.2,3 Therefore, we investigated whether ghR-34a acted in a
DICER1- and AGO2-independent manner. To this end, we constructed DICER1- and AGO2-deficient lung cancer cells (H1299)
using the CRISPR/Cas9 system (Figure 3). Guide RNAs were
designed at two locations upstream of the functional domain of
DICER1 and AGO2 (Figure 3a,d) and induced frame-shift mutations at the gene with Cas9. We have created two clones of H1299
cell lines, lacking protein expression of DICER1 or AGO2, fromeach of the guide RNA (Figure 3b,e). DICER1-deficient cells
contained lower levels of endogenous miRNA (Supplementary
Figure S6), and AGO2-deficient cells exhibited reduced siRNA
activity (Supplementary Figure S7). Next, we transfected ghR-34a
into DICER1-deficient cells and examined expression of miR-34a
target genes. ghR-34a inhibited normal H1299 and DICER1deficient cells to the same extent (Figure 3c). This result indicatedthat like pre-miR-451, ghR-34a acted in a DICER1-independent
manner. In addition, we transfected ghR-34a and miR-451-form
miR-34a into AGO2-deficient cells (Figure 3f). Because miR-451form miR-34a imitates the shape of pre-miR-451, and its activity
declined in AGO2-deficient cells, this RNA may be processed in
an AGO2-dependent manner. Surprisingly, ghR-34a was active in
AGO2-deficient cells, indicating that it does not require AGO2
for processing. To elucidate the mechanisms of interference by
ghR-34a, we performed reporter analysis. If ghR-34a inhibits theirtarget genes via conventional miRNA pathway, inhibition is dependent on 3’UTR sequence of target mRNA. Therefore, we created areporter plasmid that constructed with the AXL 3’UTR fused with
firefly luciferase gene. H1299 cells stably expressing the luciferase
reporter construct were transfected with ghR-34a and analyzed
by luciferase assay (Supplementary Figure S8a). Luciferase activity in cells transfected with ghR-34a was significantly reduced aswell as miR-34a. This result indicated that ghR-34a inhibits the1279

Molecular Therapy vol. 24 no. 7 jul. 2016-137-© The American Society of Gene & Cell Therapy

Development of Novel Short Hairpin RNA

a

b

AXL

1.6

miR-34a1.4

22+22 = 44nt

1.2

23+19 = 42nt

miR-451-form
miR-34a

1.0
0.8
0.6

**

**

0.4

1.0

Relative expression

Relative expression

1.2
miR-451

0.8
0.6

0

**

0.2

Mock Cont

34a

Normal

c

*

0.4

0.2

22+18 = 40nt

MET

1.4

Cont

0

34a

Mock Cont

miR-451-form

34aNormalCont

34a

miR-451-form

Guide/passenger

miR-451-form
miR-34a(G22/P18)

G21/P17

G20/P16

G19/P15

G18/P14

G17/P13

G16/P12

Total length

40nt

38nt

36nt

34nt

32nt

30nt

28nt

Predicted secondary
structure(CentroidFold)
4nt

6nt

4nt

6nt

4nt

6nt

−21.60−16.10−15.40−12.80−12.90−11.20G16/P12

G17/P13

G18/P14

G19/P15

G20/P16

G21/P17

G22/P18

Control

G16/P12

G17/P13

G18/P14

G19/P15

G20/P16

G21/P17

G22/P18

Mock

Control

0

0.4
0.2
0

G16/P12

0.2

**

**

G17/P13

0

**

**

0.6

G18/P14

0.2

** **

0.4

** **

G19/P15

**

**

0.6

*

0.8

G20/P16** ** ** **

0.4

**

0.8

1.0

G21/P17

**

**

1.0

Control

0.8

CDK6

1.2

G22/P18

1.0

Relative expression

Relative expression

1.2

0.6

MET

1.2

Mock

AXL

1.4

Mock

d

4nt
−22.20Relative expression

Loop length
Free energy(Kcal/mol)Figure 1 Development and characterization of short mimic miRNA. (a) Secondary structures of miR-34a, pre-miR-451, and pre-miR-451–mimicking miR-34a (miR-451-form miR-34a). The formulas show the numbers of bases in the guide sequence and passenger sequence. (b)Comparison of inhibitory activity of normal miR-34a, miR-451-form miR-34a and each form of nontargeting control RNA in H1299 cells. At 48 hours
post-transfection, the expression levels of AXL and MET were measured by real-time polymerase chain reaction (PCR) and normalized against β-actin.
The experiments were performed with a final RNA concentration of 5 nmol/l. Data are expressed as means ± SD (n = 3); *P < 0.05, **P < 0.01 compared with control. (c) The secondary structures and free energies of synthetic shortened RNA were predicted using the CentroidFold algorithm.
Black arrow indicates the direction of nucleotide deletion. (d) Comparison of inhibitory activity among miR-451-form miR-34a and deleted forms. At
48 hours post-transfection, the expression levels of AXL, MET, and CDK6 were measured by real-time PCR and normalized against β-actin. Data areexpressed as means ± SD (n = 3); *P < 0.05, **P < 0.01 compared with control. All data are representative of at least three independent experiments.

expression of AXL mRNA by acting on 3’UTR. Next, we performed AGO2 immunoprecipitation (IP) analysis. The ghR-34a
interacted with AGO2 complex (Supplementary Figure S8b)
and could directly recognize target genes of miR-34a in AGO2expressed normal cells (Supplementary Figure S8c). These
results suggested that ghR-34a inhibits miR-34a target genes using
AGO2 complex.
When subjected to AGO2 Slicer activity, pre-miR-451 produces a 30-nucleotide miR-451 species whose 3’ end is further

trimmed by poly(A)-specific ribonuclease (PARN) to yield
mature ~23-nt miR-451 (ref. 35). The final steps of the cleavage process are unrelated to the inhibitory action of miR-451.
Because ghRs were designed to have a secondary structure
similar to that of pre-miR-451, we speculated that they would
be processed by AGO2. However, contrary to our hypothesis,
ghRs were generated in an Ago2-mediated catalytic cleavageindependent manner (Figure 3f). It remains unknown whether
the 30-nt ghR undergoes PARN-mediated 3’-end trimming. Our

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© The American Society of Gene & Cell Therapy

MET

AXL

1.4

25 nmol/l

5 nmol/l

1 nmol/l

200 pmol/l 40 pmol/lc1.2

25 nmol/l

5 nmol/l

1 nmol/l

miR-34a200 pmol/l 40 pmol/l

d

n.s.

*
*

15

4

Cell number (× 10 /well)

Control

n.s.

0.8

34a

Nontreated

n.s.
n.s.

ghRNA

Control

20
miRNA

ghR-34amiR-34amiR-34aghR-34amiR-34aghR-34aghR-34amiR-34aghR-34amiR-34aghR-34amiR-34aghR-34amiR-34aghR-34amiR-34a0

Control ghRNA

0

Mock

0.2
Control miRNA

0.2

1
Relative expression levels of AXL

0.4

ghR-34a

n.s.

0.4

n.s.

n.s.

0.6

miR-34an.s.

0.8

Control ghRNA

n.s.

0.6

n.s.

Mock

0.8

1.0

Control miRNA

n.s.

n.s.

n.s.

Nontreated

1.0

Relative expression

1.2

Mock

b

1.2
n.s.

NontreatedRelative expression

1.4

ghR-34a

1.6

34a

a

Development of Novel Short Hairpin RNA

AXL

0.6
n.s.
n.s.

0.4

MET
Nontreated

0.2

miR-34a

10

5

GAPDH
0

ghR-34aControl
miRNA

0
24

48

72

96

miR-34aControl

ghR-34aghRNA

120 (hours)

Figure 2 Comparison of inhibitory activity between wild-type miR-34a and ghR-34a. (a) Comparison of inhibitory activity between wild-typemiR-34a and ghR-34a at various concentrations in H1299 cells. At 48 hours post-transfection, the expression levels of AXL and MET were measured
by real-time polymerase chain reaction (PCR). Data are expressed as means ± SD (n = 3); NS, not significant compared with control. (b) Comparisonof inhibitory activity among normal miR-34a and ghR-34a over a time course in H1299 cells. At each time point, the expression level of AXL wasmeasured by real-time PCR and normalized against β-actin. Data are expressed as means ± SD (n = 3); NS=not significant. (c) Immunoblot analyses
confirmed the inhibition of AXL and MET expression by ghR-34a. (d) Inhibitory effects of ghR-34a on proliferation of H1299 lung cancer cells. Cells
were harvested and counted at 96 hours post-transfection. Data are expressed as means ± SD (n = 3); NS, not significant; *P < 0.05 compared with
control. All data are representative of at least three independent experiments.

experiments showed that dT modification of the 3’-overhang did
not alter ghRNA activity (Supplementary Figure S9), possibly
suggesting a relatively minor biological role for exoribonucleasemediated 3’ end trimming. In addition, we examined the importance of mismatches in the +1 and +6 positions (Supplementary
Figure S9). Human Pre-miR-451 does not have a mismatch in
the +6 position, although such mismatches are conserved inother species and slightly increase inhibitory activity.30,36 In
this case, we found that the inhibitory activity hardly changed
when the +6 mismatch was replaced with a complementary base,but the inhibitory activity tended to decrease when the mismatches at both +1 and +6 were mutated to be complementary(Supplementary Figure S9).

Analysis of immune response and suppression by thepassenger strand in ghR-34a
Next, we examined whether this shortening method could successfully control inflammatory reactions. Nucleic acids outside
cells are recognized by monocytes, macrophages, and dendritic
cells, in which they induce expression of inflammatory cytokines.18 The details of the nucleic acid recognition mechanism
remain poorly characterized, but it is clear that the inflammatory reaction is stronger in response to longer nucleic acids.25,28
To address this issue, we first isolated monocytes from humanperipheral blood, introduced ghR into these cells, and measured
IL-6 and TNFα expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA)

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Development of Novel Short Hairpin RNAac
CRISPR/Cas9
Guide RNA
Guide #1
Guide #2

1

Relative expression levels of AXL

Human DICER1protein

1922 aa

NH2

COOH
RNase IIIa

b

IIIb

DICER1 deficient
H1299

#1

#2

DICER1

GAPDH

1.8

H1299

1.6

Dicer1 deficient #1

1.2
1.0

n.s.

0.8

n.s.

0.6
0.4
0.2
0

Human AGO2
Protein1

859 aa

NH2

COOH
PIWI
AGO2 deficient
H1299

#1

Mock

ghRNA
control

ghR-34a

H1299

1.6

Guide #1
Guide #2eNontreatedf

CRISPR/Cas9
Guide RNA

Relative expression levels of AXLdDicer1 deficient #2

1.4

#2

AGO2

GAPDH

Ago2 deficient #1
Ago2 deficient #2

1.4
1.2

*
*

1.0

n.s.

0.8

n.s.

0.6
0.4
0.2
0
Non
treatedMock

miR-451-form
controlmiR-451-form
miR-34aghRNA
control

ghR-34aFigure 3 Analysis of the dependence of ghR-34a on DICER1 and AGO2. (a) Functional RNase domain of DICER1 protein. The locations of thetwo guide RNAs for the CRISPR/Cas9 system are indicated by thick lines. (b) Immunoblot analysis of DICER1 in DICER1-deficient H1299 cell lines.
(c) Analysis of the dependence of ghR-34a on DICER1. Inhibitory activity of ghR-34a was measured by real-time polymerase chain reaction (PCR) in
DICER1-deficient H1299 lung cancer cell lines. At 48 hours post-transfection, the expression levels of AXL were measured by real-time PCR. Data areexpressed as means ± SD (n = 3); NS, not significant compared with control. (d) Functional PIWI domain of Human AGO2 protein. The locations of
two guide RNAs for CRISPR/Cas9 system are indicated by thick line. (e) Immunoblot analysis of AGO2 in AGO2-deficient H1299 cells. (f) Analysisof the dependence of ghR-34a on AGO2. Inhibitory activity of ghR-34a was measured by real-time PCR in AGO2-deficient H1299 lung cancer cells.
miR-451-form miR-34a used as a AGO2 dependent control. At 48 hours post-transfection, the expression levels of AXL were measured by real-time
PCR. Data are expressed as means ± SD (n = 3); NS=not significant; **P < 0.01 compared with control. All data are representative of at least two
independent experiments.

(Figure 4a,b). ghR-34a and ghR-control significantly decreased
IL-6 and TNFα production. To confirm these in vitro results, we
administered ghR into mice by intravitreal injection, isolated theeyeball, and measured IL-6 and TNFα expression by real-time
PCR (Figure 4c). As expected, ghRNA-34a decreased inductionof IL-6 and TNFα production in vivo.
Because the loading of miRNA onto AGO2 requires a double-stranded structure, normal mimic miRNA consists of two
strands, a guide chain and a passenger chain. In many cases,
the use of such mimic miRNAs is associated with gene repression mediated by the passenger chain, a potentially importantside effect.37 By contrast, ghR is a single-stranded structure that
can adopt a hairpin structure without requiring the passenger
chain seed sequence (Figure 4d). We predicted that this featureof ghR would eliminate unwanted gene repression by the passenger chain. To test this hypothesis, we identified 14 genes targetedby miR-34a* (the passenger chain of miR-34a) using microarraydata from miR-34a-transfected cells and the miRDB miRNAdatabase (mirdb.org/miRDB/) (Supplementary Figure S10).
ghR-34a-transfected cells exhibited no downregulation of these
14 genes (Figure 4e). Thus, ghR avoids two major side effects ofexisting short RNA reagents: immunogenicity and unwanted gene
repression by the passenger chain.

Tumor inhibitory effect of ghR-34a in lung cancer
mouse model
To assess the in vivo effect of ghR-34a, we used the KRASG12D
mouse lung cancer model.38 These mice, which uniformly develop
lung cancer due to activation of KRAS, are considered to be a suitable model for human lung cancer. Furthermore, because lungcancer in KRASG12D mice can be inhibited by administration of
miR-34a, this model is also suitable for evaluation of the in vivo
activity of miR-34a.14,15 For RNA drug delivery, we used chitosan,
an well-studied drug-delivery system for nucleic acid.39 We mixed1282

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Development of Novel Short Hairpin RNAabControl

0

34a

34a

44 nt

200

150

100

1.5

1.0
ghR-34a

0.5

50

30 nt
0

0
Non
treated

miRNA

ghRNA

Non
treatedmiRNA

Expression ratio of miR-34a* target 14 genes (%)

miR-34a2.0
Relative expression

Relative expression

250

miRNA

ghRNA

miRNA

ghRNA
34a

140

*

2.5

Control

e

n.s.
300

Mock

ghRNA

miRNA

Control

d

TNF-α

IL-6

0

n.d.

n.d. n.d.

Nontreated

34a

1,000

500

ghRNA

ghRNA

miRNA

miRNA

ghRNA

Mock

c

Control

0

1,500

20

miRNA

0.25

miRNA

0.50

0.25

n.d.

40

0.75

0.50

TNF-α (pg/ml)

1.00

60

ghRNA

0.75

ghRNA

1.00

1.25

Mock

1.25

**

2,000

80

1.50

Nontreated

1.50

100

Mock

1.75

**

2,500

*

Nontreated

1.75

120

IL-6 (pg/ml)

2.00
Relative expression

2.25

2.00

NontreatedRelative expression

2.25

0

TNF-α

2.50

miRNA

IL-6

2.50

120
100
80
60
40
20
0

ghRNA

miR-34aghR-34agf
ghR control

Number of nodule

Lung weight (g)

**

0.25

*

16
14

0.2

12
10

0.15
ghR-34a

8
0.1

6
4

0.05

2
0

0
ghR
control

ghR-34aghR
control

ghR-34aFigure 4 Evaluation of short mimic miRNA in vivo and in vitro. (a) Expression levels of IL-6 and TNFα in human monocytes were measured by realtime polymerase chain reaction (PCR) and normalized against β-actin at 12 hours post-transfection. ND indicates not detectable. Data are expressed
as means ± SD (n = 3). (b) Expression levels of IL-6 and TNFα in human monocytes were measured by ELISA at 12 hours post-transfection. Data
are expressed as means ± SD. n = 3; *P < 0.05, **P < 0.01. (c) Expression levels of IL-6 and TNFα were measured by real-time PCR 12 hours after
intravitreal injection. ND indicates not detectable. Data are expressed as means ± SE (n = 5); NS, not significant; *P < 0.05 compared with control.
(d) Secondary structures of miR-34a and ghR-34a. Highlighted region indicates seed sequence of passenger strand. (e) Fourteen miR-34a* target
genes were identified by microarray analysis. Expression level in the nontreated control was defined as 100%. (f) Representative HE staining of lungof KRASG12D mice treated with ghR-34a/chitosan particles. KRASG12D mice were administered with 1 mg/kg ghR-34a/chitosan particles seven times over
3 weeks. (g) GhR-34a-treated KRASG12D mice were analyzed for lung tumor development. Lung weight and number of nodules are indicated. Data
are expressed as means ± SE (n = 7); *P < 0.05, **P < 0.01, Student’s t-test.

1283

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Development of Novel Short Hairpin RNAegh-let-7a
G17/P13 30nt

G18/P14 32nt

HMGA2

b
1.6

KRAS
1.4

1.2
1.0
0.8
0.6
0.4

0.4

RLU (×106)

1.2

0.8

1.6

1.4

1.4

1.2

1.2

1.0

**

0.8

**

**

**

0
Nontreated

0.2

G19/P70.2

0G19/P90.2
G19/P15

0.4

Control

0.4

Mock

0.4

Nontreated

**

n.s.

0.8
0.6

Control miR-29b miR-29b
G17/P13
47nt
30nt

26nt

1.0

0.6

Mock

G19/P9A549 Luc

1.6

0.6

0

Control

G19/P728nt

H1299 Luc

n.s.

1.0

G19/P9

34nt

h
**

Mock

G19/P7

G19/P15

TGFB3

Non
treatedLuciferase ghRNA

30nt

**

0

let-7alet-7alet-7a
G18/P14 G17/P13 G16/P12
32nt
30nt
28nt

G19/P9let-7ag

G17/P13

Relative expression

0.6

G19/P15

Control

ghR-29b1.4

0.8

Control

Mock

43nt

d

1.0

0.2

0.2

c

**

1.2
Relative expression

Relative expression

1.4

0

28nt

f

*

*

KRAS ghRNA
G19/P9

G16/P12 28nt

Mock

a

Figure 5 Guide hairpin methods can be applied to other miRNAs and siRNAs. (a,c) Secondary structures and free energies of synthetic shortened
hsa-let-7a and hsa-miR-29b were predicted using the CentroidFold algorithm. (b,d) Comparison of inhibitory activity between wild-type and deleted
forms of hsa-let-7a and hsa-miR-29b in the A549 lung cancer cell line. At 48 hours post-transfection, expression levels of HMGA2 (a target of let-7a)
and TGFB3 (a target of miR-29b) were measured. Data are expressed as means ± SD (n = 3); NS, not significant, *P < 0.05, **P < 0.01 compared with
control. (e,g) Secondary structures of synthetic ghR-KRAS and ghR-luciferase were predicted using the Centroid algorithm. (f) Inhibitory activity ofsynthetic ghR-KRAS in H1299 cells. At 48 hours post-transfection, the level of KRAS mRNA was measured by real-time PCR and normalized against
β-actin. Data are expressed as means ± SD (n = 3); **P < 0.01. (h) Inhibitory activity of synthetic ghR-luciferase in H1299 and A549 cells stably
expressing firefly luciferase. At 72 hours post-transfection, expression was measured by luciferase assay. Relative light units (RLU) were normalizedagainst protein concentration. Data are expressed as means ± SD (n = 3); NS, not significant, *P < 0.05, **P < 0.01 compared with control. All data
are representative of at least three independent experiments.

chitosan and ghR-34a at N/P (Chitosan nitrogen to DNA phosphate) ratio of 10, yielding chitosan/nucleic acid particles with amean diameter of about 200 nm (Supplementary Figure S11).

We performed intratracheal administration of ghR-34a/chitosan
particles (0.5 mg/kg) using a Microsprayer every 4 days, a totalof seven times. In mice that received ghR-34a/chitosan particles,1284

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Development of Novel Short Hairpin RNA

a

-3′

5′-

miRNA

3′- 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 -5′

Seed

Seed

Guide (antisense)5′- 1 2 3 4 5 6 7 8 9 10 11 12 13 14

ghRNA

b

Passenger (sense)

17 16 153′-

Overhang

Target mRNA
-3′5′3′- 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 -5′

Hairpin5′- 1 2 3 4 5 6 7 8 9 10 11 12 13

ghRNA

19 18 17 16 15 14

3′-

Figure 6 Methods for designing of ghRNA from miRNA or target mRNA sequence. (a) To design miRNA-type ghRNA, the sequence of a selectedmature miRNA was applied to the mature sequence of pre-miR-451 (5’ side of stem + 4 or 6-nt loops). The passenger sequence was filled in with asequence complementary to the 5’ side of the stem, included mismatched nucleotides at the +1 and +6 position, which slightly enhance ghR function(Supplementary Figure S9). Next, the miR-451-form miRNA was deleted by one base in both directions from the connection point of the guideand passenger sequences. In many cases, the shortest effective length was about 30 nucleotides. (b) To design siRNA-type ghRNA, a ~19-nt sequencethat formed a hairpin structure at the 3’ side was identified in the target mRNA sequence. The 3’ side of the passenger sequence was filled in with asequence complementary to the 5’ side of stem. In many cases, the shortest effective length was about 26 nucleotides.

we observed a decrease in pulmonary weight due to reduction intumor size, and a meaningful decrease in the number of pulmonary surface tumors (Figure 4f,g).

mRNA, inhibited the expression of luciferase at the protein level(Figure 5g,h). These data suggest that a ghRNA can be designed
based on any mRNA.

Application to other miRNA and siRNA of shortening
technology
Next, we investigated whether this shortening method could be
generalized to other miRNAs. To this end, we designed ghRNAsof various lengths based on the sequences of let-7a and miR-29b(Figure 5a,c). The target gene inhibitory activity of 32-nt ghRlet-7a was equivalent to that of wild-type let-7a (Figure 5b).
Likewise, the inhibitory activity of 30-nt ghR-29b was equivalent
to that of wild-type miR-29b (Figure 5d). Although further analysis is necessary, these results demonstrate that our guide hairpin
shortening method is applicable to other miRNA sequences.
If a technique for shortening miRNA could be applied to siRNAs, it would be useful to many researchers. Therefore, we next
investigated whether we could design ghRNAs based on siRNAs,
based on our concept of miRNA shortening. In order to makehairpin structure, we chose the sequence that spontaneously
form a hairpin structure and assumed it ghRNA. For this purpose, we designed a ghRNA targeting protein-coding region of
KRAS mRNA, a strong driver gene of lung cancer (Figure 5e).
We then examined the knockdown activity of ghR-KRAS in A549
cells, which express an activated mutant form of KRAS. We foundthat ghR-KRAS was able to control expression of KRAS mRNA
(Figure 5f). In addition, we confirmed the knockdown ability ofghRNA using H1299 cells stably expressing the luciferase gene.
ghR-Luciferase, targeting protein-coding region of luciferase

DISCUSSION

Since the discovery of RNAi and its therapeutic potential in mammalian species, the toxicity of exogenous nucleotides and theinflammatory responses against them have attracted increasing
attention. Scientists have investigated different ways to overcome
adverse reactions to siRNAs. One approach is to chemically modify the guide strand capable of silencing the target mRNA, after
separating it from the passenger strand. However, such singlestranded siRNA or miRNA molecules are less biologically active
than the corresponding siRNA or miRNA duplexes.40–42 Ge et al.
synthesized short (approximately 40-nt) shRNAs with 19-bp or
shorter stems (sshRNAs).28 These sshRNA molecules induced
weaker inflammatory reactions, and development of sshRNAs fortreatment of hepatitis C virus infection is currently underway.43
Our study showed that 30 nt is the minimum length for nonmodified RNAs with potent interference potential. Several methods
could be used to make ghRNA more RNase-tolerant, including
fluoro-modification, methoxy-modification, and introduction of
deoxyribonucleotides at specific locations in the sequence.
Nucleic acids that imitate the shape of miR-451 have been wellcharacterized as a novel AGO2-dependent RNAi platform.30,44–46
The Slicer activity of AGO2 cleaves the 42-nt miR-451, reducing its length of 30-nt. After that, the 30-nt miR-451 is further
shortened to 23-nt by PARN; however, this process is unnecessary
for subsequent acquisition of inhibitory activity.35 Because the1285

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Development of Novel Short Hairpin RNA

structure of ghRNA was similar to that of miR-451, we anticipatedthat the activity of ghRNA would depend on processing by AGO2;
contrary to this hypothesis, however, ghRNA exerted inhibitory
activity in an AGO2-independent manner (Figure 3f). To explain
this observation, we postulate that the 30-nt ghRNA is too short
to serve as a substrate for the Slicer activity of AGO2. In addition, we do not yet know whether the 30-nt ghRNA is shortened
further from the 3’ end by PARN. Further study is required toelucidate the detailed molecular mechanisms of ghRNA processing. The processing and the suppression mechanisms of ghRNAshould be separately. AGO2-IP results indicated that ghR-34a
binds to the target mRNAs with AGO2 complex. However, ghR34a can inhibits their target genes in AGO2 deficient cell. To
make AGO2 deficient cell, the guide RNA of CRISPR/Cas9 system were designed at upstream of the MID domain of AGO2. The
sequence data of cloned AGO2 deficient cells indicated that the
MID domain, contains a binding pocket for 5’ phosphate of theguide strand, and the PIWI domain, contains a slicer domain, arecompletely deleted by frame-shift mutations (not shown data).
The functional defects of AGO2 were confirmed by siRNA activity (Supplementary Figure S7). These results suggested that ghR34a interacts with the PAZ and MID domains of AGO2 for RISC
loading, but does not require the catalytic PIWI (slicer) domain
for processing ghR-34a. Because AGO1, AGO3, and AGO4 have
PAZ, MID and PIWI domains similar to AGO2 but lack the slicer
activity, it is possible that ghR-34a can bind and act with all AGO
family similar to endogenous miRNAs.
shRNA can be divided into two types. Guide sequence thatlocated in 5’ side of loop sequence is called left-hand type or
L-type and located in 3’ side of loop sequence is called righthand type or R-type. The activity of L-type shRNA is higher
with a short loop, unlike the R-type which requires a long loop
for high activity.26,28 The form of ghRNA is an L-type, consistent with the results that the activity is higher with a short loop
(Supplementary Figure S3).
To make hairpin-form ghRNA from miRNA, we replaced
the 3’ side of miRNA guide sequence to take a hairpin structure.
The 3’ side of miRNA is dispensable for the recognition of thetarget mRNA (Figure 6a). On one hand, it is possible to makeghRNA from an existing siRNA by applying this method. Because
sequences other than the seed play a role in recognition of thetarget mRNA in siRNA, the knockdown activity of ghRNA-formsiRNA is limited. Knockdown effect on mRNA with nonperfect complementary sequence by siRNA can be regarded as themiRNA-like off-target effects.47,48 Therefore the knockdown
activity of ghRNA-form siRNA considered corresponding to themiRNA-like off-target effects. Accordingly, to increase the knockdown activity of ghRNA-form siRNA, we tested hairpin structurewith a sequence that was perfectly complementary to the target
mRNA. We then investigated whether it would be possible to
make a ghRNA by selecting a hairpin structure-forming sequence
from the target mRNA sequence (Figure 6b). In this approach, 19
perfectly complementary bases are maintained for target recognition in the ghRNA. Using these methods, we designed a ghRNA
against KRAS and luciferase gene by the methods and investigatedtheir knockdown activity (Figure 5f,h). The results suggested thata ghRNA can be designed for any mRNA. However, because thedesign technology has already been established for siRNA, but not
ghRNA, the suppression effect was weaker than that of normal
siRNA. In the future, we believe that it will be possible to design
highly active ghRNA for any mRNA.
A recent report described the development of immunestimulatory siRNAs that take advantage of the immunoreaction
induced by nucleic acids.49 In that study, the authors designed thestructure of the nucleic acid, including sequence and chemical
modifications, to be easily recognized by TLR. This new strategy
for nucleic acid drugs is intended primarily for use against infectious disease. In this study, we found that ghRNA did not induce
TNFα, whereas miRNA did (Figure 4a–c). Because TNFα canregulate tumor growth, induction of TNFα could be exploited
to control tumors. However, most cytokines induces side effects,
such as cachexia. Therefore, cytokines (including TNFα) are notgenerally used in the treatment of cancer. Therefore, preventing
induction the natural immunity is indispensable for clinical application of nucleic acid medicine.
Short oligonucleotides provide opportunities to investigate
hitherto unknown biological mechanisms that open the door
for new medicines. In addition to avoiding induction of naturalimmunity and untoward gene repression by the passenger fragment, short oligonucleotides such as ghR-34a may have additional
advantages. Because shorter molecules are generally transported
into cells at higher rates, they may be more membrane-permeable
than its naturally occurring counterpart. In addition, smallermolecules may infiltrate lesion tissue more readily because theymigrate more easily through the extracellular space. Before
nucleic acid drugs can be widely used in clinical settings, however,numerous problems remain to be solved: for example, it is important to increase their in vivo stability and develop appropriate
drug-delivery system. Nucleic acid drugs have many advantages
over conventional therapeutics, warranting further research anddevelopment efforts. We anticipate that the results of our study
will contribute to clinical application of nucleic acid drugs.

MATERIALS AND METHODS

Cell lines and transfection of synthetic oligonucleotide. Human non–smallcell lung cancer cell lines (NCI-H1299, Calu-3, A549) were purchasedfrom American Type Culture Collection (Manassas, VA) and cultured at
37 °C in the supplier’s recommended media supplemented with 10% fetal
calf serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). All
oligonucleotides were purchased from GeneDesign (Osaka, Japan) andtransfected into cells using the Lipofectamine RNAiMAX transfection
reagent (Life Technologies, Gaithersburg, MD). All hairpin RNA were
heated to 95 °C for 4 minutes and snap-cooled in an ice-water bath to eliminate dimers.28 All experiments, unless otherwise stated, were performed
with a final RNA concentration of 25 nmol/l. The sequences of all synthetic
RNA oligonucleotides in this study are show in Supplementary Table S1.
Real-time PCR for mRNA and miRNA. All samples were tested in triplicate. Total RNA from cell lines was isolated using ISOGEN (Nippon
Gene, Tokyo, Japan), and then reverse transcribed using M-MLV
reverse transcriptase (Life Technologies). Quantitation of mRNA levels was performed using the FastStart Universal SYBR Green Master(Roche Diagnostics GmbH, Mannheim, Germany). β-Actin mRNAwas used as an internal control. The primer sequences were as1286

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Development of Novel Short Hairpin RNA

follows: AXL, 5′-CTCAACCAGGACGACTCCAT-3′ and 5′-AGACCGC
TTCACTCAGGAAA-3′; MET, 5′-CAGGCAGTGCAGCATGTAGT-3′ and5′-TGTCCAACAAAGTCCCATGA-3′; CDK6, 5′-AAGTTCCAGAGCCTGGAGTG-3′ and 5′-CGATGCACTACTCGGTGTGA-3′; E2F3, 5′-ACCAAGACCACAATGGGAAT-3′ and 5′-GAGGCCAGAGGAGAAAGGTT-3′;
CCND1, 5′-CCCTCGGTGTCCTACTTCAA-3′ and 5′-AAGACCTCCTCC
TCGCACTT-3′; SIRT1, 5′-CAGTGTCATGGTTCCTTTGC-3′ and 5′-GC
ACCTAGGACATCGAGGAA-3′; TGFb3, 5′-CGTGAGTGGCTGTTGAG
AAG-3′ and 5′-GGATATCTCCATTGGGCTGA-3′; HMGA2, 5′-GAAG
CCACTGGAGAAAAACG-3′ and 5′-CTTCGGCAGACTCTTGTGAG-3′;
KRAS, 5′-GCAAGAGTGCCTTGACGATA-3′ and 5′-TTGACCTGCTGT
GTCGAGAA-3′; and β-Actin, 5′-ACTCTTCCAGCCTTCCTTCC-3′ and5′-TGTTGGCGTACAGGTCTTTG-3′. Quantitation of miRNA levels
was performed using TaqMan miRNA assays (Life Technologies). RNU6B
RNA was used as an internal control. Quantitative PCR reactions wererun on a thermocycler (MX3000P; Stratagene, La Jolla, CA) and analyzed
using MxPro (Stratagene). Relative expression of target genes was determined by the 2-ΔΔCt method.50
Western blot analysis. Cell lines were lysed in radio-immunoprecipitation

assay buffer. After boiling with sodium dodecyl sulfate loading buffer, equal amounts (10 μg) of the proteins were electrophoresed on
10% sodium dodecyl sulfate–polyacrylamide gels and transferred to
Immobilon membranes (Millipore, Bedford, MA) by semidry blotting.
5% milk powder dissolved in phosphate-buffered saline buffer containing 0.1% Tween-20 was used to block unspecific binding. Then, using
standard techniques, the membranes were probed with the following
antibodies: anti-Axl (C89E7) (Cell Signaling Technology, Beverly, MA),
anti-Met (D1C2) (Cell Signaling Technology), anti-Cdk6 (C-21) (Santa
Cruz Biotechnology, Dallas, TX), anti-DICER1 (4A6; ab82539) (Abcam,
Cambridge, UK), anti-AGO2/eif2C2 (ab32381) (Abcam) and antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (6C5) (Santa
Cruz Biotechnology). Labeling was visualized using Immobilon Westernchemiluminescent horseradish peroxidase substrate (Millipore), and signals were recorded on an LAS-3000 mini system (Fujifilm, Tokyo, Japan).

Generation of DICER1 and AGO1/AGO2 knockout cell lines. The bicis-

tronic Cas9/sgRNA expression vector pSpCas9(BB)-2A-Puro (PX459)
was a gift from Feng Zhang (Addgene plasmid # 48139).51 Two custom
sgRNAs upstream of the functional domain of each gene were designed
in silico using the CRISPR Design Tool (http://tool.genome-engineering.
org). The sequences of the sgRNA guide sequence oligos were as follows: DICER1 #1, 5′-CACCGATCTGCTGAAACTTCAACGTGG-3′ and5′-AAACCCACGTTGAAGTTTCAGCAGATC-3′; DICER1 #2, 5′-CACC
GCAGGAGAGTACATTCATCGCTGG-3′ and 5′-AAACCCAGCGATGAATGTACTCTCCTGC-3′; AGO2#1, 5′-CACCGATCCATACGTCCGTGA
ATT-3′ and 5′-AAACAATTCACGGACGTATGGATCA-3′; AGO2#2,
5′-CACCGATTCCAAATTCACGGACGTA-3′ and 5′-AAACTACGTCCGTGAATTTGGAATC-3′. sgRNA guide sequence oligos were inserted
into plasmid PX459, and the resultant plasmids were transfected into
H1299 cells using the Lipofectamine 3000 transfection reagent (LifeTechnologies). Transiently expressing cells were selected with puromycin
for 48 hours. Single clones were isolated, and frameshift mutations in target regions were confirmed by direct sequencing.
Analysis of Argonaute 2-associated mRNA and ghRNA. H1299 cells weretransfected with synthetic miR-34a or ghR-34a (with two dTTP overhang
at the 3’end) in 10-cm dishes. Coimmunoprecipitation experiments were
performed using microRNA isolation kit (human Ago2) (Wako Pure
Chemical Industries, Tokyo, Japan). The RNA was isolated from precipitates using ISOGEN reagent according to the manufacturer’s instructions.
Ago2-associated RNAs were detected using real-time PCR as describedabove. Ago2-associated ghR-34a was detected by Synthetic siRNAquantitation core kit (TAKARA Bio Inc. Shiga, Japan) according to the

manufacturer’s instructions. The sequences of ghR-34a detection primer
was as follows: 5′-TGGCAGTGTCTTAGCTGAGAC-3′.
Luciferase reporter system to analysis of RNA interference activity ofghRNA. To generate ghR-34a reporter plasmids which express the firefly

luciferase mRNA with a AXL 3’UTR, pDNA3.1-zeo was modified as follows: The firefly luciferase gene from the pGL3-Basic plasmid (Promega,
Madison, WI) was inserted into the NheI and XbaI site of pDNA3.1-zeo.
Luciferase expression construct was generated by cloning of the 3’UTRs
of AXL into the pDNA3.1-luciferase vector described above. The 3’UTRs
were cloned via PCR amplification from cDNA libraries and ligated into thecorresponding XhoI and NotI sites of pDNA3.1-zeo. The primer sequences
are: AXL 3’UTR, 5′-CTCGAGACAACCCTCCACCTGGTACT, 5′-GCGG
CCGCCTCGTTGATATACAAAGTTT. H1299 cells were transfected withthe reporter plasmid using the Lipofectamine 3000 transfection reagent
and selected with 200 μg/ml zeocin. Luciferase assays were performed 48
hours after transfection of miRNA or ghRNA using the luciferase assay
system (Promega). Firefly luciferase activity was normalized to protein
concentration of each sample.
Preparation of ghRNA/Chitosan particles and intratracheal administration to KrasG12D/+ mice. Low–molecular weight chitosan (Sigma-Aldrich,

St. Louis, MO) was dissolved overnight in 0.5% (w/v) hydrochloric acid ata final concentration of 5 mg/ml. The nucleic acid solution (10 mg/ml) was
added to diluted and filtered chitosan solution (N/P = 10), and incubatedfor 30 minutes.52 KrasG12D/+ mice (strain name: 129S/Sv- Krastm3Tyj/J) wereobtained from Jackson Laboratories. ghR-34a/chitosan particles (0.5 mg/kg) were administered intratracheally to 6-week-old KrasG12D/+ mice using
a MicroSprayer (Penn-Century, Philadelphia, PA) every 4 days, a total ofseven times. The mice were sacrificed after about 1 month, and lung weightand number of nodules were analyzed. All experiments were approved by
the Committee for Animal Research at our institution.
DNA microarray analysis. Labeled probes for Affymetrix DNA microar-

ray analysis were prepared according to the manufacturer’s instructions.
Expression profiling experiments used Human Genome U133 Plus 2.0
GeneChips (Affymetrix, Santa Clara, CA). DNA chips were scanned on an
Affymetrix GeneChip scanner, and the signals were processed to generate
standard RMA measures of expression. The data were analyzed using theAffymetrix GeneChip Command Console and the Affymetrix Expression
Console software. MiR-34a-targeted mRNAs regulated by mRNA degradation, rather than translational inhibition, were identified using the microarray data. Furthermore, target genes of miR-34a* were predicted using
the miRDB algorithm (http://mirdb.org/miRDB/). Expression of 14 miR34a* target genes were identified using microarray data from ghR-34 transfectants. The microarray data has been deposited in the Gene Expression
Omnibus database maintained by the National Center for Biotechnology
Information (accession no. GSE71423).

Isolation of monocyte from human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells were isolated using Ficoll-Paque
PLUS (GE Healthcare, Waukesha, WI). Enriched monocyte populations wereisolated by the plastic adherence method. Briefly, after 2–3 hours incubation
in a 5% CO2 incubator at 37 °C, cells were washed with warm medium, andthen adherent cells were detached, washed, and analyzed by flow cytometry for the expression of CD14. In most cases, more than 70% of the cells
obtained by this technique were CD14+. The cells were cultured in RPMI1640 medium supplemented with 10% fetal calf serum. All oligonucleotides
were transfected using the Lipofectamine RNAiMAX transfection reagent.
ELISA. Monocytes from human peripheral blood mononuclear cells were
stimulated with miRNA or ghRNA at final concentration of 25 nmol/l.
At 12 hours post-transfection, the supernatant was assayed for IL-6
and TNFα using Human IL-6 Quantikine ELISA Kit (R&D Systems,
Minneapolis, MN) and Human TNF-alpha Quantikine ELISA Kit (R&D
Systems) according to manufacturer’s instructions.

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Development of Novel Short Hairpin RNA

Intravitreal injection. Nucleic acids were dissolved in phosphate-bufferedsaline at a concentration of 100 μmol/l and injected into the vitreous cavity
in a total volume of 1 μl using a 31-gauge Hamilton micro syringe pipette.

Tumor histology. Mouse lung tissues were harvested, fixed in 10% natural-buffered formalin, and paraffin-embedded according to standard
procedures. Sections were stained by hematoxylin and eosin (H&E) andevaluated by digital light microscopy using a Keyence BZ-9000 and the
BZ-II imaging software. Tumor areas were determined using the ImageJsoftware (National Institutes of Health, Bethesda, MD).
Statistical analysis. Differences were statistically evaluated using one-way

analysis of variance followed by Scheffe’s multiple comparison test. P values < 0.05 were defined as statistically significant (*P < 0.05, **P < 0.01).
Unless otherwise stated, all experiments were performed in triplicate.

SUPPLEMENTARY MATERIAL
Figure S1. Shortening of miR-451-form miR-34a at the 5’ side of thepassenger sequence.
Figure S2. Comparison of inhibitory activity among miR-451-form
miR-34a and deleted forms in Calu-3 cells.
Figure S3. Analysis of inhibitory activity and stability in 4-nt loop
forms.
Figure S4. Comparison of inhibitory activity between wild-type miR34a and ghR-34a at various concentrations in Calu-3 cells.
Figure S5. Inhibition of proliferation by ghR-34a in Calu-3 cells.
Figure S6. Analysis of DICER1-deficient lung cancer cell lines.
Figure S7. Analysis of AGO2-deficient lung cancer cell lines.
Figure S8. Analysis of direct interactions between ghR-34a and miR34a target genes by Luciferase assay and AGO2 immunoprecipitation
assay.
Figure S9. Effect of mismatch and length of overhang on inhibitory
activity of ghR-34a.
Figure S10. Microarray analysis of miR-34a* target genes.
Figure S11. Analysis of ghRNA/chitosan particles and their inhibitory
effect on lung cancer development in model mice.
Table S1. Sequences of the RNA oligonucleotides used in this study.

ACKNOWLEDGMENTS
We thank T. Mizutani for helpful comments on synthesis of nucleic
acids. This research was supported by Grants-in-Aid for scientificresearch (B) (grant number 26293088), (C) (grant number 15K08410)
and a Grant-in-Aid for Exploratory Research from the Japan Society
for the Promotion of Science (grant number 15K15100), and by
the Strategic Research Foundation Grant-aided Project for Private
Universities from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan (MEXT) (grant number S1511011), and by
Tokyo Medical University Grants-in-Aid for research.

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Placenta 45 (2016) 37e41

Contents lists available at ScienceDirect

Placenta
journal homepage: www.elsevier.com/locate/placenta

HLA-G expression is regulated by miR-365 in trophoblasts underhypoxic conditions
Asako Mori a, Hirotaka Nishi a, *, Toru Sasaki a, Yuzo Nagamitsu a, Rie Kawaguchi b,
Aikou Okamoto b, Masahiko Kuroda c, Keiichi Isaka aabcDepartment of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan
Department of Obstetrics and Gynecology, The Jikei University School of Medicine, Tokyo, Japan
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 2 March 2016
Received in revised form
11 July 2016
Accepted 19 July 2016

Introduction: Hypoxia occurs in the ﬁrst trimester of placental development and is implicated in theregulation of trophoblast differentiation. Prolonged hypoxic conditions in the placenta are related to thedevelopment of preeclampsia. MicroRNAs (miRNAs) are noncoding, single-stranded RNAs that modulate
gene expression by targeting messenger RNA. We hypothesized that, under hypoxic conditions, trophoblasts may have a unique miRNA proﬁle that may play a critical role in placental development.
Methods: Total RNA was extracted from human trophoblast, HChEpC1b, exposed to normoxia (20% O2) or
hypoxia (2% O2) for 24 h, and the miRNA expression proﬁles were investigated using a microRNA array.
Several differential miRNAs were selected and validated using real-time reverse transcription PCR. We
identiﬁed potential targets of these miRNAs using in silico analysis. We conﬁrmed a potential target
protein by western blot analysis and luciferase assays.
Results: The expression of miR-365 was signiﬁcantly upregulated under hypoxic conditions. In silico
analysis showed that miR-365 targeted human leukocyte antigen (HLA)-G. Both hypoxic conditions andoverexpression of miR-365 inhibited the expression of HLA-G proteins. The overexpression of miR-365
also decreased the activity of the luciferase reporter containing the 30 -untranslated region (UTR) of
HLA-G with the predicted miR-365-binding site.
Discussion: HLA-G is a non-classical HLA class-Ib molecule that is expressed mainly in extravillous trophoblasts and which plays a key role in maintaining immune tolerance at the maternalefetal interface.
Our results indicate that miR-365 targets the HLA-G 30 UTR to repress its expression. The expression of
miR-365 may play an important role in human placental development and in immunoprotection of thesemiallogenic embryo.
© 2016 Elsevier Ltd. All rights reserved.

Keywords:
miRNA
miR-365
Hypoxia
Placenta
HLA-G

1. Introduction
Hypoxia occurs during the development of placenta in the ﬁrst
trimester and is implicated in trophoblast differentiation [1e4].
Intervillous blood ﬂow increases after 10 weeks of gestation andresults in exposure of trophoblast to oxygen [1]. Before this time,
low oxygen appears to prevent trophoblast differentiation toward
an invasive phenotype [1]. Prolonged hypoxic conditions in theplacenta are known to be a major factor related to the development* Corresponding author. Department of Obstetrics and Gynecology, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan.
E-mail address: nishih@tokyo-med.ac.jp (H. Nishi).
http://dx.doi.org/10.1016/j.placenta.2016.07.004
0143-4004/© 2016 Elsevier Ltd. All rights reserved.

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of preeclampsia, fetal growth restriction (FGR) and miscarriage[1e5]. Especially, preeclampsia is a syndrome characterized by the
new onset of hypertension and proteinuria during pregnancy and is
the leading cause of maternal and fetal morbidity and mortality[1e3]. In normal human pregnancies, trophoblasts inﬁltrate thedecidua during early pregnancy, and the walls of the spiral arteries
are replaced by trophoblasts [6]. The spiral arteries are consequently transformed into large-lumen vessels lined by extravillous
trophoblasts (EVTs), thereby facilitating the circulation in theplacenta. As a result, the maternal spiral arteries are converted fromhigh- to low-resistance vessels that supply nutrients and oxygen to
the fetus. Abnormally shallow EVT invasion impairs spiral artery
remodeling, and the resistance of the spiral arteries remains high,and this may be part of the mechanism by which pre-eclampsia

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A. Mori et al. / Placenta 45 (2016) 37e41

develops. This results in reduced blood ﬂow in the placenta [7e11],
which can lead to hypoxia. Hypoxia affects trophoblast gene
expression, and consequently modulates cell survival, differentiation, metabolic function, and apoptosis [12]. Placental dysfunction
may result from altered gene expression during hypoxia [13,14].
MicroRNAs (miRNAs) are noncoding ribonucleic acids of 20e24
nucleotides that usually negatively regulate gene expression bybinding to sequences in the 30 untranslated region (30 UTR) of thetarget gene's messenger RNA (mRNA) [15e17]. Such binding resultsin either translational obstruction or degradation of the mRNA [18].
To date, more than 2500 different miRNAs have been identiﬁed inhumans, each potentially controlling hundreds of target genes[15,19,20]. miRNAs play a key role in various pathological andphysiological disorders, and there is increasing evidence that they
are important regulators of placental development [18]. miRNAs
are found in human placentas, and abnormal miRNA levels havebeen reported in placentas from pregnancies with complications,such as preeclampsia, FGR and recurrent miscarriage [21e26].
We hypothesized that trophoblasts under hypoxic conditions
have a unique miRNA proﬁle that may play a critical role inplacental development. The aim of this study was to identify themiRNAs in trophoblasts that are affected by hypoxia and to determine their relationship with their target genes.
2. Material and methods

Fig. 1. Effect of hypoxia on miR-365 expression in HChEpC1b and JAR cells. Cells were
cultured for 24 h under normoxic (20% O2) or hypoxic (2% O2) conditions. Total RNA
was prepared and subjected to real-time RT-PCR analysis. Total RNA input was
normalized using miR-16 as an endogenous control. Experiments were carried out in
triplicate.

2.1. Cell lines and culture conditions
Experiments were carried out using HChEpC1b cells and JAR
choriocarcinoma cells. HChEpC1b is a human extravillous trophoblast cell line immortalized by retroviral infection of E6, E7, andhTERT. The cell line was established using primary humantrophoblast from the placenta of a pregnancy that had been electively aborted at the 7th week of gestation. HChEpC1b cells were
cultured in Roswell Park Memorial Institute (RPMI) 1640 medium(Invitrogen, Life Technologies, Carlsbad, CA, USA) containing 10%
fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 mg/ml streptomycin (Invitrogen) and incubated in 5% CO2 at 37 � C. JAR
choriocarcinoma cells were cultured in RPMI1640 containing 10%
FBS and incubated in 5% CO2 at 37 � C. Both cell types were also
cultured under hypoxic conditions using a hermetically enclosed
incubator (ESPEC N2-O2-CO2 BNP-110 incubator, TabaiESPEC, Osaka,
Japan) that was continually ﬂushed with a mixture of 5% CO2, 2% O2,
and 93% N2 for 24 h.
2.2. MicroRNA microarray proﬁling
Total RNA was extracted from HChEpC1b cells under normoxic
and hypoxic conditions using ISOGEN reagent (Nippon Gene,
Tokyo, Japan). Total RNA samples were labeled using a miRCURY
LNA microRNA Power Hy3/Hy5 Labeling Kit (Exiqon, Vedbaek,
Denmark), and miRNA expression proﬁles were assessed with amiRCURY LNA microRNA Array (Exiqon).

reactions were carried out in triplicate in 96-well plates using areal-time PCR System (Applied Biosystems). Fold changes were
determined using the 2eDDCT method [27]. Total RNA input was
normalized using miR-16 as an endogenous control. Using miR-16
provided higher stability and expression in our model comparedwith RNU24 and RNU38B.
2.4. Prediction of miRNA targets
We searched for potential target genes of the identiﬁed miRNAs
using in silico analysis (miRBLAST). We then selected the humanleukocyte antigen (HLA)-G which was the potential target of miR365 and performed western blot analysis.
2.5. Western blot analysis
Equal amounts of protein (20 mg/lane) were separated by SDSPAGE using the NuPAGE System (Invitrogen). Each protein wastransferred to polyvinylidene diﬂuoride membranes (Invitrogen) at
30 V for 60 min. These membranes were blocked using 5% ECL
Prime Blocking Reagent (GE Healthcare UK) in PBS-T overnight at
4 � C. The membranes were then washed in PBS-T, incubated withmouse monoclonal HLA-G antibody (clone MEM-G/1; Santa Cruz
Biotechnology, Santa Cruz, CA, USA) at a 1:1000 dilution overnight,
and reacted with Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (GEHealthcare UK) at a 1:10000 dilution. To verify equal protein

2.3. Real-time reverse transcription PCR analysis
The expression levels of these miRNAs were conﬁrmed using
real-time reverse transcription PCR (RT-PCR). Total RNA wasextracted from HChEpC1b cells under normoxic and hypoxic conditions. RNA purity was then assessed using an Ultrospec 3300 pro(GE Healthcare UK, Little Chalfont, UK). Reverse transcription was
performed with miRNA-speciﬁc stem-loop RT primers and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Life
Technologies, Carlsbad, CA, USA), followed by RT-PCR using speciﬁc
TaqMan Assays and a TaqMan Universal PCR Master Mix. All

Fig. 2. HLA-G protein levels in hypoxic trophoblasts. Equal amounts of trophoblast
proteins (20 mg/lane) were separated and subjected to western blot analysis under
normoxic (N) or hypoxic (H) conditions. To verify equal protein loading, membranes
were then stripped and reprobed with actin antibody.

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39

were mixed in Opti-MEM (Invitrogen) using Lipofectamine 2000
(Invitrogen). Luciferase activity was measured 48 h after transfection using the LightSwitch Luciferase Assay System (Switch Gear
Genomics, Menlo Park, CA, USA). All transfection experiments wereconducted in triplicate.
2.7. Statistical analysis
All values are presented as the mean ± SD of three independent
experiments that were performed in triplicate. Statistical analyses
were performed using the paired t-test, with P < 0.05 considered
statistically signiﬁcant.
3. Results
3.1. miRNA expression proﬁling

Fig. 3. HLA-G mRNA levels in hypoxic trophoblasts. JAR cells were transfected with a
HLA-G 30 UTR luciferase construct using Lipofectamine 2000. Cells were then exposed
to normoxic or hypoxic conditions. Cell lysates were prepared after 48 h and analyzed
using a luciferase assay.

loading, membranes were then stripped and reprobed with actin
antibody (MAB1501R; Millipore, Temecula, CA, USA). Signals weredetected using ECL Prime Western Blotting Detection Reagent (GEHealthcare UK).
2.6. Transfection and luciferase assays

We examined the miRNA expression proﬁles in HChEpC1b cells
that had been cultured for 24 h under normoxic or hypoxic conditions. We identiﬁed nine miRNAs with a >2.5-fold difference in
expression level between normoxic and hypoxic conditions. Of
these miRNAs, miR-149, miR-299-5p, miR-300, miR-365, miR-620,
and miR-3074-3p were upregulated under hypoxia, and miR-720,
miR-1260b, and miR-1280 were downregulated under hypoxia.
Each fold changes were as follows; 2.62-fold (miR-149), 3.75-fold(miR-299-5p), 3.25-fold (miR-300), 3.18-fold (miR-365), 4.24-fold(miR-620), 2.91-fold (miR-3074-3p), 0.44-fold (miR-720), 0.40fold (miR-1260b), and 0.40-fold (miR-1280).
3.2. MiR-365 expression under hypoxic conditions

A luciferase reporter vector containing the entire 30 UTR of HLA-

G (GoClone Reporter Constructs 30 UTR HLA-G) was purchased from
SwitchGear Genomics (Menlo Park, CA, USA). The luciferase assays
were carried out using a standard protocol [28]. JAR cells wereseeded in triplicate into a 96-well plate (2 � 104 cells/well) andwere incubated for 24 h at 37 � C in 5% CO2 to obtain 80% conﬂuence.
For each transfection, 100 ng of the GoClone Reporter Constructs 30
UTR HLA-G (SwitchGear Genomics) and 0e100 nM of miR-365
mimic (miCENTURY OX siMature, Exiqon), miR-365 inhibitor(miRCURY LNA Power Inhibitor), or the negative control for each

To corroborate the microarray results, we performed real-time
RT-PCR. The results conﬁrmed that miR-365 was upregulated in
HChEpC1b cells under hypoxic conditions (Fig. 1). We then performed the same experiment with JAR cells and obtained similar
results (Fig. 1).
3.3. Candidates for miR-365 target genes
We searched for potential target genes of the identiﬁed miRNAs

Fig. 4. Functional validation of HLA-G as a target of miR-365. The luciferase assay was performed using JAR cells. (A) To mimic the effect of overexpression of miR-365, JAR cells weretransfection with miR-365 siMature (25 nM) and an HLA-G 30 UTR luciferase construct (100 ng). Overexpression of miR-365 decreased HLA-G expression. (B) JAR cells weretransfected with miR-365 inhibitor (25 nM) and HLA-G 30 UTR luciferase construct (100 ng). Repression of miR-365 increased HLA-G expression. All experiments were carried out in
triplicate.

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A. Mori et al. / Placenta 45 (2016) 37e41

using in silico analysis. We speciﬁcally focused on miR-365.
Because HLA-G had been found to be a potential target gene ofmiR-365 which we identiﬁed 175 candidate target genes for. The
HLA-G expression is restricted mainly to EVTs [29e31]. HLA-G plays
a key role in maintaining immune tolerance at the maternalefetal
interface and is involved in the pathogenesis of preeclampsia [30].
3.4. HLA-G protein levels under hypoxic conditionsFirst, we investigated the expression of HLA-G in trophoblasts
under hypoxia using western blot analysis. We found that the levels
of HLA-G proteins were clearly downregulated in hypoxic
HChEpC1b and JAR cells (Fig. 2). We then performed a luciferase
reporter assay using the GoClone Reporter Constructs 30 UTR HLAG. The assay showed that the luciferase activity was greatly reduced
under hypoxic conditions (Fig. 3).

pathological and physiological mechanism of preeclampsia. However, there should be a limitation of the study since these results
were derived from choriocarcinoma cell line, not from the EVT cellline, HChEpC1b. Transfection efﬁcacy of HChEpC1b was so inefﬁcient that we had no choice but to use choriocarcinoma cell line,
JAR.
No previous study has investigated the relationship between
miR-365 and trophoblasts. However, some studies have reported a
relationship between miR-365 and some types of cancer, including
lung, colorectal, and skin cancers. Interestingly, in many of thesestudies, miR-365 is reported to inhibit cancer growth [41,42]. Our
study shows that miR-365 regulates HLA-G expression by targetingits 30 UTR in trophoblasts under hypoxic conditions. Because thedecreased expression of HLA-G causes several disorders during
pregnancy, miR-365 expression may play an important role inhuman placental development and immunoprotection of thesemiallogenic embryo.

3.5. Functional validation of HLA-G as a target of miR-365
We performed a luciferase reporter assay to demonstrate thatmiR-365 can target HLA-G. We transfected JAR cells with miR-365
mimic (siMature, miCENTURY OX siMature, Exiqon) and foundreduced luciferase activity from the HLA-G reporter. We then performed the same experiment using a miR-365 inhibitor and foundincreased luciferase activity. These results demonstrate that HLA-G
is a functional target gene for miR-365 (Fig. 4), meaning that the
HLA-G protein level under hypoxic condition is regulated by miR365 through translation interference or mRNA degradation.
4. Discussion
miRNAs have recently been identiﬁed as major determinants ofdiverse cellular functions [15,16,18,32]. Many studies have particularly suggested that miRNAs are important regulators of placental
development and that abnormal miRNA expression is related topregnancy complications such as preeclampsia which is caused byhypoxia in placenta [11,21e23,33,34]. miRNA binds to the 30 UTR ofa target gene's mRNA, destabilizing the target mRNA and inhibiting
protein translation [35]. In the present study, we identiﬁed 175
candidate target genes for miR-365 using in silico analysis. Of thesegenes, we focused on HLA-G, which is expressed especially in EVTs.
HLA-G plays a key role in maintaining immune tolerance at thematernalefetal interface and is involved in the pathogenesis ofpreeclampsia [30]. We are interested in the relationship between
preeclampsia and placental hypoxia. That is the reason why wefocused on miR-365.
HLA-G is a nonclassical HLA-Ib molecule that is characterized by
its expression in EVTs [30,31]. HLA-G has gained much attention
because of its multiple functions in the immune system [36e39].
HLA-G is responsible for inducing maternal immune tolerance,
which contributes to the maintenance of pregnancy [30]. As aresult, this protein plays a very important role in establishing anormal pregnancy. Reduced HLA-G expression in EVTs causes
various pregnancy complications, including preeclampsia which iscorrelated with placental hypoxia [39]. Hypoxia reduced the HLA-G
expression in trophoblasts [40]. HLA-G has been reported toestablish motherechild immune tolerance at the maternalefetal
interface by binding to the inhibitory receptors on natural killer
cells, thereby inhibiting their cytotoxic function [38,39].
In the current study, we found that hypoxia downregulated
HLA-G mRNA levels in trophoblasts and that miR-365 interactedwith the 30 UTR of the HLA-G mRNA. We also found that miR-365
expression was upregulated under hypoxic conditions in trophoblasts. Since miR-365 targets HLA-G to repress its expression, thissuggests that miR-365 may play an important role in theConﬂict of interest
The authors declare that they have no conﬂicts of interest
related to this study.
Acknowledgements
This work was supported by the Private University Strategic
Research Based Support Project (Grant no. S1311016) and Grantsin-Aids from the Ministry of Education, Culture, Sports, Science andTechnology of Japan (MEXT, Grant no. 24592533).
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Biochemical and Biophysical Research Communications 484 (2017) 269e277

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Repression of Smad3 by Stat3 and c-Ski/SnoN induces geﬁtinib
resistance in lung adenocarcinoma
Yojiro Makino a, 1, Jeong-Hwan Yoon b, c, 1, Eunjin Bae b, 1, Mitsuyasu Kato d,
Keiji Miyazawa e, Tatsuo Ohira a, Norihiko Ikeda a, Masahiko Kuroda b,
Mizuko Mamura b, c, f, *a

Department of Surgery, Tokyo Medical University, Tokyo, Japan
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan
Biomedical Research Institute, Kyungpook National University Hospital, Daegu, Republic of KoreadDepartment of Experimental Pathology, Graduate School of Comprehensive Human Sciences and Faculty of Medicine, University of Tsukuba, Ibaraki, Japane
Departments of Biochemistry, University of Yamanashi, Yamanashi, JapanfPhysician, Student and Researcher Support Center, Tokyo Medical University, Tokyo, Japanbca r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 January 2017
Accepted 19 January 2017
Available online 20 January 2017

Cancer-associated inﬂammation develops resistance to the epidermal growth-factor receptor (EGFR)
tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR
mutations. Stat3-mediated interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-b(TGF-b) signaling pathways play crucial regulatory roles in cancer-associated inﬂammation. However,mechanisms how these pathways regulate sensitivity and resistance to EGFR-TKI in NSCLCs remain
largely undetermined. Here we show that signal transducer and activator of transcription (Stat)3 represses Smad3 in synergy with the potent negative regulators of TGF-b signaling, c-Ski and SnoN,
whereby renders geﬁtinib-sensitive HCC827 cells resistant. We found that IL-6 signaling via phosphorylated Stat3 induced geﬁtinib resistance as repressing transcription of Smad3, whereas TGF-b enhanced
geﬁtinib sensitivity as activating transcription of Smad3 in HCC827 cells with geﬁtinib-sensitizing EGFR
mutation. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad2/3/4induced transcription of the Smad3 gene. Smad3 was found to be an apoptosis inducer, which upregulated pro-apoptotic genes such as caspase-3 and downregulated anti-apoptotic genes such as Bcl-2. Our
results suggest that derepression of Smad3 can be a therapeutic strategy to prevent geﬁtinib-resistancein NSCLCs with geﬁtinib-sensitizing EGFR mutation.
© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Cell cycle
Geﬁtinib resistanceLung cancer
Smad3
Stat3
c-Ski/SnoN

1. Introduction
Although EGFR-TKIs have remarkably improved the therapeutic
efﬁcacy for NSCLCs harboring the sensitive EGFR mutation [1], drug
resistance inevitably develops [2]. Genetic changes such as secondary mutations in EGFR (T790M) and ampliﬁcation of the MET
receptor tyrosine kinase are the main mechanisms of acquired drug

* Corresponding author. Department of Molecular Pathology, Tokyo Medical
University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo, 160-8402, Japan; Biomedical
Research Institute, Kyungpook National University Hospital, 50 SAMDUK-2GA,
Jungu, Daegu, 41944, Republic of Korea. Tel.: þ81 3 3351 6141; fax: þ81 3 3351 6173.
E-mailaddresses:
mamumik@tokyo-med.ac.jp,
mikoeyo@gmail.com(M. Mamura).
1
These authors contributed equally to this work.

resistance [3,4]. Unknown mechanisms of EGFR-TKI resistance inthe remaining cases [4] could be attributed to chronic inﬂammation, which plays critical roles in tumorigenesis [5]. It is also
possible that chronic inﬂammatory responses induce DNA damages
and mutations [6].
IL-6 is the pivotal pro-inﬂammatory cytokine in inﬂammation
including cancer-associated inﬂammation [7,8], which affects
treatment effectiveness [9]. IL-6 and EGF share Stat3 for theirsignaling pathways, which promote carcinogenesis throughinﬂammation [10,11]. In NSCLCs, correlation between elevation of
IL-6 and phosphorylation of Stat3 are signiﬁcantly correlated[12,13].
In contrast with IL-6, TGF-b is the principal immunosuppressive
cytokine secreted in the tumor microenvironment [14]. Elevation of
TGF-b is frequently observed in NSCLCs, which is correlated with

http://dx.doi.org/10.1016/j.bbrc.2017.01.093
0006-291X/© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

2.3. Immunocytochemistry
Abbreviations
EGFR
TKIs
NSCLCs
TGF-b
IL-6
STAT

epidermal growth-factor receptor
tyrosine kinase inhibitors
non-small cell lung cancers
transforming growth factor-binterleukin-6
signal transducer and activator of transcription

poor prognosis and metastasis [15]. Cytoplasmic proteins called
Smads mediate the canonical pathway for signal transduction fromthe TGF-b receptor (TbR) serine threonine kinases. Activated TbRI
phosphorylates serine residues in MH2 domains of receptorregulated Smads (R-Smads), Smad2 and Smad3, which oligomerize with the common-mediator Smad, Smad4 to regulatetranscription of the target genes [16,17]. Smad2 and Smad3 arehighly homologous molecules with distinct effects in contextdependent manners, however, detailed mechanisms of involvement of Smad2 versus Smad3 in carcinogenesis remains largely
undetermined [18].
Transmodulation between the STAT and SMAD signaling pathways has been implicated as the basis for the antagonism between
TGF-b and pro-inﬂammatory cytokines such as interferon-g, IL-1b
and TNF-a [19,20]. It has been reported that TGF-b-dependent IL-6
secretion and subsequent Stat3 activation induce EGFR-TKI resistance in NSCLCs [21]. We previously reported that Smad2 andSmad3 oppositely regulate Th17 differentiation as Stat3 cofactors[22]. However, the mechanisms whether and how Stat3-mediatedIL-6 signaling and Smad-mediated TGF-b signaling interact toregulate EGFR-TKI resistance in NSCLCs remain largely unknown.
In this study, we examined the mechanisms how R-Smads andStat3 regulate the sensitivity and resistance to geﬁtinib in HCC827
lung adenocarcinoma cells with geﬁtinib-sensitizing EGFR
mutation.
2. Materials and methods
2.1. Cell culture and transfection
Human lung adenocarcinoma HCC827 cells (ATCC) were
cultured in RPMI 1640 (Hyclone) supplemented with 10% heatinactivated FBS (Gibco) and 1% penicillin/streptomycin (ThermoScientiﬁc) with or without the combinations of human IL-6 (R&D,
10 ng/ml), TGF-b1 (R&D, 5 ng/ml) and geﬁtinib (AstraZeneca, 2 mM)
for 4 h after serum starvation and grown at 37 � C with 5% CO2. No
mutations or copy number variations of TGF-b signaling molecules
are observed in HCC827 cells (http://cancer.sanger.ac.uk/cell_lines/
sample/overview?id¼1240146). HCC827 cells were seeded a day
before transfection and transfected with Smad3 cDNA, Smad3
siRNA, Stat3 siRNA or control siRNA (GE Dharmacon) using Lipofectamine LTX & Plus Reagent (Invitrogen) following manufacture's protocol as previously described [22].
2.2. Cell cycle analysis
DNA content was determined by propidium iodide (PI; BD
Pharmingen). Brieﬂy, HCC827 cells were ﬁxed by cold 80% ethanol
overnight at �20 � C, washed twice with phosphate-buffered saline(PBS) containing 1% bovine serum albumin (BSA), and then resuspended in PI solution in PBS containing RNase A (0.1 mg/ml; SigmaAldrich). Stained samples were acquired by FACSCalibur (BDBioscience) and analyzed by FlowJo (Tree Star).

Proximity ligation assay (PLA) was performed as previously
described [22]. HCC827 cells were ﬁxed with 4% formaldehyde.
Fixed slides were permeabilized by 0.1% Triton X-100 in PBS for
staining with Duolink in situ PLA probes anti-rabbit PLUS and PLA
probe anti-rabbit MINUS or PLA probe anti-mouse PLUS and PLA
probe anti-mouse MINUS and detection reagents (Sigma-Aldrich),rabbit or mouse antibodies against Smad2, Smad3, phospho-Stat3
Y705, phospho-Stat3 S727 (Cell Signaling Technology), Stat3, cSki and SnoN (Santa Cruz). Nucleus was stained with DAPI. Slides
were observed using a confocal microscope, LSM700 (Carl Zeiss).
PLA signals were quantiﬁed using BlobFinder software (http://
www.cb.uu.se/~amin/BlobFinder/).
2.4. RNA isolation and quantitative RT-PCR
Total RNA was extracted from HCC827 cells using Trizol according to the manufacturer's instructions (Invitrogen). RNA wasreverse transcribed with a cDNA RT kit (Invitrogen). Human cDNA
was quantitated by SYBR green (Applied Biosystems) using ABI
7900 (Applied Biosystems). Each experiment was performed intriplicate. The following primers were used: GAPDH 50 - CTCAAGACCTTGGGCTGGG-30 , 50 -TCGAACAGGAGGAGCAGAGA-30 , Smad3
50 -GAGTTGAGGCGAAGTTTGGG-30 , 50 -AGTGAAAGGCAGGATGGACG30 , Bcl2 50 -AACATCGCCCTGTGGATGAC-30 , 50 -CAGGGCCAAACTGAGCAGAG-30 , Bbc3 50 -ACTCACCAAACCAGAGCAGG-30 , 50 -ACAAATCTGGCAGGGGACC-30 , Bnipl 50 -AACCAGCTCCCCAACAACTC-30 , 50 ATGTCTCAGGGCTGCTCCTA-30 , Caspase2 50 -CTTGCTGCCTAAGAGG
GGTC-30 , 50 - CTCACACACCGGAAAAGGGA-30 , Caspase3 50 -CTCTGGT
TTTCGGTGGGTGT-30 , 50 -TCCAGAGTCCATTGATTCGCT-30 , Cidec3 50 50 -CACAGAGGTACGCACTCAGAGCCAGGGGATGAGAAA-30 ,GACA-30
2.5. Luciferase reporter assay
The 2000 bp promoter region of Smad3 was generated by PCRfrom genomic DNA using the primers: 50 -GATCACGCGTTTGGGTTCAAATTCCAGCTC-30 and 50 -GATCCTCGAGGCAGCAGAAGTTTGGGTT
TC-30 . The product was veriﬁed by sequencing and was subcloned
into pGL3 Basic ﬁreﬂy luciferase construct (Promega) using Mlu1
and XhoI sites. The promoter construct was transfected in various
combination with Stat3, Flag-tagged Stat3Y705F, Stat3 S727A(Addgene, submitted by J. Darnell), Flag-tagged Smad2/3/4, Flagtagged c-Ski, SnoN, TGIF or empty pcDNA3 plasmid in
HCC827 cells using Lipofectamine (Invitrogen). Twenty-four hours
after transfection, HCC827 cells were lysed using lysis buffer for themeasurement using luciferase assay kit (Promega) by luminometer.
Each experiment was performed in triplicate.
2.6. Chromatin immunoprecipitation (ChIP)
Chromatin was prepared from HCC827 cells. Immunoprecipitation was performed with antibodies against Smad2, Smad3, trimethyl histone H3 Lys4 and tri-methyl histone H3 Lys27 (Cell
Signaling Technology), Smad4, c-Ski, SnoN and STAT3 (Santa Cruz)using ChIP kit (Cell Signaling Technology) according to the manufacturer's protocol. Immunoprecipitated DNA released from thecross-linked proteins was quantitated by real time PCR using theprimers: �1700 to �1534 50 -AATGCCAAGTAAGGCACAGG-30 50 CTCCCTTCCACTTGCTGCTA-30 , �1559 to �1410 50 -TCTCTAGCAG50 -GCAGCTTGTCAGGGGTTGT-30 ,�1196
CAAGTGGAAGG-30
0
to �1003 5 -TAGCCTGATAGGGAGGCTGA-30 50 -CCGGAGAGGACTCGAGAAGT-30 , �524 to �372 50 -GAGCTTTTCTGAACCCCTCA-30 50 -AC
�220
to
�28
50 CGGACTCCTGGGGACT-30 ,-154-

Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

CTGGGAAGGAGGCTGCAC-30 50 -AAACTTTGCTGGCCTGGAT-30 �107
50 -CGAto
þ63
50 -CAGAGGAGGAGGAGGAGGAG-30
GACTCCAAGTGGCAGTAG-30 and was normalized to input DNA.
Each experiment was performed in triplicate.
3. Results
3.1. Stat3-mediated IL-6 signaling induces geﬁtinib resistance in
HCC827 lung adenocarcinoma cells

271(Fig. 3B, lower), which was epigenetically inactive with trimethylated histone H3 lysine 27 (Fig. 3C, right). PLA showed that TGF-b
inhibited, whereas IL-6 enhanced the interactions between Stat3
and c-Ski/SnoN (Fig. 3D) in accordance with Stat3 phosphorylation
status (Fig. 1B). These results indicate that phosphorylated Stat3
cooperates with c-Ski and SnoN to repress Smad-induced transcription of the Smad3 gene.
3.4. Smad3 induces apoptosis and restores IL-6-induced geﬁtinib
resistance

We used HCC827 lung adenocarcinoma cells with EGFR746E750A deletion to examine the mechanisms how IL-6 and its
downstream Stat3 signaling induce geﬁtinib resistance in thisstudy. TGF-b and geﬁtinib induced apoptosis with synergistic proapoptotic effect, whereas IL-6 suppressed TGF-b/geﬁtinib-induced
apoptosis (Fig. 1A). IL-6 enhanced phosphorylation of Stat3 attyrosine 705 (pY705) and serine 727 (pS727), whereas TGF-b andgeﬁtinib suppressed Stat3 phosphorylation (Fig. 1B). Consistent
with the previous report [23], we conﬁrmed that Stat3 deletion by
RNAi accelerated, whereas Stat3 overexpression inhibited TGF-b/
geﬁtinib-induced apoptosis (Fig. 1C, Supplementary Fig. 1). Thus,
Stat3-mediated IL-6 signaling induced resistance to TGF-b/geﬁtinib
in HCC827 cells.
3.2. Stat3-mediated IL-6 signaling represses Smad3 in HCC827 cells
We next examined the status of TGF-b receptor-regulated Smads(R-Smads), Smad2 and Smad3 in HCC827 cells treated with TGF-b
and geﬁtinib with or without IL-6. We found that IL-6 abolished theexpression of Smad3 protein, whereas Smad2 remained expressed(Fig. 2A). IL-6 completely inhibited the expression of Smad3 mRNA
(Fig. 2B), indicating that IL-6 repressed the transcription of the
Smad3 gene. By contrast, TGF-b and geﬁtinib upregulated Smad3
mRNA (Fig. 2B). Luciferase reporter spanning 2 kilobase upstreamof the ﬁrst exons of the Smad3 genes showed that IL-6 repressed,
whereas TGF-b and geﬁnitib induced the Smad3 gene promoter
activity (Fig. 2C). Canonical TGF-b signaling molecules, R-Smads:
Smad2/3 and common Smad: Smad4 synergistically induced the
Smad3 gene promoter activity (Fig. 2D). By contrast, Stat3 repressed
the Smad3/4-induced Smad3 gene promoter activity (Fig. 2E).
Consistently with the phosphorylation status of Stat3 (Fig. 1B) andthe expression levels of Smad3 (Fig. 2A), Stat3 mutations at Y705 or
S727 completely abolished this repression (Fig. 2E), indicating that
Stat3 phosphorylation at Y705 and S727 is essential for Stat3induced repression of the Smad3 gene. Overexpression of Stat3
signiﬁcantly suppressed, whereas Stat3 knockdown upregulated
the expression of Smad3 protein in HCC827 cells (Fig. 2F). These
results show that Smad-mediated TGF-b signaling induces,whereas Stat3-mediated IL-6 signaling represses the transcriptionof the Smad3 gene.
3.3. c-Ski and SnoN cooperate with Stat3 to repress Smad3
We sought to determine how Stat3 represses the Smad3 gene.
We screened the representative Smad co-repressors: Ski/SnoN and
TGIF [17,24e26] for Stat3 co-repressors, and found that c-Ski and
SnoN, but not TGIF showed synergy with Stat3 to repress the
Smad3/4-induced Smad3 gene promoter activity (Fig. 3A). c-Ski and
SnoN are the potent negative regulators of TGF-b signaling [24e26].
ChIP showed that Smad2/3/4 were bound to the same site in the
Smad3 promoter (�1700 to �1534) upon TGF-b stimulation (Fig. 3B,
middle), which was epigenetically active with methylated histone
H3 lysine 4 (Fig. 3C, left). By contrast, Stat3, c-Ski and SnoN werebound to the Smad3 promoter (�1559 to �1410), overlapped or
proximal to the Smad2/3/4 biding site upon IL-6 stimulation-155-

We examined the physiological relevance of IL-6/Stat3-induced
repression of Smad3 to geﬁtinib resistance by overexpression or
knockdown of Smad3 in HCC827 cells. Forced expression of Smad3
signiﬁcantly induced, whereas knockdown of Smad3 by RNAi
signiﬁcantly inhibited spontaneous and geﬁtinib/TGF-b-induced
apoptosis of HCC827 cells (Fig. 4A). IL-6-induced geﬁtinib resistance was completely abolished by forced expression of Smad3(Fig. 4A, right third row). Smad3 overexpression upregulated proapoptotic genes such as Caspase3, Caspase2 and Cidec (cell deathinducing DNA fragmentation factor-like effector c), while
repressed anti-apoptotic genes such as Bcl2 (b-cell leukemia/lymphoma 2 protein), Bbc3 (BCL2 Binding Component 3) and Bnipl(BCL2 Interacting Protein Like) (Fig. 4B). By contrast, Smad3
knockdown showed the opposing patterns (Fig. 4B). These data
show that Smad3 enhances geﬁtinib/TGF-b-induced apoptosis andrestores IL-6-induced geﬁtinib resistance by upregulating proapoptotic genes and downregulating anti-apoptotic genes in
HCC827 cells.
4. Discussion
Various mechanisms lead to inevitable development of EGFR TKI
resistance including the secondary mutations to a drug-resistantstate, activation of alternative signaling pathways, impairment of
EGFR-TKI-mediated apoptosis and histologic transformation orepithelial-mesenchymal transition (EMT) [27]. Chronic inﬂammatory responses in tumor microenvironment not only induce drug
resistance but also induce DNA damages and mutations [6,21,23].
Thus, it is important to investigate the inﬂammatory mechanisms
to induce EGFR TKI resistance. The representative proinﬂammatory cytokine in cancer-associated inﬂammation, IL-6 isthe potent inducer of EGFR TKI resistance in NSCLCs [23]. TGF-b isessential for erlotinib resistance, EMT and increased activation of
IL-6 axis in resistant H1650 cells [21]. Unlike resistant NSCLCs, TGFb exerted synergistic pro-apoptotic effect with geﬁtinib in case ofhighly EGFR TKI-sensitive HCC827 cells [28], in which both TGF-b
and geﬁtinib suppressed Stat3 phosphorylation at Y705 and S727 in
the absence of IL-6. The most signiﬁcant difference between highly
EGFR TKI-sensitive HCC827 cells and resistant NSCLC cell lines is IL6-induced repression of Smad3 in HCC827 cells. By contrast, Smad3
remained expressed and highly phosphorylated in resistant
H1650 cells with augmented IL-6 secretion [23]. It has been reported that high expression of Smad3 and proﬁlin-2 is linked topoor survival rate in lung cancer patients [29]. Because TGF-b
switches from tumor suppressor at early stages of carcinogenesis to
pro-oncogene at later stages of disease leading to metastasis [16],our ﬁndings suggest that combined anti-inﬂammatory therapies
with EGFR-TKI at early stages may prevent cancer progression.
Although aberrant TGF-b signaling such as the mutations/deletion of Smad4 and the TGF-b type II receptor is frequent incancers, the mutations/deletion of Smad2 and Smad3 are rarely
found [30]. Loss of Smad2 or Smad3 by post-transcriptional modulations has been reported in various cancers [18]. However, precise molecular mechanisms how their expression is selectively

Fig. 1. IL-6 and STAT3 render HCC827 adenocarcinoma cells geﬁtinib-resistant. (A) HCC827 cells were treated with various combinations of geﬁtinib, IL-6 and TGF-b. (B) STAT3
phosphorylated at Y705 or S727 was detected by PLA. Signals were quantiﬁed using BlobFinder software (scale bars: 10 mm, nucleus: black, cytoplasm: white, n ¼ 10 ﬁelds). Data aremean þ s.d. (C) HCC827 cells were transfected with STAT3 cDNA, STAT3 siRNA or control siRNA and treated with various combinations of geﬁtinib, IL-6 and TGF-b. DNA contents
were determined by PI staining using ﬂowcytometry (A, C). Data are representative of three independent experiments.

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Fig. 2. STAT3-mediated IL-6 signaling represses Smad3 in HCC827 adenocarcinoma cells. HCC827 cells were treated with geﬁtinib and TGF-b in the presence or absence of IL-6. (A)Expression of Smad2 and Smad3 proteins in HCC827 cells was determined by PLA. (B) The expression of Smad3 mRNA relative to GAPDH in HCC827 cells was determined byquantitative RT-PCR. (C) HCC827 cells transfected with the Smad3 promoter reporter construct were treated with geﬁtinib and TGF-b in the presence or absence of IL-6. (D)HCC827 cells were transfected with the Smad3 promoter reporter construct with the indicated combinations of Smad2, Smad3 and Smad4. (E) HCC827 cells were transfected with
the Smad3 promoter reporter construct with the indicated combinations of Smad3, Smad4, STAT3, STAT3 mutants: Y705F or S727A. Fireﬂy luciferase activity was measured with aluminometer and normalized to b-galactosidase activity. Each experiment was performed in triplicate. (F) Smad3 protein in HCC827 cells transfected with STAT3 cDNA, STAT3 siRNAor control siRNA was detected by PLA. Signals were quantiﬁed using BlobFinder software (scale bars: 10 mm, nucleus: black, cytoplasm: white, n ¼ 10 ﬁelds). Data are mean þ s.d.
Data are representative of three independent experiments.

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Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

Fig. 3. Phosphorylated STAT3 represses the transcription of the Smad3 gene in synergy with c-Ski and SnoN. (A) HCC827 cells were transfected with the Smad3 promoter reporter
construct with the indicated combinations of Smad3, Smad4, STAT3, c-Ski, SnoN and TGIF. Fireﬂy luciferase activity was measured with a luminometer and normalized to bgalactosidase activity. Bindings of Smad2/3/4, STAT3, c-Ski and SnoN to (B) the Smad3 proximal promoter region and (C) trimethylated histone H3 Lys4 (H3K4me3) and trimethylated histone H3 Lys 27 (H3K27me3) in HCC827 cells were determined by ChIP. Each experiment was performed in triplicate. (D) Protein interactions between STAT3 and c-Ski,
STAT3 and SnoN in HCC827 cells treated with or without TGF-b or IL-6 were detected by PLA. Signals were quantiﬁed using BlobFinder software (scale bars: 10 mm, nucleus: black,cytoplasm: white, n ¼ 10 ﬁelds). Data are mean þ s.d. Data are representative of three independent experiments.

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regulated remain largely unknown. Downregulation of either of RSmads could be one of the mechanisms to distinguish their distinct
functions [18]. Smad3 is downregulated through decreased transcription in lung epithelial cells [31] or through decreased geneactivation as well as increased protein degradation in glomerularmesangial cells [32]. Here, we show that IL-6-Stat3 signaling represses transcription of Smad3, thereby inducing geﬁtinib/TGF-b
resistance in HCC827 cells. Similar and synergistic effect of geﬁtinibwith TGF-b on HCC827 cells might be attributed to induction of
TGF-b by geﬁtinib [33]. Pro-inﬂammatory cytokine signaling
pathways antagonize TGF-b through upregulation of inhibitory
Smad, Smad7 [19,20]. Recent report shows that direct crosstalk275

between Stat3 and Smad3 antagonizes TGF-b [34]. Ski and theclosely related SnoN are oncogenes, which act as transcriptional corepressors in TGF-b signaling through interaction with Smads[24e26]. We show that c-Ski/SnoN cooperate with Stat3 to antagonize TGF-b by repressing Smad2/3/4-induced transcription of the
Smad3 gene. Future studies are required to clarify the relevance ofrepression of Smad3 to the interactions of c-Ski/SnoN, Stat3 andSmads in inﬂammation and carcinogenesis.
Stat3 activated through IL-6/JAK1 promotes cell survival anderlotinib resistance in EGFR-addicted NSCLC cell lines [23]. TGF-b
also plays pivotal roles in cell survival [16e18]. Smad3 sensitizes
hepatocytes to apoptosis through down-regulation of Bcl-2 [35] orFig. 4. Smad3 enhances geﬁtinib-induced apoptosis of HCC827 cells. (A) HCC827 cells were transfected with Smad3 cDNA, Smad3 siRNA or control siRNA and treated with geﬁtinibwith or without TGF-b or IL-6. DNA contents were determined by PI staining using ﬂowcytometry. (B) The expression of Caspase2, Caspase3, Cidec, Bcl2, Bbc3 and Bnipl mRNA
relative to GAPDH in HCC827 cells transfected with Smad3 cDNA, Smad3 siRNA or control siRNA was determined by quantitative RT-PCR. Each experiment was performed intriplicate. Data are mean þ s.d. Data are representative of three independent experiments.

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Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

through cleavage of Bcl-2-associated death promoter (BAD) protein[36]. TGF-b/Smad3 induces apoptosis through inducing and activating caspase-3 [37]. Our ﬁndings that Smad3 signiﬁcantly prevented IL-6-induced geﬁtinib resistance in HCC827 cells as
upregulating caspase3 and downregulating Bcl2 are consistent
with these previous reports. Stat3 also targets similar set of
apoptosis-related genes [38]. Involvement of crosstalk between
Smads and Stat3 in regulation of apoptosis in IL-6-induced geﬁtinib
resistance is yet to be determined.
In summary, present study elucidated the novel mechanism
how IL-6/Stat3 induce geﬁtinib resistance in highly sensitive
HCC827 cells. IL-6 enhanced phosphorylation of Stat3 at Y705 andS727, which interacted with the Ski family of proto-oncoproteins, cSki and SnoN to repress Smad2/3/4-induced transcription of the
Smad3 gene. Repression of Smad3 resulted in resistance to
geﬁtinib-induced apoptosis, whereas forced expression of Smad3
restored IL-6-induced geﬁtinib resistance. Smad3 induced
apoptosis by upregulating pro-apoptotic genes as downregulating
anti-apoptotic genes. Our ﬁndings suggest that derepression of
Smad3 might prevent inﬂammation-induced geﬁtinib-resistance atearly stages of NSCLCs with geﬁtinib-sensitizing EGFR mutation
while they retain sensitivity to tumor suppressor effects of TGF-b.
Declaration of interest
NI receives support from AstraZeneca. Other authors declare nocompeting interests.
Acknowledgements
The work was supported by Basic Science Research Program
through the National Research Foundation of Korea(2015R1A1A3A04001051), the Pioneer Research Center Program
through the National Research Foundation of Korea funded by the
Ministry of Science, ICT and Future Planning (2015-001937 and2015-001923) to MM, Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B03931785) to JHY and NRF(2014R1A1A2057903) to EB, supported in part by Grants-in-Aid forscientiﬁc research (B: 26293088), a Grant-in-Aid for Exploratory
Research (15K15100) from Japan Society for the Promotion of Science (JSPS) Strategic Research Foundation Grant-aided Project forPrivate Universities from the Ministry of Education, Culture, Sports,
Science and Technology of Japan to MK.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2017.01.093.
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Biochemical and Biophysical Research Communications 485 (2017) 209e214

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

PRG4 expression in myxoid liposarcoma maintains tumor cell growththrough suppression of an antitumor cytokine IL-24
Kosuke Oikawa a, b, *, Anna Mizusaki a, Masakatsu Takanashi b, Takashi Ozaki a, c,
Fuyuki Sato a, Masahiko Kuroda b, Yasuteru Muragaki aabc

Department of Pathology, Wakayama Medical University, 811-1 Kimiidera, Wakayama-shi, Wakayama 641-8509, Japan
Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
Department of Clinical Laboratory, Kinan Hospital, 46-70 Shinjo, Tanabe-shi, Wakayama 646-8588, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 7 February 2017
Accepted 9 February 2017
Available online 10 February 2017

PRG4 is one of the downstream molecules of the myxoid liposarcoma (MLS)-speciﬁc fusion oncoproteins
TLS-CHOP and EWS-CHOP. Exogenous PRG4 expression increases the tumorigenicity of cells injected innude mice. The molecular functions of PRG4 in tumorigenesis and/or tumor progression of MLS cells,
however, still remain unclear. In this report, we demonstrated that siRNA-mediated knockdown of PRG4
suppressed the growth of the MLS-derived cell lines 1955/91 and 2645/94. In addition, PRG4 knockdown
promoted adipocytic differentiation in 1955/91 cells. Thus, PRG4 may play essential roles in MLS cell
growth and have potential as a therapeutic target. On the other hand, our previous study has revealed
that TLS-CHOP suppresses expression of an anti-tumor cytokine IL-24, contributing to tumor cell survival. In this study, we found that double knockdown of PRG4 and IL-24 did not inhibit MLS cell growth,and single knockdown of PRG4 remarkably increased IL-24 expression. These results suggest that thegrowth inhibitory effect of PRG4 knockdown is caused by induction of IL-24 expression, and PRG4 maycontribute to maintain MLS cell growth through repression of IL-24 expression.
© 2017 Elsevier Inc. All rights reserved.

Keywords:
Myxoid liposarcoma
TLS-CHOP
FUS-DDIT3
PRG4
IL-24

1. Introduction
All of known human myxoid liposarcomas (MLSs) are associatedwith chromosomal translocations t (12; 16) (q13; p11) and t (12;22) (q13; q12), which generate fusion oncogenes comprising the 50
parts of the translocated in liposarcoma (TLS) gene, also known as
the fused in sarcoma (FUS) gene, in 16p11 and the Ewing's sarcoma(EWS) gene in 22q12, respectively, and the 30 part of the CCAAT/
enhancer binding protein homologous protein (CHOP) gene, also
called the DNA-damage-inducible transcript 3 (DDIT3) gene or thegrowth arrest- and DNA damage-inducible gene 153 (GADD153) gene,in 12q13 [1e3]. The resultant fusion oncogenes TLS-CHOP and EWSCHOP (also known as FUS-DDIT3 and EWS-DDIT3, respectively) areconsidered to encode MLS speciﬁc abnormal transcription factors
and regulate expression of downstream molecules that are
involved in tumorigenesis and/or tumor progression of MLS. Our
previous study showed that the proteoglycan 4 (PRG4) gene, named* Corresponding author. Department of Pathology, Wakayama Medical University, 811-1 Kimiidera, Wakayama-shi, Wakayama 641-8509, Japan.
E-mail address: k-oikawa@wakayama-med.ac.jp (K. Oikawa).
http://dx.doi.org/10.1016/j.bbrc.2017.02.055
0006-291X/© 2017 Elsevier Inc. All rights reserved.

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the downstream of the liposarcoma-associated fusion oncoprotein 54(DOL54) gene in the report, is one of the downstream genes of TLS/
EWS-CHOP [4]. PRG4 is expressed in the malignant component ofhalf of MLS cases [5], and increases the tumorigenicity of cells
injected subcutaneously into nude mice [4]. In addition, PRG4 istransiently expressed early during nomal adipocytic differentiation,suggesting that abnormal constitutive expression of PRG4 may playa role for inhibition of adipocytic differentiation and contribute totumorigenesis and/or tumor progression of MLS [4]. The molecularfunctions of PRG4 in MLS, however, are still not fully understood.
Interleukin-24 (IL-24) was originally described as melanoma
differentiation-associated gene 7 (MDA-7). Expression of IL-24
protein is detected in cells of the immune system and normal human melanocytes, and a progressive loss of IL-24 expression isobserved during melanoma progression, suggesting an inverse
relation between IL-24 expression and the development of melanocytes to various stages of melanoma [6e8]. On the other hand,ectopic expression of IL-24 induces growth arrest and apoptotic cell
death in human malignant cells from diverse origins including MLS,but causes minimal lethality toward normal cells [9e12]. Furthermore, adenoviral vector-mediated delivery of IL-24 was evaluated210

K. Oikawa et al. / Biochemical and Biophysical Research Communications 485 (2017) 209e214

in a phase I clinical trial in patients, and was shown to be safe andclinically efﬁcacious [13]. Thus, IL-24 is expected to be a potential
therapeutic candidate for treatment of various malignant tumors.
We have previously found that knockdown of TLS-CHOP induces asigniﬁcant increase in the expression of IL-24 and cell death in MLSderived cells, but double knockdown of TLS-CHOP and IL-24 does
not suppress the growth of the cells [11]. These results suggest that
TLS-CHOP suppresses IL-24 expression, contributing to MLS cell
growth. Molecular mechanism for suppression of IL-24 expression
by TLS-CHOP, however, remains unclear.
In this report, we investigated a novel function of PRG4 in MLS.
Our results suggest that PRG4 may play a key role for MLS cell
growth and is a candidate therapeutic target for MLS therapy. Inhibition of IL-24 expression by PRG4 may be one of importantmechanisms for MLS cell growth.

2. Materials and methods
2.1. Cell culture and small interfering RNA (siRNA) transfection
The myxoid liposarcoma-derived cell lines 1955/91 and 2645/94
were kindly provided from Prof. David Ron (University of Cambridge), and were grown in Dulbecco's modiﬁed Eagle's medium(DMEM) (Sigma-Aldrich Corporation, St. Louis, MO) containing 10%
fetal bovine serum (FBS).
For siRNA transfection, cells were transfected with 20 nM ofchemically-synthesized siRNAs using Lipofectamine RNAiMAX
Transfection Reagent (Invitrogen, Thermo Fisher Scientiﬁc, Waltham, MA) and Opti-MEM I Reduced Serum Medium (Invitrogen).
The nucleotide sequences of TLS-CHOP, IL-24, and negative control
siRNAs were previously described [11], and PRG4 siRNA sequence isshown in Fig. 1A.
Cell quantiﬁcation was performed as previously described [14].

Fig. 1. Repression of PRG4 expression by TLS-CHOP and PRG4 siRNAs in MLS-derived cell lines suppressed cell growth. (A) Schematic structure of PRG4 mRNA and its encoded
domains. Open box represent the coding region of the PRG4 mRNA. The target sites of the newly designed siRNAs speciﬁc for PRG4 are also shown. SO, Somatomedin B-like domains;
59X8AA repeats, 59X 8AA repeats of K-X-P-X-P-T-T-X; HX, Hemopexin-like repeats. (B) Western blot analysis of TLS-CHOP and PRG4 expression in 1955/91 and 2645/94 cells at 48 h
after siRNA transfection. a-tubulin is shown as a loading control. (C) Representative phase-contrast images of 1955/91 and 2645/94 cells at 48 h after siRNA transfection. (D) Growth
curves of 1955/91 and 2645/94 cells after siRNA transfection. The cells in 12-well culture plates were counted at several time points using a haemocytometer. Bars, SD.

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2.2. Western blot analysis
Protein samples were prepared as previously described [15].
Equal amounts of the protein samples (10 mg/lane) were separated
by SDS-PAGE and transferred to Hybond ECL Nitrocellulose Membranes (Amersham, GE Healthcare UK Limited, Buckinghamshire,UK). Membranes were then probed with speciﬁc antibodies. The
signals were visualized by ECL Prime Western Blotting Detection
Reagent (Amersham) and detected with the Chemiluminescence
CCD Imaging System (AE9300 Ez-Capture MG) (ATTO Corp., Tokyo,
Japan). We have previously generated anti-TLS-CHOP monoclonal
antibody (clone 14) [16]. Monoclonal Anti-a-Tubulin Clone B-5-1-2(T-5168; Sigma-Aldrich) and Anti-PRG4 Polyclonal Antibody(AB2200; Merck Millipore Corporation, Merck KGaA, Darmstadt,
Germany) were purchased.
2.3. Adipogenesis assay
One-day preconﬂuent MLS-derived cells grown in DMEM containing 10% FBS were transfected with TLS-CHOP, PRG4 and negative
control siRNAs respectively at a ﬁnal concentration of 20 nM.
Twenty-four hours after the transfection, the cells were grown to
conﬂuence. Then, the medium was replaced with DMEM containing 10% FBS, 10 mg/ml insulin, 2.5 mM dexamethasone, and 0.5 mM
3-isobutyl-1-methylxanthine, and the cells were incubated for
48 h. After that, the medium was changed and the cells weremaintained in DMEM containing 10% FBS and 10 mg/ml insulin for
48 h. The cells were then washed with PBS three times. Oil Red O
solution (0.5% in isopropanol, Sigma-Aldrich) was diluted 3:2 withwater and ﬁltrated, and the cells were incubated in the diluted Oil
Red O solution for 20 min. Finally, the cells were washed ﬁve times
with 60% isopropanol, and were observed using light microscopy.
2.4. Quantitative real-time PCR analysis
RNA isolation was performed as previously described [17]. First
strand cDNA was synthesized from 1 mg of total cellular RNA with
Random Primers (Invitrogen) and SuperScript III Reverse Transcriptase (Invitrogen). Then, Real-time PCR analysis was performed
using the Thermal Cycler Dice Real Time System Single (Takara Bio
Inc., Shiga, Japan) with iQ SYBR Green Supermix (Bio-Rad

211

Laboratories Inc., Hercules, CA). The nucleotide sequences of thespeciﬁc primers for IL-24 and b-actin were previously described[11,14]. Reaction mixtures were denatured at 95 � C for 3 min andthen were subjected to 45 PCR cycles at 95 � C for 10 s and 60 � C for
30 s. IL-24 mRNA levels were normalized to b-actin signals. The PCR
reactions were performed in triplicate.
2.5. Statistical analysis
For statistical analysis, we used two-tailed Student's t-test. We
considered that P values less than 0.05 were statistically signiﬁcant.
3. Results
3.1. PRG4 knockdown induces growth suppression of MLS-derived
cell lines
To investigate the molecular functions of PRG4 in MLS cells, weﬁrst synthesized three different siRNAs speciﬁc for PRG4, andselected the most powerful siRNA (hereafter called PRG4 siRNA)
among them (Fig. 1A). On the other hand, the TLS-CHOP siRNA had
been previously designed and selected [11]. Western blot analysis
showed that PRG4 is expressed in MLS-derived cell lines, 1955/91
and 2645/94, and both TLS-CHOP and PRG4 siRNAs repressed PRG4
expression in the MLS-derived cells (Fig. 1B). These results are
consistent with the previous report that has identiﬁed PRG4 as oneof the downstream molecules of TLS-CHOP [4]. Furthermore, PRG4siRNA as well as TLS-CHOP siRNA suppressed cell growth andinduced cell death of the MLS cells (Fig. 1C and D). On the other
hand, negative control siRNA did not affect cell growth, indicatingthat the growth suppressive effects were not simply caused by thecytotoxicity of the siRNA transfection reagent or process. Thus,
PRG4 expression may be essential for maintaining growth of the
MLS cells.
3.2. PRG4 knockdown promotes adipocytic differentiation
Previous studies have shown that TLS-CHOP has inhibitory activity on adipogenesis [18,19]. On the other hand, PRG4 is transiently expressed only at the early stage of normal adipocytic
differentiation, providing a notion that the unscheduled induction

Fig. 2. Adipocytic differentiation of 1955/91 cells was promoted by PRG4 knockdown. After transfection of TLS-CHOP and PRG4 siRNAs, 1955/91 and 2645/94 cells were treated withthe reagent for adipocytic differentiation, and then detected lipid droplets by Oil Red O staining (see Materials and methods for detail). Scale bars ¼ 10 mm.

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K. Oikawa et al. / Biochemical and Biophysical Research Communications 485 (2017) 209e214

of PRG4 expression may interfere with the differentiation andcontributes sarcomagenesis of MLS [4]. Thus, we next examined
whether knockdown of TLS-CHOP and PRG4 allows the MLSderived cell lines to differentiate into adipocytes. In the general
protocol for adipogenesis assay, cells are required to become
conﬂuent at the ﬁrst process. Thus, to prevent the cell growth inhibition by TLS-CHOP and PRG4 siRNAs, we performed siRNAtransfection only one day before the cells reached conﬂuence. One
day after transfection, we started to treat the cells with the reagentsfor induction of adipocytic differentiation, and then assessed
accumulation of lipid droplets in the cells by Oil Red O staining. In
1955/91 cells, we observed Oil Red O positive cells in TLS-CHOP and
PRG4 siRNA-transfected cells (Fig. 2A). However, the positive cells
in the PRG4 siRNA-transfected cells were far less than that in the
TLS-CHOP siRNA-transfected cells. Because transient induction of
PRG4 during adipocytic differentiation seems to play a role for
adipogernesis [4], these results may be reasonable. We rarely

observed the positive cells in negative control siRNA-transfected
cells and non-transfected control cells (Fig. 2, upper panels). On
the other hand, we did not observed Oil Red O positive cells at all inthe 2645/94 cells (Fig. 2, lower panels). Thus, we suspect that
intracellular conditions of 1955/91 and 2645/94 cells are different
for adipogenic mechanism.
3.3. Cell growth suppression by PRG4 knockdown is due toinduction of IL-24 expression
Our previous study showed that double knockdown of TLSCHOP and an anti-tumor cytokine IL-24 does not suppress MLS
cell growth, indicating that IL-24 knockdown blocks the growth
suppressive effects by the single knockdown of TLS-CHOP [11].
Therefore, we next examined the effects of double knockdown of
PRG4 and IL-24 in MLS-derived cells, and found that the double
knockdown did not inhibit the growth of the cells (Fig. 3AeC). On

Fig. 3. Growth arrest of MLS cells by PRG4 knockdown is caused by IL-24 expression. (A) Representative phase-contrast images of 1955/91 and 2645/94 cells at 48 h after
transfection with PRG4 siRNA and/or IL-24 siRNA, or negative control siRNA. (B) Cell numbers of 1955/91 and 2645/94 cells at 48 h after siRNA transfection. The cells in 12-wellculture plates were counted at several time points using a haemocytometer. Bars, SD; **P < 0.01; ***P < 0.001. (C) Western blot analysis of PRG4 expression in 1955/91 and
2645/94 cells at 48 h after siRNA transfection. a-tubulin is shown as a loading control. (D) Induction of the IL-24 mRNA expression in 1955/91 and 2645/94 cells by PRG4 siRNA. 48 h
after siRNA transfection, total RNA sample was prepared from the cells and subjected to quantitative real-time PCR analysis. Data were normalized to the mRNA level of non-treatedcells that was arbitrarily set to 1 in the graphical presentation. Bars, SD; *P < 0.05; **P < 0.01; ***P < 0.001.

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the other hand, we have previously shown that single knockdownof IL-24 has no effect on MLS cell growth [11]. These results prompt
us to determine the IL-24 expression levels in the cells. Quantitative
real-time PCR analysis revealed that PRG4 knockdown in MLS cells
induced a signiﬁcant increase in IL-24 expression (Fig. 3D). Thus,
we considered that PRG4 contributes to maintain MLS cell growththrough repression of IL-24 expression.
4. Discussion
Our previous study has revealed that PRG4 increases tumorigenicity of MLS, and has suggested that unscheduled expression of
PRG4 inhibits adipocytic differentiation [4]. In this study, we havefound a novel function of PRG4 that represses IL-24 expression,
contributing to evasion of MLS cell death (Fig. 4A). Thus, PRG4
expression seems to be essential for MLS cells. In our previous
study, however, the PRG4 mRNA expression was detected only inhalf of MLS cases [5]. How do MLS cells survive without PRG4
expression? Although it might be possible that the intracellularconditions of PRG4-expressing MLS cells are different from that of
the MLS cells without PRG4 expression, further studies are required
to solve the question.
Our results also provided new evidence that constitutive
expression of PRG4 inhibits adipogenesis. However, although
repression of PRG4 expression in 1955/91 cells induced generationof lipid droplets, that in 2645/94 cells did not (Fig. 2). 1955/91 and
2645/94 cells carry type 1 and type 2 of TLS-CHOP variants,
respectively (Fig. 1B) [11]. A major structural difference between
type 1 and type 2 variants is presence or absence of one of the RNAbinding domains, RGG box 1 (Fig. 4B). In previous studies, type 1
showed a weak inhibitory activity on adipogenesis of a preadipocyte cell line, 3T3-L1, but type 2 induced a profound inhibitionof adipogenic conversion of a preadipocyte cell line, ST-13 [18,19].

Fig. 4. Representations of PRG4 function and TLS-CHOP variants. (A) Model ofmechanisms involving PRG4 in MLS cells. (B) Schematic structures of type 1 and type 2
variants of TLS-CHOP protein. RGG1, the RNA binding domain RGG box 1.

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Thus, type 2 may have stronger activity to alter the cells to be
difﬁcult to differentiate into adipocytes. Otherwise, it is also
possible that the difference in the differentiation potential between
1955/91 and 2645/94 cells only depends on the variation between
individual established cell lines. In any case, our results support thenotion that PRG4 inhibits adipocytic differentiation.
Previous studies have shown that PRG4 expression is observed
not only in MLS but also in diverse sarcomas such as Low Grade
Fibromyxoid Sarcoma (LGFMS), Malignant Fibrous Histocytoma(MFH), Ewing Tumor (ET), Synovial Sarcoma (SS), and Extraskeletal
Myxoid Chondrosarcoma (EMC) [5,20]. Thus, we suspect that PRG4
may play an important role for growth and survival of various
sarcoma cells. Although additional studies are needed, our results
will contribute to shed light on a novel mechanism involving PRG4
in various sarcoma cells.
Disclosures
No conﬂicts of interest are declared by the authors.
Acknowledgements
This study was supported by Grant-in-Aid for Scientiﬁc Research(C) from the Japan Society for the Promotion of Science (Grant
Number 25460480 to K. Oikawa).
Transparency document
Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.bbrc.2017.02.055.
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miR-125b-1 and miR-378a are predictive biomarkers
for the efficacy of vaccine treatment against
colorectal cancer
Hironori Tanaka,1 Shoichi Hazama,1,2
Michihisa Iida,1 Ryouichi Tsunedomi,1Hiroko Takenouchi,1Masao Nakajima,1 Yukio Tokumitsu,1 Shinsuke Kanekiyo,1 Yoshitaro Shindo,1 Shinobu Tomochika,1
Yoshihiro Tokuhisa,1 Kazuhiko Sakamoto,1 Nobuaki Suzuki,1 Shigeru Takeda,1 Shigeru Yamamoto,1Shigefumi Yoshino,3 Tomio Ueno,1 Yoshihiko Hamamoto,4 Yusuke Fujita,4 Hiroaki Tanaka,5 Ko Tahara,6
Ryoichi Shimizu,7 Kiyotaka Okuno,8 Koji Fujita,9 Masahiko Kuroda,9 Yusuke Nakamura10 and Hiroaki Nagano1
1
Department of Gastroenterological, Breast and Endocrine Surgery, Yamaguchi University Graduate School of Medicine; 2Department of Translational
Research and Developmental Therapeutics against Cancer, Yamaguchi University School of Medicine; 3Oncology Center, Yamaguchi University Hospital;
4
Division of Electrical, Electronic and Information Engineering, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Ube;
5
Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka; 6Department of Surgery, Kure-Kyosai Hospital, Kure;
7
Department of Surgery, Ogori Daiichi General Hospital, Yamaguchi; 8Department of Surgery, Kinki University Faculty of Medicine, Osaka-Sayama;
9
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; 10Department of Medicine and Surgery, The University of Chicago, Chicago,USA

Key words
Biomarker, colorectal cancer, miR-125b-1, miR-378a, peptide vaccine
Correspondence
Hiroaki Nagano, Department of Gastroenterological,
Breast and Endocrine Surgery, Yamaguchi University
Graduate School of Medicine, 1-1-1 Minami-Kogushi, Ubecity, Yamaguchi 755-8505, Japan.
Tel: +81-836-22-2264; Fax: +81-836-22-2263;
E-mail: hnagano@yamaguchi-u.ac.jp
Funding Information
Research program of the Project for Development of
Innovative Research on Cancer Therapeutics (P-DIRECT),(Grant/Award Number: ‘11039020’) The Japan Agency for
Medical Research and Development (AMED), (Grant/Award Number: ‘15cm0106085h0005’) Leading Advanced
Projects for Medical Innovation (LEAP), (Grant/Award
Number: ‘16am0001006h0003’).
Received May 10, 2017; Revised August 24, 2017;
Accepted August 27, 2017
Cancer Sci 108 (2017) 2229–2238

Many clinical trials of peptide vaccines have been conducted. However, these vaccines have provided clinical benefits in only a small fraction of patients. The purpose of the present study was to explore microRNAs (miRNAs) as novel
predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer. First, we carried out microarray analysis of pretreatment cancer tissues in aphase I study, in which peptide vaccines alone were given. Candidate miRNAs
were selected by comparison of the better prognosis group with the poorer
prognosis group. Next, we conducted microarray analysis of cancer tissues in aphase II study, in which peptide vaccines combined with chemotherapy were
given. Candidate miRNAs were further selected by a similar comparison of prognosis. Subsequently, we carried out reverse-transcription PCR analysis of phase II
cases, separating cancer tissues into cancer cells and stromal tissue using laser
capture microdissection. Treatment effect in relation to overall survival (OS) andmiRNA expression was analyzed. Three miRNA predictors were negatively associated with OS: miR-125b-1 in cancer cells (P = 0.040), and miR-378a in both cancer
cells (P = 0.009) and stromal cells (P < 0.001). Multivariate analysis showed that
expression of miR-378a in stromal cells was the best among the three predictors(HR, 2.730; 95% CI, 1.027–7.585; P = 0.044). In conclusion, miR-125b-1 and miR378a expression might be considered as novel biomarkers to predict the efficacy
of vaccine treatment against colorectal cancer.

doi: 10.1111/cas.13390

C

olorectal cancer is the third most commonly diagnosed
cancer in males and the second most commonly diagnosed
cancer in females, with an estimated 1.4 million cases and693 900 deaths occurring in 2012.(1) In the past decade,chemotherapy and monoclonal antibody treatment have
improved the prognosis of patients with mCRC. However,most of these patients die because the cancer develops tolerance to these agents. There is, therefore, a great need fordevelopment of additional treatment.
Immunotherapy has been used to treat mCRC and has thepotential to eradicate the disease by activating immune
responses.(2) Peptide vaccines are one form of cancer vaccinethat induces tumor-specific T-cell responses using tumor-associated antigen-derived peptides. Many phase I and II clinical

trials using peptide vaccines have been conducted and thesevaccines have provided clinical benefits in a small fraction ofpatients.(3,4) Currently, immune checkpoint blockade therapies
are a very promising cancer immunotherapy. For example,
antibodies blocking PD-1 and PD-L1 have shown durable
responses in a number of different advanced malignancies.(5,6)
Additionally, several recent studies suggest alternative targeting strategies for indoleamine 2,3-dioxygenase blockade at thelevel of gene expression.(7,8) Furthermore, combination
immunotherapy with peptide vaccines and immune-checkpoint
blockade therapies that are designed to concurrently activate
tumor-specific immune responses and inactivate immunosuppression in the tumor microenvironment may lead to the induction of stronger antitumor responses.(9,10)© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.
This is an open access article under the terms of the Creative Commons Attrib
ution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used forcommercial purposes.

Cancer Sci | November 2017 | vol. 108 | no. 11 | 2229–2238-168-

Original Article
miRNA biomarkers for CRC vaccine

www.wileyonlinelibrary.com/journal/cas

We previously reported a phase Ι and II study in which fiveepitope peptides were applied to advanced-stage colon cancer
patients.(11,12) However, active immunotherapy using epitope
peptides requires preservation of the host immune system.
Hence, it is desirable to identify predictive biomarkers for theselection of patients who are likely to respond well, and toeffectively induce specific CTL to epitope peptides.
miRNAs are endogenously expressed non-coding RNAs, 18–
25 nucleotides in length, which regulate gene expression at the
post-transcriptional level.(13) miRNAs play roles as crucial
modulators of various biological processes such as proliferation, tumor initiation and development in CRC.(14) However,miRNAs are also critical regulators of immune responses,(15)and their aberrant expression or dysfunction in the immune
system has been associated with cancer. For example, p53
modulates the tumor immune response by regulating PD-L1
through miR-34 in non-small-cell lung cancer.(16) In anotherexample, miR-20b, -21 and -130b are upregulated in advanced
CRC and inhibit PTEN expression, resulting in PD-L1 overexpression.(17) In addition, some miRNAs have been mentioned
as possible biomarkers for immunotherapy.(18–20)
Cancer tissues consist of cancer cells and surrounding stromal cells, including inflammatory cells, immunocompetentcells, endothelial cells, and fibroblasts. Stromal cells in thetumor microenvironment play an important role in cancer
metastasis.(21) Cancer stroma interacts with cancer tissues
through miRNAs, creating a niche for the cancer cells.(22) A
study showed that high levels of miR-21 in the stroma predict
short disease-free survival in stage II CRC.(23) In a separate
study, miR-21 suppression with antisense inhibitors was shown
to prevent fibroblast-to-myofibroblast transdifferentiation in
response to TGF-b.(24)The purpose of the present study was to explore miRNAs
as novel predictive biomarkers for the efficacy of
immunotherapies before their treatment. We previously
reported that three miRNAs might be useful biomarkers.(25)As the cellular origin is crucial for determination of miRNAs,in the present study, we used LCM to separate cancer cells
and stromal cells for miRNA analysis. Here we demonstrate
the results of miRNA microarray analysis and qRT-PCR in
CRC cancer and stromal cells. We suggest that miR-125b-1
and miR-378a may serve as novel predictive biomarkers for
active immunotherapies.
Materials and Methods
Summary of the phase I study. Detailed protocol of the phaseI study was described previously.(11) Briefly, patients were eligible for enrolment when: (i) they had histologically confirmed
CRC without indication for surgical resection; (ii) they had
failed to respond to prior standard chemotherapy or could not
tolerate the standard therapy; and (iii) when they had thehuman leukocyte antigen (HLA)-A*2402-allele by DNA typing.
Good Manufacturing Practice grade peptides of RNF43721,(26) TOMM34-299,(27) KOC1 (IMP-3)-508,(28) VEGFR11084,(29) and VEGFR2-169,(30) which are peptides that arerecognized by HLA-A*2402-restricted CTL, were used. Doseescalation was carried out in three patient cohorts using dosesof 0.5 mg, 1 mg, and 3 mg for each peptide. Each peptide wasgiven to patients s.c. into the thigh or axilla regions on days 1,
8, 15, and 22 in a 28-day treatment course. The determinedrecommended dose was 3.0 mg, which was followed by carrying out a single injection of a cocktail of the five peptides.
The cocktail of the five peptides at a dose of 3 mg was given

to six patients. Among 18 HLA-A*2402-matched CRC patients
who were enrolled in this study, eight primary CRC tissues
were available for miRNA microarray analysis.
Summary of the phase II study. To evaluate clinical benefitsof cancer vaccination treatment, we conducted a phase II trial,
non-randomized, HLA-A status double-blind study. Detailed
protocol of this phase II study was described previously.(12)Briefly, the therapy consisted of a cocktail of five peptides in
addition to oxaliplatin-containing chemotherapy. The peptides
used in this study were recognized by HLA-A*2402-restricted
CTL; however, the HLA-A status of all enrolled patients was
masked. The cocktail of 3 mg each of five peptides was mixed
with 1.5 mL incomplete Freund’s adjuvant and given s.c. into
the thigh or axilla regions weekly for 13 weeks, then biweekly
after 14 weeks. Patients were eligible for enrolment when they
were ≥20 years old with a histologically confirmed advanced
CRC, were na€ıve for chemotherapy, had adequate functions ofcritical organs, and had a life expectancy of ≥3 months.
Between February 2009 and November 2012, 96 CRC patientswere enrolled under the masking of their HLA-A status.
Among these 96 patients, 26 cases were available for miRNA
microarray analysis, and 68 primary CRC tissues were available for qRT-PCR analysis.
Sample collection. In the phase I trial, eight primary CRC tissues from 18 patients were obtained by surgery before treatment. Tissues were snap-frozen in liquid nitrogen and stored at
80°C. In the phase II trial, 26 primary CRC tissues wereobtained from 96 patients and were stored as frozen tissues; 16
samples were from the HLA-A*2402-matched group and 10
samples were from the HLA-A*2402-unmatched group. Sixtyeight primary CRC tissues were obtained and stored as FFPE
tissues; 34 samples from each of the HLA-A*2402-matchedand HLA-A*2402-unmatched groups. Primary CRC lesions
were resected in advance of the vaccine treatment studies at
Yamaguchi University Hospital and affiliated hospitals. Written informed consent was obtained from all patients. This
study was approved by the Institutional Ethics Review Boardsof Yamaguchi University and was conducted in accordancewith the Declaration of Helsinki (H20-102, Clinical Trial
Registration: UMIN000001791).
Microarray analysis. Total RNA was extracted from frozen
tissue using a mirVana™ miRNA Isolation Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. Concentrations and purities of total RNAs were assessedusing a spectrophotometer and RNA integrity was verified
using an Agilent RNA 6000 Nano Kit (Agilent Technologies,
Santa Clara, CA, USA). OD260/OD280 ratios of each sample
were between 2.00 and 2.10, which were accepted as adequate
for microarray analysis. The miRNA array analysis was carriedout with 1-lg samples of total RNA using the miRCURY
LNA™ microRNA Array 6th generation (Exiqon, Vedbaek,
Denmark) and the GenePix4000B (Molecular Devices, Sunnyvale, CA, USA). Relative intensity of each hybridization signal was evaluated using the Microarray Data Analysis Tool
Ver3.2 (Filgen, Nagoya, Japan).
Laser capture microdissection. FFPE tumor specimens weresectioned into 15-lm-thick slices, and stained with H&E toenable visualization of histology.(31) LCM was carried out using
the LMD system LMD6000 (Leica Microsystems, Tokyo, Japan)
to microdissect cancer cells and stromal cells from CRC tissues.
At least 30 mm2 of cancer cells and 45 mm2 of stromal cells
were collected from each FFPE specimen. Depending on thepercentage of cancer cells and stromal cells in the specimen, thisgenerally required from two to four sections.

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Original Article
Tanaka et al.

www.wileyonlinelibrary.com/journal/cas
qRT-PCR. Total RNA was extracted from LCM-captured
material using a QIAGEN miRNeasy FFPE Kit (Qiagen,Tokyo, Japan) according to the manufacturer’s protocol. Concentrations and purities of the total RNAs were assessed as
described for microarray analysis using a spectrophotometer.
Expression of miRNAs in both cancer cells and stromal cells
was verified by qRT-PCR analysis using TaqMan MicroRNA
Assays (Life Technologies). cDNA was synthesized in a 7.5lL reaction volume from total RNA (10 ng) using the
TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Waltham, MA, USA). Reactions were incubated at
16°C for 30 min and then at 42°C for 30 min, and were
inactivated at 85°C for 5 min. Each cDNA was analyzed in
duplicate by qRT-PCR using sequence-specific primers
for hsa-miR-125b-1 (ID002378), hsa-miR-224 (ID002099),
hsa-miR-486 (ID001278), hsa-miR-147b (ID002262), hsa-miR196b (ID002215), rno-miR-422b (ID001314), or RNU6B(ID001093) in an Applied Biosystems 7300 real-time PCR
instrument (Life Technologies). miR-378a was previously
known as miR-422b, and rno-miR-422b has the same sequence
as miR-378a (ACUGGACUUGGAGUCAGAAGGC). Eachindividual assay was carried out in a 20-lL reaction volume
with 1.33 lL cDNA, 1.0 lL TaqMan MicroRNA Assay,
10 lL TaqMan™ Universal PCR Master Mix II (Applied Biosystems) with No AmpErase UNG and 7.67 lL nuclease-freewater (Applied Biosystems). Reactions were incubated for
10 min at 95°C, and then for 55 cycles with denaturation for
15 s at 95°C and annealing/extension for 60 s at 60°C. Data
were analyzed using SDS software v1.3.1 (Life Technologies).
Threshold and baseline settings were set according to protocol
recommendations.
In situ hybridization. ISH was carried out on 5-lm-thick sections of FFPE samples using the RiboMap Kit (Ventana Medical Systems, Tucson, AZ, USA) on the Discovery ULTRA
automated ISH instrument (Ventana Medical Systems). Probes
were 50 DIG-labelled ISH LNAs (miRCURY-LNA detection
probes; Exiqon). Sequences of probe for miR-125b-1 and miR378a were/5DigN/AGCTCCCAAGAGCCTAACCCGT and/
5DigN/CCTTCTGACTCCAAGTCCAGT, respectively.
Sequence of the scramble probe for a negative control was/
5DigN/GTGTAACACGTCTATACGCCCA. The ISH steps
after the deparaffinization step were carried out based on thestandard protocol provided in the manufacturer’s RiboMap
application note. Briefly, after treatment with proteinase K, theslides were hybridized with the detection probe for 8 h at
52°C. The digoxigenins were detected with a polyclonal antiDIG antibody and an alkaline phosphatase-conjugated second
antibody (Ventana Medical Systems). Signal was detectedusing the BlueMap NBT/BCIP substrate kit (Ventana Medical
Systems). The sections were counterstained with Kernechtrot
as a marker stain and covered with a glass coverslip.
Statistical analysis. In microarray analysis, expression signals
were calculated by log2 transformation of the normalized data,
and differentially expressed miRNAs were detected by using
the fold-change value and the Fisher index using Microsoft
Excel 2010 according to a previously reported formula.(32) In
qRT-PCR, relative quantities of miRNA levels were determined using the 2-DDCT method after normalization withRNU6B as a standard reference.(33) Differences between
groups were estimated using the Mann–Whitney U-test. Categorical variables were compared by using the chi-squared andFisher’s exact tests. The cut-off value was determined by Youden’s index for each ROC analysis. OS was measured inmonths from the first vaccination to the day of patient death
Cancer Sci | November 2017 | vol. 108 | no. 11 | 2231

from any cause. Survival curves were analyzed by the Kaplan–
Meier method and the log–rank test, and a Cox’s proportional
hazards model was used to estimate the HR and the 95% CI
for the treatment effect in relation to OS and miRNA expression, other biomarkers, and prognostic clinical information.
Statistical analyses were done with JMP V11 (SAS InstituteInc.). P < 0.05 was considered statistically significant.
Results
Selection of candidate miRNAs to predict the efficacy of vaccination. We first carried out microarray analysis of the expres-sion profiles of 1425 miRNAs using eight frozen tissues frompatients enrolled in a phase I study. We classified the patients
into two groups of four patients each, a responder group and a
non-responder group. Responders were defined according to thefollowing criteria: (i) having a PFS period of more than
150 days; or (ii) having an OS period of longer than 300 days
without any additional chemotherapy. Non-responders were
defined according to the following criteria: (i) having a PFS
period shorter than 90 days; and (ii) having an OS period
shorter than 200 days. Subsequently, on the basis of the relative
comparison of hybridization signals of individual miRNAs, weselected the top 30 miRNAs showing significant differences in
expression levels between the two groups (Table S1). Next, 26
frozen tissues from the phase II study were analyzed using amiRNA microarray. In this analysis, we examined expression
levels of the 30 candidate miRNAs and their association withclinical outcome. The expression levels of six miRNAs weresignificantly associated with prognosis in the HLA-A*2402matched groups. There was no difference in prognosis in HLAA*2402-unmatched groups according to the expression levelsof these six miRNAs. Therefore, these miRNAs had the potential to be predictive biomarkers for the efficacy of the vaccine
treatment. Expression levels of these six miRNAs and survival
analysis are shown in Table 1.
Validation of candidate miRNA expression and association of
the miRNA expression levels with clinical outcome. We carriedout qRT-PCR analysis of the expression of these six miRNAs in
68 FFPE tissues from the phase II study. The FFPE specimens
were separated into cancer cells and stromal cells by LCM.
miRNA expression levels were examined in these two types ofcells, and association of miRNA expression levels with clinicaloutcome was evaluated. Characteristics of these 68 patients arelisted in Table 2. No difference in any characteristic was foundbetween HLA-A*2402-matched and HLA-A*2402-unmatchedgroups. Expression levels of the six miRNAs and survival analysis of the patients according to low or high expression of thesemiRNAs are shown in Table 3. Three predictors were negatively
associated with OS in the HLA-A*2402-matched group only:
miR-125b-1 in cancer cells (P = 0.040), and miR-378a in bothcancer cells (P = 0.009) and stromal cells (P < 0.001). In addition, expression of miR-125b-1 (P = 0.089) and miR-486(P = 0.092) in stromal cells tended to be negatively associatedwith OS. Overall survival of the HLA-A*2402-matched and unmatched groups of patients according to high or low expression of miR-125b-1 or miR-378a is shown in the form of Kaplan
Meier curves in Figure 1. These data suggest that miR-125b-1
and miR-378a have the potential to be negative predictive
biomarkers for the efficacy of the vaccine treatment.
Univariate and multivariate analysis of the association of 125b1 and miR-378a expression with OS. Results of univariate andmultivariate analyses are shown in Table 4. Expression of
miR-378a in both cancer cells (HR, 2.853; 95% CI 1.281–© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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Table 1. Microarray analysis of candidate miRNA expression and phase II study patient survival analysis (log–rank test)
Expression in cancer tissue
miR name

Cut-off value
Mean

SD

Median

689.1

666.5

526.5

196b-5p

400

HLA-A*2402

Group

n

P-value

Matched

Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High

7
9
5
5
9
7
5
5
9
7
5
5
11
5
6
4
8
8
5
5
11
5
5
5

0.001

Unmatched
147b

483.6

185.2

460.0

500

Matched
Unmatched

378a6225.62216.96336.76500

Matched
Unmatched486-5p

345.3

71.0

339.0

365

Matched
Unmatched224-5p

343.2

290.8

272.2

270

Matched
Unmatched125b-1

604.0

312.0

518.0

550

Matched
Unmatched

0.366
0.040
0.366
0.066
0.515
0.006
0.998
0.011
0.657
0.021
0.092

HLA, human leukocyte antigen; miRNA and miR, microRNA; SD, standard deviation.

Table 2. Clinical characteristics of patients in the phase II study
whose tissues were analyzed for miRNA using qRT-PCR
Characteristics

HLA-A*2402
matched (n = 34)

Sex
Male
FemaleAge (years)
Mean
SD
Range
Unresectable site
Liver
Lung
Dissemination
Bone
Lymph node
Other
No. involved organs
One
Two
Three
Primary tumor site
Colon
RectumHLA-A*2402
unmatched (n = 34)

P-value

14
20

19
15

0.225

68
8
47–82

64
8
38–77

0.080

19
11
4
0
5
3

26
10
3
2
8
1

0.073
0.793
0.690
0.151
0.355
0.303

27
6
1

22
8
4

0.302

22
12

25
9

0.431

HLA, human leukocyte antigen; SD, standard deviation.

6.788: P = 0.010) and stromal cells (HR, 3.984; 95% CI
1.661–9.243: P = 0.003) was significantly associated with OS.
The expression of miR-378a in stromal cells was confirmed to

be an independent predictor of OS by multivariate regression
analysis (HR, 2.730; 95% CI, 1.027–7.585; P = 0.044).
miR-125b-1 and miR-378a localization in tumor tissue. For
specific identification of miR-125b-1 and miR-378a expression
in tissue sections, we carried out ISH in tissue sections frompatients with a poor prognosis. miR-125b-1 was highly
expressed in both cancer and stromal cells (Fig. 2a). Within
stromal cells, fibroblast-like cells (Fig. 2b) and mononuclear
cells (Fig. 2c) showed strong miR-125b-1 signals. A weakmiR-378a signal was observed in both cancer and stromal cells(Fig. 2f). Within stromal cells, fibroblast-like cells showed
weak miR-378a signals (Fig. 2g). Such weak signals might bea result of the affinity of LNA probes for miR-378a.
Discussion

Findings of the present study showed that miR-125b-1 andmiR-378a are negative predictive biomarkers for the efficacy
of vaccine treatment against colorectal cancer. To our knowledge, this is the first study to systematically investigate
miRNA biomarkers for cancer immunotherapy and to focus on
separate analysis of cancer cells and stromal cells within cancer tissue. Previous miRNA microarray analysis by our group
showed that miR-196b, miR-378a, and miR-486 are possible
predictive biomarkers. However, it should be noted that the
sample size was small and that the precise origins of thesemRNAs are unknown. In the present study, we used qRT-PCR
in an increasing number of samples and used LCM to separate
the cancer cells and stromal cells. In addition, we carried out
ISH for specific identification of the miRNAs.
First, our results indicated that high miR-125b-1 expression
in cancer cells was significantly associated with poor prognosis© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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Table 3. qRT-PCR analysis of candidate miRNA expression and phase II study patient survival analysis (log–rank test)
Expression in cancer cells
miR nameMean
196b-5p

135.079 9 10SD2

169.617 9 10Median2

Cut-off value

63.862 9 10

2108.588 9 10

2

HLA-A*2402

Group

n

P-value

Matched

Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High

24
10
22
12
28
6
27
7
15
19
13
21
18
16
16
18
20
14
18
16
27
7
23
11

0.652

Unmatched147b

1.237 9 1021.254 9 1020.834 9 102

1.527 9 102

Matched
Unmatched

378a

61.995 9 10265.306 9 10240.041 9 10227.856 9 102

Matched
Unmatched486-5p5.450 9 102

20.571 9 1022.299 9 1022.290 9 102

Matched
Unmatched224-5p101.859 9 102

113.0851 9 102

61.063 9 102

68.359 9 102

Matched
Unmatched125b-10.786 9 1021.690 9 1020.289 9 1020.498 9 102

Matched
Unmatched

0.120
0.210
0.046
0.009
0.777
0.165
0.798
0.172
0.512
0.040
0.429

Expression in stromal cells
miR name

196b-5p

Mean

SD

MedianCut-off value

HLA-A*2402

Group

n

P-value

33.801 9 102

40.004 9 102

18.673 9 102

22.450 9 102

Matched

Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High
Low
High

22
12
18
16
17
17
17
17
23
11
23
11
19
15
17
17
16
18
13
21
20
14
18
16

0.311

Unmatched147b

0.599 9 1020.535 9 1020.424 9 1020.413 9 102

Matched
Unmatched

378a

43.009 9 10244.441 9 10228.802 9 102

43.080 9 102

Matched
Unmatched486-5p7.569 9 1028.741 9 1024.725 9 1024.766 9 102

Matched
Unmatched224-5p19.249 9 102

19.626 9 102

13.315 9 102

10.984 9 102

Matched
Unmatched125b-12.311 9 1022.371 9 102

1.554 9 1021.690 9 102

Matched
Unmatched

0.218
0.290
0.515
0.001
0.215
0.092
0.124
0.525
0.990
0.089
0.136

HLA, human leukocyte antigen; miRNA and miR, microRNA; SD, standard deviation.

in HLA-matched patients. Previous studies have shown thatmiR-125b plays crucial roles in immune system development
and immunological host defense.(34) Upregulated expression of
miR-125b may enhance type I interferon expression in airway

epithelial cells in eosinophilic chronic rhinosinusitis with nasal
polyps,(35) and promote classical activation of macrophages.(36,37) miR-125b-1 was also found to be specificallyupregulated in patients with ulcerative colitis,(38) and may

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Fig. 1. Overall survival (OS) according to miRNA expression. OS of the HLA-A*2402-matched (left side) and -unmatched groups (right side) ofphase II study patients according to high or low expression of miR-125b-1 or miR-378a. (a) According to miR-125b-1 in cancer cells. (b) According
to miR-125b-1 in stromal cells. (c) According to miR-378a in cancer cells. (d) According to miR-378a in stromal cells. Solid line shows high miRNA
expression and dashed line shows low miRNA expression. Statistical analysis was carried out using the log–rank test. HLA, human leukocyte antigen; miRNA and miR, microRNA.

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Table 4. Univariate and multivariate analyses of associations between clinical data and overall survival
Univariate analysis
95% CI

FactorCut-off

CRP
NLR
CEA
CA19-9No. involved organs
Relative expression of miR-378a in cancer cells
Relative expression of miR-378a in stromal cells
Relative expression of 125b-1 in cancer cells
Relative expression of 125b-1 in stromal cells

Multivariate analysis

>1
>3
>16
>21
One or more
>27.856 9 102>43.080 9 102>0.498 9 102
>1.690 9 102

HR

95% CI
P-value

1.537
1.298
1.972
2.010
2.487
2.853
3.984
2.586
1.950

Lower

Upper

0.619
0.542
0.871
0.907
0.960
1.281
1.661
0.925
0.876

3.525
2.939
4.784
4.514
5.759
6.788
9.243
6.286
4.279

0.338
0.544
0.105
0.085
0.060
0.010
0.003
0.068
0.100

HR

1.888
2.730

P-value
Lower

Upper

0.695
1.027

5.031
7.585

0.207
0.044

CA19-9; carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; CI, confidence interval; CRP, C-reactive protein; HR, hazard ratio; NLR, neutrophil:lymphocyte ratio.

increase risk of CRC through nuclear factor kappa B (NF-jB)
activation.(39,40) However, miR-125b negatively regulates
TNF-a expression in neonatal monocytes,(41) and inhibits
proper TNF-a production in response to LPS.(42) Hence, miR125b-1 might play the role of an immune suppressor byincreasing immune exhaustion,(43) and resulting in the inhibition of the antitumor immune system. Expression of miR125b-1 in cancer cells seems to be more important than that instromal cells (Fig. 1a,b), although strong signals were also
observed in stromal cells (Fig. 2b,c). miR-125b-1 might beproduced by cancer cells and/or stromal cells, and cross-talkby miRNAs between cancer cells and stromal cells might ultimately work as an immune suppressor.
Second, our results indicated that high expression levels of
miR-378a in both cancer cells and stromal cells were significantly associated with poor prognosis in HLA-matchedpatients. Previous reports showed that miR-378 was downregulated in CRC(44,45) and that miR-378a expression might play
an important role as a tumor suppressor gene.(46) In contrast,
miR-378a was reported to inhibit cytotoxicity of human NK
cells(47) and enhance tumor growth and angiogenesis throughrepression of the expression of two types of tumor suppressors,
Sufu and Fus-1.(48) Hence, after the establishment of tumor
microenvironments, the function of miR-378a was thought tobe as an immune suppressor. Regarding expression of miR378a, there were some discrepancies in miRNA expression
using ISH, miRNA microarray, and qRT-PCR. Expression
level of miR-378a was higher than that of miR-125b-1 in
miRNA microarray and qRT-PCR analyses. However, theopposite phenomenon was observed in ISH analysis. The miR378a signal was weak in the cancer cells and stromal cells,
and miR-125b-1 was highly expressed in both cell types. This
discrepancy might mean that there were some problems withthe affinity of the ISH probe for miR-378a.(49)
In multivariate analysis, miR-378a in stromal cells showed
the best performance among the three predictors for selection ofpatients who are likely to respond well to peptide vaccines(Table 4). We additionally considered whether the combinationof miR-125b-1 and miR-378a gave a better predictive power.
Because the high expression of miR-125b-1 and miR-378a
might be negative predictive factors, we classified 68 patients
into three groups according to the number of predictive factors(i.e. high expression of miR-125b-1 or miR-378a in cancer or
stromal cells). The OS of each group was shown in the form of

Kaplan–Meier curves in Figure S1. There was a significant difference between the “0 factor” group and the “2 factors” group.
Hence, the combination of miR-125b-1 and miR-378a in cancer
cells and stromal cells might have effective prognostic power.
The link between miR-125b-1 and miR-378a withimmunotherapy is unclear, and it is very important to identify
the target genes of these two miRNAs. Hence, we carried out
RNA sequencing using 22 cancer tissues from a phase II study(data not shown). We classified the 22 samples into threegroups according to the expression of each miRNA by qRTPCR analysis: the high expression group in both cancer andstromal cells, the low expression group in both cell types, andthe group of others. Difference in target gene expression
between the “high expression group” and the “low expression
group” was assessed by log2 ratio and Ingenuity Pathways
Analysis (IPA) (Qiagen).(50) Regarding miR-125b-1, receptor
activator of NF-jB (RANK) was downregulated in the “highexpression group”. RANK, a member of the TNF receptor
family, and its ligand RANKL play important roles in the
immune system. T cells activated as a result of RANKL
expression stimulate dendritic cells, expressing RANK, toenhance their survival and enhance induction of T-cell
response.(51,52) RANK is a target gene of miR-125b-1 by IPA(Qiagen), and miR-125b-1 might work as an immune suppressor regulating RANK. Regarding miR-378a, RNF125 was
downregulated in the “high expression group”. RNF125, E3
ubiquitin ligase, is positive regulator of T-cell activation.(53)miR-378a was considered to regulate RNF125 according to
IPA and could also be an immune suppressor. Thus, miR125b-1 and miR-378a might be therapeutic targets.
It must be mentioned that the mean survival time of the“HLA matched-miRNA high” group appeared to be shorter
than that of the “HLA unmatched-miRNA high” group(Fig. 1). However, there was no significant difference between
their survival curves (Fig. S2). Furthermore, we investigatedside-effects in the “HLA matched-miRNA high” group. The
incidence of severe AE in each group of miR-378a is shownin Table S2. There was no significant difference in the incidence of AE between the “HLA matched-miRNA high” groupand other groups. It is quite unlikely that vaccine treatment led
to deleterious effects in “HLA matched-miRNA low” patients.
It is known that the behavior of cancer cells and the development of tumor tissues are modulated by cross-talk throughmiRNAs between cancer cells and stromal cells such as

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Fig. 2. In situ hybridization detecting miR-125b-1
and miR-378a. (a) miR-125b-1 is highly expressed incancer cells and stromal cells. (b) Arrows indicate
miR-125b-1-positive fibroblast-like cells. (c) Arrows
indicate miR-125b-1-positive mononuclear cells. (d)Arrows indicate miR-125b-1-positive cancer cells. (e)Scramble probe. (f) A miR-378a signal was observedin both cancer cells and stromal cells. (g) Arrows
indicate miR-378a-positive cancer cells. (h) Arrows
indicate miR-378a-positive fibroblast-like cells. (i)Scramble probe. Bar, 200 lm. miR, microRNA.

fibroblasts, endothelial cells, pericytes, immune cells, and local
and bone marrow-derived stromal stem cells.(54) Although several studies have reported the importance of miRNAs such as
miR-21,(23) miR-17-92a and the miR-106b-25 cluster(22) incancerous stromal cells, the present study is the first to regard
the role of miR-125b-1 and miR-378a in colorectal cancer
cells and stromal cells for predictive markers of immunotherapy.
In conclusion, miR-125b-1 and miR-378a expression might
be considered novel biomarkers to predict the efficacy of
immunotherapy in CRC. Although our results are preliminary
and the functions of each of the miRNAs in immune responses
are still unclear, the applicability of these miRNAs should be
confirmed in a future large-scale study as biomarkers to selectpatients who can expect a better outcome with the vaccine
treatment.
Acknowledgments
This study was carried out as a research program of the Project forDevelopment of Innovative Research on Cancer Therapeutics(P-DIRECT; 11039020), The Japan Agency for Medical Research andDevelopment (AMED; 15cm0106085h0005). This study was supported
in part by a grant for Leading Advanced Projects for Medical Innovation (LEAP; 16am0001006 h0003) from the Japan Agency for Medical
Research and Development. The authors would like to thank Dr
Takuya Tsunoda and Dr Koji Yoshida, Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The
University of Tokyo, for their excellent advice and cooperation and for© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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providing all of the peptides. The authors thank Ms Naoko Okayama
for technical support.

Disclosure Statement
Yusuke Nakamura is a stockholder and a scientific advisor of
OncoTherapy Science, Inc. The other authors declare no potential conflicts of interest for this article.

Abbreviations
AE
CA19-9
CEA
CI
CRC
CRP
DIG
FFPE

adverse event
carbohydrate antigen 19-9
carcinoembryonic antigen
confidence intervalcolorectal cancer
C-reactive protein
digoxigenin
formalin-fixed paraffin-embedded

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HLA
HR
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LCM
LNA
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miRNA
NLR
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PD-1
PD-L1
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qRT-PCRRNF125
ROC
TGF-b
TNF

human leukocyte antigen
hazard ratioin situ hybridization
laser capture microdissection
locked nucleic acidmetastatic CRC
microRNA
neutrophil lymphocyte ratio
overall survival
programmed death-1
PD-ligand 1
progression-free survival
phosphatase and tensin homolog
quantitative RT-PCR
ring finger protein 125
receiver operating characteristictransforming growth factor beta
tumor necrosis factor

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Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this article:
Table S1. Top 30 miRNAs upregulated or downregulated in a responder group compared with a non-responder group, as assessed using microarray analysis.
Table S2. Frequency of severe adverse events (CTCAE version 3.0) in four groups classified according to miR-378a expression and HLA-A status.
Fig. S1. Overall survival (OS) according to number of predictive factors. OS of the HLA-A*2402-matched (left side) and -unmatched groups (rightside) of phase II study patients according to the number of predictive factors (high expression of miR-125b-1 and/or miR-378a). (a) Predictive factors in cancer cells. (b) Predictive factors in stromal cells. Dotted line shows the survival curve of patients with 0 factors. Solid line shows the survival curve of patients with one factor and the dashed line shows the survival curve of patients with two factors. Statistical analysis was doneusing the log–rank test. HLA, human leukocyte antigen.
Fig. S2. Overall survival (OS) according to miRNA expression and HLA-A status. (a) According to miR-125b-1 in cancer cells. (b) According tomiR-125b-1 in stromal cells. (c) According to miR-378a in cancer cells. (d) According to miR-378a in stromal cells. Red line shows HLAA*2402-matched group and black line shows -unmatched group of phase II study patients. Solid line shows high miRNA expression and thedashed line shows low miRNA expression. Statistical analysis was done using the log–rank test. HLA, human leukocyte antigen; miR, microRNA.

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Exp. Anim. 66(2), 125–136, 2017

—Original—

An improved Red/ET recombineering system andmouse ES cells culture conditions for the generationof targeted mutant mice
Katsuyoshi Kumagai1), masakatsu TaKanashi2), shin-ichiro OhnO2),
masahiko KurOda2), and Katsuko sudO1)1)Pre-Clinical Research Center, University-related Facilities, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-

ku, Tokyo 160-8402, Japan2)Department of Pathology, Tokyo Medical University, Tokyo, Japan

Abstract: Targeted mutant mice generated on a C57BL/6 background are powerful tools for analysisof the biological functions of genes, and gene targeting technologies using mouse embryonic stem(ES) cells have been used to generate such mice. Recently, a bacterial artificial chromosome (BAC)
recombineering system was established for the construction of targeting vectors. However, gene
retrieval from BACs for the generation of gene targeting vectors using this system remains difficult.
Even when construction of a gene targeting vector is successful, the efficiency of production oftargeted mutant mice from ES cells derived from C57BL/6 mice are poor. Therefore, in this study, we
first improved the strategy for the retrieval of genes from BACs and their transfer into a DT-A plasmid,
for the generation of gene targeting vectors using the BAC recombineering system. Then, we attempted
to generate targeted mutant mice from ES cell lines derived from C57BL/6 mice, by culturing inserum-free medium. In conclusion, we established an improved strategy for the efficient generationof targeted mutant mice on a C57BL/6 background, which are useful for the in vivo analysis of genefunctions and regulation.
Key words: C57BL/6, ES cells, gene retrieval, gene targeting, KSR

Introduction
Targeted mutant mice are powerful tools for analysisof the biological functions of genes, and many investigators have generated knockout (KO), conditional knockout (cKO) and knockin (Ki) mice by the gene targeting
technology using mouse embryonic stem (Es) cells.
Construction of a gene targeting vector is required forthe generation of targeted mutant mice. recently, several recombinase-based systems have been developedthat have significantly simplified the construction of genetargeting vectors [33, 34]. A bacterial artificial chromo-

some (BaC) recombineering system involving dna
cloning and engineering has also been developed for theconstruction of such targeting vectors [33]. The first stepin the construction of a gene targeting vector from a BaC
clone is gene retrieval into a dT-a plasmid by red/ET
recombination. However, the efficiency of gene retrieval from BaC clones remains poor, and the toxicity ofeukaryotic genomic sequences causes instability of theplasmid in Escherichia coli [5, 16, 31].
The C57BL/6 strain of mice is one of the best characterized for gene targeting, and was also the reference
strain for establishing the mouse genome sequence [29,(Received 30 August 2016 / Accepted 26 October 2016 / Published online in J-STAGE 25 November 2016)
Address corresponding: K. Sudo or K. Kumagai, Pre-Clinical Research Center, University-related Facilities, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
Supplementary Tables and Figures: refer to J-STAGE: https://www.jstage.jst.go.jp/browse/expanim©2017 Japanese association for Laboratory animal science

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30]. This strain is widely used in a variety of biomedical
fields, and in fact, the JAX Mice Database (http://jaxmice.jax.org/) includes many gene-targeted mouse lines
with the C57BL/6 genetic background, including thoseexpressing Cre recombinase, which is used for generating tissue-specific KO mice. Furthermore, large-scale
mouse mutagenesis projects were organized by the International Knockout mouse Consortium to select inbred
C57BL/6 strain-derived Es cells for producing mutationsof all the protein-coding genes in the mouse [3, 10].
Thus, the genetic background of the C57BL/6 strain isextremely useful for the generation of targeted mutant
mice. however, for reasons that are not entirely clear, itremains difficult to acquire chimeric mice with confirmed
germline competency on this background [2, 38, 42, 43].
a one of the technology that has been used to overcome
this problem is the straightforward and efficient clusteredregularly interspaced short palindromic repeat (CrisPr)/CrisPr-associated (Cas) system for genome editing both in vivo and in vitro [6, 11, 13, 19, 27, 36]. Thissystem has been demonstrated to cause double-strand
breaks in mammalian cells, and to be useful for the generation of KO mice [41]. However, it was still difficult
to produce cKO mice and Ki mice of the inserted cdna,
as off-target mutagenesis frequently occurs in the genomic dna when using the CrisPr/Cas9 system [12,17, 35, 44].
We here describe an improved method to generatetargeted mutant mice on a C57BL/6 background. The
method involves improved gene retrieval from a BaC
clone for the construction of a gene targeting vector, useof a lower voltage for electroporation, and use of a serum-free medium (Ksr medium), which is widely available for culture. These improvements enabled more efficient generation of targeted mutant mice on a C57BL/6
genetic background and are expected to promote betteruse of the publicly available C57BL/6 strain Es cellresources by the biomedical research community.
Materials and Methods
Generation of the gene retrieval vector
a gene retrieval vector was constructed by isolatinga genomic region of the Wapl gene from the BaC clone
rP23-478g5 (Life Technologies Japan, Tokyo, Japan)
and transferring it into the pmCs.dT-a plasmid (addgene). another gene retrieval vector was constructedwith a genomic region of the Pgr gene that was isolated

from the BaC clone rP23-23L9 (Life Technologies
Japan). The two end homologies (5′ homology arm, Noti/
SacII fragment; 3′ homology arm, Sacii/Saci fragment)
were amplified by polymerase chain reaction (PCR) using the appropriate primer pairs (supplementary Tables1) and each BaC clone as the template. The two amplified sequences were ligated into the pMCS.DT-A plasmidusing Ligation high, ver.2 (Toyobo, Osaka, Japan). Ligation between the two end homologies and the pmCs.
DT-A plasmid was confirmed by PCR using the primer
pairs shown in supplementary Table s1. Then, eachplasmid dna was linearized by digestion with Sacii,
and the phosphates were removed from the ends of the
dna to prevent religation of the linearized plasmid dna
using calf intestinal alkaline phosphatase (Takara Bio,shiga, Japan). These plasmids were then used as the gene
retrieval vectors.
Construction of the Wapl gene cKO targeting vector
For construction of the Wapl gene cKO targeting vector, one loxP site was inserted upstream of exon 3 andanother was inserted downstream of exon 4 to generate
a frameshift by Cre-mediated deletion of exon 3 and 4
in the targeted Wapl gene region. The detailed protocol
for the generation of this vector is explained in supplementary methods.
Animals
C57BL/6J and C57BL/6n mice were purchased fromCharles river Laboratories, Japan (Kanagawa, Japan).
iCr and pseudopregnant recipient mice were purchasedfrom sankyo Labo service Corporation (Tokyo, Japan).
The animals were housed in cages (five animals per cage)and provided access to water and food (FR-1; Funabashi
Farm, Chiba, Japan) ad libitum. The animal rooms weremaintained in a temperature-controlled (21°C–25°C) andlight-controlled (12L:12D cycle, lights on at 0800 h)
environment. all of the mice were bred and maintained
under specific-pathogen-free conditions. All animal careand procedures that were performed in this study conformed to the guidelines for animal experiments of Tokyo medical university, and they were approved by the
animal research Committee of Tokyo medical university.
Derivation and culture of mouse ES cells
Es cells derived from C57BL/6J mice were established using standard methods [32], with some modifica--179-

PRODUCTION OF TARGETED MUTANT MICE

tions in the duration of culture. Blastocysts collected at
3.5 days postcoitus (dpc) were cultured on 96-well tissue
culture plates with a mouse embryonic fibroblast (MEF)
feeder layer in Ksr medium, which consisted of highglucose DMEM (GIbco, Frederick, MD) supplemented
with 15% Knockout-serum-replacement (gibco), 2 mmglutamine (gibco), 1 mm sodium pyruvate (gibco), 0.1
mm non-essential amino acids (gibco), 0.1 mm 2-mercaptoethanol (nakalai Tesque, Kyoto, Japan) and 1,000
U/ml LIF (Merck Millipore, Darmstadt, Germany). After the emergence of primary Es cells, the cells werepassaged three more times in Ksr medium, and thenfrozen in liquid nitrogen for cryopreservation when theyreached 70%–80% confluence (Supplementary Fig.
s1a).
In the case of ES cells cultured on a MEF feeder
layer in serum-containing medium (Figs. 2A and 3A),
the medium consisted of high-glucose dmEm supplemented with 15% serum (gibco), 1 mm sodium pyruvate, 0.1 mm non-essential amino acids, 0.1 mm 2-mercaptoethanol, and 1,000 U/ml LIF. Mouse ES cells werepassaged in serum-containing medium, when theyreached 70%–80% confluence.
Chromosome number analysis
Chromosome number analysis of the Es cells was
performed as described previously [39]. Well-spread
metaphases were identified by microscopy, and the chromosome numbers were counted on the micrographs.
Alkaline phosphatase staining and immunocytochemical
analysis
ES cells were fixed with 4% paraformaldehyde inphosphate buffer solution (Wako, Osaka, Japan). alkaline phosphatase activity was detected using a commercial Es Cell Characterization Kit (merck millipore), as
described previously [26]. immunostainings for ssEa1
and Oct-4 were performed using the Es cell sample
marker kit (Merck Millipore). The FITC-conjugated goatanti-mouse IgM antibody, AP128F (Merck Millipore)
was used as the secondary antibody for the detection of
SSEA1, and the Cy3-conjugated goat anti-mouse IgM
antibody, aP128C (merck millipore) was used as thesecondary antibody for the detection of Oct-4. stained
ES cells were observed under a fluorescence microscope(KEYEnCE, Osaka, Japan).

127

Isolation of ES clones with homologous recombination
by electroporation
Es cells (4 × 107) in hEPEs-buffered salt buffer weretransfected with 120 µg of the linearized targeting vector in a 4-mm electrode gap cuvette (1 × 107 cells, 30 µgof linear dna, in a total volume of 700 µl/cuvette) byelectroporation using Bio-rad gene Pulser (Bio-rad,hercules, Ca). at 10 min after the transfection, the Es
cells were plated onto a feeder layer. selection with 300µg/ml of g418 (gibco) was started 24 h after the electroporation. after 7–9 days of selection, Es cell colonies
were picked up onto a 96-well tissue culture plate witha feeder layer. all of the colonies were replicated forcryopreservation and subjected to PCR and Southernblot analysis.
Validation of homologous recombination
PCr-based screening of the target Wapl gene Es cellclones was performed using the internal screening_F4
primer (TgCCTgCTTgCCgaaTaTCa) and the external screening_r4 primer (TggaCCaCaaTCaCCaCgTT) to detect the 2.6-kb PCr fragment. PCr-positive
clones were confirmed by the presence of the 5′ loxP and
3′ loxP sites (Supplementary Table S2). Moreover, thePCR-positive clones were subjected to Southern blot
analysis using the 3′ external probe (500 -bp) and to Scaidigestions for confirmation of 3′ integration. The Neo
insert was confirmed by Xhoi digestion of genomic dna(10 µg) and using neo internal probe (804 -bp). afterdigestion, the genomic dna was separated by electrophoresis on a 0.8% agarose gel and blotted on to a nylon
membrane (Pall corporation, Washington, nY) before
hybridization with the dig high Prime dna Labelingand detection starter Kit ii (roche applied science,
Mannheim, Germany). Blotting with the 3′ external
probe yielded a 5.0-kb fragment corresponding to thewild-type allele, and a 6.6-kb fragment corresponding
to the mutant allele. Furthermore, blotting with the Neointernal probe yielded a 6.2-kb fragment corresponding
to the mutant allele.
The 3′ external probe was generated from a 500-bp
PCR fragment using the ex3_7_F primer (TGATGTCACagCCaagaaTT) and ex3_7_r primer (CaaaaCTaTTagCTgCaagT). The neo probe was generated
from an 804-bp PCR fragment using the Neo_F1 primer(TCagaagaaCTCgTCaagaag) and neo_r1
primer (aTgggaTCggCCaTTgaaCa).

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Generation of chimeric mice
Es cell clones containing the target Wapl gene were
aggregated for the generation of chimeric mice. The cellclones were placed on a MEF feeder layer 2 days before
embryo manipulation. The propagated Es cells weredissociated with 0.05% trypsin just before the aggregation. Eight-cell-stage-embryos were collected at 2.5 dpc
from female iCr mice that had been superovulated byintraperitoneal (i.p.) injection of 5.0 IU of equine chorionic gonadotropin (serotropin; asKa Pharmaceutical,
Tokyo, Japan) at 1600 h, followed by injection of 5.0 IU
of human chorionic gonadotropin (gonadotropin 3000;
asKa Pharmaceutical) 48 h later and then mated naturally with male iCr mice. The zona pellucida was removed by treatment with acid Tyrode’s solution (sigmaAldrich, St. Louis, MO). Fifteen to twenty ES cells were
aggregated with each zona-free embryo and cultured
overnight in micro-drops of KsOm medium (arK resource, Kumamoto, Japan) under mineral oil at 37°C,
5% CO2, and 95% humidity. The chimeric embryos weretransferred at 2.5 dpc into the uterine horns of pseudopregnant female iCr mice (sankyo Labo service Corporation). Chimeric mice were identified 20 days after
birth by their black eyes and their black coat color. Chimeric males showing germline transmission were crossedwith C57BL/6 females.
Statistics
data are expressed as average values ± sEm. The
student’s t-test was used to statistically analyze the differences between two groups. a P-value of less than 0.05
was considered to indicate a significant difference betweween two group.
Results
Gene retrieval from a BAC clone by Red/ET recombination
For the construction of a conditional Wapl gene targeting vector, we first retrieved a region of the Wapl genefrom a BaC clone and inserted it into the dT-a cassetteby Red/ET recombination (Fig. 1A). Gene retrieval from
BaC clones are often unsuccessful, because of plasmid
instability in E. coli owing to the presence of an unidentified promoter that produces a toxic peptide from eukaryotic genomic sequences [5, 16, 31]. Therefore, thechallenge was how to maintain maximum plasmid stability in E. coli after gene retrieval. a previous study
showed that the number of proteins synthesized is dra-

matically reduced in E. coli when cultured at low temperatures [20]. moreover, static culture effectively suppressed the growth of ground beef microflora whileallowing the healthy growth of E. coli [4]. Thus, weanalyzed the culture conditions (shaking or static culture
and high or low temperatures) that are the most suitablefor obtaining optimal plasmid stability in E. coli aftergene retrieval (Fig. 1A). Furthermore, to identify thegene retrieval clone, we performed PCr analysis. a 530bp fragment including the targeted Wapl gene was detected upon both shaking and static culture at 37°C and
30°C by PCr 1. in contrast, a 588-bp fragment was
detected in the static culture at 30°C by PCR 2 (Fig. 1B,
supplementary Table s1).
We next attempted to validate the gene retrieval ofanother gene, as a confirmatory experiment: genomic
dna from the Pgr gene was retrieved from a BaC clone
and transferred into a dT-a cassette by red/ET recombination (Fig. 1C). In the case of the Pgr gene, a 539-bp
fragment was detected in static culture at 30°C by PCr
3. Furthermore, a 524-bp fragment was detected instatic culture at 30°C by PCR 4 (Fig. 1D, Supplementary Table s1). These results showed that gene retrieval
was accomplished for both genes by static culture at
30°C.
Efficient gene retrieval from a BAC clone by static culture
at a low temperature
To examine the efficiency of gene retrieval from a
BaC clone by red/ET recombination, we compared therate of gene retrieval between shaking and static culture
at high and low culture temperatures. For the case of theWapl gene, following culture at 37°C after colony harvesting, the gene was not retrieved from any of the 500
colonies after either shaking or static culture (0.0%;
Table 1). By contrast, following shaking culture at 30°C
after colony harvesting, the gene was retrieved from twocolonies out of the 50 colonies tested (4.0%; Table 1).
interestingly, the gene was retrieved from 40 colonies
out of 50 colonies after static culture (80.0%; Table 1).
next, for the Pgr gene, following culture at 37°C aftercolony harvesting, the gene could not be retrieved fromany of the 100 colonies after either shaking or static
culture (0.0%; Table 1). in contrast, following static
culture at 30°C after colony harvesting, the gene was
retrieved from 44 colonies out of 50 colonies (88.0%;
Table 1), although no positive colonies (0.0%; Table 1)
were obtained with shaking culture. These results indi--181-

PRODUCTION OF TARGETED MUTANT MICE

129

Fig. 1. strategy for gene retrieval using the red/ET recombineering system. (a) Exon 3 and exon 4 of the Wapl
gene were isolated from an rP-478g5 BaC clone and transferred to a dT-a cassette using the red/ET
recombineering system; gene retrieval was attempted by shaking or static culture at 37°C or 30°C. (B)genotype analysis by PCr. subcloning by shaking culture at 37°C and 30°C yielded a 530-bp fragment
amplified by PCR 1. On the other hand, subcloning by static culture at 30°C yielded a 588-bp fragment
amplified by PCR 2. M, marker; B, BAC clone used as a negative control. (C) Exon 1 of the Pgr gene wasisolated from an rP-23L9 BaC clone and transffered to the dT-a cassette using the red/ET recombineering system; gene retrieval was attempted by shaking and static culture at 37°C and 30°C. (d) PCr analysisof the genotype. Subcloning by static culture at 30°C yielded a 539-bp fragment amplified by PCR 3 and a
524-bp fragment amplified by PCR 4. M, marker; B, BAC clone used as a negative control.

cated that static culture at 30°C markedly improved theefficiency of gene retrieval from a BAC clone in E. coli.

Electroporation of constructs into C57BL/6 strain ES
cells for the generation of conditional Wapl KO miceusing our newly established Es cell lines (supplementary Fig. S1A-G), we next attempted to generate

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K. Kumagai, ET AL.
Table 1. Efficiency of gene retrieval by Red/ET recombination
Target gene

Culture temperature

Culture method

no. of colonies tested

no. of colonies withthe target gene (%)a

Wapl

37°C
37°C
30°C
30°C

shaking
staticshaking
static

500
500
50
50

0 (0.0)b,d0 (0.0)b,e2 (4.0)c,d40 (80.0)c,e

Pgr

37°C
37°C
30°C
30°C

shaking
staticshaking
static

100
100
50
50

0 (0.0)f,h
0 (0.0)f,i
0 (0.0)g,h
44 (88.0)g,i

aNo. of colonies with the target gene/no. of colonies tested. Fisher exact test was used for pairwise comparisons,as indicated by superscripts: b,f,hP=1.0000; c,e,g,iP=0.0000; dP=0.0081.

Fig. 2. generation of C57BL/6-strain Es cells containing a homologous recombination. (a) Experimental design to examine
the optimal voltage for electroporation. (B) number of Es colonies after electroporation. The average number was 33
for C57BL/6n Es colonies and 40 for C57BL/6J Es colonies after electroporation at 250 V, and 341 for C57BL/6n
Es colonies and 328 for C57BL/6J Es colonies after electroporation at 220 V. *P<0.01. (C) rate of homologous recombination in the Es cells after electroporation. The average rate was 0.00% for C57BL/6n Es colonies and C57BL/6J
Es colonies after electroporation at 250 V, 0.47% for C57BL/6n Es colonies and 0.31% for C57BL/6J Es colonies
after electroporation at 220 V. *P<0.01. (d) The structure of the murine Wapl gene and the targeted locus are shown.
Genotyping by Southern blot analysis using the 3′ external probe on Scai-digested genomic dna, and internal neo
probe on Xhoi-digested genomic dna. The blue arrowheads indicate band sizes of 5.0-kb (wild-type) and 6.6-kb(target) in the ScaI-digested genomic DNA using the external 3′ probe. The orange arrowhead shows a fragment of
6.2-kb (target) in the Xhoi-digested genomic dna detected using the neo internal probe. (E) southern blot analysis
of genomic DNA to confirm homologous recombination in the ES cells lines on a C57BL/6 strain background.

genetically modified mice on a C57BL/6J background.
Specifically, we attempted to generate conditional Wapl
gene KO mice on a C57BL/6J background, and towards
that objective, we selected C57BL/6J ES-#2, whichyielded the largest number of 100% F0-chimeric mice,
which confirms germline transmission, among the four
newly established Es cell lines (supplementary Table
S3). For comparison, we also generated, in parallel, the

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PRODUCTION OF TARGETED MUTANT MICE
Table 2. generation of germline-competent chimeric mice by induction of homologous recombination in Es cells

no. of mice with a rate of gLTd inrate of gLTd in
low contribution
high contribution
high contribution(< 70%)(> 70% chimerism)
F0-chimeric miceof Es cellsc (%)b F0-chimeric mice

Es culture
mediumno. oftransferred
chimeric
embryos

no. of total
pups (%)ano. of colored
F0- chimericmice (%)b

B6nEs

serum medium
Ksr medium

140
245

12 (8.6)e38 (15.5)e3 (25.0)f
30 (78.9)f0 (0.0)g
28 (73.7)g

0/3
0/2

0
25/28

B6JEs

serum medium
Ksr medium

142
143

18 (12.7)h29 (20.3)h

5 (27.8)i
22 (75.9)i0 (0.0)j
17 (58.6)j

0/5
0/5

0
16/17

Es cell line

ano. of total pups/no. of transferred chimeric embryos. bNo. of F0 chimeras/no. of total pups. cmice that show coat color contribution of

the aggregated Es cells in the host mouse. dGLT, germline transmission. Fisher exact test was used for pairwise comparisons, as indicatedby superscripts: eP=0.0588; fP=0.0012; g,hP=0.0000; iP=0.1099; jP=0.0021.

same mice using Egr-101, an Es cell line derived from
C57BL/6n [14].
For the generation of cKO mice, electroporation of
the construct is an important step, and the optimum electric voltage is known to vary among Es cell lines. We
first tested 250 V, which is the voltage recommended for
the TT2 Es cell line [45], as well as 220 V, which isrecommended for the A2lox ES cell line [15] (Fig. 2A).
after 7–10 days of selection with g418 following theelectroporation, the number of C57BL/6 strain Es colonies obtained after electroporation at 250 V was foundto be significantly lower than that after electroporation
at 220 V (Fig. 2B). Moreover, although we could notconfirm the occurrence of homologous in any of the
C57BL/6 strain Es cells after electroporation at 250 V,
successful homologous recombination was confirmed
after electroporation at 220 V (Figs. 2C–2E). These results suggest that the number of C57BL/6 strain Es
colonies obtained improves by electroporation at 220 V
for obtaining homologous recombination in Es cells. We
therefore set the voltage for electroporation to 220 V forthe subsequent experiments.
Efficient generation of targeted mutant mice
For the generation of targeted mutant mice via homologous recombination in C57BL/6 strain cells, we
attempted to generate targeted mutant mice with a highcontribution of Es cells. however, mice with a highcontribution of Es cells could not be obtained from homologous recombination in C57BL/6 strain cells (Table2). Therefore, another challenge was to establish an
optimal method for the maintenance of Es cells withhomologous recombination, for generating targeted mutant mice. Es cells need to be maintained in both an
undifferentiated and germline-transmitted state for thegeneration of targeted mutant mice. Therefore, we examined various media to identify the most suitable forobtaining germline-transmitted chimeric mice. it has
been reported that differentiation is more restrained in
Ksr medium than in fetal bovine serum-containing medium [8]. Therefore, the four newly established cell lines
were cultured in Ksr medium, which was found to be
the suitable stem cell medium for stably maintaining the
C57BL/6J Es cells in an undifferentiated (supplementary Figs. S1B–E) and germline-transmitted (Supplementary Figs. S2F and 2G) state. First, we attempted
homologous recombination in Es cells obtained in Ksr
medium before electroporation and cells cultured in Ksr
medium after electroporation (Fig. 3A, #1). However,
only a small number of Es colonies were obtained andnone of them had undergone homologous recombination(Figs. 3B and 3C). Thus, KSR medium before electroporation affects not only the survival rate of cells after
electroporation. We suggest that the electroporation
conditions affect the number of C57BL/6 strain Es
colonies that are obtained, which is lower in serumcontaining medium than in KSR medium (Fig. 2B). We
therefore analyzed homologous recombination in Es
cells obtained in serum-containing medium before electroporation and those obtained by culturing in Ksr
medium after electroporation (Fig. 3A, #2). Our results
showed that the number of C57BL/6 strain Es colonies
obtained after culturing in serum-containing medium
was significantly higher than that obtained by culturing
in KSR medium (Fig. 3B); furthermore, we confirmedthat homologous recombination had occurred in these
ES cells (Fig. 3C). Thus, the number of C57BL/6 strain
Es colonies increased with the use of serum-containing
medium before electroporation (Fig. 3B). Taken together, these results suggest that culturing the cells in serum-

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K. Kumagai, ET AL.

Fig. 3. generation of targeted mutant mice. (a) Experimental design for the culturing of Es cells, including the use of the Ksr
medium, before electroporation. (B) The number of Es colonies after electroporation. The average number was 36 for
C57BL/6N ES colonies and 26 for C57BL/6J ES colonies by #1, and 323 for C57BL/6N ES colonies and 327 for C57BL/6J
ES colonies by #2. *P<0.01. (C) The rate of homologous recombination in the Es cells after electroporation. The averagerate was 0.00% for C57BL/6N ES colonies and C57BL/6J ES colonies by #1, 0.42% for C57BL/6N ES colonies and 0.41%
for C57BL/6J ES colonies by #2. *P<0.01. (D) Photographs of an F0-chimeric mouse (green arrowhead) derived from
C57BL/6N ES cells and an F0-chimeric mouse (black arrowhead) derived from C57BL/6J ES cells. Analysis of germline
transmission by coat color analysis after natural mating between a male mouse with a high contribution C57BL/6n Es cells(green arrowhead) and a wild-type C57BL/6N female mouse (white arrowhead), and between a 100% F0-chimeric male
mouse (black arrowhead) and a wild-type C57BL/6J female mouse (red arrowhead). Generation of F1 mice with a blackcoat color (orange arrowheads and blue arrowheads), confirming germline transmission in a male mouse with a high contribution of C57BL/6N ES cells and in 100% F0-chimeric male mice derived from each of the C57BL/6 ES cell lines. (E)Southern blot analysis of genomic DNA to confirm homologous recombination in the F1 mice.

containing medium before electroporation improves
obtainment the number of C57BL/6 strain Es colonies
for obtaining homologous recombination in Es cells.
interestingly, not a single mouse with a high contribution of Es cells was generated from the transferred embryos aggregated with the Es cells cultured in serum
medium, despite the generation of a certain number ofcolored F0-chimeric mice (Table 2). In contrast, in thecase of embryos aggregated with the Es cells cultured
in KSR medium, colored F0-chimeric mice were efficiently generated, most of which were mice with a highcontribution of ES cells (Fig. 3D, Table 2). Furthermore,

germline transmission was confirmed in most of the micewith a high contribution ES cells (Figs. 3D, 3E, Table2). In contrast, germline transmission was not confirmed
in any of the F0 mice derived from C57BL/6J ES cellswith less than 100% chimerism and C57BL/6n Es cellswith less than 70% chimerism (Table 2).
moreover, we investigated the generation of targetedmutant mice for the 4 targeted genes by using C57BL/6n
Es cells (supplementary Table s4, represented as athrough d). although the recombination frequencies tothe homologous target loci of the surviving Es cells
after selection with g418 were in the range of 0.6% to-185-

PRODUCTION OF TARGETED MUTANT MICE

5.5% in four independent experiments, homologous recombination in Es cells was obtained by culturing inserum-containing medium before electroporation and in
Ksr medium after electroporation in all experiments(Supplementary Table S4). Furthermore, in all the experiments performed by the aggregation method, thenumber of F0-chimeric mice obtained was between 7
and 12, mice with greater than 70% chimerism wereobtained at a rate of 41.7% to 66.7%, and germlinetransmitting-mice were successfully obtained at a rateof 16.6% to 55.6% (supplementary Table s4). These
results suggest that there is a high possibility that
C57BL/6 strain Es cells can be germline-transmitted byculturing in serum-containing medium before electroporation and in Ksr medium after electroporation.
Discussion
The red/ET recombineering system has been introduced as an efficient construction of a targeting vector
for the generation of targeted mutant mice. however, as
there is a loxP site in the BaC backbone, although aloxP-neo-loxP cassette is first inserted into the BAC, it
is difficult to remove the genomic region of neo-loxP
using the 706-Cre plasmid, when constructing conditional gene targeting vectors in a BaC. Therefore, wefirst attempted gene retrieval into a DT-A cassette froma BaC to remove the loxP site within the BaC backbone.
however, gene retrieval was unsuccessful owing to thetoxicity of eukaryotic genomic sequences that are unstable in E. coli. in a recent study, a toxic peptide froma eukaryotic genomic sequence was found to be produced
by an unidentified promoter of a high-copy-number vector in E. coli [5, 16, 31]. Therefore, a low-copy-number
vector was selected to reduce the possible toxic effects
in E. coli. moreover, it was reported that successful gene
retrieval could be achieved by retransformation of the
dna isolated by mini-preparation; however, many
points remain unclear regarding the details of this method [7]. On the other hand, we successfully achieved gene
retrieval in an efficient manner, by static culture at 30°C.
Therefore, our findings suggested that static culture at alow culture temperature prevents the production of
toxic peptides from eukaryotic genomic sequences in E.
coli, enabling efficient gene retrieval, and it may inhibit the expression of genes that promote self-ligation/
end-joining of retrieval plasmid. Furthermore, previous
studies showed that the loxP-oligo and loxP-neo cassette133

can be simultaneously inserted into the BaC before retrieval, and hence it is possible to construct conditional
gene targeting vectors in 7 days [33]. however, as therate of loxP insertion into the BaC was 0%–6.2%, repeated trial and screening by PCr analysis would be
required, resulting in high costs, which is unfavorable
as a method for the construction of conditional genetargeting vectors. in this study, we demonstrated that our
method takes 15 days, but requires fewer samples forscreening by PCr analysis, as well as fewer trials andat a lower cost, and highly stable and hence useful forthe construction of conditional gene targeting vectors.
Therefore, our newly established method was able toovercome the problems of the previous methods.
KO mice generated by gene targeting technology areoften used to analyze the biological functions of genes.
nearly two decades have passed since Es cell lines derived from C57BL/6 mice were first established and theirusefulness has frequently been reported [21, 24], although developmental defects in these mice have been
noted [18], and the cells often become unstable aneuploidcells and deteriorate under standard culture conditions[18]. much fewer KO mice have been generated from
C57BL/6 Es cell lines compared with from the widelyused 129 ES cell lines [22]. In recent studies, modifications of the culture conditions of C57BL/6 Es cells havebeen proposed, one of which is the use of Ksr medium
for culturing of the Es cells [8]. moreover, the use of
two inhibitors, targeting ErK and gsK3 has been reported to prevent the differentiation of Es cells [46, 47].
Furthermore, the use of three inhibitors, targeting the
FGF receptor, ERK, and GSK3, which were originallyused to culture rat Es cells [25], has been reported to beeffective for the maintenance of germline-competent
C57BL/6 Es cells [23, 37]. however, the conditions ofelectroporation and of subsequent culture remained to
be fully optimized for C57BL/6 Es cells. in this study,
we first established four germline-competent C57BL/6J
Es cell lines. Then, we attempted to generate germline
chimeric mice in an efficient manner. However, electroporation at 250 V, which is the recommended voltage
for TT2 cells, did not enable the satisfactory maintenanceof Es cells, and homologous recombination was unsuccessful in these cells. On the other hand, electroporation
at 220 V improved both the survival of the cells after
electroporation and the homologous recombination efficiency. Furthermore, we used KSR medium for culturing the Es cells after electroporation. inhibitors do no

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K. Kumagai, ET AL.

need add to the medium in this strategy, and it is possible to prepare the medium at low cost, enabling thegeneration of a sufficient number of chimeric mice.
new technologies have recently been proposed forgenerating genetically engineered mice on a C57BL/6
background without the use of Es cells, including theuse of zinc-finger nucleases (ZFNs) [28], transcription
activator-like effector nucleases (TaLEns) [40], and
CrisPr/Cas9. using these methods, targeted mutant
mice can be generated by direct injection of the enzymes
into the pronuclei and/or cytoplasm of the fertilized embryos [41]. however, the gene targeting region is restricted to the ZFNs and TALENs that can be constructed [28, 40]. Furthermore, off-target mutagenesis is an
important critical issue when the CrisPr/Cas9 technology is used [12, 17, 27, 35, 44]. a recent study reported
that although the frequency of off-target mutagenesis
was reduced with the use of the CrisPr/Cas9 technology, the risk still exists, along with the risk of deficientgene disruption by off-target mutagenesis [9]. On theother hand, our improved conventional method can beused for artificial modification and for gene targeting of
any genetic region, and allows complete genetic disruption while avoiding off-target mutagenesis, with the useof a minimum number of animals. a recent study hasreported the construction of a targeting vector for thegeneration of Ki mice using the CrisPr/Cas9 technology [1]. Therefore, our findings indicate that an improved
method of gene retrieval from a BaC clone is required
for the stable generation of targeted mutant mice.
in conclusion, we have established an improved strategy based on a conventional technique, to generate
stable germline chimeric mice using C57BL/6-strain Es
cell lines. First, efficient gene retrieval from a BAC clone
into the dT-a cassette was accomplished using red/ET
recombination by static culture at 30°C. Then, a combination of electroporation at 220 V and culture in Ksr
medium after the electroporation enabled efficient generation of targeted mutant mice on a C57BL/6 background. This strategy is expected to improve the efficient
generation of targeted mutant mice for the analysis ofgene functions.

versity Foundation Cancer Research Fund (K.K.). We
thank asako Oohara for her assistance with the animalcolony maintenance, and drs. naoto Kubota and Takayoshi sasako of the department of diabetes and metabolic diseases, graduate school of medicine, The university of Tokyo, for their technical assistance and advice
regarding the experiments.

Acknowledgments
This work was supported in part by a grant-in-aid forYoung scientists (B) (K.K.) from the Japan society for
the Promotion of science, and the Tokyo medical uni-

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International Journal of Gynecological Pathology
36:582–592, Lippincott Williams & Wilkins, Baltimore
Copyright r 2017 by the International Society of Gynecological Pathologists

Original Article

Downloaded from https://journals.lww.com/intjgynpathology by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3OY3Hg5ow9MQ6avsAyjaw7urwNXv13Tk+y9oMswJ0I/+OSeEDqxNBiA== on 04/08/2020

Vascular Smooth Muscle Cell Maturation Stage and Ki-67
Index are Diagnostic Biomarkers for Pathologic Grade ofOvarian Teratoma
Masako Hongo-Kohama, M.D., Atsushi Kurata, M.D., Hirotsugu Hashimoto, M.D.,
Koji Fujita, C.T., Hajime Horiuchi, M.D., Toshitaka Nagao, M.D., and Masahiko Kuroda, M.D.

Summary: Although immature teratoma of the ovary is a rare disease, its pathologic
grading, especially between low-grade (grade 1) and high-grade (grade 2 or 3) immature
teratomas, is important for optimal therapy and prognosis. This grading, however, iscurrently solely dependent on quantitation of neuroepithelial components as judged bysubjective assessments. As we have recently successfully studied the maturation of vascularsmooth muscle cells (SMCs) in other organs using an h-caldesmon to a-smooth muscle
actin (a-SMA) ratio, we decided to use this ratio to investigate a potential link between
teratoma grade and SMC maturation, in combination with Ki-67 index. Sixteen immature
teratomas along with 5 mature teratomas of the ovary were studied and stained with
antibodies to CD31, a-SMA, h-caldesmon, and Ki-67. The number of vascular SMCs
calculated using the a-SMA/CD31 ratio did not diﬀer between teratoma grades (except
between grade 0 and 3), whereas the number of mature vascular SMCs calculated using theh-caldesmon/CD31 ratio and maturation state calculated using the h-caldesmon/a-SMA
ratio reduced signiﬁcantly as teratoma grade progressed from 0 to 3. Furthermore, theseparameters were signiﬁcantly lower in high-grade than in low-grade immature teratomas(Po0.05). Ki-67 labeling index, regardless of germ cell layer, also signiﬁcantly increased
with teratoma grade (Po0.05). These results suggest that not only the number ofneuroepithelial elements but also vascular immaturity and proliferating cell counts arebiomarkers for ovarian teratoma grading. Thus, assessment of the maturity of vascular
SMCs may serve as a valuable diagnostic tool for assessing teratoma maturity. Key Words:
Immature teratoma—Maturity—Smooth muscle cells—h-Caldesmon—Ki-67.

Ovarian germ cell tumors represent 20% to 25% of
all ovarian neoplasms, but only 3% to 5% of them
are malignant (1). Teratoma is a subtype of germ celltumors, composed of elements from at least 2 of 3
germ cell layers, namely the ectoderm, mesoderm,and endoderm (2). Immature teratoma is a malignant
form of teratoma (3), which includes both matureand immature tissue, the latter being most commonly
immature neural elements (2,4). Immature teratomaof the ovary accounts for approximately 20% to 45%
of malignant ovarian germ cell tumors and 1% of all
ovarian teratomas (2,5,6).
Although immature teratoma of the ovary is a raredisease, it often occurs in young women of reproductive age (3,5). Therefore, surgical procedures

From the Departments of Molecular Pathology (M.H.-K., A.K.,
K.F., M.K.); Anatomic Pathology (T.N.), Tokyo Medical University; and Department of Diagnostic Pathology, NTT Medical
Center Tokyo (H. Hashimoto, H. Horiuchi), Tokyo, Japan.
The authors declare no conﬂict of interest.
Address correspondence and reprint requests to Atsushi Kurata,
MD, Department of Molecular Pathology, Tokyo Medical
University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.
E-mail: akurata@tokyo-med.ac.jp.

DOI: 10.1097/PGP.0000000000000373

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VASCULAR SMCS IN OVARIAN TERATOMA
should be performed to preserve fertility in combination with comprehensive staging (6). Norris et al.
(3) found that the pathologic grade of the tumor,
based on the quantity of neuroepithelial components,
was the best predictor of recurrence and survival rate
after local tumor resection. Currently, chemotherapy
has dramatically improved prognosis, and the recommended treatment for high-grade (grades 2 and 3)
immature teratomas is combination adjuvant chemotherapy. However, chemotherapy is not recommended for those with low-grade (grade 1) andstage I immature teratomas as they are not regarded
as suﬃciently aggressive (5,7,8).
Grading of immature teratomas is thus, very
important; however, it is currently based on thesubjective judgment of the pathologists’ eye. The
histology of neuroepithelial components is ambiguous, thus grading reproducibility between pathologists is not suﬃciently high for accurate diagnosis (8).
The measurement of serum a-fetoprotein levels andimmunohistochemical expression of Oct4 have been
proposed (9,10), but no consensus has yet beenreached as, for example, a mixed germ cell tumor may
inﬂuence a-fetoprotein elevation (5).
We have recently reported that the maturity ofvascular smooth muscle cells (SMCs) can be estimated by the immunohistochemical expression ratioof h-caldesmon/a-smooth muscle actin (a-SMA) in
coronary and carotid arteries (11,12). Therefore, wehypothesized that this characteristic expression ofvascular h-caldesmon may also reﬂect the maturity ofteratoma tissues. Furthermore, to the best of ourknowledge, Ki-67, which speciﬁcally reacts with thenuclei of proliferating cells (13), has not been
investigated as a potential biomarker for assessing
the maturity of teratomas, although a Ki-67 labelingindex has been reported in other ovarian germ celltumors and testicular seminomas (14,15). Thus, thepresent study was designed to assess the immunohistochemical expression ratios of vascular SMC hcaldesmon to CD31 and to a-SMA, as well as Ki-67
labeling index, and to investigate their relationshipwith ovarian teratoma grade.
MATERIALS AND METHODSPatients and Tissues
The protocol of the present study was approved by
the committee of human study of Tokyo Medical
University and NTT Medical Center Tokyo. A total of
16 cases of surgically resected immature teratoma wereidentiﬁed from the databases at these 2 institutions

583

TABLE 1. Histologic grade, age at diagnosis, history ofgravidity and parity, maximal diameter of the tumor, andstage of the tumor investigated in 21 patients with ovarian
teratoma
Histologic
grade
0
0
0
0
0
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3

Age atdiagnosis(y)History ofgravidity andparity (G/P)

40
36
35
25
20
35
22
31
26
37
20
20
25
24
23
18
32
33
33
22
30

0/0
0/0
1/0
0/0
0/0
NR/0
0/0
NR/0
0/0
0/0
0/0
1/0
NR/0
0/0
NR/0
NR/0
0/0
0/0
0/0
0/0
0/0

MaximalStage ofdiameter of thethetumor (cm)
tumor
6
7
4
7.5
9
13
4.8
3.5
3.5
16
15
15
12.6
14.5
17
20
17.3
14
15
10
22

Ia
Ia
Ia
Ia
Ia
Ic
Ia
Ic
Ia
Ic
Ia
Ia
Ia
Ia
Ic
Ia
IIb
Ic
Ic
Ic
Ia

NR indicates not recorded.

between 2003 and 2015. An additional 5 surgicallyresected, mature, teratomas were selected. As shownin Table 1, the selected patients’ age range was 18
to 40 y (mean 28.0 ± 6.52 y). The maximal diameterof these tumors was from 3.5 to 22 cm (mean
11.7 ± 5.46 cm). All patients were nulliparous, andtheir teratomas were limited to stage I with 1
exception. The diagnoses of these 21 specimens had
been performed by at least 2 experienced pathologists.
The grading was based on the criteria of Thurlbeck
and Scully (16), as modiﬁed by Norris et al. (3).
Brieﬂy, grade 0, 1, 2, and 3 correspond to none, r1, 2
or 3, and Z4, respectively, of the number of low
magniﬁcation (40  ) ﬁelds containing neuroepithelium in any slide. Our 20 study specimens consisted of
5  grade 0 (mature teratoma), 5  grade 1, 6  grade
2, and 5  grade 3. We reviewed each specimen and 1
tissue slide for each immature teratoma was selectedthat included dense neuroepithelium and randomly
selected 1 from each mature teratoma.

Immunohistochemistry
The surgically resected specimens had previously
been ﬁxed in 10% formalin solution for 24 hours andembedded in paraﬃn. Immunohistochemistry was
performed on the selected paraﬃn-embedded sections,
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M. HONGO-KOHAMA ET AL.
ratio), h-caldesmon+ tubules to CD31+ tubules(h-caldesmon/CD31 ratio), and h-caldesmon+ tubules
to a-SMA+ tubules (h-caldesmon/a-SMA ratio) was
calculated in the diﬀerent 3 ﬁeld each. Further, theaverage a-SMA/CD31 ratio, h-caldesmon/CD31 ratio,
and h-caldesmon/a-SMA ratio was calculated in eachspecimen.
As for assessment of immunostaining for Ki-67,
neural, stromal, and epithelial tissue areas were randomly
selected from each tissue using light microscopy (200  ).
Then, at least 1000 cell nuclei were counted using publicdomain software, Fuji, which is open-source software
Image J and is focused on biological image analysis (17).
Out of these nuclei, nuclei that positively stained brown
for Ki-67 were counted. Then, the ratio of Ki-67+ nuclei
to all the nuclei was calculated and indicated bypercentage as the Ki-67 labeling index. In counting,neuroepithelium (neural tube) was excluded from neuraltissue, as were hair follicles and intestinal epithelium from
epithelial cells, as their immunopositivity by Ki-67 was
obviously enhanced.

4-mm thick, using the avidin-biotin-peroxidase complex according to standard methods. The monoclonal
antibody to CD31 (clone JC70A, working dilution
1:200; Dako, Glostrup, Denmark) was used to
identify endothelial cells, a-SMA (clone 1A4, working
dilution 1:400; Dako) was used to identify all SMCs,
h-caldesmon (clone h-CD, working dilution 1:50;
Dako) was used to identify SMCs beyond intermediate diﬀerentiation, and Ki-67 (clone MIB-1, working
dilution 1:200; Dako) was used to identify proliferating cells. Deparaﬃnized and hydrated sections weretreated with 0.3% hydrogen peroxide in methanol for
10 minutes to block endogenous peroxidase activity.
For immunostaining of CD31, h-caldesmon, and Ki67, sections were autoclaved in Tris-EDTA buﬀer (pH9.0) at 1211C for 10 minutes to expose antigens andwere cooled for 30 minutes. After rinsing in 0.1 mol/L
phosphate buﬀered saline (PBS, pH 7.4), the sections
were incubated with aﬃnity-puriﬁed primary antibodies at room temperature (a-SMA: for 1 h; CD31,
h-caldesmon, and Ki-67: for 2 h). Thereafter, they
were incubated with Histostar (Ms+Rb) for Humantissue (Medical & Biological laboratories, Nagoya,Japan) for 45 minutes. The peroxidase reaction
was performed using DAB liquid (Dako). Finally,
nuclear counterstaining was performed with Mayer
hematoxylin.

Statistical and Scattergram Analyses
Comparisons of average values for data including
age, maximal diameter of the tumor, and immunohistochemical results among diﬀerent tumor grades
were performed by the Tukey-Kramer test using MS
Excel 2000 (Microsoft Corp., Tokyo, Japan) software
packages. As immunohistochemical results, a-SMA/
CD31 ratio, h-caldesmon/CD31 ratio, and h-caldesmon/a-SMA ratio were analyzed, along with Ki-67
labeling index in neural, stromal, and epithelial tissue
areas. The level of signiﬁcance was set at Po0.05.
Furthermore, scattergrams were constructed using
representative immunohistochemical results to identify suitable cut-oﬀ values.

Assessment
Light microscopy was performed with a Carl Zeiss
HAL 100 instrument and a W-PI 10  /23 ocular lens
was used to analyze the tissues. Three diﬀerent ﬁelds(100  ) in each tissue where vessels were identiﬁed
were randomly selected. Digital images of immunohistochemistry in stainings for CD31, a-SMA, andh-caldesmon (the same ﬁelds) were captured by the
NY-D5000 super system (Microscope Network; Nikon, Tokyo, Japan) and were printed by a true-color
printer (IPSiO SP C420; Ricoh, Tokyo, Japan). In
each ﬁeld, CD31+ cells were regarded as endothelial
cells, and all the tubular structures in which endothelial cells were identiﬁed for more than half theircircumference were counted as vessels. Structures
where tubules were collapsed were excluded from thecounting. In the same ﬁelds as counting for CD31+
cell, a-SMA+ tubular structures and h-caldesmon+
tubular structures were also counted. In these countingof SMCs, densely stained tubular structures werecounted as 1, whereas faintly stained tubular structures were counted as 0.25. Then, the ratio of
a-SMA+ tubules to CD31+ tubules (a-SMA/CD31

RESULTSClinical Findings
As shown in Figure 1A, average age was not
signiﬁcantly diﬀerent between any 2 tumor gradegroups. In contrast, maximal diameter of the tumor
was signiﬁcantly greater (Po0.05) in grade 2 or 3
than in grade 0 or 1 (Fig. 1B).
Immunohistochemistry for CD31, a-SMA, andh-Caldesmon in Vasculature
CD31 immunostained all cells regarded as endothelial cells in all specimens (Figs. 2A, D). However,collapsed capillaries were occasionally observed

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VASCULAR SMCS IN OVARIAN TERATOMA

A 45
40
35
Age (y.o.)

30
25
20
15
10
5
0
grade 0B

grade 1

grade 2

grade 3h-caldesmon/a-SMA ratios, like h-caldesmon/
CD31 ratios, tended to decrease as tumor grades
progressed from 0 to 3. Average h-caldesmon/a-SMA
ratios ± SDs in grade 0, 1, 2, and 3 were 0.730 ±
0.147, 0.410 ± 0.215, 0.140 ± 0.107, and 0.139 ±0.062, respectively. Among these grades, average hcaldesmon/a-SMA ratio was signiﬁcantly higher(Po0.01) in grade 0 than in grade 2 or 3; signiﬁcantly
higher (Po0.05) in grade 0 than in grade 1 and also
higher in grade 1 than in grade 2 or 3 (Fig. 4C).

*
**

25

*
*

Maximal diameter (cm)

585

20
15
10
5
0

grade 0

grade 1

grade 2

grade 3

* Indicates P < 0.05, ** indicates P < 0.01 by Tukey-Kramer test.

FIG. 1. Comparison of average age at diagnosis (A) and average
maximal diameter of the tumor (B) among ovarian teratomas ofdifferent histologic grades. *Po0.05, **Po0.01 by Tukey-Kramer
test.

especially in tissues in grade 3, but were not counted
because they did not form tubules (Fig. 3).
a-SMA immunostained all the SMCs aroundvessels, seemingly pericytes and media, along withother stromal SMCs and myoﬁbroblasts in all specimens (Figs. 2B, E); however, SMCs around the small
vessels were sporadically absent in tissues in grade 3.
Average a-SMA/CD31 ratios ± SDs in grade 0, 1, 2,
and 3 groups were 0.977 ± 0.020, 0.932 ± 0.048,
0.935 ± 0.024, and 0.837 ± 0.139, respectively.
Among these groups, the average a-SMA/CD31 ratio
was signiﬁcantly diﬀerent (Po0.05) in grades 0 and 3(Fig. 4A).
In contrast, h-caldesmon tended to immunostain
pericytes and media of the vessels less strongly inhigher tumor grades (Figs. 2C, F). Average hcaldesmon/CD31 ratios ± SDs in grade 0, 1, 2, and3 groups were 0.713 ± 0.146, 0.386 ± 0.213, 0.131 ±0.099, and 0.115 ± 0.050, respectively. Among thesegroups, average h-caldesmon/CD31 ratio was signiﬁcantly higher (Po0.01) in the grade 0 group than inthat of any other grade; and was signiﬁcantly higher(Po0.05) in grade 1 than in grade 2 or 3 (Fig. 4B).

Ki-67 Labeling Index in Neural, Stromal, andEpithelial Tissues
Representative photomicrographs of neural, stromal, and epithelial tissues immunostained by Ki-67 in
grade 0 and grade 3 teratomas are shown in Figure 5.
Ki-67 immunolabeled <5% of neural tissues (excluding neuroepithelium) in all the grade 0 specimens
and 3 out of 5 specimens in grade 1. In contrast, Ki67+ neural tissue was as high as 14% in several grade
2 specimens. In grade 3, Ki-67 positivity was >14%
in all the 5 specimens and was >20% in 3 out of 5
specimens. Average Ki-67 labeling index ± SDs inneural tissues excluding neuroepithelium in grade 0,
1, 2, and 3 were 1.27 ± 0.87, 3.44 ± 4.38,
12.83 ± 5.66, and 27.18 ± 10.78, respectively. Amongthese groups, the average Ki-67 labeling index was
signiﬁcantly higher (Po0.01) in grade 3 than in any
other grade, and was signiﬁcantly higher (Po0.05) in
grade 2 than in grade 0 (Fig. 6).
In stromal tissues, Ki-67 positivity fell below 2% in
all 5 grade 0 specimens and ranged from 0.73% to8.21% in grade 1, and <14% in all the specimens ingrade 0 to 2. However, in grade 3, Ki-67 immunostained >14% in 4 out of 5 specimens, and further,it immunostained >20% in 1 specimen (Fig. 5).
Average Ki-67 labeling index ± SDs in stromal
tissues in grade 0, 1, 2, and 3 were 0.80 ± 0.70,
4.34 ± 2.91, 9.42 ± 2.53, and 17.29 ± 4.89, respectively. Among these groups, average Ki-67 labelingindex was signiﬁcantly higher (Po0.01) in grade 3
than in any other grade and was signiﬁcantly higher(Po0.01) in grade 2 than in grade 0 (Fig. 6).
In counting Ki-67+ cells in epithelial tissues, we
intended to assess the same morphology among diﬀerent
grade groups. As most of the squamous epithelium inimmature teratomas was not well stratiﬁed, comparedwith that in mature teratomas, counting Ki-67+ cells insquamous epithelium was deemed inappropriate. Therefore, counting Ki-67+ cells in columnar epithelium, thatwas not so morphologically varied between diﬀerent
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M. HONGO-KOHAMA ET AL.

A

D

B

E

C

F

FIG. 2. Ovarian teratomas with grade 0 (A–C: same fields) and grade 3 (D–F: same fields) shown by immunohistochemistry for CD31 (A, D),
a-SMA (B, E), and h-caldesmon (C, F) (200  ). The numbers of vessels positive for CD31 and a-SMA are not different between grade 0 and
3, but h-caldesmon+ vessels in grade 3 are obviously lower than that in grade 0.

teratoma grades, was performed. The counting wasmostly performed in pseudostratiﬁed columnar epithelium, but in areas where it was absent, counting
was performed in simple columnar epithelium. Ki-67

immunolabeled <5% of epithelial tissues in all the grade
0 and 4 out of 5 grade 1 specimens. It exceeded 14% in 3
out of 6 grade 2 specimens and 20% in 3 out of 5 grade 3
specimens (Fig. 5). Average Ki-67 labeling index ± SDs

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VASCULAR SMCS IN OVARIAN TERATOMA

A

A

587

α-SMA/CD31 ratio
*

1
0.8
0.6
0.4
0.2
0

grade 0

grade 1B

grade 2

grade 3h-caldesmon/CD31 ratio
**
**

1
**
0.8

*
*

0.6

B

0.4
0.2
0

grade 0

grade 1C

grade 2

grade 3h-caldesmon/α-SMA ratio
**
**

1

*
*

0.8
*

0.6
0.4
0.2
0

FIG. 3. Collapsed vessels, not forming tubules, in grade 3 teratoma
visualized by hematoxylin eosin staining (A) and immunohistochemical staining for CD31 (B) (200  ).

in epithelial tissues in grade 0, 1, 2, and 3 groups were2.05 ± 1.02, 5.70 ± 7.78, 15.50 ± 6.88, and 24.39 ±10.79, respectively. Among these groups, average Ki-67
labeling index was signiﬁcantly higher (Po0.01) in grade
3 than in grade 0 or 1, and was signiﬁcantly higher(Po0.05) in grade 2 than in grade 0 (Fig. 6).

Scattergram Analyses
Scattergrams were constructed using immunohistochemically determined h-caldesmon/CD31 ratios andKi-67 labeling indices in 3 diﬀerent germ cell layers(Fig. 7). This analysis demonstrated that the hcaldesmon/CD31 ratio cut-oﬀ value required to
diﬀerentiate low-grade (grade 1) from high-grade(grade 2 or 3) immature teratomas is 0.24 to 0.35
with 1 exception (1 specimen with grade 1 showed
0.061). In the same manner, cut-oﬀ values of Ki-67
labeling indices are: 5.28% to 5.98% in neural tissue

grade 0

grade 1

grade 2

grade 3

* Indicates P < 0.05, ** indicates P < 0.01 by Tukey-Kramer test.

FIG. 4. Comparison of a-SMA/CD31 ratio (A), h-caldesmon/
CD31 ratio (B), and h-caldesmon/a-SMA ratio (C) based on
immunohistochemical analysis of ovarian teratomas with different
histologic grades. The a-SMA/CD31 ratio in grade 0 is significantly
higher than that in grade 3. h-caldesmon/CD31 ratio along with hcaldesmon/a-SMA ratio is significantly higher in grade 0 than thatin any other grade, and significantly higher in grade 1 than in grade
2 or 3. *Po0.05, **Po0.01 by Tukey-Kramer test.

with 1 exception (1 specimen with grade 1 showed
10.34%), 5.32% to 6.80% in stromal tissues with 1
exception (1 specimen with grade 1 showed 8.21%),and 3.94% to 10.24% in epithelial tissue with 1
exception (1 specimen with grade 1 showed 19.48%).

DISCUSSION
In the present study, we immunohistochemicallyassessed vascular SMC maturation using a panel ofantibodies, including h-caldesmon, combined with a
Ki-67 labeling index across each of the 3 germ cell
layers in teratomas. We report diﬀerences in these
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A

D

B

E

C

F

FIG. 5. Immunohistochemistry for Ki-67 in neural (A, D), stromal (B, E), and epithelial (C, F) tissues in ovarian teratomas with grade 0(A–C) and grade 3 (D–F) (200  ). In any germ cell layer, Ki-67 immunostains more nuclei in grade 3 than in grade 0 teratomas.

parameters among diﬀerent histologic grades for theﬁrst time. We demonstrate that the immaturity ofvascular SMCs, as reﬂected by decreased h-caldesmon/CD31 and h-caldesmon/a-SMA ratios, as wellas increased numbers of proliferating cells, as shownby a higher Ki-67 labeling index in any germ cell
layer, was paralleled by a higher histologic grade ofteratoma. These results may provide the basis for anovel grading system and improve our understandingof the nature of teratomas.

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Ki-67 labelling index in neural tissue
**
**
**
40

*

35
30
25
% 20
15
10
5
0
grade 0B

grade 1

grade 2

grade 3

Ki-67 labelling index in stromal tissue
**

40

**

35

**

30
**

25
% 20
15
10
5
0
grade 0C

grade 1

grade 2

grade 3

Ki-67 labelling index in epithelial tissue
**
40

**

35

*

30
25
% 20
15
10
5
0
grade 0

grade 1

grade 2

grade 3

* Indicates P < 0.05, ** indicates P < 0.01 by Tukey-Kramer test.

FIG. 6. Comparison of Ki-67 labeling index in neural (A), stromal (B), and epithelial (C) tissue among ovarian teratomas of different
histologic grades. In any germ cell layer, Ki-67 labeling index tends to increase as the grade of teratoma progresses. *Po0.05, **Po0.01 by
Tukey-Kramer test.

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M. HONGO-KOHAMA ET AL.

A

B

C

FIG. 7. Scattergrams of immunohistochemically determined hcaldesmon/CD31 ratios versus Ki-67 labeling index in 3 different
germ cell layers and plotted for 4 different grades of teratoma. Neuraltissue (A), stromal tissue (B), and epithelial tissue (C).

In our assessment of vasculature in teratomas, CD31
was helpful to immunostain virtually all the endothelium. Other authors have also reported that vascularproliferation within mature and immature teratomas
was immunolabeled by endothelial markers such as
CD34 and factor VIII-related antigen (18). Furthermore, in the present study, a-SMA immunostained all
the SMCs around vessels. Indeed, it has long been
pointed out that a-SMA immunolabels all SMCs alongwith pericytes and myoﬁbroblasts (19,20). Other authors
have also demonstrated that pericytes of vessels withinmature and immature teratoma were positive for aSMA (18). In the present study, the average a-SMA/
CD31 ratio was not signiﬁcantly diﬀerent between any 2
teratoma grades with 1 exception: it was signiﬁcantly
reduced in grade 3 compared with grade 0, as reﬂectedby the observation that small vessels devoid of SMCs
were sporadically observed in grade 3 tissues. This may
be due to the fact that grade 3 teratomas are the mostimmature, thus they contain tumorous vessels because
generally the structure of the vessel wall is abnormal andpericytes are tenuous within tumors (21).
It is noteworthy that the vascular h-caldesmonpositivity ratio decreased gradually from grade 0, 1, 2
to 3 teratomas. Average h-caldesmon/CD31 and hcaldesmon/a-SMA ratios were signiﬁcantly higher ingrade 0 and 1 than in grade 2 and 3. Caldesmon, ﬁrst
identiﬁed in chicken gizzard (22), is an actin-binding
protein that also interacts with myosin, tropomyosin,
and calmodulin. h-caldesmon, one of its isoforms,
has been reported to be expressed exclusively in
SMCs (23). Expression of h-caldesmon has beenreported to increase as SMCs diﬀerentiate but
decrease or diminish in immature SMCs (24). This
is in stark contrast to a-SMA, which is expressed evenin immature SMCs and myoﬁbroblasts (20). We havepreviously shown that the maturity of vascular SMCs
can be estimated by a higher immunohistochemical
expression ratio of h-caldesmon in coronary andcarotid arteries (11,12). The results of the present
study indicate that maturity of teratomas can also bedemonstrated by the maturity of vascular SMCs.
We have further revealed that the Ki-67 immunohistochemical positivity ratio is proportional toteratoma grade and that growth progressed regardlessof the type of germ cell layer. Of particularimportance is the ﬁnding that the Ki-67 labelingindex was <14% in almost all cases and types ofgerm cell layer in grade 0 and 1 teratomas, whereas it
almost exclusively exceeded 20% in grade 3 teratomas; 14% and 20% positivity have previously been
proposed as cut-point in breast cancer and neuroendocrine tumors (25–27). Because the Ki-67 antigen
is expressed in all active parts of the cell cycle, that is,
G1, S, G2, and mitosis, but not in G0 (13), its labelingindex is widely used as a proliferation marker inhuman malignancies including general ovarian germ
cell tumors (including 2 immature teratomas) andtesticular seminomas (14,15). In contrast, comparison
between atypical polypoid adenomyoma and myoinvasive endometrioid adenocarcinoma has demonstrated that the Ki-67 labeling index was unreliablebecause of variable expression rates both in epithelial

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VASCULAR SMCS IN OVARIAN TERATOMA
and stromal components (28). The present study is theﬁrst to investigate the Ki-67 labeling index according
to grading and type of germ cell layer, and suggeststhat this index is indeed useful for grading teratomas.
Clinical diﬀerentiation between grade 1 and 2
immature teratomas is pivotal because chemotherapy
is prescribed to the latter, whereas it can be avoided
in the former (5,7,8). In the present study, averageh-caldesmon/CD31 and h-caldesmon/a-SMA ratios
were signiﬁcantly diﬀerent, but average Ki-67 labeling index in each of the 3 germ cell layers analyzed
was not signiﬁcantly diﬀerent between grade 1 and 2
immature teratomas. Therefore, immunohistochemistry of vascular SMCs using h-caldesmon may besuperior to Ki-67 labeling index in practice. Scattergram analyses reveal that the cut-oﬀ value of hcaldesmon/CD31 ratio required to diﬀerentiate grade
1 and 2 immature teratomas is 0.24 to 0.35 with 1
exception. The h-caldesmon/CD31 ratio may be asuperior diagnostic tool than Ki-67 labeling indices inneural and stromal tissues where the range of the cutoﬀ was narrow (5.28%–5.98% and 5.32%–6.80%,
respectively) with 1 exception each. However, the Ki67 labeling index may be of more value in epithelial
tissue where its cut-oﬀ value has a wider range(3.94%–10.24%) with 1 exception, although the Ki67 labeling index was a long way from the cut-oﬀ
value in this instance.
The signiﬁcance of the present study is not limited toroutine diagnostic tools. Transformation of a metastatic
immature teratoma into a mature teratoma occurs after
the administration of cisplatin-based chemotherapy.
This phenomenon, ﬁrst reported by DiSaia et al. (29)in 1977 under the name of chemotherapeutic retroconversion, is a common ﬁnding in immature teratomas
with distant metastases (30). Growth of mature
teratoma has also been reported under the nameGrowing Teratoma Syndrome (31). Although we havereported this peculiar phenomenon, that is, decreased
Ki-67 labeling index in proportion to maturity ofteratoma tissues (30), investigation of vascular maturity
may further elucidate the mechanisms of chemotherapeutic retroconversion. Moreover, the results of thepresent study may contribute to regenerative medicine
through assessment of maturity of immature teratomas,
because pluripotency is partly veriﬁed by formation of
immature teratomas in human embryonic, and induced
pluripotent stem cells transplanted subcutaneously into
immunodeﬁcient mice (32).
In conclusion, we have conducted an immunohistochemical study of SMC markers and Ki-67 on
teratoma tissues of diﬀerent maturity, and revealed

591

that levels of vascular SMC immaturity and increased
Ki-67 labeling index were proportional to theimmaturity of teratoma tissue. These results maylead to the development of powerful, new diagnostic
tools and signiﬁcantly improve our understanding ofteratoma maturation.
Acknowledgment: The authors thank Shin-Ichiro Ohno,
Akira Saito, and Fumihide Kurosawa for their skillful
technical assistance.

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16. Thurlbeck WM, Scully RE. Solid teratoma of the ovary. A
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17. Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an opensource platform for biological-image analysis. Nat Methods
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RESEARCH ARTICLE

The Journal of Clinical Investigation

Cardiac myofibroblast engulfment of dead cells
facilitates recovery after myocardial infarction
Michio Nakaya,1 Kenji Watari,1 Mitsuru Tajima,1 Takeo Nakaya,2,3 Shoichi Matsuda,1 Hiroki Ohara,1 Hiroaki Nishihara,1
Hiroshi Yamaguchi,4,5 Akiko Hashimoto,1 Mitsuho Nishida,1 Akiomi Nagasaka,1 Yuma Horii,1 Hiroki Ono,1 Gentaro Iribe,6
Ryuji Inoue,7 Makoto Tsuda,8 Kazuhide Inoue,8 Akira Tanaka,2 Masahiko Kuroda,3 Shigekazu Nagata,4,5 and Hitoshi Kurose1
Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan. 2Department of Pathology, Jichi Medical University, Tochigi, Japan.

1

Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan. 4Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 5Core Research for Evolutional

3

Science and Technology, Japan Science and Technology Agency, Tokyo, Japan. 6Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical
Sciences, Okayama, Japan. 7Department of Physiology, School of Medicine, Fukuoka University, Fukuoka, Japan. 8Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences,
Kyushu University, Fukuoka, Japan.

Myocardial infarction (MI) results in the generation of dead cells in the infarcted area. These cells are swiftly removed byphagocytes to minimize inflammation and limit expansion of the damaged area. However, the types of cells and molecules
responsible for the engulfment of dead cells in the infarcted area remain largely unknown. In this study, we demonstrated thatcardiac myofibroblasts, which execute tissue fibrosis by producing extracellular matrix proteins, efficiently engulf dead cells.
Furthermore, we identified a population of cardiac myofibroblasts that appears in the heart after MI in humans and mice.
We found that these cardiac myofibroblasts secrete milk fat globule-epidermal growth factor 8 (MFG-E8), which promotes
apoptotic engulfment, and determined that serum response factor is important for MFG-E8 production in myofibroblasts.
Following MFG-E8–mediated engulfment of apoptotic cells, myofibroblasts acquired antiinflammatory properties. MFG-E8
deficiency in mice led to the accumulation of unengulfed dead cells after MI, resulting in exacerbated inflammatory responses
and a substantial decrease in survival. Moreover, MFG-E8 administration into infarcted hearts restored cardiac function andmorphology. MFG-E8–producing myofibroblasts mainly originated from resident cardiac fibroblasts and cells that underwent
endothelial-mesenchymal transition in the heart. Together, our results reveal previously unrecognized roles of myofibroblasts
in regulating apoptotic engulfment and a fundamental importance of these cells in recovery from MI.

Introduction

Myocardial infarction (MI) is a major disease worldwide (1,2). Following MI, cardiomyocytes receiving blood supply from
occluded blood vessels die rapidly due to the shortage of oxygenand nutrients (3, 4). These dead cells release noxious intracellular contents that induce secondary cell death and inflammatory responses (5–7). Therefore, dead cells generated following
MI are swiftly engulfed to prevent the expansion of the damaged area. Leakage of cellular contents from dead cells triggers
the recruitment of inflammatory cells to the infarcted area (5).
These inflammatory cells, such as macrophages and dendritic
cells, have long been considered responsible for engulfment(5, 8). However, the cell types that mediate the engulfment ofdead cardiomyocytes generated following MI and the molecular mechanisms underlying engulfment remain unknown.
Reparative responses also occur at the infarcted area (9). To
prevent cardiac rupture caused by the loss of cardiomyocytes, thearea occupied by dead cardiomyocytes is rapidly filled with collagen and extracellular matrix proteins produced by myofibroblasts.
Myofibroblasts have been reported to initiate protein secretion
Conflict of interest: The authors have declared that no conflict of interest exists.
Submitted: July 17, 2015; Accepted: October 20, 2016.
Reference information: J Clin Invest. 2017;127(1):383–401. doi:10.1172/JCI83822.

only when they receive signals from activated inflammatory cells(10). In addition, myofibroblasts are not normally present, but rather appear in damaged tissues following differentiation from several cell types in response to inflammation (11–13). In failed hearts,
cardiac myofibroblasts differentiate from 5 reported cell types:
bone marrow–derived cells, pericytes, cells that have undergone
epithelial-mesenchymal transition (EMT), those that have undergone endothelial-mesenchymal transition (EndMT), and resident
fibroblasts (12, 14). However, the functional differences between
myofibroblasts with different origins remain to be clarified (12–14).
We report that cardiac myofibroblasts execute the engulfment of dead cells generated following MI, a task previously
believed to be performed only by infiltrating inflammatory
cells. Engulfment by myofibroblasts was mediated by milk fat
globule-epidermal growth factor 8 (MFG-E8), a protein known
to promote apoptotic engulfment. MFG-E8 binds to both phosphatidylserine on dead cells and the integrin receptor αvβ5 on
phagocytes, acting as a bridging molecule (15, 16). MFG-E8 was
produced by myofibroblasts, and MFG-E8 production in thesecells involved the TGF-β/serum response factor (SRF) pathway.
MFG-E8–positive myofibroblasts were also found in infarcted
areas of patients with MI. MFG-E8–deficient mice displayed an
exacerbated inflammatory response and reduced survival after
MI due to the accumulation of dead cells. Importantly, MFG-E8
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Figure 1. Cardiac myofibroblasts engulf dead cells ex vivo. (A) Ex vivo phagocytosis assay of apoptotic cells. Cardiac macrophages and myofibroblasts isolated
from WT mice that underwent MI were exposed to apoptotic cells (green) (n = 3). The numbers of engulfed apoptotic cells per cardiac macrophage (Mac) andmyofibroblast (Myo) are shown. Images were taken at 12–15 randomly selected fields. Arrows indicate engulfed apoptotic cells. Scale bar: 100 μm. Original
magnification, ×40. (B) mRNA expression levels of Il6 or Tgfb1 in myofibroblasts treated with LPS after apoptotic engulfment. Cardiac myofibroblasts cocultured
with (+) or without (−) apoptotic thymocytes (Apo) for 2 hours were treated with (+) or without (−) LPS at 1 μg/ml for 24 hours (n = 4). (C) αSMA-positive myofibroblasts did not express CD45. The cells collected from infarcted mouse hearts as myofibroblasts were harvested by treatment with accutase and immediately
stained with antibodies for αSMA and CD45. Error bars represent the mean ± SEM. (A) **P < 0.01, unpaired 2-tailed Student’s t test. (B) ***P < 0.001, 1-way
ANOVA followed by Newman-Keuls analysis.

administration promoted the restoration of cardiac function andmorphology after MI, suggesting that MFG-E8 is a new therapeutic target for the treatment of MI.

Results

Cardiac myofibroblasts efficiently engulf dead cells. In the infarcted area after MI, many dead cells are rapidly generated. We considered that the recruited inflammatory cells are not sufficientfor the removal of dead cells. Therefore, we hypothesized thatmyofibroblasts can engulf dead cells following MI in cooperation
with macrophages. To test this hypothesis, we examined whether
cardiac myofibroblasts isolated from infarcted hearts can engulf
apoptotic cells ex vivo. We performed permanent occlusion of theleft anterior descending artery to induce MI and isolated cardiac
macrophages (Supplemental Figure 1A; supplemental material
available online with this article; doi:10.1172/JCI83822DS1) (17)and myofibroblasts from mouse hearts 3 days after infarction. The
lack of surface-marker protein specific for myofibroblasts led us
to establish a method to selectively collect myofibroblasts fromthe infarcted hearts. Specifically, the infarcted mouse hearts were384

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digested by collagenase/trypsin, and the digested cardiac cells
were allowed to attach to the plates overnight. The attached cells
included macrophages and myofibroblasts (positive for α smoothmuscle actin [αSMA]) as well as other cardiac cells (Supplemental Figure 1B). Notably, cardiac myofibroblasts seemed to be moredifficult than cardiac macrophages to collect using our isolation
method from infarcted hearts because, as revealed by our immunohistochemical analysis, the number of cardiac myofibroblasts
was the same as that of cardiac macrophages in the infarcted area
(Supplemental Figure 1C). When the overnight-attached cells
were cultured in 10% FBS/DMEM for more than 6 days, almost
all of the cells on the plates were positive for αSMA and SM22α,
2 myofibroblast marker proteins (18, 19) (>97.9% and >93.8%,
respectively) (Supplemental Figure 1, D and E), indicating that thecells were primarily composed of cardiac myofibroblasts. This isprobably because only myofibroblasts were able to grow rapidly inthe culture medium.
Isolated cardiac macrophages and myofibroblasts wereallowed to engulf fluorescently labeled apoptotic cells, and we
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Figure 2. Cardiac myofibroblasts engulf dead cells in vivo. (A) Representative LV sections from WT mice that underwent MI were double stained with TUNEL(green) and anti-αSMA antibody (red) (n = 4). The area indicated by a white square on the merged image is enlarged. Kymograph along the white dashed line in
the enlarged image is shown. The area indicated by a white square on the enlarged image was further enlarged, and a 3D image in a white square on the enlarged
image is shown. White arrows indicate myofibroblasts containing an apoptotic cell. Scale bars: 30 μm. (B) Transmission electron micrographs of engulfment ofan apoptotic cell by a myofibroblast. The areas in yellow and red boxes within a myofibroblast (orange dotted line) are enlarged in the middle and right panels.
Well-developed rough-surfaced endoplasmic reticulum and intracellular actin filaments (arrowheads) can be seen in the myofibroblast. MN, myofibroblast nucleus; AN apoptotic nucleus; rER, rough-surfaced endoplasmic reticulum; Mt, mitochondria. Scale bar: 1 μm. (C) Quantification of engulfed TUNEL+ apoptotic cellsby macrophages (CD68+) or myofibroblasts (αSMA+) on the sections from mouse hearts 3 days after MI (n = 4). The number of engulfed apoptotic cells per square
millimeter of CD68+ macrophage or αSMA+ myofibroblast area is shown. Scale bars: 50 μm. (D) Representative infarcted LV sections from WT mice with EGFP-labeled cardiomyocytes were stained with anti-αSMA antibody (red) (n = 4). Scale bars: 30 μm. (E) Transmission electron micrographs of engulfment of a necrotic
cell by a myofibroblast. The areas in black and red dotted boxes are enlarged in middle and right panels. Necrotic nucleus (NN) was observed in the myofibroblast.
Arrowheads indicate single-membrane structure. Scale bar: 1 μm. Error bars represent the mean ± SEM. (C) **P < 0.01, unpaired 2-tailed Student’s t test.
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Figure 3. MFG-E8 expression is increased in the heart after MI. (A) mRNA expression levels of apoptotic cell recognition–related genes in hearts ofsham-operated mice (white bars, n = 3) and in infarcted (black bars, n = 5) or remote areas (gray bars, n = 5) of mouse hearts 3 days after MI. (B) Western
blot images of the long (black arrowhead) or short (white arrowhead) form of MFG-E8 in LV after sham (Sh) operation or MI. Western blot data were quantified (n = 3). Error bars represent the mean ± SEM. (A) *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Newman-Keuls analysis.
(B) *P < 0.05; **P < 0.01, unpaired 2-tailed Student’s t test.

myofibroblasts. As previously reported (20), when phagocytes
engulfed apoptotic cells, the apoptotic cells underwent a phasebright to phase-dark transition. We also used this transition
to distinguish engulfed apoptotic cells from those attached tophagocytes (Supplemental Figure 2A). Indeed, nearly all of thephase-dark apoptotic cells labeled with pH-sensitive pHrodo dyes
— which are fluorescent only in acidic environments — exhibited
fluorescence, indicating that these cells were internalized andfused with lysosomes (acidic organelles) in phagocytes (Supplemental Figure 2B). The fluorescence uptake and phase-bright tophase-dark transition revealed that cardiac macrophages effectively engulf apoptotic cells and, importantly, cardiac myofibroblasts also phagocytosed apoptotic cells. The engulfment abilityof cardiac fibroblasts was about two-thirds of that of cardiac macrophages (Figure 1A). In addition, engulfment assays, which were
performed for 6, 12, 18, and 24 hours, revealed that about 80%of the isolated cardiac myofibroblasts had the capacity to engulf
dead cells (Supplemental Figure 2C).
Necrotic and necroptotic cells were detected in addition to
apoptotic cells in the infarcted area (4, 5, 21). We determined
whether myofibroblasts engulf necrotic cells ex vivo. Fluorescently labeled L929 cells that underwent necroptosis (22)were used as prey in the engulfment assay. Fluorescence sig386

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nal was frequently localized in cardiac myofibroblasts, indicating that these cells also efficiently internalized necroptotic
cells (Supplemental Figure 2D).
Cardiac myofibroblasts acquire antiinflammatory properties
upon engulfment. Because cardiac myofibroblasts engulf deadcells in a manner similar to that of macrophages, we examined whether myofibroblasts had other engulfment-related
properties characteristic of macrophages. It was reported that
inflammatory cytokine production (e.g., IL-6) in macrophages
is reduced after the engulfment of apoptotic cells, whereas antiinflammatory cytokine production (e.g., TGF-β) in macrophages
is increased (23). We found that the production of IL-6 (encodedby Il6) induced by LPS treatment in myofibroblasts was significantly attenuated by the engulfment of apoptotic cells (Figure
1B), whereas the LPS–induced production of TGF-β (encoded by
Tgfb1) was increased following engulfment (Figure 1B). These
results suggested that myofibroblasts attenuated inflammationin the infarcted area in a manner similar to that of macrophages
and acquired antiinflammatory properties. We then costained
the cardiac myofibroblasts with antibodies against CD45 — ahematopoietic marker — and αSMA to determine whether thesecardiac myofibroblasts have a hematopoietic origin, similar to
macrophages. The costaining revealed that the cardiac myofi-

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Figure 4. MFG-E8 is expressed in cardiac myofibroblasts after MI. (A and B) MFG-E8 expression colocalizes with vimentin-positive (A) or αSMA-positive(B) myofibroblasts in LV 3 days after MI (n = 4). White squares on merged images mark the magnified areas. Arrows indicate merged cells. Scale bars: 50
μm (lower magnification); 20 μm (higher magnification). (C) Cardiac myofibroblasts from WT or MFG-E8 KO mice were costained with anti–MFG-E8 andanti-vimentin or anti-αSMA antibodies (n = 4). Numbers in quadrants indicate the percentages of cells. (D) Cardiac myofibroblasts were stained with anti–
MFG-E8 antibody without (−) or with saponin (+) permeabilization (n = 3). The staining profile (purple) and its control (without primary antibody; green)
are shown. (E) Staining of heart sections from non-MI patients and patients with MI by anti–MFG-E8 antibody (n = 4). Arrows indicate MFG-E8 signals.
Percentages of MFG-E8–positive cells in each area are shown in the graph. Scale bars: 30 μm. (F) Serial heart sections of patients with MI stained withanti–MFG-E8 antibody or anti-αSMA antibody. Arrows indicate double-positive myofibroblasts. Scale bar: 10 μm. Error bars represent the mean ± SEM.
(E) **P < 0.01, unpaired 2-tailed Student’s t test.

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Figure 5. MFG-E8–dependent engulfment by cardiac myofibroblasts has a protective effect on heart after MI. (A) Cardiac myofibroblasts from WT and
MFG-E8 KO mice were exposed to fluorescently labeled apoptotic cells in the absence (−) or presence of recombinant MFG-E8 (rMFG-E8). The number ofengulfed apoptotic cells per myofibroblast was determined (n = 4). Scale bars: 100 μm. (B) The staining profile of cardiac myofibroblasts with PE–antiintegrin αv antibody is shown in red (n = 3). The blue histogram represents the control. (C) Integrin β5 (red) and vimentin (green) staining of infarcted areaof heart sections from WT mice 3 days after MI (n = 3). A square on the first column marks the areas shown at a higher magnification. White arrows
indicate vimentin-positive myofibroblasts merged with integrin β5. The percentages of integrin β5–positive fibroblasts in remote or infarct area of hearts
after MI is shown in the graph. Scale bars: 50 μm (lower magnification); 20 μm (higher magnification). (D) Decreased survival rate of MFG-E8 KO mice
(sham, n = 8; MI, n = 38) compared with that of WT mice (sham, n = 11; MI, n = 36) after MI. Kaplan-Meier survival analysis using a log-rank test. ###P <0.001. (E) PicroSirius red staining of the cardiac sections of WT and MFG-E8 KO mice at day 3 after MI. The collagen volume fraction (CVF) was determinedby counting collagen-depositing areas (WT, n = 4; KO, n = 5). Scale bars: 100 μm. Error bars represent the mean ± SEM. (A) *P < 0.05; ***P < 0.001, 1-way
ANOVA followed by Newman-Keuls analysis. (C and E) ***P < 0.001; ##P < 0.01, unpaired 2-tailed Student’s t test.

broblasts did not express CD45 (Figure 1C), although we could
not exclude the possibility that the cardiac myofibroblasts lost
CD45 expression after differentiation. We also confirmed thatthese cardiac myofibroblasts do not express MHC class II (Supplemental Figure 3), ruling out the possibility that they function
as antigen-presenting cells.
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Cardiac myofibroblasts efficiently engulf apoptotic cells in vivo.
We then examined whether cardiac myofibroblasts engulf apoptotic cells in vivo. Heart sections were prepared from mice 3 days
after MI and double stained with TUNEL (green) and an antibody
against αSMA (red). In this double staining, unengulfed apoptotic cells display green fluorescence, but when apoptotic cells are

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Figure 6. MFG-E8–dependent engulfment by cardiac myofibroblasts influences the conditions of heart after MI. (A) TUNEL-positive nuclei in the borderzone of hearts in WT (n = 4) and MFG-E8 KO (n = 3) mice at day 3 after MI. The percentages indicate the ratio of TUNEL-positive nuclei to DAPI-positive
nuclei. Scale bars: 100 μm. (B) Representative LV sections from WT mice that underwent MI were double-stained with anti-TNNI3 (green) and anti-αSMA(red) antibodies (n = 5). The numbers of TNNI3-positive cardiomyocytes per square millimeter of αSMA-positive area in infarcted hearts of WT and
MFG-E8 KO mice are shown in the graph. Scale bar: 20 μm. (C) mRNA expression levels of inflammatory genes 3 days after sham operation or MI in thehearts of WT or MFG-E8 KO mice (sham, n = 4; MI, n = 5). (D) Echocardiographic measurements of WT mice (n = 7) or MFG-E8 KO mice (n = 5) at 4 weeks
after MI. Ejection fraction (EF) and fractional shortening (FS) are shown. (E) Ratio of heart weight (HW) to body weight (BW) of WT mice (n = 7) or MFG-E8
KO mice (n = 5) at 4 weeks after MI. (F) Representative heart sections of WT mice (n = 6) or MFG-E8 KO mice (n = 5) at 4 weeks after MI stained with
Masson’s trichrome. The ratios of the fibrotic area to the LV area were quantitatively estimated and are shown in the graph. Scale bars: 1 mm. Error bars
represent the mean ± SEM. (A, B, D–F) *P < 0.05; **P < 0.01; #P < 0.05, unpaired 2-tailed Student’s t test. (C) *P < 0.05; ***P < 0.001; #P < 0.05; ##P <0.01, 1-way ANOVA followed by Newman-Keuls analysis.

engulfed, they appear yellow as an overlay of green and red (24–26). Confocal microscopy showed yellow signal inside αSMA-positive cells at the infarct and border areas of infarcted hearts,
suggesting that cardiac myofibroblasts engulfed apoptotic cells
in these areas (Figure 2A). However, TUNEL- or αSMA-positive
signals were rarely observed in remote areas of infarcted hearts(Supplemental Figure 4).
To confirm that cardiac myofibroblasts engulf apoptotic
cells in vivo, we examined myofibroblasts in infarcted mouseheart tissue sections by electron microscopy. Myofibroblasts
are characterized by a well-developed rough endoplasmic reticulum and intracellular actin filaments (27). Many myofibroblasts were detected in the infarcted area, some of which hadinternalized an apoptotic nucleus (Figure 2B). These results
indicated that cardiac myofibroblasts take up apoptotic cells.
We then compared the contributions of cardiac myofibroblasts
and cardiac macrophages to the clearance of dead cells in MI. As
shown in Supplemental Figure 1C, the number of cardiac myofibroblasts was almost the same as the number of cardiac macrophages in
the infarcted area. TUNEL-positive signals in cardiac macrophages(CD68-positive cells) or myofibroblasts (αSMA-positive cells) in theinfarcted area were counted, and we found that the number of apoptotic cells engulfed by myofibroblasts in the infarcted hearts was
about 40% of that engulfed by macrophages (Figure 2C). These datasuggested that the contribution of cardiac myofibroblasts for engulfment was about two-fifths that of cardiac macrophages (Figure 2C).
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Figure 7. MFG-E8 is produced in myofibroblasts derived from resident cardiac fibroblasts or cells that underwent EndMT. (A) Schematic representationof the experimental design. LV sections from infarcted MFG-E8 KO mice (CD45.2+) transplanted with the bone marrow cells of WT mice (CD45.1+) were
stained with anti-CD45.1 (green) and anti–MFG-E8 (red) antibodies (n = 3). Scale bar: 50 μm. FACS dot plot of peripheral blood cells for recipient-type
CD45.2-APC and donor-type CD45.1-FITC is shown. (B and C) LV sections from WT mice on day 3 after MI were stained with anti–MFG-E8 (red) andanti-PDGFRβ (green) (B) or anti–MFG-E8 (red) and anti–Wt-1 (green) (C) antibodies (n = 3). White squares on the merged images (first columns) mark theareas shown at a higher magnification. Higher-magnification images are shown on the right side of lower-magnification images. Scale bars: 50 μm (lower
magnification); 20 μm (higher magnification). (D) LV sections from WT mice on day 3 after MI were stained with anti–MFG-E8 (red), anti-CD31 (green),
and anti-vimentin (blue) antibodies. The first column is the merged image of MFG-E8, CD31, and vimentin. The magnified views of the white boxed region
on the low-magnification merged images are displayed in a vertical row. The insets (lower panels) show the details of the MFG-E8, vimentin, and CD31
immunoreactive myofibroblasts. Scale bars: 50 μm (lower magnification); 20 μm (higher magnification). White arrows indicate merged cells. (E) Cardiac
myofibroblasts isolated from WT mice on day 3 after MI were stained with anti–MFG-E8, FITC-anti-αSMA, and PerCP/Cy5.5-anti-CD31 antibodies andanalyzed by flow cytometry (n = 4). Representative data are shown (A–E).

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Figure 8. MFG-E8 is produced in myofibroblasts via the SRF-dependent pathway. (A) Acta2, Adam12, and Mfge8 mRNA expression levels in rat neonatal
cardiac fibroblasts treated with TGF-β1 (24, 48, and 72 hours, n = 5). (B) Acta2 and Mfge8 mRNA expression levels in rat neonatal cardiac fibroblasts treatedwith TGF-β1 (10 ng/ml) in the absence or presence of CCG-1423 (1 or 10 μM, n = 5 or 6). (C) Acta2, Mfge8, and Srf mRNA expression levels in HUVECs treatedwith or without TGF-β2 (10 ng/ml) for 72 hours in the presence of siRNA against SRF (n = 4 or 5). (D and E) Acta2 and Mfge8 mRNA expression levels in cardiac
myofibroblasts, which were treated with or without CCG-1423 (1 or 10 μM) for 24 hours (D) or treated with siRNA against SRF (E) (n = 4 or 5). Decreased Srf
mRNA expression in myofibroblasts treated with siRNA against SRF was confirmed in E (n = 4 or 5). Error bars represent the mean ± SEM. (A–D) *P < 0.05;**P < 0.01; ***P < 0.001, 1-way ANOVA followed by Newman-Keuls analysis. (E) *P < 0.05; ***P < 0.001, unpaired 2-tailed Student’s t test.

To determine the identity of cells engulfed by myofibroblasts,
we specifically labeled cardiomyocytes with EGFP using the adenoassociated virus 9 (AAV9) vector system with the cardiac troponin
T (cTnT) promoter to drive cardiomyocyte-specific expression (28,29) (Supplemental Figure 5, A and B). Confocal microscopy showedthat αSMA-positive cells were located in the border areas of infarcted mouse hearts (Figure 2D), but not in remote areas (Supplemental Figure 5B). Furthermore, some EGFP signal was detected inside
αSMA-positive cells (Figure 2D), demonstrating that myofibroblasts
engulf dead cardiomyocytes in MI. This was confirmed by FACS
analysis; in the intact mouse heart, platelet-derived growth factor
receptor-α–positive (PDGFR-α–positive) cells were negative for EGFP(Supplemental Figure 5, C and D). However, 3 days after MI, a fraction of PDGFR-α–positive cells — most of which were presumed to be
myofibroblasts — expressed EGFP (Supplemental Figure 5, C and D).

We next investigated whether necrotic cardiomyocytes are
engulfed by myofibroblasts in vivo. Electron microscopy revealed
that myofibroblasts in the infarcted area contained a degradednucleus (Figure 2E), presumably derived from necrotic cardiomyocytes that were abundant in the infarcted area (Supplemental
Figure 6A). The phagosome-containing degraded nucleus was asingle-membrane structure of about 4 μm, indicating that the phagosome was different from autophagosome and autolysosome in
mammalian cells (0.5–1.5 μm) (30). We also investigated whether necroptotic cardiomyocytes are engulfed by myofibroblasts
in vivo. Cardiomyocytes were infected with EGFP fused withreceptor-interacting protein 3 (RIP3), which plays an importantrole in promoting necroptosis (31). RIP3 overexpression increases
the probability of necroptosis in cardiomyocytes (32). In infarcted hearts of mice with cardiomyocytes labeled with RIP3-EGFP,
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Figure 9. MFG-E8 administration after MI improves cardiac function in vivo. (A) Schematic representation of MFG-E8 intramyocardial injection. (B)
TUNEL-positive nuclei in border zone of PBS- or MFG-E8–administered mice (3.2 μg) (sham, n = 5; MI, n = 7) 3 days after MI. (C) MFG-E8 intramyocardial
injection (1.6 or 3.2 μg) decreased expression of the upregulated inflammatory genes at the infarct (Inf), not remote (Rem), areas of hearts 3 days after MI(n = 4 each). (D) Temporal changes in echocardiographic parameters (LV end-diastolic internal diameter [LVIDd], LV end-systolic internal diameter [LVIDs],
interventricular-septal thickness at end-diastole [IVSTd], ejection fraction, and fractional shortening) of PBS-treated (sham, n = 7, black-dotted lines; MI,n = 10, black lines) or MFG-E8–treated mice (sham, n = 7, red-dotted lines; MI; n = 7, red lines) 2, 6, 8, and 10 weeks after MI. (E) Hemodynamic parameters(dP/dtmax, -dP/dtmin, and Tau) of PBS-treated (sham, n = 3; MI, n = 7) or MFG-E8–treated mice (sham, n = 5; MI, n = 7) 10 weeks after MI. (F) Heart weight
to body weight ratio of PBS-treated (sham, n = 7; MI, n = 10) or MFG-E8–treated mice (sham, n = 7; MI, n = 9) 10 weeks after MI. (G) Representative Masson’s trichrome–stained heart sections of PBS-administered (n = 6) or MFG-E8–administered mice (n = 5) 10 weeks after MI. Scale bars: 1 mm. Error bars
represent the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA followed by Newman-Keuls analysis.

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Figure 10. Schematic model depicting origins and functions of MFG-E8–producing myofibroblasts appearing at
the infarcted area after MI. Resident fibroblasts and cells
that underwent EndMT differentiate into MFG-E8–producing myofibroblasts upon inflammation. MFG-E8 was
produced during the differentiation via the SRF-dependent
pathway. The myofibroblasts not only produce extracellularmatrix, but also engulf dead cells via an MFG-E8/integrin
αvβ5–dependent pathway, and attenuate inflammation,
similarly to macrophages.

we observed EGFP fluorescence inside αSMA-positive cells at theborder areas, suggesting that necroptotic cardiomyocytes were
engulfed by cardiac myofibroblasts (Supplemental Figure 6B).
These results provide in vivo evidence that myofibroblasts engulf
apoptotic and necrotic cardiomyocytes in MI.
MFG-E8 is upregulated in hearts after MI. To identify the molecules involved in the engulfment of dead cells by cardiac myofibroblasts after MI, we examined mRNA expression of several molecules that are involved in the recognition of apoptotic cells (7) in theinfarcted area. Three days after MI, infarcted mouse hearts were
surgically removed and subdivided into “infarcted areas” and “noninfarct remote areas.” RNA from each area was isolated. Real-timereverse-transcription PCR (RT-PCR) analysis revealed that mRNAlevels of Mfge8, a secreted protein that promotes engulfment (15, 16),and its corresponding receptor on phagocytes, integrin receptor αvβ5(Itgav, Itgb5) (15), were both increased in the infarcted areas, but notin the remote areas (Figure 3A). MER (Mertk), a phagocytic receptor
that recognizes apoptotic cells via GAS6, was also induced selectively in infarcted areas after occlusion (Figure 3A), whereas Gas6
expression was not increased. Conversely, the expression of TIM4
(Timd4) and stabilin 2 (Stab2) was decreased in the infarcted area
(Figure 3A). BAI-1, another phagocytic receptor, was not detected ineither intact or infarcted hearts.
Among the molecules selectively upregulated in infarcted
areas, Mer was recently reported to be expressed in macrophagesthat infiltrate the infarcted area and mediate the removal of deadcells (8). Thus, we focused on the MFG-E8/integrin αvβ5 pathway
of engulfment. Consistent with the RT-PCR result, the protein
levels of 2 isoforms of MFG-E8 (long and short forms) (16) greatly increased, peaking 4 days after MI surgery, and then gradually
decreased (Figure 3B). These results prompted us to investigate
whether MFG-E8 mediates engulfment by cardiac myofibroblasts.
MFG-E8 expression is induced in myofibroblasts after MI inmice and humans. We examined whether cardiac myofibroblasts
express MFG-E8 after MI in the heart. Immunohistochemical
staining of heart sections with an antibody against MFG-E8 at
1 or 3 days after occlusion revealed that MFG-E8–positive cells
were mainly found in the infarct and border areas where abun-dant cell death had occurred (Supplemental Figure 7, A–C). The
infarct areas were characterized by sparse DAPI signal (Supplemental Figure 7A–C). In contrast, MFG-E8–positive cells were
not detected in remote areas where few dead cells were generated(Supplemental Figure 7, A–C). Myofibroblasts are characterizedby vimentin (pan-fibroblast marker, fibroblast and myofibroblast
marker) and αSMA expression (19). Immunohistochemical staining revealed that vimentin- and αSMA-immunoreactive cells
exhibited positive immunostaining for MFG-E8, implying thatmyofibroblasts produce MFG-E8 (Figure 4, A and B). Consistent
with these results, isolated cardiac myofibroblasts were positive
for MFG-E8 (Supplemental Figure 7D). In contrast, inflammatory cells recruited to infarcted areas (i.e., infarct and border areas),
including CD68-positive macrophages (Supplemental Figure 8A)
and Gr-1–positive neutrophils (Supplemental Figure 8B) as well as
α-actinin–positive cardiomyocytes (Supplemental Figure 8C), did
not exhibit MFG-E8 immunoreactivity.
A previous report demonstrated that MFG-E8–positive signals can be detected via immunohistochemical analysis of splenic macrophages that do not produce MFG-E8 by themselves,
as MFG-E8 secreted from follicular dendritic cells is trappedby integrin receptors on the macrophages (25). Therefore, we
stained the cells collected by our procedure to selectively collectfibroblasts (both resident fibroblasts and myofibroblasts) frominfarcted hearts with anti–MFG-E8 antibody with or withoutsaponin treatment. As shown in Figure 4C, nearly 90% (WT,
20.6% + 68.3% = 88.9%; MFG-E8 KO, 85.0% + 1.9% = 86.9%)of the collected cells isolated by the procedure were vimentin
positive. We then coimmunostained the myofibroblasts isolated
from WT and Mfge8–/– mice (herein referred to as MFG-E8 KO)with anti–MFG-E8 antibody in combination with anti-vimentin
or anti-αSMA antibodies after saponin permeabilization andanalyzed them using FACS. Cardiac myofibroblasts (αSMA-positive cells) made up approximately 55% (WT, 21.8% + 34.2% =56.0%; MFG-E8 KO, 1.0% + 52.2% = 53.2%) of the collected
cells, and 40% (WT, (21.8%/[21.8% + 34.2%]) × 100%) of themyofibroblasts were MFG-E8 positive (Figure 4C). MFG-E8–
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blasts were isolated from MFG-E8 KO mice (Figure 4C). FACS
analysis also revealed that isolated cardiac myofibroblasts were
only stained with anti–MFG-E8 antibody after saponin treatment(Figure 4D), ruling out the possibility that MFG-E8 was trappedby integrin receptors. We also confirmed that MFG-E8–positive
cells were negative for CD21/CD35, a typical follicular dendritic
cell marker (33) (Supplemental Figure 8D). Consistently, immunohistochemical analysis demonstrated that CD21/CD35-positive cells were not found in the infarcted hearts (Supplemental
Figure 8E). These results demonstrated that a portion of cardiac
myofibroblasts produced MFG-E8 after MI.
We next examined whether MFG-E8–producing cells wereobserved in the hearts of patients with MI. MFG-E8–expressing cells selectively appeared in the interstitium of the infarcted
area in MI patients (Figure 4E). We then examined whether these
MFG-E8–positive cells are myofibroblasts. We used serial heart
sections of patients with MI stained for MFG-E8 and αSMA staining. The staining revealed that MFG-E8–positive cells expressed
αSMA (Figure 4F), suggesting that MFG-E8–producing myofibroblasts play a role in the pathogenesis of human MI.
Myofibroblasts engulf apoptotic cells via MFG-E8. We assessed
the ability of myofibroblasts from WT and MFG-E8 KO mice toengulf apoptotic cells. We found that myofibroblasts from WT
mice efficiently phagocytosed apoptotic cells, but myofibroblasts
isolated from MFG-E8 KO mice exhibited severely decreased
engulfment (Figure 5A). This diminished engulfment was recovered by MFG-E8 addition in a concentration-dependent manner(Figure 5A), demonstrating that engulfment by cardiac myofibroblasts depends on MFG-E8.
We then investigated whether cardiac myofibroblasts express
integrin αvβ5, a receptor for MFG-E8. FACS analysis of cells stainedwith an antibody against integrin αv revealed that the receptor was
expressed in cardiac myofibroblasts (Figure 5B). Furthermore,immunohistochemical analysis demonstrated that most vimentin-positive signals in infarcted areas were found in integrin β5-positive cells, indicating that myofibroblasts expressed integrin β5 (Figure
5C). These cells were not detected in remote areas of infarcted hearts(Supplemental Figure 9). These results suggested that MFG-E8/
integrin αvβ5 mediated engulfment by cardiac myofibroblasts.
Hyperinflammation caused by accumulation of dead cells after
MI in MFG-E8 KO mice leads to deterioration of cardiac function andmorphology. To investigate the physiological importance of engulfment by cardiac myofibroblasts via MFG-E8 in MI, we assessed
the survival of WT and MFG-E8 KO mice after the infarction. In
WT mice, the survival rate 28 days after MI was 72.2% (n = 36), but
the rate in MFG-E8 KO mice (n = 38) was remarkably decreased
to 26.3% (Figure 5D), indicating that MFG-E8 plays a critical rolein the healing process after MI. Evans blue dye and 2,3,5-triphenyl tetrazolium chloride (TTC) staining of heart sections from
WT and MFG-E8 KO mice 3 hours after MI revealed that the sizeof area at risk (AAR) and infarct area were similar between the 2groups (Supplemental Figure 10A). H&E and PicroSirius red staining of heart sections revealed that wall thickening (Supplemental
Figure 10B) and fibrosis (Figure 5E) were attenuated in MFG-E8
KO mice 3 days after MI, suggesting an increase of cardiac rupture after MI in the KOs. Consistent with these observations, thenumber of myofibroblasts (Supplemental Figure 10C) and the394

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induction of actin alpha (Acta2), which encodes αSMA, and thegenes encoding integrin αVβ5 at the infarcted area were reduced in
MFG-E8 KO mice (Supplemental Figure 10D).
The difference in the survival rate between WT and MFG-E8
KO mice was evident during days 3–5 after MI (Figure 5D). We
compared the condition of the WT versus MFG-E8 KO mousehearts 3 days after infarction to explore the role of MFG-E8–mediated engulfment by cardiac myofibroblasts. We first performed a
TUNEL assay of infarcted heart sections of WT and MFG-E8 KO
mice to test for defects in apoptotic engulfment. The assay revealed
that the number of TUNEL-positive cells was increased in theinfarcted heart tissues of MFG-E8 KO mice (Figure 6A). Consistent
with this result, the ability of cardiac myofibroblasts to engulf deadcardiomyocytes was decreased in MFG-E8 KO mice (Figure 6B
and Supplemental Figure 11A). The addition of MFG-E8 to cardiomyocytes did not influence their death (Supplemental Figure 11B),
suggesting that MFG-E8 does not contribute to cardiomyocyte cell
death, as in the case of splenocytes (24). Unengulfed apoptotic
cells undergo secondary necrosis, leading to the leakage of theirintracellular contents and excessive inflammation (7). We thenexamined the expression levels of inflammatory genes in heart tissues of WT and MFG-E8 KO mice after MI. RT-PCR experiments
demonstrated that the inflammatory genes Il1b, Cxcl2, Tnf, Il6, andMmp9 were upregulated by MI and that the upregulation was significantly higher in MFG-E8 KO mice (Figure 6C and Supplemental Figure 12A). However, the induction of antiinflammatory genes
in the heart after MI was similar between WT and MFG-E8 KO
mice (Supplemental Figure 12B). These results demonstrated thatthe accumulation of apoptotic cells and inflammatory responses
after MI were augmented in the MFG-E8 KO mice due to a defectin engulfment by cardiac myofibroblasts.
Hyperinflammation of the infarcted area leads to deteriorationof cardiac function after MI (5, 9). We therefore examined cardiac
function, morphology, and infarct size 28 days after MI. Echocardiography and hemodynamic measurements demonstrated thatcardiac function and morphology were deteriorated in MFG-E8
KO mice (Figure 6D and Supplemental Figure 13), while there wasno difference between nonoperated WT and MFG-E8 KO micein terms of cardiac function (Supplemental Table 1). In addition,
heart weight and infarct area after MI were markedly increased in
MFG-E8 KO relative to those in control mice (Figure 6, E and F).
MFG-E8–producing myofibroblasts are derived from resident
fibroblasts and cells that have undergone EndMT. It has been reported that cardiac myofibroblasts are derived from the following 5cell types during inflammation (14, 34): bone marrow–derived
cells, pericytes, cells that have undergone EMT and EndMT, andresident fibroblasts. As shown in Figure 4C, 40% of cardiac myofibroblasts (αSMA-positive cells) were MFG-E8 positive 3 days
after MI. This result indicates that MFG-E8 is a new determinantthat functionally characterizes a subpopulation of myofibroblasts.
Therefore, we sought to determine the origin of MFG-E8–producing myofibroblasts. We first examined whether bone marrowcells differentiated into MFG-E8–producing myofibroblasts. Bonemarrow cells obtained from WT mice (CD45.1 allotype) weretransplanted into lethally irradiated MFG-E8 KO mice (CD45.2
allotype), and 2 weeks after the transplantation, the engraftment
efficiency of these cells in the mice was determined to be more

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than 95% (Figure 7A). After MI, CD45.1-positive cells appearedin the infarcted area, but MFG-E8–positive myofibroblasts were
not observed (Figure 7A). Further, to assess whether MFG-E8–
producing myofibroblasts were derived from pericytes, infarcted
heart sections were coimmunostained with antibodies against
MFG-E8 and PDGFRβ (35). However, PDGFRβ failed to colocalize
with MFG-E8 (Figure 7B). Furthermore, we tested to determine
whether MFG-E8–producing myofibroblasts originated from cells
that underwent EMT, identified by Wt1 expression (36, 37). As
reported previously, myofibroblasts positive for Wt1 were found in
the infarcted area (36), but these cells did not produce MFG-E8(Figure 7C). In addition, the upregulation of other EMT markers— namely, T-box 18 (Tbx18) and Snai1 (37, 38) — in infarcted areas
was comparable between WT and MFG-E8 KO mice, suggestingthat MFG-E8 does not contribute to EMT (Supplemental Figure
14A). Cells expressing S100A4 (fibroblast-specific protein-1), a
proposed marker for a subset of myofibroblasts (39), were similarly negative for MFG-E8 (Supplemental Figure 14B).
Conversely, triple immunohistochemical staining for CD31,
a marker for myofibroblasts that underwent EndMT (39, 40),
MFG-E8, and vimentin, revealed that some cells are triple positive,
indicating that a portion of MFG-E8–producing myofibroblasts were
derived from EndMT cells (CD31-positive) (Figure 7D). Consistently, FACS analysis of cardiac myofibroblasts using CD31, MFG-E8,
and αSMA antibodies demonstrated that approximately 20% of
MFG-E8–producing myofibroblasts were derived from EndMT cells(Figure 7E). Previous reports (14, 34) suggested that myofibroblasts
that were not derived from bone marrow–derived cells, pericytes,
or cells that underwent EMT or EndMT were generated from resident cardiac fibroblasts because a specific marker for myofibroblasts
differentiated from resident fibroblasts has not yet been identified.
Therefore, we reasoned that the remaining MFG-E8–positive myofibroblasts (approximately 80%) originated from resident cardiac
fibroblasts. Taken together, these results suggested that MFG-E8–
positive myofibroblasts were derived mainly from cardiac-resident
fibroblasts and from cells that underwent EndMT.
MFG-E8 expression in myofibroblasts is induced by the SRFdependent pathway. Despite the importance of MFG-E8 in apoptotic
engulfment by macrophages (24, 25), the molecular machinery regulating MFG-E8 production in phagocytes has long been obscure.
We set out to investigate the mechanism underlying MFG-E8 production by myofibroblasts. Resident cardiac fibroblasts were foundto be the main source of MFG-E8–positive cardiac myofibroblasts.
We thus examined whether MFG-E8 was induced in resident cardiac fibroblasts by TGF-β treatment, which transdifferentiates theminto myofibroblasts (41, 42). Because it is difficult to acquire a sufficient number of resident cardiac fibroblasts from mice for in vitro
experiments, we used a transdifferentiation system using rat neonatal resident cardiac fibroblasts (41). As reported previously (41),treatment of rat neonatal resident cardiac fibroblasts with TGF-β1
induced the expression of Acta2, which encodes αSMA, and a disintegrin and metalloproteinase 12 (ADAM12), another myofibroblast marker protein (43) (Figure 8A), indicating that the resident
fibroblasts were transdifferentiated. In the TGF-β1–stimulated resident fibroblasts, Mfge8 expression was induced (Figure 8A) witha time course similar to those of Acta2 and Adam12 (Figure 8A).
Because Acta2 expression induced by TGF-β treatment was report-

ed to depend on SRF (41, 44), we examined whether SRF is also
involved in MFG-E8 production. RT-PCR analysis demonstrated
that pretreatment of resident fibroblasts with CCG-1423 (45), an
inhibitor of SRF, attenuated Acta2 expression induced by TGF-β1
in a concentration-dependent manner (Figure 8B). Similar to Acta2
expression, TGF-β–induced Mfge8 expression was significantly
attenuated by CCG-1423 treatment in resident cardiac fibroblast(Figure 8B), suggesting that SRF mediated TGF-β–induced Mfge8
expression in these cells.
We further examined whether MFG-E8 was also induced
in EndMT cells during their differentiation into myofibroblasts.
It was reported that HUVECs undergo EndMT by TGF-β2 stimulation and they can be used as a cell model for myofibroblasts
derived from cells that have undergone EndMT (46). We thus
examined whether MFG-E8 expression was increased in HUVECs
stimulated by TGF-β2. The stimulation induced Acta2 and Adam12
expression in the cells, similarly to TGF-β1–treated resident fibroblasts, indicating that they acquired myofibroblast-like properties(Figure 8C and Supplemental Figure 15A). In the TGF-β2–stimulated HUVECs, Mfge8 expression was induced, and the expression was inhibited by treatment with siRNA against SRF (Figure8C). These results indicated MFG-E8 was induced in HUVECs by
TGF-β via SRF, suggesting that myofibroblasts differentiated fromresident fibroblasts and EndMT cells share common TGF-β/SRF–
dependent machinery for MFG-E8 production.
To examine whether mechanisms of MFG-E8 production invitro apply in vivo, we treated myofibroblasts isolated from infarcted mouse hearts with CCG-1423 or siRNA against SRF and evaluated Mfge8 expression. CCG-1423 (Figure 8D) and siRNA (Figure8E) treatment significantly attenuated Mfge8 expression as well as
Acta2 expression. We also confirmed that the expression of TGF-β1
and TGF-β2 was selectively increased in infarcted areas (Supplemental Figure 15B). These results suggested that MFG-E8 production in myofibroblasts is mediated by the TGF-β/SRF pathway.
MFG-E8 administration improves the conditions of hearts after
MI. We revealed a protective role of engulfment by cardiac myofibroblasts via MFG-E8 against MI. Because MFG-E8 is a secreted
protein, it can be administered to infarcted mouse hearts. Therefore, we examined whether MFG-E8 treatment after MI leads torecovery of infarcted hearts. Ultrapurified recombinant mouse
MFG-E8 protein was intramyocardially administered to WT mice
immediately after surgery to induce MI (Figure 9A). Evans blue/
tetrazolium chloride staining revealed that the areas at risk andthe infarct size were comparable between PBS-treated mice and
MFG-E8–treated mice during the initial phase after MI (Supplemental Figure 16A). However, 3 days after surgery, we found fewer TUNEL-positive cells in the infarcted areas of MFG-E8–treated mice, suggesting that dead cells were efficiently removed by
MFG-E8 treatment (Figure 9B). Furthermore, MFG-E8 administration significantly attenuated the upregulation of inflammatory
genes (Il1b and Cxcl2) that occurred after surgery to induce MI in
a concentration-dependent manner (Figure 9C). Consistent withthese results, MFG-E8 administration improved survival rates
after MI, although the difference was not statistically significant(Supplemental Figure 16B). The favorable effects by MFG-E8
administration were not so prominent in normal hearts (Figure 9,
B and C), suggesting a critical role of MFG-E8 in MI. On the othjci.org-213-

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did not induce the mobilization of myofibroblasts in tissues otherthan the heart, confirming that the effects of MFG-E8 were heart
specific (Supplemental Figure 17). These results demonstrated
that MFG-E8 treatment promoted the engulfment of dead cells in
the infarcted area, resulting in the reduction of secondary necrosis
and inflammatory responses in the area.
We next examined whether the effects of MFG-E8 administration on infarcted hearts were mediated by the MFG-E8/integrin
engulfment pathway. It was previously reported that the MFG-E8
D89E mutant failed to bind integrins and inhibited the engulfmentof apoptotic cells by masking exposed phosphatidylserine (24). We
administered recombinant MFG-E8 D89E protein to mice after
MI. The survival rate after MI was reduced by MFG-E8 D89E treatment, although the difference was not statistically significant (P =0.30, Supplemental Figure 18A). The number of TUNEL-positive
cells in infarcted areas of the heart in MFG-E8 D89E–treated mice
was increased (Supplemental Figure 18B) along with the degreeof inflammation in the infarcted area (Supplemental Figure 18C),
indicating that the salutary effect of MFG-E8 on the infarcted heart
involves MFG-E8–mediated engulfment.
We next assessed whether cardiac morphology and function
in infarcted mice were improved by MFG-E8 treatment. MFG-E8
treatment did not affect the morphology and function of normalmouse hearts (Figure 9, C–G, and Supplemental Figure 16D).
However, echocardiographic measurements performed on thehearts of PBS-treated and MFG-E8–treated mice at 2, 6, 8, and10 weeks after MI showed that MFG-E8 treatment significantly
attenuated the deterioration of cardiac morphology and function after MI (Figure 9D). Consistently, catheter-based measurements 10 weeks after MI indicated that the intramyocardialadministration of MFG-E8 improves cardiac function (Figure9E). Significant decreases in heart weight and infarct area werealso observed in MFG-E8–administered mice 10 weeks after MI(Figure 9, F and G). In contrast, treatment with MFG-E8 D89E
reduced cardiac function and increased infarct area and heart
weight after MI (Supplemental Figure 18, D–G). These findings
indicated that MFG-E8 treatment improved the condition of theheart after MI by promoting the engulfment of dead cells, suggesting that MFG-E8 could be a new therapeutic target for thetreatment of MI in the future.

Discussion

To date, dead cells generated in MI were believed to be removed
only by infiltrating immune cells, such as macrophages and dendritic cells recruited to the infarcted area (5, 8). Our study identified myofibroblasts as responsible for the removal of dead cells in
MI (Figure 10). Cardiac engulfment was found to be dependent
on MFG-E8. Myofibroblasts engulf dead cells and acquire antiinflammatory properties upon engulfment, similar to macrophages.
Both macrophage-like properties of cardiac myofibroblasts greatly
contribute to limiting the size of the infarcted area. In fact, in theinfarcted area of MFG-E8 KO mice, the number of unengulfed
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mation was induced, which led to a decrease in the number ofmyofibroblasts at the infarcted area as well as decreases in wall
thickness and survival rate after MI (47, 48) (Figure 5, Figure 6, andSupplemental Figure 10). Based on evidence that macrophagesthat infiltrate the infarcted area contribute to the clearance of deadcells by using Mer, a phagocytic receptor (8), we hypothesized that
infiltrated macrophages and myofibroblasts cooperate with eachother to remove dead cells by using different molecules in theinfarcted heart. In the infarcted area, an abundance of dead cells
is rapidly generated. Thus, it is reasonable to assume that myofibroblasts differentiated from resident fibroblasts, which originally reside near dead cardiomyocytes, actively participate in theremoval of dead cells. In our study, we used MFG-E8 KO mice forexperiments because MFG-E8 is expressed in myofibroblasts, but
not cardiomyocytes or inflammatory cells, in infarcted areas of theheart. However, to understand the precise contribution of myofibroblasts to the elimination of dead cells in MI via MFG-E8–mediated engulfment, myofibroblast-specific MFG-E8 conditional KO
mice would be useful. Similarly to what occurs in MI, we assume
that myofibroblasts that appear in other damaged tissues also
actively contribute to elimination of dead cells and attenuation of
inflammation. In fact, we found that MFG-E8 is also expressed in
myofibroblasts in a mouse model of liver injury (Supplemental Figure 19). It will be interesting to examine whether myofibroblasts inother tissues have the ability to engulf apoptotic cells and express
engulfment-related molecules, including MFG-E8.
Myofibroblasts have long been known to facilitate fibrosis. In
the current model of tissue repair after injury, myofibroblasts are
believed to produce extracellular matrix proteins only after theyreceive cytokines released from macrophages that recognize
dead cells and “danger signals,” such as inflammatory mediators
leaked from dead cells at injury sites (49). However, the findingthat cardiac myofibroblasts appear after MI, engulf dead cells,
and acquire macrophage-like antiinflammatory properties uponengulfment raises the possibility that engulfment by myofibroblasts itself triggers the production of extracellular matrix proteins without the assistance of macrophages. In this regard, it
will be of great value to examine whether engulfment by cardiac
myofibroblasts influences the production of extracellular matrix
proteins to reassign the role of myofibroblasts in tissue repair
and subsequent fibrosis development. It was recently reported
that the autophagy machinery in macrophages is recruited tophagosomes containing dead cells and regulates the expressionof proinflammatory genes, a process that requires the autophagy-related molecule ATG-7 (50). If this also occurs in cardiac
myofibroblasts, it could modulate the production of extracellular matrix proteins. Interestingly, Atg7 expression was increased
in infarcted hearts, an effect that was attenuated in MFG-E8 KO
mice, raising the possibility that MFG-E8 influences autophagy(Supplemental Figure 20).
Myofibroblasts have been considered to have diverse functions because they originate from multiple cell types (11–13).
However, only a few proteins have been reported to be involved
in determining functional differences between cardiac myofibroblasts. The results of the present study indicate that MFG-E8 was
produced only by a population of myofibroblasts differentiatedfrom resident fibroblasts and cells that had undergone EndMT

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among the 5 cellular origins of cardiac myofibroblasts. Therefore,
MFG-E8 can be potentially used as a new marker that characterizes cardiac myofibroblasts that appear after MI. MFG-E8 expression may endow a special engulfment activity to a subset of cardiac myofibroblasts. Future studies examining the engulfment
ability of non–MFG-E8–producing cardiac myofibroblasts will
clarify this point and elucidate functional differences in a variety
of myofibroblasts that appear in MI.
So far, none of the proteins involved in the removal of deadcells following MI have been reported as potential therapeutictargets for the treatment. Here, we demonstrated that MFG-E8
administration substantially improved cardiac function after MI.
However, further optimization in MFG-E8 administration methods remains to be examined in detail to maximize its effects on
infarcted hearts. MFG-E8 treatment can also be used in combination with other emerging candidates for treatments that target
molecules other than those involved in engulfment (51).

Methods

Mice. Eight- to ten-week-old male WT (C57BL/6) (Charles River) and
MFG-E8 KO mice (backcrossed for more than 7 generations on the
C57BL/6 background) (24) were used in the study.
MI. Mice were anesthetized with pentobarbital (50 mg/kg) andsubjected to permanent occlusion of the left coronary artery.
Culture and isolation of cardiac cells. At 3 days after operation,
the ventricles of MI-operated mice were digested with PBS containing 0.1% collagenase A (Roche Diagnostics) and 0.1% trypsin(Sigma-Aldrich) at 37°C with agitation. Cells were placed in plates
containing 10% FBS/DMEM overnight, and the culture medium
was then changed to remove the unattached cells. The attached cells
were cultured for more than 6 days and used as myofibroblasts in
our in vitro experiments. We used myofibroblasts from passage 1 (P1)throughout our study. Myofibroblasts were transfected with siRNAusing Lipofectamine 2000 (Invitrogen). All validated siRNAs werepurchased from Ambion. The target sequence for human SRF was
5′-UGAUGUACCCUAGCCCGCATT-3′, while that for mouse Srf
was 5′-GCCUUUUCACGGUUUCUUUTT-3′. Silencer Select Negative Control no. 1 siRNA was used as control in each experiment(Thermo Fisher Scientific).
Neonatal cardiac fibroblasts and cardiomyocytes were isolated
from the heart ventricles of 2-day-old Sprague-Dawley rats (Kyudo
Laboratories). The ventricles were digested with Liberase Research
Grade (Roche Diagnostics). The digested cells were plated on dishes
and incubated for 1 hour at 37°C, and adherent and nonadherent cells
were harvested as cardiac fibroblasts and cardiomyocytes, respectively.
HUVECs purchased from ATCC were grown in endothelial cell
growth medium-2 (Lonza) on collagen I–coated plates (BD Biosciences). The cells were transfected with siRNA targeting human SRF
or control scrambled siRNA using Lipofectamine 2000. At 24 hours
after transfection, the HUVECs were starved with human endothelial serum-free medium-2 (Invitrogen) for 24 hours. These cells werethen cultured in human endothelial SFM with or without TGF-β2(R&D Systems) for 3 days.
Production of AAV9-cTnT-EGFP. HEK293 cells were cotransfectedwith an AAV transgene plasmid (pENN-PI-cTnT-eGFP-WPRE-rBG)
and 2 helper plasmids (pXR9 and pXX6-80) using polyethylenimine“Max” (Polysciences). The AAV transgene plasmid pENN-cTnT-PI-

eGFP-WPRE-rBG was purchased from Penn Vector Core. The AAV
serotype helper plasmid pXR9 and the adenoviral helper plasmid
pXX6-80 were gifts from J. E. Rabinowitz (Temple University, Philadelphia, Pennsylvania, USA). AAV was purified with ultracentrifugation as previously reported, with some modifications (29). The virus
titer was determined by real-time RT-PCR (52).
In vivo phagocytosis assay. Virus carrying the cDNA for EGFP was
diluted in PBS to adjust the viral titer (cTnT-EGFP-AAV9 = 1 × 1012
copies/μl), and 20 μl of the viral solution was injected in 3-day-old
mice (by subcutaneous injection) with 29-gauge needles (BD Biosciences). Eight to ten weeks after injection, mice were subjectedto the MI operation. For FACS analysis, at 3 days after operation,
hearts were digested using a cocktail of collagenase type II (CLS2)(Worthington) and elastase (ESL) (Worthington) in PBS at 37°C
with agitation. After digestion, cells were stained with eFluor780
dye (1:1000, 65-0865; eBioscience) and phycoerythrin-conjugated(PE-conjugated) anti-CD11b (1:200, M1/70; BioLegend), and allophycocyanin-conjugated (APC-conjugated) anti–PDGFRα (1:200,
5A5; BioLegend) antibodies were added. Stained cells were sortedwith a FACSAria III (BD Biosciences).
For immunohistochemical analyses, the hearts of WT, MFG-E8
KO, and AAV9-cTnT-EGFP–administered mice 3 days after MI were
fixed in 4% PFA, embedded in OCT compound (Sakura FineTek),and cut into 6-μm–thick sections using a cryostat (Leica Microsystems). The sections were labeled with Cy3-conjugated anti-αSMA(1:400, 1A4; Sigma-Aldrich), anti-GFP (1:300, ab13970; Abcam), andanti-cardiac troponin I3 (TNNI3) (1:200, ab56357; Abcam) antibodies. Images were captured using a confocal microscope (LSM700,
Zeiss). Over 1200 αSMA-positive cells were evaluated, and engulfment capacity was determined by dividing the number of engulfed
apoptotic cells by the area of αSMA-positive cells, averaged from 5 to
11 randomly selected fields.
Labeling of necroptotic cardiomyocytes with AAV. AAV9-expressing
RIP3-EGFP under the control of the cTnT promoter was administeredintramyocardially to 7-week-old WT mice (4 × 1012 copies/mouse).
Mice expressing RIP3-EGFP specifically in cardiomyocytes (10 weeksold) were subjected to MI. Three days after the operation, their hearts
were fixed in 4% PFA. Cryostat sections were labeled with Cy3-conjugated anti-αSMA (1:400) and anti-GFP (1:300) antibodies.
Electron microscope. The infarct and border areas of infarcted
hearts 3 days after MI were dissected and immersed in 2% PFA and
2% glutaraldehyde buffered with 0.1 M phosphate buffer at 4°C overnight. Samples were post-fixed with 2% OsO4 in 0.1 M phosphatebuffer at 4°C for 2 hours. Tissue samples were then dehydrated in a
graded series of ethanol and embedded in Quetol-812 (Nisshin EM).
Ultrathin sections were prepared on an ultramicrotome (EM UC7; Leica) and stained with 2% uranyl acetate and lead citrate. Sections werevisualized by transmission electron microscopy (Tecnai 20, FEI Co.).
RT-PCR. Total RNA of various tissues was extracted from WT or
MFG-E8 KO mice using Isogen (Nippon Gene) and purified using an
RNeasy Plus Mini Kit (QIAGEN). Total RNA was isolated from neonatal rat
cardiac fibroblasts, mouse myofibroblasts, and HUVECs using an RNeasy
Micro Kit (QIAGEN). RT-PCR was carried out using a One Step PrimeScript RT-PCR Kit (Takara Bio) on a 7500 Fast Real-Time PCR System(Applied Biosystems). The following TaqMan probes were purchased from
Applied Biosystems: Mfge8 (Mm00500549_m1), Mfge8 (Rn00563082_
m1), Mfge8 (Hs00170712_m1), Itgav (Mm00434486_m1), Itgb5
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The Journal of Clinical Investigation(Mm00439825_m1), Tnfα (Mm00443258_m1), Il1b (Mm00434228_m1),
Acta2 (Rn01759928_g1), Acta2 (Mm00725412_s1), Acta2 (Hs00426835_g1), Adam12 (Rn01479221_m1), Adam12 (Mm00475719_m1), Srf(Mm00491032_m1), Tgfb1 (Mm01178820_m1), Tgfb2 (Mm00436955_
m1), Ctgf (Mm01192932_g1), and eukaryotic 18S rRNA (Hs03003631_
g1). Other primer sequences used for real-time RT-PCR included thefollowing: Il6 (forward: 5′-GGGACTGATGCTGGTGACAA-3′, reverse:
5′-TGCCATTGCACAACTCTTTTCT-3′, probe: 5′-TCACAGAGGATACCACTCCCAACAGACCTG-3′); macrophage inflammatory protein-2 (Cxcl2) (forward: 5′-CGCTGTCAATGCCTGAAGAC-3′, reverse:
5′-CCTTGAGAGTGGCTATGACTTCTG-3′, probe: 5′-TCCAGAGCTTGAGTGTGACGCCCC-3′); Gas6 (forward: 5′-CCTGCCAGAAGTATCGGTGATT-3′, reverse: 5′-CCCGTTTACCTCCAGAGTCATG-3′, probe:
5′-CACCCGTCACTGCGTTCTACCGC-3′), Mertk (forward: 5′-CTGCATGTTGCGGGATGAC-3′, reverse: 5′-ACTTCACAGGCATTTTGGCAAT-3′, probe: 5′-CTGCGTGGCAGACTTTGGCCTCTC-3′); Timd4(forward: 5′-CCCAGAGCTGCTTCCAACA-3′, reverse: 5′-CAGGCAGAGTTTCTGATTCTGTAGTG-3′, probe: 5′-ACACTAGCCACCACTGCCTTCAGTACAGCA-3′); Stab2 (forward: 5′-CACTGACCGGAGTTCTCATTCC-3′, reverse: 5′-ACCAATTTTCAGGTTCCATGCT-3′, probe:
5′-TCGGAGCCCACGGCAATCTTCAC-3′); matrix metalloproteinase-9 (Mmp9) (forward: 5′-GGCCCCAGGAGTCTGGATA-3′, reverse:
5′-AATAGGCTTTGTCTTGGTACTGGAA-3′, probe: 5′-ACCCACGTCAGCGGGCTTCTCC-3′); Wt1 (forward: 5′-GAGCAACCACGGCACAGG-3′, reverse: 5′-GCTGACCGGACAAGAGTTGG-3′, probe:
5′-CTCCAGATACACGCCGCACATCCTGAATG-3′); Tbx18 (forward:
5′-CTACGGACTCTCACCTTTGAAGATA-3′, reverse: 5′-TGAGGATGTGTAGCAGGGACA-3′, probe: 5′-CCCAAAGCAAGGCAACACAAGTTCTTCAGC-3′); Snai1 (forward: 5′-CCGCCGGAAGCCCAACTA-3′, reverse: 5′-TGAGGGTGGGCAGCGAAG-3′, probe:
5′-CGTGTGTGGAGTTCACCTTCCAGCAGC-3′); Atg7 (forward:
5′-CTCACCAGATCCGGGGTTTTC-3′, reverse: 5′-GAATCCTTCTCGCTCGTACTGA-3′, probe: 5′-TTCGATAATGTTCTTCCTGTCAGCCTGGCA-3′); Becn1 (forward: 5′-CAAGCTCAAGAAAACCAATGTCTTC-3′, reverse: (5′-CAGGCAGCATTGATTTCATTCCA-3′, probe:
5′-CCTTCCACATCTGGCACAGCGGACAGT-3′); and Gapdh (forward:
5′-TGCCCCCATGTTTGTGATG-3′, reverse: 5′-GGCATGGACTGTGGTCATGA-3′, probe: 5′-ACCACCAACTGCTTAGCCCCCCTG-3′)
The results were normalized to GAPDH or 18S rRNA.
Flow cytometry. Isolated cardiac myofibroblasts were harvested
with accutase treatment and subsequently stained with the following
antibodies: PE–anti-CD45 (1:200, 30-F11, BioLegend), APC–antiCD68 (1:200, FA-11, BioLegend), FITC–anti-αSMA(1:200, 1A4; Sigma-Aldrich), peridinin chlorophyll protein (PerCP)/eFluor710–anti–
MHC class II (1:200, 46-5321-80; eBioscience), anti-SM22α antibody(1:200, ab14106; Abcam), APC–anti-PDGFRα (1:200, APA5; BioLegend), and PE–anti-integrin αv (1:200, RMV-7; BioLegend) antibody.
For αSMA and SM22α staining, cells were permeabilized with 0.5%
saponin before staining. For MFG-E8 labeling, cells were incubatedwith PerCP/Cy5.5–anti-CD31 (1:100, 390; BioLegend) and PE–antiCD21/CD35 (1:100, 7E9; BioLegend) antibodies, followed by permeabilization with 0.5% saponin. Cells were then labeled with anti–
MFG-E8 (1:200, 2422; MBL), anti-vimentin (1:100, GP53; Progen),and FITC-conjugated anti-αSMA (1:200, 1A4; Sigma-Aldrich) antibodies. Alexa Fluor 647–conjugated anti-hamster IgG, Alexa Fluor
488–conjugated anti-guinea pig IgG (both from Jackson ImmunoResearch), or Alexa Fluor 488–conjugated anti-rabbit IgG (Invitro398

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were analyzed using FACSCalibur or FACSAria III.
Western blot analysis. Hearts of mice were homogenized in alysis buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1.0% NP-40,
0.5% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF) containing protease inhibitors (Nacalai). Lysed tissues were subjected toWestern blotting using anti–MFG-E8 (1:2,000, 18A2-G10; MBL)
followed by HRP–anti-hamster IgG secondary antibody (1:5,000,
Santa Cruz Biotechnology Inc.), and bands were visualized using
the ECL System (PerkinElmer).
Immunohistochemistry and immunocytochemistry. Hearts of WT
or MFG-E8 KO mice, livers of CCl4-injected mice, and spleens of WT
mice were excised. The cryostat sections were fixed in acetone andincubated overnight at 4°C with the following primary antibodies:anti–MFG-E8 (1:200, 18A2-G10; MBL), anti-vimentin (1:100, GP53;
Progen), Cy3-conjugated anti-αSMA (1:400, 1A4; Sigma-Aldrich),anti-CD31 (1:100, 550513; BD Biosciences), FITC-conjugated anti–
Gr-1 (1:200, RB6-8C5; BioLegend), anti-CD68 (1:200, MCA1957; AbD
Serotec), anti–PDGFR-β (1:100, APB5; eBioscience), anti–Wt-1 (1:200,
sc-192; Santa Cruz Biotechnology Inc.), biotin-conjugated anti-CD21/
CD35 (1:200, 7E9; BioLegend), and anti-S100A4 (1:100, Ab-8; Neomarkers). Integrin β5 (1:200, ab15459; Abcam) staining was performedon 6-μm–thick sections of MI-operated hearts fixed in 4% PFA at 4°C.
Images were captured and analyzed using an epifluorescence microscope (BZ-9000; Keyence) and a confocal microscope (A1Rsi; Nikon).
Paraffin-embedded heart sections of patients who had undergone MI were deparaffinized and autoclaved in 10 mM citrate buffer(pH 6.0) at 121°C for 10 minutes for antigen retrieval. The sections
were treated with 0.3% H2O2/MeOH solution for 10 minutes andsubsequently blocked with PBS containing 10% normal goat serum.
Next, the sections were stained by mouse monoclonal anti-human
MFG-E8 antibody (1:200) (53) and rabbit anti-αSMA antibody (1:200,
EPR5368; Epitomics) and visualized using a biotin-streptavidin-peroxidase kit (LSAB 2 kit; DAKO). Quantitative analysis was performed
using ImageJ (NIH) software. All stained sections were observed
under a light microscope (BX50; Olympus), fluorescence microscope,or confocal microscope (FV10i; Olympus).
Myofibroblasts cultured in an 8-well slide chamber were fixed in acetone and incubated overnight at 4°C with anti-SM22α (1:200, ab14106;
Abcam) and anti–MFG-E8 (1:200, 18A2-G10; MBL) antibodies.
TUNEL staining with an αSMA- or CD68-specific antibody. Hearts,
lungs, livers, spleens, and kidneys were excised from MI-operated
mice and fixed in 4% PFA at 4°C. Sections were stained with Cy3-conjugated anti-αSMA (1:400, 1A4; Sigma-Aldrich) or anti-CD68 (1:200,
MCA1957GA; AbD Serotec) antibody at 4°C. TUNEL staining (Millipore) was performed according to the manufacturer’s instructions.
Over 70 engulfed apoptotic cells were evaluated, and engulfment
capacity was determined by dividing the number of these cells by thearea of αSMA-positive (or CD68) cells, averaged from 8 to 10 randomly selected fields.
Bone marrow transfer experiment. We lethally irradiated (10 Gy)
recipient male MFG-E8 KO mice. Bone marrow cells were isolated from male WT mice by flushing the tibias and femurs. Recipient MFG-E8 KO mice were administered donor bone marrow cells
through the orbital vein. Reconstitution efficiency was assessed bystaining of blood leukocytes prepared from the transplanted mice with

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FITC–anti-CD45.1 (1:200, A20; BioLegend) and APC–anti-CD45.2(1:200, 104; BioLegend) after 5 weeks. Percentage of chimerism in the
blood was determined for all monocytes.
Phagocytosis assay of apoptotic cells in cardiac macrophages andcardiac myofibroblasts. At 3 days after operations, hearts were excisedfrom MI-operated WT mice and digested using a cocktail of 4.4 mg/
ml Dispase II (Sigma-Aldrich), 2.5 mg/ml trypsin, and 1 mM EDTA
in PBS at 37°C with agitation. After digestion, cells were stained with
eFluor 780 viability dye for 30 minutes. The following antibodies werethen added, and the reaction was allowed for 30 minutes: FITC antiLy-6C (1:100, HK1.4; BioLegend), PE anti-Ly-6G (1:200, 1A8; BioLegend), PerCP/Cy5.5 anti-CD11b (1:200, M1/70; BioLegend), and APC
anti-F4/80 (1:100, BM8; BioLegend). The stained cells were analyzed,
and cardiac macrophages (Ly-6G–/CD11b+/Ly-6Clo/F4/80hi) were
sorted by FACSAria III. Sorted macrophages (5 × 104 cells/well) andadult mouse cardiac myofibroblasts (1 × 104 cells/well) were cultured
in an 8-well slide chamber coated with poly-l-lysine (Sigma-Aldrich).
For the phagocytosis assay, thymocytes were collected from female
C57BL/6 mice, labeled with 5-chloromethylfluorescein diacetate(CMFDA), pHrodo Red succinimidyl ester (P36600; Thermo Fisher
Scientific), or CellTrace Far Red dye (C34564; Thermo Fisher Scientific), and treated with 10 μM dexamethasone at 37°C for 4.5 hours to
induce apoptosis (26). CMFDA-labeled apoptotic thymocytes were
cocultured with cardiac macrophages or cardiac myofibroblasts at
37°C, after which the slides were washed vigorously with PBS to
remove the attached apoptotic thymocytes. The slides were fixed in
1% PFA and mounted in FluorSave mounting medium (Calbiochem).
Phagocytosis was evaluated under a phase-contrast microscope asreported previously (20). Over 150 myofibroblasts were evaluated,
and the phagocytosis index was determined as the number of apoptotic cells engulfed per macrophage (or myofibroblast).
Phagocytosis assay of necroptotic cells in cardiac myofibroblasts. To
prepare necroptotic cells, L929 cells (Riken BioResource Center) werelabeled with CMFDA under serum-free conditions and then treatedwith 20 μM Z-Val-Ala-Asp(OMe)-CH2F (Z-VAD-FMK) (a gift from M.
Sodeoka and K. Dodo [Riken]) at 37°C for 2 hours in DMEM supplemented with 10% FBS prior to addition of human TNF-α (10 ng/ml)(Peprotech). Floating L929 cells were harvested as necroptotic cells
4.5 hours after TNF-α stimulation. CMFDA-labeled necroptotic L929
cells (2 × 105 cells/well) were cocultured with cardiac myofibroblasts(2 × 104 cells/well) in an 8-well chamber slide for 3 hours at 37°C.
Phagocytosis was evaluated by phase-contrast microscopy.
Recombinant MFG-E8 preparation and administration. Recombinant MFG-E8 (WT) and MFG-E8 (D89E) were prepared as previously
reported (16). Mice were intramyocardially administered 20 μl (2 doses of 10 μl each) MFG-E8 or MFG-E8 D89E solution or PBS immediately after ligation of the left anterior descending coronary artery,using 29-gauge needles (BD Biosciences).
Echocardiographic analysis and hemodynamic measurement. Transthoracic ultrasound cardiography and hemodynamic measurements
were performed on mice under anesthesia after sham or MI operation
as previously reported (54).
Histological analysis. Infarct hearts were fixed in 10% neutral-buffered formalin and embedded in paraffin. Ventricular sections were stained with Masson’s trichrome, H&E, and PicroSirius
red and observed under a microscope. The infarct/left ventricular(LV) area, collagen volume fraction at the infarct area, and myocar-

dial wall thickness of the infarct area were analyzed using a BZ-II
analyzer (Keyence). Wall thickness was determined at 3 points of
the LV for each group.
Assessment of infarct size. The initial infarct size was assessed 3
hours after MI using Evans blue dye and TTC staining as previously
reported (54). The necrotic area (TTC negative) was represented as apercentage of AAR (area not stained with Evans blue dye), while the
AAR was represented as a percentage of the LV area.
Statistics. We used GraphPad Prism 6.0 software for all statisticalanalyses. Data represent the mean ± SEM of at least 3 independent
experiments. Two-tailed Student’s t tests were used for 2-group comparisons, and ANOVA with the Student–Newman-Keuls test was used
for multiple-group comparisons. Differences were considered significant at P < 0.05. No statistical method was used to predetermine
sample size. Intergroup differences in survival rate were tested by the
log-rank test. The experiments were randomized, and the researchers
were not blinded to allocation during experiments and outcome analysis, except for histological experiments, echocardiographic analysis,
and hemodynamic measurements.
Study approval. All animal experiments were performed using
approved protocols and in accordance with the guidelines of Kyushu
University. Mice were housed in accordance with the Institutional
Animal Care and Use Committee regulations at Kyushu University.
This study using human autopsy specimens was performed under
the informed consent of the participants, following the ethicalguideline of Tokyo Medical University.

Author contributions

M Nakaya conceived the project, designed the project, performed experiments, and analyzed and interpreted data. KW,
MT, SM, H Ohara, HN, M Nishida, YH, and H Ono performedsome in vivo and in vitro experiments and helped with datainterpretation. KW, MT, TN, H Ohara, HN, YH, H Ono, and AT
performed animal surgery. AH purified MFG-E8 protein. TN
and MK analyzed human samples. HY, AN, AT, GI, MT, RI, KI,
and SN provided key reagents and contributed to the scientificdiscussion. M Nakaya and HK wrote the paper.

Acknowledgments

We thank K. Fujita (Tokyo Medical University) for his expert
assistance. This study was supported by grants from the Ministryof Education, Culture, Sports, Science, and Technology of Japan(MEXT) (25713007 to M Nakaya and 25253011 to HK); Grantin-Aid for Scientific Research on Innovative Areas (Homeostatic regulation by various types of cell death, and High definition physiology project) MEXT (15H01383 to M Nakaya and22136008 to M Nakaya, GI, and RI); Kyushu University Interdisciplinary Programs in Education and Projects in Research
Development (P&P) (to M Nakaya); the Japan Heart Foundation/Novartis Grant for Research Award on Molecular and Cellular Cardiology 2014, the Takeda Science Foundation, the Naito
Foundation, the Mochida Memorial Foundation for Medical andPharmaceutical Research, the Suzuken Memorial Foundation,
the Astellas Foundation for Research on Metabolic Disorders,the Kanae Foundation for the Promotion of Medical Science,
the Kaibara Morikazu Medical Science Promotion Foundation,
MSD Life Science Foundation, and the Nakajima Foundation
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The Journal of Clinical Investigation(to M Nakaya); the Platform Project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery,
Informatics, and Structural Life Science) the MEXT and theJapan Agency for Medical Research and development (AMED);
and the Funding Program for Next Generation World-Leading Researchers (NEXT Program) (to MT). We appreciate thetechnical support from the Research Support Center, Graduate
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Novel form of miR-29b suppresses bleomycininduced pulmonary fibrosis
Yuko Yamada1, Masakatsu Takanashi1, Katsuko Sudo2, Shinobu Ueda1, Shinichiro Ohno1, Masahiko Kuroda1*
1 Department of Molecular Pathology, Tokyo Medical University, Shinjuku, Tokyo, Japan, 2 Animal
Research Center, Tokyo Medical University, Shinjuku, Tokyo, Japan* Kuroda@tokyo-med.ac.jp

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OPEN ACCESS
Citation: Yamada Y, Takanashi M, Sudo K, Ueda S,
Ohno S-i, Kuroda M (2017) Novel form of miR-29b
suppresses bleomycin-induced pulmonary fibrosis.
PLoS ONE 12(2): e0171957. doi:10.1371/journal.
pone.0171957

Abstract
MicroRNA 29b (miR-29b) replacement therapy is effective for suppressing fibrosis in amouse model. However, to develop clinical applications for miRNA mimics, the side effectsof nucleic acid drugs have to be addressed. In this study, we focused on miRNA mimics
in order to develop therapies for idiopathic pulmonary fibrosis. We developed a singlestranded RNA, termed “miR-29b Psh-match,” that has a unique structure to avoid problems
associated with the therapeutic uses of miRNAs. A comparison of miR-29b Psh-match anddouble-stranded one, termed “miR-29b mimic” indicated that the single-stranded form wassignificantly effective towards fibrosis according to both in vivo and in vitro experiments.
This novel form of miR-29b may become the foundation for developing an effective therapeutic drug for pulmonary fibrosis.

Editor: Bernard Mari, Institut de Pharmacologie
Moleculaire et Cellulaire, FRANCE
Received: November 11, 2016
Accepted: January 28, 2017
Published: February 24, 2017
Copyright: © 2017 Yamada et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.
Funding: This research was supported in part by
Grants-in-Aid for Scientific Research (B:26296088) and (C: 16K09376) and a Grant-in-Aid
for Exploratory Research (15K15100) from Japan
Society for the Promotion of Science (JSPS) andsupported in part by Strategic Research
Foundation Grant-aided Project for Private
Universities from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.

Introduction
Idiopathic pulmonary fibrosis (IPF) is characterized as progressive fibrosis and alveolar complex, and is known as an intractable disease due to its high lethality [1]. Pulmonary fibrosis ischaracterized by the excessive deposition of collagen and other extracellular matrix proteins
within the lung interstitium, which is mediated by transforming growth factor beta 1 (TGFβ1) through its downstream signaling pathway [2]. In recent years, small molecule inhibitors
such as nintedanib and pirfenidone have been developed for clinical use [3, 4]; however, it isdifficult to cure severe IPF cases with these drugs.
In this study, we focused on miRNA replacement therapy in order to develop a novel classof IPF therapeutic. MicroRNAs (miRNAs) are one class of non-coding RNA, and are key
biological regulators that control gene expression by suppressing translation of, and by destabilizing, target mRNAs by RNA interference [5]. One miRNA can regulate multiple targeted
mRNAs, including mRNAs involved in the same biological pathways. Therefore, modulating amiRNA, in principle, allows for an entire gene network to be influenced, and for complex
disease phenotypes to be modified [6]. Many studies have shown the therapeutic efficacy ofmiRNA inhibitors and mimic miRNAs, efforts to restore or increase the function [7].
Nucleic acid drugs have not been employed yet in practical therapeutic applications due tothe activation of an innate immune response through Toll-like receptor 3 (TLR3) [8].

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Novel miRNA replacement therapy against IPF

Competing interests: The authors have declaredthat no competing interests exist.

Furthermore, double-stranded RNA is easily modified by enzymes such as RNase and lacks invivo stability.
It is necessary to develop a drug delivery system (DDS) because nucleic acid drugs are otherwise unable to reach targeted organs or tissues efficiently due to their instability. Recently, toovercome this limitation of nucleic acid drugs, we developed three different types of synthetic
single-stranded RNA, termed “nkRNA”, “PnkRNA”, and “PshRNA”. All three of these RNAs
have unique single-stranded RNA structures [9, 10]. The single-stranded structure imparts
greater in vivo stability to the RNAs and also makes it difficult for TLR3 to recognize them.
Hence, using such single-stranded RNAs makes it possible to avoid side effects due to theimmune response triggered by double-stranded RNAs [11].
Aberrant expression of miRNAs has been regarded as a common feature of fibrotic diseases.
The miR-29 family has been found to play an important role to maintain the extra cellularmatrix (EM) through the regulation of genes such as Collagen 1a1 (Col1a1) and Collagen 3a1(Col3a1) [12, 13]. It was previously reported that miR-29 replacement inhibited tissue fibrosis
and exhibited therapeutic effects on the heart [13], kidney [14–16], liver [17–19], lung [12, 16],and also on systemic sclerosis [20] in mouse models. In addition, recent studies have suggested
that miR-29b exerts therapeutic effects in a bleomycin-induced pulmonary fibrosis mousemodel [21].
We have developed novel types of miR-29b mimics that resemble “Pnk-RNA” and “PshRNA”. This novel platform structure of RNA can be adapted not only to miR-29b but to all
miRNAs. We named this novel mimic RNA “miR-29b Psh-match”. Furthermore, we confirmed that the miR-29b Psh-match shows an enhanced therapeutic effect compared with traditional double-stranded miR-29b (miR-29b mimic) in bleomycin-induced pulmonary
fibrosis model mice. Therefore, our data indicated that administration by inhalation of themiR-29b Psh-match may be useful for therapeutics treatment of pulmonary fibrosis.

Materials and methods
Ethics statement
Our experimental procedure was approved by the Animal Experimentation Ethics Committeeof Tokyo Medical University. The approval number is S-27027.

Single-stranded miR-29b
We developed the single-stranded miR-29b called miR-29b Psh-match (Fig 1). This RNA contains no mismatch sequence. It is made of a ‘sense strand’ and an ‘antisense strand’ for the target gene sequence from the central portion in the alignment. This construct contains a cassetteregion for bearing a loop structure. The cassette region has the nucleotide residue proline (P).

Animals
We used eight-week-old male C57BL/6J mice (Japan SLC, Hamamatsu, Japan) in our experiments. The mice were housed in the animal facility of Tokyo Medical University. The mice
were subject to a constant 12 h light / 12 h dark cycle in a temperature- and humidity-controlled room. Drinking water and food were made freely available. All animals were sacrificed
with an overdose of a combination anesthetic cocktail (0.3 mg/kg body weight of medetomidine, 4 mg/kg body weight of midazolam, and 5 mg/kg body weight of butorphanol) by i.p.
and their lungs collected. The health status of all mice was monitored daily. Mice that showed
greater than 20% weight loss were closely monitored. If other humane endpoints became
worse, in addition to weight loss, they were immediately sacrificed. The other humane

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Novel miRNA replacement therapy against IPF

Fig 1. Construction of miR-29b Psh-match and miR-29b mimic. (A) miR-29b mimic. The double-stranded
miR-29b is made of both a ‘sense strand’ and an ‘antisense strand’, and contains a mismatch. (B) miR-29b
Psh-match. The single-stranded miR-29b contains no mismatches, and is made of both a ‘sense strand’ andan ‘antisense strand’ derived from the target gene sequence in its central portion. It also contains a cassettearea that adopts a loop structure. The cassette area has the nucleotide residue proline (P).
doi:10.1371/journal.pone.0171957.g001

endpoint criteria were dyspnea, markedly reduced mobility, debilitating diarrhea, jaundice,anemia, abnormal neurological signs, bleeding from any orifice, and self-induced trauma.

Inhalation study in mouse
Mice were administered with two doses of 20 μl of 1% (w/v) methylene blue in physical saline
through their noses while anesthetized with 40% isoflurane. The mice were sacrificed 120 min
after the treatment and their lungs resected under anesthesia in order to observe the existenceof the dye in the lungs.

Bleomycin-induced pulmonary fibrosis in a mouse model
Pulmonary fibrosis was induced in mice using bleomycin (Sigma-Aldrich). Following anesthetization with 40% isoflurane, mice were subject to nasal injections of 0.01 U/body bleomycin
for 3 days. A control group of mice received similar injections of an identical volume of saline.
Mice were sacrificed on days 7, 14, 21, and 28 following bleomycin treatment, and their lungs
were collected for further experiments.

miR-29b administration to pulmonary fibrosis mouse model
Sixty micromoles of either miR-29b Psh-match-, mimic-, or scrambled-RNA was administered intranasally to mice. We administered either miR-29b Psh-match, mimic-, or scrambleRNA to mice on days 8, 9, and 10 after the initial bleomycin treatment, and sacrificed them on
day 15. We also administered either miR-29b Psh-match- or scramble-RNA on days 4, 8, and15, after the initial bleomycin treatment before the mice were sacrificed on day 22. Lastly, we
administered either miR-29b Psh-match, mimic, or scramble-RNA on days 8, 9, and 10 following initial bleomycin treatment, and sacrificed the mice on day 28 before collecting their lungs
to determine hydroxyproline content.

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Novel miRNA replacement therapy against IPF

In vitro experiments
NIH/3T3 cells were maintained in DMEM medium containing 10% bovine serum albumin,
100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma). Cells were transfected with 100 nMof either of two types of miR-29bs (Psh-match and mimic) or with scramble RNA using HiPerFect transfection reagent (Qiagen). At the time of RNA transfection, 5 ng recombinant TGF-β(Peprotech, NJ) was also added. Cells were harvested 24 hours after transfection for RNA
extraction.

TLR signaling activation assay
TLR signaling activation by miR-29b mimic and Psh-match was detected using an NF-κB activation assay kit (Promega). Twenty-four hours after transfecting HKE293 cells with an IκB
reporter plasmid that drives overexpression of either TLR3 or TLR7, these cells were transfected a second time with 100 nM of either miR-29b mimic- or miR-29b Psh-match-RNA,along with 10 μg/ml of either poly (I:C) (Novus Biological, CO) or Imiquimod (Novus Biological). Cells were harvested 6 h after the miRNA transfection and assayed using the Promega kit
according to the manufacturer’s instructions.

Real-time PCR
Total RNA was extracted using the ISOGEN reagent (Nippon Gene, Tokyo). One hundred
nanograms of RNA from each sample was used to generate cDNA using a cDNA synthesis kit(Transcriptor First Strand cDNA Synthesis Kit, Roche). Real-time PCR analysis for Col1a1
and Col3a1 mRNA was performed using the Roche LightCyclergaba 96 system. Primers forreal-time PCR were: Col1a1 sense 5’-CATGTTCAGCTTTGTGGACCT-3’ and antisense 5’GCAGCTGACTTCAGGGATGT-3’, and Col3a1 sense 5’-TCCCCTGGAATCTGTGAATC-3’
and antisense 5’-TGAGTCGAATTGGGGAGAAT-3’.

Histology
Lung tissue specimens from mice were fixed in buffered formalin, embedded in paraffin, andsectioned into 4 μm-thick slices for staining with hematoxylin and eosin. Masson’s trichrome
staining and Sirius Red staining were also performed. The Ashcroft score was calculated aspreviously described [22–24].

Hydroxyproline measurement
Hydroxyproline levels in mouse lungs were determined using a hydroxyproline colorimetricassay kit (Biovision, CA). Data were calculated as hydroxyproline content per unit wet weightof lung tissue.

Statistical analysis
Statistical analysis was performed using Student’s t-test and the Tukey-Kramer test. A p valueof <0.05 was considered statistically significant. Graphs show the mean value ± standard errorof the mean (s.e.m.).

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Novel miRNA replacement therapy against IPF

ResultmiR-29b Psh-match suppresses collagen expression in TGF-βstimulated fibroblasts without activating TLRs
To determine whether the unique structured form of miR-29b Psh-match is better able to suppress the expression of its target genes than the miR-29b mimic, we transfected these RNAs
into NIH/3T3 cells activated with recombinant TGF-β (rTGF-β) and performed real-time
PCR to determine the expression of Col1a1 mRNA 24 h after transfection. These data showedthat Col1a1 mRNA levels in NIH/3T3 cells activated by rTGF-β were elevated compared tountreated cells. In contrast, miR-29b Psh-match decreased Col1a1 mRNA expression in TGFβ-treated cells when compared with similarly treated cells transfected with either scrambled
control RNA or miR-29b mimic (Fig 2A). From these results, we concluded that miR-29b Pshmatch suppresses Col1a1 mRNA expression to a greater extent than the miR-29b mimic.
The activation of TLR signaling leads to the activation of the NF-κB transcription factor. T
he activation of either TLR3 by a stimulus such as viral dsRNA, or TLR7 by a stimulus such asssRNA, leads to the elevated expression of inflammatory cytokines, a reaction mediated by thetranscription factor NF-κB. Therefore, to determine whether miR-29b Psh-match induced theactivation of TLRs signaling pathways, we performed an NF-κB activation assay using luciferase cDNA under the control of a κB response element. Our data indicates that miR-29b Pshmatch-RNA induced luciferase expression at a low level. Moreover, cells overexpressing either
TLR-7 or TLR-3 were compared with cells subjected to each TLR ligand treatment (Fig 2B).

Bleomycin-induced pulmonary fibrosis in mice
We went on to determine whether miR-29b Psh-match can suppress the expression of the
fibrosis-related gene Col1a1 in pulmonary fibrosis model mice. In order to generate

Fig 2. miR-29b protects fibrotic responses in NIH/3T3 cells induced with TGF-β without activating TLRs. (A) Col1a1 mRNA
expression in NIH/3T3 cells was reduced after transfection with miR-29b Psh-match compared with cells treated only with TGF-β. The
degree of reduction in COL1A1 expression induced by miR-29b Psh-match transfection was greater than that induced by either miR-29b
scrambled control or miR-29b mimic (Fig 2A) (*P<0.05). (B) miR-29b Psh-match does not induce TLR signaling activity. TLR signaling
activity was measured using an NF-κB reporter in which luciferase cDNA was placed under the control of the κB response element. The
dsRNA and ssRNA induce the activation of NF-κB through TLR3 and TLR7, respectively.
doi:10.1371/journal.pone.0171957.g002

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Novel miRNA replacement therapy against IPF

Fig 3. Bleomycin-induced pulmonary fibrosis in mouse lungs. (A) Images of lungs from mice that were either subject to nasal
administration of methylene blue (right) or not (left). (B) Fold changes in Col1a1 mRNA showed a peak one week after bleomycin
administration. (*P<0.05) (C) Histological staining by HE and Masson’s trichrome showed pronounced diffuse fibrosis in the lungs three
weeks after bleomycin administration.
doi:10.1371/journal.pone.0171957.g003

pulmonary fibrosis in C57BL/6J mice, we administered bleomycin intranasally. To determine whether bleomycin can be introduced into the lungs by nasal administration. We
administered methylene blue intranasally. We observed blue-stained bronchial tubes andareas peripheral to them 120 min after dye administration (Fig 3A). These results demonstrated that nasal administration was successful for delivering reagents to the lungs. To
determine that when expressed at the peak of expression levels for the related genes by bleomycin stimulation and occurred the most remarkable histological changes in their lung tissues, we determined the expression of Col1a1 mRNA in the lung of, and performed
Masson’s trichrome staining of lung tissue from, mice sacrificed on days 7, 14, 21, and 28
following bleomycin treatment. These results indicate that the fold change in Col1a1 mRNA
peaked on day 7 following bleomycin treatment (Fig 3B). Our histological examination
using hematoxylin eosin staining (H&E) and Masson’s trichrome staining revealed pronounced diffuse fibrosis and lymphocyte infiltration in the lungs of mice on day 21 following bleomycin administration (Fig 3C). Based on these results, we concluded that Col1a1
mRNA expression in the lungs of bleomycin-induced model mice occurred on day 7, andthat morphological changes first appeared on day 21.

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Novel miRNA replacement therapy against IPF

Comparison of therapeutic effects of miR-29b Psh-match and miR-29b
mimic
We compared the therapeutic effects of miR-29b match type and mature type in bleomycininduced mice. To determine fibrosis suppression in lungs induced by miR-29b Psh-match andmiR-29b mimic, we administered either miR-29b or scramble RNA to bleomycin-induced
mice. We could not detect the administered miR-29b mimic and Psh-match in lungs and inany organs by real-time qPCR. Because the results of our inhalation study using nasally administered dye indicated that we could detect the dye in bronchi (Fig 3A), we concluded that themiR-29b mimic and Psh-match arrived at the lungs and that both miR-29bs had no effect on
the survival of bleomycin-treated mice. Our real-time qPCR data showed that Col1a1 mRNA
expression in the lungs of miR-29b Psh-match-treated mice decreased relative to mice treatedwith either PBS, scrambled control RNA, or miR-29b mimic (Fig 4A, P<0.05). Histologicalanalysis revealed that the lungs of mice that had been administered with mature type miR-29b

Fig 4. miR-29b Psh-match suppresses pulmonary fibrosis in mice to a greater degree than miR-29b mimic. (A) In bleomycininduced pulmonary fibroblast model mice, quantitative real-time PCR data indicates an increase in Col1a1 mRNA expression.
Expression of Col1a1 decreased in the lungs of mice administered with miR-29b Psh-match relative to mice administered with either
PBS, miR-29b scrambled control, or miR-29b mimic (*P<0.05). (B) HE and Masson’s trichrome staining showed the pronounced diffuse
fibrosis on the lungs in the bleomycin-treated mice. Administration of miR-29b Psh-match suppressed pulmonary fibrosis in an
established mouse model of bleomycin-induced pulmonary fibrosis (*P<0.05). High magnification images of the lungs of miceadministered with either miR-29b mimic or Psh-match mice are shown in the frames. (C) Hydroxyproline content was measured as
micrograms of hydroxyproline per weight in grams of the left lung’s tissues in mice.
doi:10.1371/journal.pone.0171957.g004

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Novel miRNA replacement therapy against IPF

or match-one showed an improvement of fibrosis. Although alveolar thickening was observedin the lungs of mice administered with miR-29b mimic, the lungs of mice administered with
miR-29b Psh-match showed the most improvement for the pulmonary fibrosis condition (Fig
4B). The infiltration of many lymphocytes was observed in the lungs of mice administeredwith miR-29b mimic but not in mice administered with Psh-match (Fig 4B). According to ourdata, we concluded that miR-29b Psh-match had no side effects compared with miR-29b
mimic.
We determined the amount of hydroxyproline in the lungs of the mice to measure matrix
collagen levels. At day 28, hydroxyproline was lowest in the lung of miR-29b Psh-match—
treated mice compared to mice treated with PBS, miR-29b scrambled control, or miR-29b
mimic (Fig 4C).

miR-29b Psh-match decreases collagen expression in bleomycininduced pulmonary fibrosis mice
The lungs of mice treated with bleomycin showed an increase in the expression of Col1a1 andCol3a1 mRNAs on day 21, as measured by real-time PCR. Neither PBS nor scrambled control
RNA reduced the extent of this increase in collagen expression. miR-29b Psh-match caused
the expression of Col1a1 and Col3a1 mRNAs to decrease with the PBS control and scrambled
control RNA-treated mice (Fig 5A, P<0.05). The data from HE staining and Masson’s trichrome staining showed apparently diffused fibrosis in the lungs of bleomycin-treated mice.
In contrast, lung tissues from miR-29b Psh-match—treated mice showed improvement for
fibrosis. The Ashcroft score showed a significant improvement for fibrosis in the lungs of miceadministered with miR-29b Psh-match (Fig 5B). Our results indicate that miR-29b Psh-matchdecreased collagen expression and improved pulmonary fibrosis.

Discussion
It has been reported that dysregulation of miRNAs has been implicated in the development ofvarious human diseases [25, 26]. The miR-29 family, comprising miR-29a, 29b, and 29c, is atranscriptional target of TGF-β. Upregulating the expression of these three miRNAs induces
dysregulation of the immune response and promotes fibrosis [12, 13]. Although the underlying molecular mechanism is still unclear, activation of the TGF-β/Smad signaling pathway
plays a crucial role in the development of idiopathic pulmonary fibrosis (IPF) [2, 27, 28].
Therefore, miR-29 replacement therapy represents a promising approach for IPF treatment. In
this study, we presented the development of miR-29b replacement therapy as a new treatment
method for pulmonary fibrosis by intranasal administration of the nucleic acid drug.
Concerning the synthetic RNA platform, we recently developed novel types of synthetic
RNA, named nkRNA and PnkRNA, that exhibit sequence-specific gene silencing throughRNAi without activating TLRs or RIG-I-like receptor (RLR) signaling [11]. This novel class ofsynthetic RNAs shows an enhanced RNAi effect and greater in vivo stability compared withclassical siRNA [9]. These results indicate that nkRNA and PnkRNA are a suitable technology
platform for molecularly targeted therapy [9, 29]. We sought to develop a novel miRNA mimic
similar to nkRNA and PnkRNA, and in the process, generated miR-29b Psh-match, which hasa characteristic secondary structure. First, we found that miR-29b Psh-match showed significant suppression of the expression of the target genes Col1a1 and Col3a1 compared with themiR-29b mimic (Fig 2). These data suggest that miR-29b Psh-match shows more effective
RNAi effect compared with the miR-29b mimic. We speculate that miR-29b Psh-match is better able to attract and load the Argonaute protein complex compared with the miR-29b
mimic. Furthermore, in pulmonary fibrosis model mice that were administered with miR-29b

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Novel miRNA replacement therapy against IPF

Fig 5. (A) Suppression of the expression of miR-29b-targeted genes by miR-29b Psh-match and (B) histological detection offibrosis in mouse lungs. (A) Bleomycin treatment of mice increased Col1a1 and Col1a3 mRNA expression in their lungs. Administration of
miR-29b Psh-match decreased Col1a1 and Col1a3 expression to a greater extent compared to mice treated with PBS or miR-29bscrambled control (P<0.05). The administration of miR-29b Psh-match to bleomycin-induced pulmonary fibrosis model mice decreased theAshcroft score (*P<0.05). (B) Histological staining using HE, Masson’s trichrome, and Sirius Red revealed pronounced diffuse fibrosis in thelungs of bleomycin-treated mice. The administration of miR-29b match type appeared to suppress pulmonary fibrosis.
doi:10.1371/journal.pone.0171957.g005

Psh-match, the expression of fibrosis-related genes, the amounts of hydroxyproline in collagen, and the Ashcroft scores were all markedly decreased (Fig 5). We hypothesize that the hairpin structure of miR-29b Psh-match imparts a higher biological stability in vivo and thus
makes it less likely to be degraded by the time it reaches the target cells. DDS is the most problematic issue facing the clinical use of nucleic acid drugs. We recently confirmed the therapeutic effect of TGF-β siRNA using IPF mice models [9]. In that study, we administered naked
siRNA to mice by intratracheal instillation. In general, naked siRNA cannot be taken up by
normal cells because of the charge of the cell membrane. Even if siRNA is taken up by the target cells, the siRNA has to undergo endosomal escape [30] in order to exert any effect on target
gene expression. In the case of IPF, TGF-β secreted by alveolar macrophages plays a crucialrole in accelerating acute and chronic inflammation following development of fibrosis in the

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Novel miRNA replacement therapy against IPF

lung. Alveolar macrophages are active phagocytes and can therefore take up siRNA without
DDS. In considering the systemic delivery of nucleic acid therapies, there are many problems
to be overcome in order for clinical applications to succeed. However, when targeting alveolar
macrophages with nucleic acid drugs, DDS is not required. It is difficult to deliver either small
molecules or antibodies to alveolar macrophages by systemic administration. Therefore, for
topical drug administration of miRNAs and siRNAs to alveolar macrophages, administration
by inhalation is ideal.
Up to the present, some drugs have been developed for treating IPF that have been
approved for clinical use. Two new representative drugs are pirfenidone and nintedanib [31,32]. Pirfenidone is an oral anti-fibrotic drug that exerts its anti-fibrotic effects by downregulating the TGF-β signaling pathway [33]. Based on Phase III trials, pirfenidone improves diseaseprogression, lung function, and progression-free survival in patients with IPF [4]. Nintedanib
is a small molecule tyrosine kinase inhibitor, targeting vascular endothelial growth factor
receptor, fibroblast growth factor receptor, and platelet derived growth factor receptor. In a
recent Phase III study, nintedanib reduced the decline in lung function in patients with IPF[3]. However, nintedanib inhibits physiological remodeling and formation of blood vessels.
Therefore, it shows side effects that included gastrointestinal disturbance and impaired liverfunction.
As for miR-29b, this miRNA is tightly regulated by many signaling molecules such as Myc
and HDAC. In cancerous tissue, miR-29 expression was mostly decreased, and it contributed
to the invasive and metastatic phenotype of cancer cells [34, 35]. In the environment of the
fibrotic lesion, decreased expression of the miR-29 family caused activation of the TGF-β signaling pathway. Therefore, it is logical to supply the miR-29 family back to these cells to suppress TGF-β. Additionally, miR-29 is a natural product, so we anticipate fewer side effects
compared with pirfenidone and nintedanib.
Taken together, we conclude that the single-stranded miR-29b Psh-match exerts anenhanced therapeutic effect compared with previous double-stranded ones. Hence, singlestranded miR-29b Psh-match may be an effective therapeutic drug for pulmonary fibrosis.

Acknowledgments
We thank Akio Ishikawa for his technical assistance. This research was supported in part by
Grants-in-Aid for scientific research (B: 26296088) and (C: 16K09376) and a Grant-in-Aid for
Exploratory Research (15K15100) from Japan Society for the Promotion of Science (JSPS) andsupported in part by Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Author Contributions
Conceptualization: MK.
Data curation: YY MK.
Formal analysis: YY.
Funding acquisition: MK MT SU.
Investigation: YY MT KS SU.
Methodology: MK SO.
Project administration: MK.
Resources: MK.

PLOS ONE | DOI:10.1371/journal.pone.0171957 February 24, 201710 / 12-229-

Novel miRNA replacement therapy against IPF

Supervision: MK.
Validation: MT.
Visualization: YY.
Writing – original draft: YY MT MK.
Writing – review & editing: MK YY.

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www.nature.com/scientificreports

OPEN

Received: 28 February 2017
Accepted: 6 November 2017
Published: xx xx xxxx

Repair of the TGFBI gene in humancorneal keratocytes derived froma granular corneal dystrophy
patient via CRISPR/Cas9-induced
homology-directed repair
Yukako Taketani1, Kohdai Kitamoto1, Toshihiro Sakisaka1, Mikiko Kimakura1, Tetsuya
Toyono1, Satoru Yamagami2, Shiro Amano3, Masahiko Kuroda4, Tara Moore5,6, Tomohiko
Usui1 & Yasuo Ouchi7
Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple
discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused
by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome
5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing forthe correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homologydirected repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specificguide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An
R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected bydelivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDRdonor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% inhomozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy
for GCD2; this method may also be applicable to other heredity corneal diseases.
Granular corneal dystrophy (GCD) is a bilateral, progressive, genetic, and non-inflammatory disease characterised by multiple granular deposits in the corneal stroma. Using the IC3D classification1, GCD has two subtypes,
both of which are classified as Category 1, i.e., causal point mutations have been identified in the transforming
growth factor-beta-induced (TGFBI) gene, located on chromosome 5q312. TGFBI, also called keratoepithelin or
Big-h3, is 68-kDa protein found in the extracellular matrix of human tissues. It is particularly abundant in thecornea.
There are two clinical types of GCD, GCD1 and GCD2. Although originally described in a family from the
Italian region of Avellino, the R124H mutation associated with GCD2 is occurs in unrelated individuals in all
populations studied and is the most common type in Asia, including Japan3,4. In GCD2, discrete grey-white
granular deposits (hyaline) with snowflake, star, or disk shapes are detected in the corneal stroma at an earlyage5 and amyloid deposits are observed in elder patients in deeper stroma6. GCD2 has a diffuse anterior stromalhaze between the typical granular opacity. The haze may be caused by amyloid deposits, which are thought tobe similar to Gelatinous drop-like CD, instead of the linear opacity seen in the early stage of lattice dystrophy6,7.
Compared to heterozygous patients, homozygous patients may have an onset under 10 years old, and demonstrate a more rapid progression. These progressive corneal opacities cause a loss of visual acuity. To avoid visual
impairment in GCD2, phototherapeutic keratectomy (PTK) is a major treatment option. However, multiple
1

Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. 2Nihon University
Itabashi Hospital, Tokyo, Japan. 3Inoue Eye Hospital, Tokyo, Japan. 4Department of Molecular Pathology, Tokyo
Medical University, Tokyo, Japan. 5Biomedical Sciences Research Institute, Centre for Molecular Biosciences, Ulster
University, Coleraine, Northern Ireland. 6Avellino Labs, Menlo Park, CA, USA. 7Department of Mucosal Immunology,
School of Medicine, Chiba University, Chiba, Japan. Correspondence and requests for materials should be addressed
to T.U. (email: tomohiko-tky@umin.ac.jp) or Y.O. (email: o-yasuo@chiba-u.jp)

1

SCIENTIfIC REPORTS | 7: 16713 | DOI:10.1038/s41598-017-16308-2-232-

www.nature.com/scientificreports/
opacities usually recur within several years8. Compared with PTK, corneal clarity can be retained for longer durations using keratoplasty, but opacity eventually occurs via the gradual invasion of host corneal cells, especially inhomozygous patients9. Thus, the development of a radical treatment is needed. GCD2 is typically associated withan R124H (histidine replacing arginine) point mutation in the TGFBI gene; accordingly, a gene therapy approach
may be effective.
CRISPR/Cas9 (clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associatedprotein)-mediated genome editing has been increasingly applied to repair mutated genome sequences10. Thisversatile tool for genome engineering enables the induction of site-specific double-strand breaks (DSBs) using
guide RNAs (gRNAs)11–15. DSBs can be repaired by two major pathways, non-homologous end joining andhomology-directed repair (HDR). In the presence of exogenous donor DNA as a repair template, DSBs canbe repaired precisely via the HDR pathway. This technique is useful for codon replacements or reporter insertions16,17. For small genetic modifications, such as point mutations, the application of single-stranded oligodeoxynucleotides (ssODNs) as HDR templates shows higher editing efficiency than that of plasmid donors18. Here,
we report the first CRISPR-mediated HDR using cultured corneal keratocytes derived from an R124H GCD2
patient. The results of this study have important clinical implications given the lack of effective treatment options
for GCD2.

Results

Gene targeting strategy and construction for CRISPR/Cas9-mediated HDR of an R124H mutation. To develop an efficient strategy to repair the genetic mutation in GCD using CRISPR/Cas9, we usedhuman cultured corneal keratocytes derived from an R124H GCD2 patient as a model system. The TGFBI R124H
mutant keratocytes have a monoallelic point mutation at Arg124 (GCA→ACA) in Exon 4 of TGFBI (Fig. 1a). To
repair mutant R124H cells, we designed an R124H mutation-specific gRNA based on a public algorithm (Fig. 1b).
Then, the designed gRNAs were computationally evaluated for potential off-target effects using the E-CRISP algorithm. The gRNA with the lowest off-target risk was selected for subsequent analyses.
For the HDR repair template, we synthesized a 100-nucleotide (nt) donor repair template ssODN with a novel
BsiWI restriction site (Fig. 1b). The substitutions ensured that the sequence of the wild-type donor template wasresistant to CRISPR/Cas9 cleavage by the R124H mutation-specific gRNA, and the BsiWI restriction site allowed
the tracking of HDR by restriction fragment length polymorphism (RFLP) (Fig. 1b). A pair of annealed oligos
encoding a target sequence of R124H mutation-specific gRNA was cloned into the px458 vector, which enabled
bicistronic expression of Streptococcus pyogenes Cas9 (spCas9) and green fluorescence protein (GFP) (Fig. 1c).

CRISPR/Cas9-mediated HDR of an R124H mutation in human corneal keratinocytes.

The
CRISPR plasmid expressing spCas9/gRNA was co-transfected into primary R124H mutant human corneal keratinocytes with the ssODN as a donor template. After 7 days, single GFP-expressing cells were harvested, added toindividual wells of a 96-well plate, and clonally expanded. Then, the presence of a novel BsiWI restriction site was
examined by RFLP-based genotyping. Genomic PCR products for wild-type alleles were not cleaved by BsiWI(Fig. 2a). However, genomic PCR products for several transfected colonies were cleaved by BsiWI, suggestingtarget site alterations by HDR (Fig. 2a). We also confirmed the genomic sequences of the PCR products (Fig. 2b).
The sequence of wild-type cells had CGC, specifying arginine, at the 124th amino acid, and R124H mutant
cells had CAC at this position. Neither is expected to be cleaved by BsiWI; however, gene-edited cells have CGT,
which is expected to be cut at CGTAC. In an RFLP assay, we detected cells with heterozygous and homozygous
editing, as shown in Fig. 2c.

Efficiency of Cas9-mediated genome editing of the TGFBI R124H mutant gene. To examine theediting efficiency of the R124H mutant TGFBI gene, genomic DNA was extracted from the clonally expanded
cells in 96-well plates and examined by RFLP-based genotyping. Owing to the low growth rate and viability offlow cytometry-sorted primary keratinocytes, not all cells were sufficiently expanded by single-cell cloning in
96-well plates. Cell growth and gene editing efficiency are summarised in Fig. 2c. Thirty-eight out of 192 clones
were sufficiently expanded and examined by RFLP. Among all examined clones, 20.6% exhibited monoallelic
TGFBI correction and 41.3% showed biallelic correction. Accordingly, 62% showed clear TGFBI R124H allele
correction derived from the HDR template.
Analysis of off-target cleavage by R124H mutation-specific gRNA. To evaluate off-target effects
mediated by the gRNA, a T7 endonuclease (T7EN1) cleavage assay was used to assess off-target cleavage. Since
we rigorously designed and selected a TGFBI-specific gRNA to reduce the risk of off-target effects, only 3 potential off-target sites were found for the gRNA (Fig. 3a,b). We could not find any potential off-target sites (OTS)
with mismatches of less than 3 nt. The 3 potential OTS had mismatches of more than 4 nt with the TGFBI gRNA
(Fig. 3b). In the T7EN1 cleavage assay, we did not detect any off-target effects at the 3 OTS (Fig. 3c).

Discussion

Current therapeutic modalities for GCD, i.e. PTK and keratoplasty, are invasive and are associated with frequent
recurrence. The correction of TGFBI mutations in the local cornea may be a radical treatment for GCD patients,
minimizing progression and the recurrence of corneal opacities. In this study, we successfully repaired point
mutations in R124H mutant cells using CRISPR/Cas9 and HDR in vitro, without detectable off-target effects.
The CRISPR/Cas 9 system is an efficient tool for genome engineering and disease treatment. Kaminski et al.
successfully eliminated HIV genomes in human T cells ex vivo19; they reported a low editing efficiency in primaryculture, even using a lentivirus delivery system19. Similarly, the low transfection and growth efficiency in thisstudy (Fig. 2c) may be attributed to the use of primary culture cells. However, in general, plasmid transfection
may be safer than viral transfection in vivo. Fortunately, despite the low growth rate, our results reveal that the2

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Figure 1. Gene targeting strategy for CRISPR/Cas9-mediated HDR of a TGFBI R124H mutation. (a) Schematic
diagram of the TGFBI mutation in GCD2 in humans. (b) In GCD2, the 124th protein position is histidine (H),instead of arginine (R). The recognition sight of donor single-strand DNA is also shown. (c) Linear structure of
the plasmid transfected into R124H mutant cells. The plasmid (px 458) includes guide RNA targeting R124H
mutant cells, Cas 9 protein sequences, and EGFP. TGFBI, transforming growth factor β-induced; GCD2,
granular corneal dystrophy; HDR, homology-directed repair.

efficiency of CRISPR/Cas9 in gene correction was higher compared with those of previous studies15. It is reported
that the efficacy of HDR is generally not high, however, the efficiency of HDR using asymmetric donor DNA ismuch higher (maximum 60%) than that of conventional HDR20. In this study, the efficacy of HDR using ssODN
was greater than 60%. The reason for the high efficiency of HDR in our study is unclear, but may be explained by
the unique characteristics of the DNA repair ability of corneal epithelial cells. Previously, Mallet et al.21 demonstrated that DNA damage in human corneal epithelial cells by ultraviolet radiation could be repaired faster thanthat in epidermal keratinocytes. This suggests that there are corneal-specific mechanisms in DSB repair. This issue
should be evaluated in future studies.
CRISPR/Cas9 itself has some probability of causing off-target mutations22,23. CRISPR RNA-guide endonucleases tolerate single and double mismatches in their sequences at the gRNA interface in bacterial cells22 andhuman cells23. Wu et al.24 reported that only 2 out of 12 samples had off-target mutations when they co-injected
Cas9 mRNA and a single gRNA into mouse zygotes with dominant mutations in Crygc that cause cataracts.
Additionally, off-target mutations were detected at 1 of 10 potential OTS in the two samples. Thus, although
off-target effects are an important safety issue for clinical use, they can be greatly reduced by a cautious gRNA
sequence design25,26. Moreover, according to previous studies, gRNA does not cleave nonspecific targets with

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Figure 2. Correction of the mutation in TGFBI R124H mutant keratocytes using CRISPR-mediated HDR. (a)Result of an RFLP analysis of edited R124H cells. TGFBI exon 4 was amplified by PCR, and the products weretreated with the BsiWI restriction enzyme. The lane with three bands was edited heterozygously and the lane
with two bands was edited homozygously. (b) DNA sequences of PCR products amplified from the TGFBI geneof wild-type cells, a heterogeneous R124H mutant, and a repaired allele by HDR after transfection of Cas9 guide
RNA and ssDNA. Two peaks were observed in the sequence of the R124H heterogeneous mutant, while the baseof HDR-repaired cells was corrected to T. (c) Editing efficiency of CRISPR/Cas9-mediated HDR of an R124H
mutation. RFLP, restriction fragment length polymorphism TGFBI, transforming growth factor β-induced;
HDR, homology-directed repair.
mismatches of 3 nt or more23,27. In our study, based on these findings, we made highly specific gRNAs using an
off-target prediction tool. A T7 Endonuclease 1 cleavage assay was performed to examine off-targets effects, but
the three predicted OTS were not detected in any sample (Fig. 3c).
In ocular tissues, several reports have demonstrated successful gene editing using the CRISPR/Cas9 system28–31. Wu et al. corrected a genetic disease in mice that show early-onset cataracts using non-homologous end
joining and HDR24. The gene correction was conducted at the embryonic stage and cataracts occurred in 10 out
of 12 mice. Wang et al.32 and Bakondi et al.30 successfully edited retinal genes by electroporation, and Hung et al.28
also successfully edited retinal genes using a virus delivery system. In the cornea, Courtney et al. reported theeffectiveness of DNA cleavage by CRISPR/Cas9 for the treatment of cornea dystrophy caused by a KRT12 mutation29. To our knowledge, this study is the first to demonstrate in vitro gene correction in mutant human primarycorneal cells using CRISPR/Cas9 and HDR. The cornea is an excellent tissue for the application of genome editing

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Figure 3. Off-target sites and T7 endonuclease cleavage assay of potential off-target loci. (a) Ranked list of
potential off-target loci for R124H mutation-specific gRNA. Number of mismatched bases, PAM sequence,
chromosomes, and target gene are indicated. (b) Sequence alignments of R124H mutation-specific gRNAand potential off-target loci. (c) Potential off-target sites in edited cells were amplified by PCR. After T7
endonuclease treatment, no off-targets effects were found at any site. N/A: not applicable.

therapy owing to its accessibility and high amenability to naked plasmid DNA transfection via intrastromal injection33. Thus, gene editing is a radical GCD treatment and the in vivo application of this system is ideal for clinicalsettings in which conventional treatments are limited. In the future, it is necessary to develop safer and more
efficient methods to modify local corneal genes in vivo.
In conclusion, we used CRISPR-Cas9-mediated HDR to correct the R124H mutation. Our data suggest thatthe approach is highly specific, with no observed off-target effects. Given the lack of effective treatment options
for GCD2, this gene editing system is a potentially radical treatment for TGFBI-related corneal dystrophy andcan be used to protect corneal opacities. The in vivo application of this system is an important future challenge.

Methods

Cell culture.

Primary human corneal keratocytes of a GCD2 patient with a heterozygous TGFBI mutation(R124H) were isolated from a surgical specimen during deep anterior lamellar keratoplasty. Ethics approval forthis work was obtained from the Institutional Review Board of the Inoue Eye Hospital and informed consent was
obtained from the patient. All tissues were provided form Inoue Eye Hospital and no tissues were procured fromprisoners. All the experimental methods were carried out in accordance with the guidelines verified and approvedby the Ethics Committee of The University of Tokyo.
The cell culture method was described previously34–36. Briefly, the corneal epithelium was removed from thestroma of the surgical specimen by scraping with a razor blade. A stromal button was incubated overnight at
37 °C in basal medium, i.e. DMEM/F12 medium supplemented with B27 (Invitrogen, Carlsbad, CA, USA) containing 0.02% collagenase (Sigma-Aldrich, St. Louis, MO, USA). Subsequently, the digested tissue and cells weredispersed by pipetting and centrifuged at 800 × g for 5 min. After removing the supernatant, the keratocytes were

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Column1

Forward

Reverse#15′-ATGTCAGAAGTCCCGCTGTG-3′5′-TGATGGGGTCAGAGGGCATA–3′#25′-GCAGCAAAGCACTCAAGAGG-3′5′-CAAACTTCTGCCTGGGCATC-3′#35′-CTTCCTGCTCTGTGTTTAGCCA-3′

5′-ACCTCCAAGTTGAGCAGTGTC-3′

Table 1. Primer set used for the T7 endonuclease cleavage assay of potential off-target loci.

resuspended in 1.0 mL of basal culture medium and seeded in culture dishes. The medium was changed every 2days until the cells reached confluence. Second-passage cells were used in the subsequent transfection and otherassays.

gRNA design and CRISPR-Cas9 construct.

Single gRNA targeting the R124H mutation site of thehuman TGFBI gene was designed using the CRIPSR design tool (publically available at http://crispr.mit.edu/,
http://www.e-crisp.org/E-CRISP/). To construct the CRISPR-Cas9 plasmid targeting the human TGFBI gene,the complementary oligonucleotides hTGFBI gRNA-F and hTGFBI gRNA-R were phosphorylated using T4PNK(TAKARA, Kusatsu, Japan), annealed, and cloned into pSpCas9 BB-2A-GFP (PX458, plasmid #48138; Addgene,
Cambridge, MA, USA) via the BbsI restriction sites. To utilize HDR to edit the human TGFBI R124H mutation, a
100-nt ssODN (hTGFBI R124H HDR ssODN) was designed to target the R124H mutation site.
The oligonucleotide sequences were as follows:
hTGFBI gRNA-F: 5′-CACCACTCAGCTGTACACGGACCACA-3′,
hTGFBI gRNA-R: 5′-AAACTGTGGTCCGTGTACAGCTGAGT-3′,
and hTGFBI R124H HDR ssDNA: 5′-GAGACCCTGGGAGTCGTTGGATCCACCACCACTCAGCTGTACACGGACCGTACGGAGAAGCTGAGGCCTGAGATGGAGGGGCCCGGCAGCTTCACCATCT-3′.

Transfection and cloning. CRISPR-Cas9 constructs (2.5 µg per well) and ssODN (1 µg per well) were trans-

fected into R124H primary cells using FuGENE (Promega, Madison, WI, USA) according to the manufacturer’s
instructions and the cells were incubated for an additional 48 h. Images were obtained by fluorescence microscopy (BZ-9000; Keyence, Osaka, Japan). The cells expressing GFP were single-cell-sorted by FACS (Aria III,
Becton-Dickinson, Franklin Lakes, NJ, USA) at 1 week after transfection. The sorted cells were then clonallyexpanded and analysed as described below.

Indel analysis by restriction fragment length polymorphism (RFLP).

Total DNA was extracted
from cells using the Nucleospin Kit (Takara Bio Inc.). Polymerase chain reaction (PCR) using specific primer
sets (Forward: 5′-GTTGAGTTCACGTAGACAGGC-3′, Reverse: 5′-GACTCCCATTCATCATGCCCA-3′) was
performed to amplify the DNA using the KOD FX Kit (KOD FX; Toyobo, Osaka, Japan) with the following temperature profile: 94 °C for 2 min, followed by 40 cycles of 98 °C for 10 s and 55 °C for 30 s, and 72 °C for 2 min. The
PCR products were treated with the restriction enzyme BsiWI (New England Biolabs, Ipswich, MA, USA). One
microgram of DNA was treated with 1 unit of enzyme and NE Buffer 2.1 at 55 °C for 15 min. The samples wereanalysed by electrophoresis on a 5% polyacrylamide TBE gel.

DNA sequencing analysis. The target site (exon 4 of TGFBI) was amplified by PCR with primers (Forward:

5′-GTTGAGTTCACGTAGACAGGC-3′, Reverse: 5′-GACTCCCATTCATCATGCCCA-3′) targeting the
genomic DNA of R124H-edited cells. After the purification of PCR products, the sequence of samples was analysed using a contract genome sequencing service. (Eurofins Genomics Inc., Tokyo, Japan).

T7 Endonuclease I cleavage assay.

The genome editing efficiency was investigated using a T7 endonuclease I cleavage assay. Genomic regions surrounding the target sites and potential off-target sites of gRNAs were
amplified by PCR using Takara ExTaq (Takara Bio Inc.). Two hundred nanograms of gel-purified PCR products
was re-suspended in NEB Buffer 2, and a hybridization reaction was performed using a thermocycler (BioRad,
Hercules, CA, USA) with the following settings: 95 °C for 5 s, 95–85 °C at −2 °C/s, 85 °C for 30 s, 85–25 °C at−0.1 °C/s, 25 °C for 30 s, followed by maintenance at 4 °C. Five units of T7 endonuclease I (New England Biolabs)
were added to digest the re-annealed DNA. After 2 h of incubation at 37 °C, DNA products were loaded on a 2%
agarose gel and visualised after staining with ethidium bromide. Primers are listed in Table 1.

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Acknowledgements

This work was supported by a grant-in-aid for Scientific Research from the Japanese Ministry of Education,
Culture, Sports, Science and Technology. We would like to thank Azusa Nakamura, assistant researcher, for her
assistance with the experiments.

Author Contributions

Y.T. and K.K. did experiments and prepared all figures. Y.T. also wrote the initial draft of the manuscript. T.S.
prepared human tissue. M.K. and T.T. supported experiments. S.Y., S.A., M.K. and T.M. contributed to datacollection and interpretation, and critically reviewed the manuscript. T.U. and Y.O. designed the study, and wrote
the manuscript as corresponding authors. The final version of the manuscript was approved by all authors.

Additional Information

Competing Interests: Dr. Tomohiko Usui has had financial support from Santen Pharmaceutical Co., Ltd.,
Senju Pharmaceutical Co., Ltd., and Alcon, Inc. The other authors declare no competing financial interests.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps andinstitutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
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© The Author(s) 20177

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Biochemical and Biophysical Research Communications 495 (2018) 2227e2234

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Toll-like receptor 2 activation implicated in oral squamous cell
carcinoma development
Naoki Ikehata a, Masakatsu Takanashi b, Takafumi Satomi a, Masato Watanabe a,
On Hasegawa a, Michihide Kono a, Ai Enomoto a, Daichi Chikazu a, Masahiko Kuroda b, *ab

Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan
Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 7 December 2017
Accepted 18 December 2017
Available online 19 December 2017

Recent studies have revealed that Toll-like receptors (TLRs) are highly expressed and activated in many
types of cancer. Physiologically, TLR2 recognizes bacteria and other microorganisms in the oral cavity;
however, the role of TLR2 in oral squamous cell carcinoma (OSCC) is unclear. In this study, we demonstrated that TLR2 is highly expressed in OSCC in comparison with adjacent non-malignant tissue. TLR2
was also expressed in OSCC-derived cell lines, and its expression was activated by ligands derived frombacteria and mycoplasma. Furthermore, to elucidate the mechanism of OSCC progression via TLR2 signaltransduction, we focused on microRNAs (miRNAs) that are induced by TLR2 activation. Interestingly,
ligand activation of TLR2 induced the expression of miR-146a and we found that downregulation of
caspase recruitment domainecontaining protein 10 (CARD10) mRNA in OSCC-derived cell lines. Moreover, knockdown of CARD10 induced resistance to cisplatin-induced apoptosis in OSCC cells. Theseﬁndings suggest that the activation of TLR2 by bacterial components can enhance the progression of
OSCC and may be implicated in acquired resistance to cisplatin-induced apoptosis through regulation of
the miR-146a pathway.
© 2017 Published by Elsevier Inc.

Keywords:
Oral squamous cell carcinoma
Toll-like receptors
microRNA-146a
CARD10Cisplatin resistance

1. Introduction
The oral cavity is constantly exposed to numerous and varied
microbial species. It has been reported that chronic periodontitis is
an independent risk factor for head and neck cancer [1], and oral
cancer surface bioﬁlms contain signiﬁcantly increased levels of
aerobes and anaerobes at the tumor sites [2]. Furthermore, Binder
Gallimidi et al. reported that chronic infection of periodontal
pathogens Porphyromonas gingivalis and Fusobacterium nucleatum
promote OSCC progression via direct interaction with cancerous
oral epithelial cells, through the activation of epithelial TLRs [3]. We
therefore hypothesized that bacteria contribute to the progressionof OSCC, and focused on TLRs, which speciﬁcally recognize thepresence of invading microorganisms in the host.
TLRs are pattern recognition receptors that play important roles
in innate immune responses, as well as the subsequent induction ofadaptive immune responses [4]. TLR family induces well-* Corresponding author.
E-mail address: kuroda@tokyo-med.ac.jp (M. Kuroda).
https://doi.org/10.1016/j.bbrc.2017.12.0980006-291X/© 2017 Published by Elsevier Inc.

-239-

characterized signalling cascades that result in activation of thedownstream transcription factor NF-kB [5,6]. Recently, several TLRs
were shown to be expressed not only on immune cells but also on avariety of cancer cells [7], and many studies reported that TLRs may
also play an important role in carcinogenesis and tumor progression via activation of the NF-kB pathway [8e13]. Among the TLRs,
TLR2 and TLR4 play crucial roles in the recognition of various
pathogens derived from bacteria [14]. TLR2 forms a heterodimer
with its coreceptors TLR1 or TLR6, and together can recognize lipoproteins, lipoteichoic acid, and peptidoglycan molecules derived
from Gram-positive bacteria [15]. TLR4 which is necessary forrecognizing lipopolysaccharides (LPS) was reported to promotetumor development in head and neck squamous cell carcinoma[16] and be involved in the resistance to cisplatin-induced
apoptosis in OSCC [17]; however, the precise roles of TLR2 in
OSCC progression remain unclear.
Micro RNAs (miRNAs) are small noncoding RNAs (19e22 nucleotides in length) that regulate protein-coding gene expression
by binding to the 30 -untranslated regions (UTRs) of target
messenger RNAs (mRNAs), typically resulting in translational
repression or the cleavage of mRNA in a sequence-speciﬁc manner

2228

N. Ikehata et al. / Biochemical and Biophysical Research Communications 495 (2018) 2227e2234[18]. Studies demonstrated that miRNAs are expressed at highlevels in numerous human cancers and play crucial roles in oncogenesis, cancer progression, and metastasis [19]. miRNAs alsofunction as pivotal regulators and fundamental players in molecular networks that regulate TLR-signalling pathways [20] but it isunknown whether miRNAs induced by TLR-activating signals
contribute to the progression of OSCC.
In this study, we ﬁrst analysed the expression patterns of TLR2
and its coreceptors TLR1 and TLR6 in clinical OSCC tissue specimens, as well as their signalling pathways in OSCC cultured cells. To
determine whether TLR2 signalling contributes to the progressionof OSCC, we focused on miRNAs that were induced by TLR2 activation and analysed the function of these miRNAs and their target
mRNAs. We anticipate that our results will lead to the identiﬁcationof bacteria involved in the malignant progression of OSCC.

TLR2 antibody (ab9100, Abcam) or anti-TLR1 antibody (NBP 177244, Novus Biologicals) or anti-TLR6 antibody (sc-5657, Santa
Cruz Biotechnology).
2.4. Fluorescence immunocytostaining
After ﬁxation with acetone and blocking with 1% goat serum,
cells were incubated with anti-TLR2 antibody (ab9100, Abcam) or
anti-TLR1 antibody (NBP 1-77244, Novus Biologicals) or anti-TLR6
antibody (sc-5657, Santa Cruz Biotechnology). And then, samples
were incubated with Alexa Fluor 594 IgG (Life Technologies) or
Alexa Fluor 594 IgG.
2.5. Western blotting
Cells were lysed with TNE Buffer. Ten mg of proteins wereseparated by SDS-polyacrylamide gel electrophoresis and thentransferred onto PVDF membranes. After blocking, the membranes
were incubated with the following antibodies: anti-TLR2 antibody(ab108998, Abcam), anti-TLR1 antibody (ab22057, Abcam), antiTLR6 antibody (sc-5657, Santa Cruz Biotechnology), anti-caspase3 antibody (D3R6Y, Cell Signaling Technology), or anti-GAPDH
antibody (D16H11, Cell Signaling Technology). Proteins weredetected by ECL Select Western Blotting Detection System (GEHealthcare Life Sciences).

2. Materials and methods
2.1. Clinical specimens of oral squamous cell carcinoma
Twenty-three pairs of primary tumor and adjacent nonmalignant tissue specimens were obtained from patients with
OSCC who underwent surgery at the Tokyo Medical University
Hospital from 2014 to 2016. Patients' background information andclinicopathological characteristics are presented in Table 1. This
study was approved by the Ethics Committee at the Tokyo Medical
University (approval ID: 2842). All patients signed informed consent forms.

2.6. Luciferase reporter gene assay
HSC3 and HSC3-M3 cells were transfected with 10 mg of plasmid
containing pGL 4.32 (luc2P/NFkВ-RE/Hygro; Promega). Aftertransfection, the cells were added Pam3CSK4 (Novus Biologicals) or
FSL-1 (Invivogen). The cells were harvested, and luciferase assays
were performed using the Luciferase Assay System (Promega).

2.2. Cell lines
The human OSCC-derived cell lines, HSC3 (Riken Cell Bank),
HSC3-M3 (JCRB Cell Bank) were cultured in 10% FBS contained
Minimum Essentials Medium Eagle (Sigma-Aldrich), SCC9 and
SCC25 (American Type Culture Collection) were cultured in 10% FBS
contained DMEM-F12 HAM (Sigma-Aldrich).

2.7. Micro RNA microarray analysis
Microarray analysis were performed using the 3D-Gene miRNA
microarray platform (Toray). At each 24-hr time point after the
addition of Pam3CSK4 (100 ng/mL) or FSL-1 (100 ng/mL) to cells,
total RNA was isolated using Isogen reagent (Nippon Gene) and2.3. Immunohistochemistry
Acetone ﬁxed OSCC frozen sections were incubated with antiTable 1
Clinicopathological characteristics of the OSCC patients.
Patient no

Age

Sex

Location

Tumor

Nodes

Metastasis

Stage

Differentiation

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

71
72
70
39
64
67
64
83
69
64
67
85
73
78
63
85
50
73
68
46
74
50
71

M
M
M
M
F
M
M
M
F
F
M
F
F
F
M
M
F
M
M
M
F
F
M

Gingiva
Oral ﬂloor
Oral ﬂloor
Gingiva
Gingiva
TongueGingiva
TongueGingiva
TongueBuccal mucosa
Buccal mucosa
TongueGingiva
Oral ﬂloor
Gingiva
Oral ﬂloor
Gingiva
Gingiva
TongueGingiva
Buccal mucosa
Oral ﬂloor

2
4a
4a
4a
4a
1
4a
2
2
4a
2
4a
1
2
2
2
4a
4a
3
4a
2
2
2

1
2c
0
0
2b
0
0
0
2b
1
0
2b
0
2b
0
0
0
2b
2b
1
0
1
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

III
IVA
IVA
IVA
IVA
I
IVA
II
IVA
IVA
II
IVA
I
IVA
II
II
IVA
IVA
IVA
IVA
II
III
II

Well
Moderate
Moderate
Well
Moderate
Moderate
Well
Well
Well
Poor
Well
Poor
Well
Moderate
Moderate
Well
e
e
e
Well
ModerateeModerate-240-

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subjected to microarray analysis.
2.8. Real-time quantitative RT-PCR (qRT-PCR) analysis
qRT-PCR was performed using TaqMan MicroRNA assays(Applied Biosystems) for miR-146a (Assay ID: 000468) and a FastStart Essential DNA Green Master (Roche Diagnostics) for mRNA.
Nucleotide sequences of the speciﬁc mRNA primers were as follows: CARD10 forward (50 -CTGTGGGAGCGAATCGAGG-30 ) andreverse (50 -CAGCGCAAGATGTCCATCA-30 ); and b-actin forward (50 ACTCTTCCAGCCTTCCTTCC-30 ) and reverse (50 -TGTTGGCGTACAGGTCTTTG-30 ).
2.9. Transfection with miRNA and siRNA
A miRNA-146a-5p mimic, a non-target control, and a siRNAtargeting CARD10 (GeneDesign) were used in this study. Oligonucleotides (100 nM) were transfected into HSC3 and HSC3-M3 cells
using Lipofectamine RNAi MAX reagent (Invitrogen). The miRNAand siRNA used in this study were as follows: hsa-miR-146a sense(50 -UGAGAACUGAAUUCCAUGGGUU-30 ) and antisense (50 -CCUCUGAAAUUCAGUUCUUCAG-30 ); non-target control sense (50 AACGCGCUAGUAGGGAUUGtt-30 ) and antisense (50 -CAAUCCCUACUAGCGCGUUtt-30 ); and CARD10-speciﬁc siRNA sense (50 -GCUAUandantisense(50 GAGGCCUUCCUGGAtt-30 )UCCAGGAAGGCCUCAUAGCtt-30 ).
2.10. Cell viability assay
Twenty-four hours after the seeding of 5 � 103 HSC3 and HSC3M3 cells, 100 nM of CARD10 siRNA was transfected into the cells.
Twenty-four hours after the transfection, 5 mМ cisplatin (Wako
Pure Chemicals) was added to the cells. From 24 h to 72 h after the
addition of cisplatin, cell viability was measured by CellTiter-Glo 2.0
assay (Promega).
2.11. Statistical analysis
Statistical analysis were performed using the Student's t-test
and Tukey-Kramer test. A p-value of less than 0.05 was considered
to indicate a statistically signiﬁcant difference between two groups.
Values in the graphs are indicated as the mean ± standard
deviation.
3. Results
3.1. TLR2 is expressed in OSCC tissues and cell lines
To determine the potential roles of TLRs in the regulation oftumor development, we ﬁrst assessed the expression of TLR2 andits coreceptors TLR1 and TLR6 in 23 paired OSCC tissues (primarytumor and adjacent non-tumor tissues from the same patient) byimmunohistochemistry methods. Higher expression levels of TLR2
and TLR1/TLR6 were detected in OSCC tumor tissue compared withadjacent non-malignant tissue (Fig. 1a). We also observed theexpression of TLR2 and TLR1/TLR6 in four OSCC cell lines byimmunocytochemistry (Fig. 1b) and western blotting (Fig. 1c).
These results indicate possible involvement of these molecules in
OSCC tumor progression. Elevated expression levels of TLR2 protein
were observed in HSC3, HSC3-M3, and SCC25 cells. HSC3 and HSC3M3 cells showed similar TLR2 protein expression levels, as well as
its coreceptors TLR1 and TLR6. Therefore, we used HSC3 and HSC3M3 cells for further in vitro examinations because the same conditions could be used for the functional analysis of both TLR2 andTLR1/TLR6. Because the HSC3-M3 cell line was established from

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2229

HSC3 cells as a highly metastatic cell line [21], we also used this cellline to analyse whether TLR2 affected the acquisition of malignant
potential. SCC9 expressed low levels of TLR2 protein, and the proliferation of SCC25 was lower than the other cell lines, so these celllines were excluded from further analyses.
3.2. TLR2 and TLR1/6 are activated by their ligands
NF-kВ is the major transcriptional factor induced by the activation of TLR signalling. Activation of both TLR2/TLR1 and TLR2/
TLR6 heterodimers by a stimulus, such as bacterial lipoprotein,leads to the activation of NF-kВ via TLR signal transduction.
Therefore, to conﬁrm whether TLR2 and TLR1/TLR6, which are
expressed in OSCC cells, induce activation signalling pathways by
their ligands, we performed an NF-kВ activation assay using aplasmid vector containing the NF-kВ response element. Pam3CSK4and FSL-1 are speciﬁc ligands for TLR2/TLR1 and TLR2/TLR6 heterodimers, respectively. Increased luciferase activities wereobserved for both ligands in a concentration-dependent manner(Fig. 2a). Increased luciferase activation was also observed in thehighly metastatic cell line HSC3-M3 compared with HSC3. These
data demonstrate that TLR2/TLR1 and TLR2/TLR6 receptors playfunctional roles in OSCC cells.
3.3. Activation of TLR2 induces the expression of miR-146a in OSCC
To reveal the mechanisms of OSCC progression via TLR2 signaltransduction, we focused on miRNAs, which are induced by TLR2
activation. We performed miRNA microarray analysis andcompared the miRNA expression proﬁles in OSCC cell lines. Our
strategy for miRNA selection is presented in Fig. 2b. First, we
screened 389 miRNAs that were previously reported as associatedwith cancer. Then, we screened miRNAs that were upregulated
upon TLR2 ligand stimulation (log2 ratio >1.5). The numbers of
upregulated miRNAs are shown in Fig. 2c, and we identiﬁed miR146a-5p as upregulated at all the time points measured, by both
ligands, and in both cell lines (Fig. 2d). To validate our miRNA
microarray results, we analysed the expression levels of miR-146a5p induced by ligand stimulation of TLR2 in the two cell lines. The
expression level of miR-146a-5p was signiﬁcantly higher at all thetime points with both ligands in both cell lines, except for 6 h and48 h after Pam3CSK4 stimulation in HSC3-M3 cells (Fig. 2e). FSL-1
appeared to increase the expression level of miR-146a-5p
compared with Pam3CSK4 in both cell lines. In addition, thehighly metastatic cell line HSC3-M3 induced miR-146a-5p
expression more robustly than the HSC3 cell line at 24 h after the
addition of FSL-1.
3.4. Identiﬁcation of putative target genes regulated by miR-146a5p
To identify putative target genes regulated by miR-146a-5p, we
conducted in silico analysis. Our strategy for the selection of miR146a-5p target genes is shown in Fig. 3a. First, we screened miR146a-5p target genes that included a predictive miR-146a-5p
binding site in their 30 -UTR using the TargetScan database andidentiﬁed 224 candidate genes. Among these genes, we focused on
6 candidate genes that have tumor suppressive functions (Fig. 3b).
To investigate whether these genes were targeted by miR-146a-5p,
we analysed their mRNA expression levels by miR-146a-5p transfection. Furthermore, we found that the mRNA expression level of
CARD10 was signiﬁcantly repressed in miR-146a-5p transfected
cells compared with untreated or miR-control transfected cells inboth cell lines (Fig. 3c). Next, we analysed whether CARD10
expression was inhibited by the activation of TLR2. Expression

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N. Ikehata et al. / Biochemical and Biophysical Research Communications 495 (2018) 2227e2234

Fig. 1. TLR2 and TLR 1/TLR6 expression in OSCC tissues and cell lines. (a) Immunohistochemistry for TLR2 and TLR1/TLR6 in OSCC tissues and adjacent nonmalignant epithelia. (b)Immunocytochemistry for TLR2 and TLR1/TLR6 in OSCC cell lines. Nuclei were visualized by staining with DAPI (blue), and TLR2/TLR1/TLR6 were visualized by staining with theirrespective antibodies (red). Controls were visualized by staining with omission of the primary antibody. (c) TLR2 and TLR1/TLR6 proteins were analysed by western blotting. The
ratios of TLR2, TLR1, and TLR6 to total GAPDH are shown in the graph.

levels of CARD10 mRNA were signiﬁcantly downregulated by FSL-1
stimulation in HSC3-M3 cells (Fig. 3d). These results indicate thatthe activation of TLR2 by their ligands induced the expression of
miR-146a-5p and downregulated CARD10 in OSCC cells.
3.5. Effects of CARD10 silencing on anticancer drug resistance in
OSCC cell lines
CARD10 is known to regulate caspase-induced apoptosis [22].
Therefore, we investigated whether the silencing of CARD10 could
protect OSCC cells from cisplatin-induced cells death. CARD10siRNA was transfected into OSCC cell lines, which were then treatedwith cisplatin for 24 he72 h. As shown in Fig. 4a, HSC3 and HSC3M3 cells treated with cisplatin presented with alteredmorphology, with many cells undergoing apoptosis. However, theeffect of cisplatin was partially abolished when OSCC cells weretransfected with CARD10 siRNA. Cell viability assays demonstrated
that silencing CARD10 signiﬁcantly rescued cisplatin-induced cells
death compared with untransfected controls in both cell lines(Fig. 4b). Next, we investigated whether the inhibition of cisplatininduced cells death by CARD10 silencing was associated withdecreased apoptosis. Western blotting demonstrated lower levelsof cleaved-caspase3 protein expression in CARD10 siRNA transfectants in both cell lines, and these results were also observed inmiR-146a-5p transfectants (Fig. 4c). Taken together, these results
demonstrated that silencing of CARD10 induced resistance tocisplatin-induced apoptosis in OSCC cells.

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2231

Fig. 2. TLR signalling activation and upregulation of miR-146a-5p induced by TLR2 ligands in OSCC cells. (a) NF-kВ transcriptional activity in HSC3 and HSC3-M3 cells with
Pam3CSK4 and FSL-1. After transfection with a luciferase reporter vector containing an NF-kB response element that drives transcription of the luciferase reporter gene, each cellline was treated with each ligand. (b) Strategy for the selection of potential miRNAs associated with OSCC progression by a miRNA arrayebased approach. (c) Number of upregulated
miRNAs in OSCC cell lines induced by ligand stimulation from miRNA array-based approach. (log2 ratio > 1.5) (d) miR-146a-5p expression levels in OSCC cell lines induced bystimulation with TLR2 ligands. Graphs show fold changes (log2 ratios) of miR-146a-5p induced by ligand stimulation. The dashed line indicates the threshold. (e) Validation of miR146a-5p expression by qRT-PCR.

4. Discussion
The innate immune response is activated by TLR signalling andresponds to invading microorganisms, including bacteria and fungithat may be introduced via the oral cavity. We wondered whether
oral cancers and tumor progression were affected by innate immune activation systems. A recent study reported that TLRs are
expressed on many types of cancer cells and play pivotal roles incarcinogenesis and tumor progression [23]. In the case of oral, head,and neck SCC, TLR4 stimulation with LPS was shown to confer
resistance to tumor lysis by natural killer cells [16] as well as-243-resistance to cisplatin-induced apoptosis [17]. In regards to TLR2, it
was reported that TLR2 was expressed on the keratinocytes ofdysplastic epithelia and OSCC [24]; however, the functional role of
TLR2 in OSCC progression has not been elucidated. In this study, we
found that TLR2 was highly expressed in OSCC tissue comparedwith adjacent non-malignant epithelia (Fig. 1a). TLR2 was also
expressed in OSCC cell lines (Fig. 1b and c), and was activated withbacteria and mycoplasma-derived agonists (Fig. 2a). In addition to
the recognition of pathogen-associated molecular patterns(PAMPs), TLRs have evolved to recognize endogenous signals
released from dying cells, namely, damage-associated molecular

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N. Ikehata et al. / Biochemical and Biophysical Research Communications 495 (2018) 2227e2234

Fig. 3. Regulation of CARD10 mRNA expression by miR-146a overexpression and TLR2 activation in OSCC cells. (a) Strategy for the identiﬁcation of putative target genes
regulated by miR-146a-5p. (b) Candidate target genes of miR-146a-5p. (c) CARD10 mRNA expression after transfection with miR-146a-5p. b-actin expression was used fornormalization. (d) CARD10 mRNA expression after the addition of FSL-1. b-actin expression was used for normalization.

patterns (DAMPs) including high-mobility group box 1 (HMGB1)[25]. Importantly, HMGB1 is directly involved in cancer cell invasion and metastasis [26]. Further investigation is necessary to
determine the effect of DAMPs on OSCC progression, but our results
suggest that TLR2 is functionally active on OSCC cells, similar to
TLR4 activity in cancer cells.
How does TLR2 expression affect OSCC? To shed light on therelationship between TLR2 expression and OSCC, we ﬁrst examined
the miRNA expression proﬁle of TLR2-expressing cell lines using
miRNA microarray (Fig. 2b and d), and found that CARD10 was thetarget of miR-146a in OSCC cells (Fig. 3a and c). CARD10 is a scaffold
protein and is also referred to as caspase-recruitment domain(CARD) membrane-associated protein 3 (CARMA3), which belongs
to the CARMA protein family [27]. Each member shares similar
structures: an N-terminal CARD, followed by a coiled-coil domain, alinker region, a PDZ domain, a Src homology 3 (SH3) domain, and a
GUK-like domain [28]. The CARD domain is found in several molecules that regulate apoptosis [22]. This domain mediates the assembly of larger protein complexes by its association with different
individual CARD domains, and CARDeCARD interactions play animportant role in signalling apoptosis [29]. When cell stress, such astreatment with anti-cancer drugs, induces apoptosis, cytochrome cis released from mitochondria. The released cytochrome c binds to
the Apaf-1/CARD complex, which induces caspase 9 activation [30];however, down-regulation of CARD10 by TLR2 activation is unable
to initiate apoptosis signalling.
Therefore, CARD10 may function as a proapoptotic molecule bymediating the assembly of larger protein complexes in during thedevelopment of OSCC. Therefore, we analysed the function of

CARD10, focusing on its proapoptotic potential using cisplatin.
Cisplatin-induced cell death is known to occur via apoptosis [31]and is broadly used for the treatment of many malignancies [32].
Interestingly, as demonstrated in Fig. 4, cisplatin-induced cell death
and apoptosis were signiﬁcantly inhibited by CARD10 silencing.
These results indicated that the suppression of CARD10 induces
resistance to cisplatin-associated cell death and apoptosis in OSCC
cells. This is the ﬁrst report demonstrating that OSCC progression iscaused by the suppression of CARD10 mediating the miR-146asignalling pathway. The detailed molecular mechanism of miR146a expression by TLR2 remains unclear; it is possible that thelevel of miR-146a expression can be a sensitive marker for cisplatin
activity.
In this study, we compared the function of two OSCC cell lines,
HSC3 and HSC3-M3. HSC3 was derived from human tongue cancer[33] and HSC3-M3 is a highly metastatic cell line established from
HSC3 [21]. We selected these cell lines to analyse whether TLR2
function affects the acquisition of malignant potential. There wasno difference in protein levels of TLR2 or its coreceptors TLR1 andTLR6 between the HSC3 and HSC3-M3 cell lines (Fig. 1c). However,
the activation of NF-kВ (Fig. 2a), induction of miR-146a-5p (Fig. 2e),and suppression of CARD10 (Fig. 3d), which are all caused by TLR2
activation, were signiﬁcantly higher in the HSC3-M3 cells. Theseﬁndings indicate that OSCC cells have increased sensitivity to TLR2
as a result of acquiring malignancy. If the activity of the TLR2 signalling pathway can be evaluated, it may provide further insight
into OSCC prognosis.
In conclusion, our results indicated that TLR2 was highly
expressed in human OSCC tissue and functional in OSCC cell-244-

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2233

Fig. 4. Effect of CARD10 siRNA transfection on cisplatin resistance in OSCC cells. (a) Morphological changes in HSC3 and HSC3-M3 cells incubated with or without cisplatin or
CARD10 siRNA. (b) CARD10 siRNA transfected cells were treated with 5 mM cisplatin. Cell viability was measured from 24 h to 72 h after cisplatin treatment. (c) Caspase-3 protein
expression in CARD10 siRNA transfected cells or miR-146a-5p transfectants performed at 72 h after treatment with 5 mM cisplatin.

cultures. In addition, sensitivity to TLR2 increased as a result of
acquiring malignant phenotypes in OSCC cells. TLR2 activation bybacterial pathogens induced miR-146a-5p expression and thesubsequent suppression of CARD10, and consequently, resulted in
growth-promotion and resistance to cisplatin-induced cell death
and apoptosis in OSCC cells. In Supplemental Fig. 1, we present
putative mechanisms of OSCC progression via TLR2 activation.
Overall, these results support our hypothesis that microbial infection contributes to OSCC progression.

Appendix A. Supplementary data
Supplementary data related to this article can be found athttps://doi.org/10.1016/j.bbrc.2017.12.098.
Transparency document
Transparency document related to this article can be foundonline at https://doi.org/10.1016/j.bbrc.2017.12.098.
References

Conﬂicts of interest
The authors have no conﬂict of interest.

Acknowledgements
We thank Koji Fujita for his technical assistance. This research
was supported by a grant-in-aid from The Ishidsu Shun Memorial
Scholarship, Japan, and in part by Grants-in-Aid for scientiﬁc
research (B: 17H04067) and (C: 16K09376) and by the Development
program of microRNA measurement technology foundation inbody ﬂuid from Japan Agency for Medical Research and Development, AMED (17ae0101016s0904) and supported in part by Strategic Research Foundation (s1511011) Grant-aided Project forPrivate Universities from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.

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[30] S.B. Bratton, G.S. Salvesen, Regulation of the Apaf-1-caspase-9 apoptosome,
J. Cell Sci. 123 (2010) 3209e3214.
[31] V.M. Gonzalez, M.A. Fuertes, C. Alonso, J.M. Perez, Is cisplatin-induced cell
death always produced by apoptosis? Mol. Pharmacol. 59 (2001) 657e663.
[32] S. Dilruba, G.V. Kalayda, Platinum-based drugs: past, present and future, Canc.
Chemother. Pharmacol. 77 (2016) 1103e1124.
[33] K. Rikimaru, H. Toda, N. Tachikawa, N. Kamata, S. Enomoto, Growth of themalignant and nonmalignant human squamous cells in a protein-free deﬁned
medium, Vitro Cell Dev. Biol.: J. Tissue Cult. Assoc. 26 (1990) 849e856.

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Regular Article
LYMPHOID NEOPLASIA

Role of exosomes as a proinﬂammatory mediator in thedevelopment of EBV-associated lymphoma
Hiroshi Higuchi,1,* Natsuko Yamakawa,1,* Ken-Ichi Imadome,2,* Takashi Yahata,3 Ryutaro Kotaki,1 Jun Ogata,1 Masatoshi Kakizaki,4 Koji Fujita,5Ryouichi Horie,10 Toshiki Watanabe,11 Toshio Kitamura,12 Kiyoshi Ando,7 Takao Kashiwagi,13 Toshimitsu Matsui,13 Akinao Okamoto,14
Hiroshi Handa,15 Masahiko Kuroda,5 Naoya Nakamura,16 and Ai Kotani1,17,18
Department of Hematological Malignancy, Institute of Medical Science, Tokai University, Isehara, Kanagawa, Japan; 2Department of Infectious Diseases, National
Center for Child Health and Development, Setagaya-ku, Tokyo, Japan; 3Research Center for Cancer Stem Cell and 4Department of Gastroenterology, Tokai
University School of Medicine, Isehara, Kanagawa, Japan; 5Department of Molecular Pathology, Tokyo Medical University, Shinjuku-ku, Tokyo, Japan; 6Department
of Intractable Diseases, Institute of National Center for Global Health and Medicine, Toyama, Shinjuku-ku, Tokyo, Japan; 7Department of Hematology andOncology, Tokai University School of Medicine, Isehara, Kanagawa, Japan; 8Malaysia-Japan Institute of Technology, Universiti Teknologi Malaysia, Kuala Lumpur,
Malaysia; 9Department of Biotechnology, Kulliyyah of Science, International Islamic University Malaysia, Kuantan, Malaysia; 10Department of Hematology, Schoolof Medicine, Kitasato University, Sagamihara, Kanagawa, Japan; 11Department of Medical Genome Sciences, Graduate School of Frontier Sciences, and 12Divisionof Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan; 13Department ofInternal Medicine, Nishiwaki Municipal Hospital, Nishiwaki, Hyogo, Japan; 14Department of Hematology, Fujita Health University School of Medicine, Toyoake,
Aichi, Japan; 15Department of Medicine and Clinical Science, Gunma University, Maebashi, Gunma, Japan; 16Department of Pathology, Tokai University School of
Medicine, Isehara, Kanagawa, Japan; 17Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan; and
18
AMED-PRIME, Japan Agency for Medical Research and Development, Tokyo, Japan1

Epstein-Barr virus (EBV) causes various diseases in the elderly, including B-cell lymphoma
such as Hodgkin’s lymphoma and diffuse large B-cell lymphoma. Here, we show that EBVl EBV-coding miRNAs
acts in trans on noninfected macrophages in the tumor through exosome secretion andare transferredaugments the development of lymphomas. In a humanized mouse model, the different
from infected into
noninfected cells byformation of lymphoproliferative disease (LPD) between 2 EBV strains (Akata and B95-8)
exosome to regulate
was evident. Furthermore, injection of Akata-derived exosomes affected LPD severity,
the function for the
possibly through the regulation of macrophage phenotype in vivo. Exosomes collected
tumorigenesis.
from Akata-lymphoblastoid cell lines reportedly contain EBV-derived noncoding RNAsl Production of EBVsuch as BamHI fragment A rightward transcript (BART) micro-RNAs (miRNAs) and EBVcoding miRNAs will be
encoded RNA. We focused on the exosome-mediated delivery of BART miRNAs. In vitro,
an excellent diagnostic
marker to separate
BART miRNAs could induce the immune regulatory phenotype in macrophages characpatients with EBV1
terized by the gene expressions of interleukin 10, tumor necrosis factor-a, and arginasediffuse large B-cell
1, suggesting the immune regulatory role of BART miRNAs. The expression level of anlymphoma into 2
EBV-encoded miRNA was strongly linked to the clinical outcomes in elderly patients withgroups.
diffuse large B-cell lymphoma. These results implicate BART miRNAs as 1 of the factors
regulating the severity of lymphoproliferative disease and as a diagnostic marker for EBV1 B-cell lymphoma. (Blood.
2018;131(23):2552-2567)
KEY POINTSIntroduction
Epstein-Barr virus (EBV) is an oncogenic human g-herpes virusthat causes various diseases such as B-cell lymphoma, T-cell andnatural killer cell lymphoma, gastric cancer, nasopharyngeal
carcinoma, and some autoimmune diseases.1,2 Among these
diseases, B-cell lymphoma is 1 of the common forms in EBVrelated cancer because of the strong tropism of EBV to B cells.
EBV contains several oncogenes that encode proteins; thesegenes include latent membrane protein (LMP) and EBV nuclear antigen (EBNA). Once B cells are infected, expression ofthese genes induces immortalization and aberrant proliferationof the cells, leading to the development of lymphoblastoid cell2552

lines (LCLs).1-4 Clinical studies have demonstrated that EBV1 cases
show a poorer prognosis than EBV2 cases in Hodgkin’s lymphoma
and diffuse large B-cell lymphoma (DLBCL) of the elderly.5,6Therefore, the establishment of a novel therapeutic strategy thatspeciﬁcally treats EBV1 B-cell lymphoma is required.
Presently, we found signiﬁcant differences in the survival rate,
macrophage inﬁltration, and EBV-positive tumor cells between
mice infected with the Akata and B95-8 strains of EBV, regardlessof the similar transforming ability of the strains in vitro, suggesting that these EBV strains have a different ability to form atumor microenvironment.

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Jun Lu,6 Kazuaki Yokoyama,1 Kazuki Okuyama,1 Ai Sato,7 Masako Takamatsu,1 Natsumi Kurosaki,1 Syakira Mohamad Alba,1,8 Azran Azhim,9Akata was originally isolated from Burkitt lymphoma. B95-8 wasoriginally isolated from infectious mononucleosis and is maintained in marmoset LCLs. Hence, they are completely different
strains, and so display several differences. We focused on thedeletion in BamH1 fragment A rightward transcript (BART) region on the B95-8 genome as the most outstanding difference.
Interestingly, 40 micro-RNAs (miRNAs) were reported to cluster
and were transcribed from the BART region (BART miRNAs).7
miRNAs are small noncoding RNAs approximately 22 nucleotides in length that posttranscriptionally regulate the deadenylation, translation, and decay of their target messenger RNAs(mRNAs).8,9

Results
Differential formation of lymphoproliferative
diseases in vivo between Akata and B95-8 strains
The functional human immune system, including T cells, B cells,
and natural killer lymphocytes, was reconstituted in nonobese
diabetic (NOD)/shi-scid, interleukin (IL)-2Rg null mice (NOG mice)that received hematopoietic stem cell transplants.17 These humanized mice recapitulate the human lymphoproliferative disease(LPD) induced by EBV infection.18 To date, a variety of EBV strains
have been isolated from patients. Although all these strains equivalently transform human B ells in vitro, the potential for tumorigenicity in vivo involving nontumor cells is not well understood.

All mice infected with Akata died within 12 weeks with massive
inﬁltration of EBV-encoded RNA (EBER)1 cells evident in the
spleen (Figure 1A-B). In contrast, approximately 70% of mice
infected with B95-8 survived (Figure 1A). These surviving mice
had few EBER1 cells in the spleen (Figure 1B) and did not show
symptoms of LPD, such as elevated viral load or body weight loss
(data not shown). Pathological examination indicated increased
inﬁltration of both CD681 and CD1631 cells (markers for M1 andM2 macrophages, respectively) in the spleens of mice infectedwith Akata compared with those infected with B95-8 (Figure 1C).
Cells were counted in 16 ﬁelds of 3 Akata-infected mice and
20 ﬁelds of 3 B95-8-infected mice. Notably, the distribution of
CD681 and CD1631 cells seemed not to overlap. CD681 cells
were sparse in the EBER1 tumor cell-rich area, whereas numerous CD1631 cells were located surrounding the tumor cellrich area. Thus, the Akata and B95-8 strains showed different
potentials for lymphomagenesis in vivo, despite having a similar
titer in vitro. Consistent with increased inﬁltration of lymphoma
cells and macrophages, increased expression of IL-10 was detected in the spleens of mice infected with Akata (Figure 1D),suggesting that infection with Akata could induce immune suppressive environment.

EBV1 lymphoma-derived exosomes cause severe
LPD in humanized mice model
Recently, the involvement of the exosome, 1 of the extracellular
vesicles, has been reported in the development and metastasis
of various tumors.11-15 The exosome is composed of a lipid bilayer with a diameter of 50 to 200 nm and is a carrier of biologicalmolecules, including proteins, lipids, and nucleic acids. Importantly, EBV1 B-cell lymphoma cells secrete exosomes.10,16 To
investigate the involvement of exosomes in the differential
formation of LPD between the Akata and B95-8 strains, we intravenously injected exosomes into B95-8-infected humanizedmice.
Exosomes were isolated from the culture supernatants of Akataand B95-8-LCL cultures (Akata and B95-8 exosomes, respectively), using the differential centrifugation protocol. 21 Thismethod isolates and puriﬁes exosomes from the supernatant ofB-cell culture without EBV.10 EBV-infected cell-derived exosomeswith a diameter of 10 to 100 nm were observed by electron
microscopy (Figure 2A). The mice infected with B95-8 were injected with either Akata or B95-8 exosomes intravenously at
8 weeks postinfection. Surprisingly, the injection of Akata exosomes caused severe LPD in 6 of the 8 mice within 3 to 7 weeks
after injection, but the injection of B95-8 exosomes did not do soin 5 mice (Figure 2B). Pathological analysis showed that the inﬁltration of EBER1 lymphoma cells in the spleen of B95-8-infected
mice was recovered to a level that was comparable to that of
the Akata-infected mice by the injection of Akata exosomes(Figure 2C). Furthermore, the inﬁltration of both CD681 andblood® 7 JUNE 2018 | VOLUME 131, NUMBER 23

EBV CODING miRNAs REGULATE MACROPHAGE

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EBV1 B-cell lymphoma is composed of a proportion of tumor
cells and a large proportion of nontumor cells including immune
cells, which is termed the inﬂammatory niche. This suggeststhat the inﬂammatory niche is necessary for the survival and growthof tumor cells. Although it is unclear whether BART miRNAs
affect the formation of the tumor microenvironment, recently,
Pegtel et al10 showed that EBV1 lymphoma cells secreted BART
miRNAs through extracellular vesicles called exosomes and thatmonocyte-derived dendritic cells selectively incorporate the exosomes. Exosomes are cell-derived vesicles that function as communicators of various molecules, such as proteins, mRNAs, and miRNAs,
from donor cells to recipient cells.11-13 In tumor studies, tumorderived exosomes supported tumor development and metastasis
through various mechanisms.11-15 It was reported that EBV1 lymphoma cells secrete exosomes and deliver EBV-derived noncoding
RNA, such as BART miRNAs and EBV-encoded RNA (EBER),10,16
suggesting that tumor-derived exosomes could affect the development of EBV1 lymphoma. Exosome-mediated secretion of
miRNAs also appears to be critical for the formation of the metastatic niche.15 In this study, we demonstrate the essential role of
BART miRNAs for the formation of an inﬂammatory niche in EBV1
B-cell lymphoma. We compared lymphoma forming capacity
between 2 EBV strains, Akata and B95-8, using a humanized micemodel, and observed different lymphoma formation. Furthermore, we showed the role of exosomes for the development of
EBV1 B-cell lymphoma in vivo, possibly through regulation ofmacrophage inﬁltration. Exosome-mediated delivery of BART
miRNAs was critical for the induction of the immune regulatory
phenotype in macrophages in vitro. Furthermore, analysis of
EBV1 DLBCL in elderly patients revealed the strong correlation
between the expression levels of BART miRNAs and clinicaloutcomes of the lymphoma. These results suggest that BART
miRNAs could be a promising diagnostic tool as well as a novel
therapeutic target of EBV1 lymphoma.

To clarify the differences in tumorigenicity between EBV strains,mice were inoculated by tail vein injection with a low (1 3 103) 50%
transforming dose (supplemental Figure 1, available on the Blood
Web site) of either the Akata or B95-8 EBV strain. Both are type I
strains. Akata was isolated from EBV1 Burkitt lymphomas, in which
EBV exhibits the type I form of latency. B95-8 was isolated from
infectious mononucleosis and maintained in infected marmoset
LCL, which shows the type III form of latency.19,20

A

B

EBER
60

B95-8

80

Ratio of positive cells (%)

Survival rate (%)

B95-8

Akata

100
HE

60
40

Akata

P = 0.001

EBER

20

0

10

20

30

C

CD68
B95-8

Ratio of positive cells (%)

Ratio of positive cells (%)

10
8
6
4
2

Mouse #1

20
10

5

*

4
3

Akata
B95-8

2
1
0

0

D

Akata
B95-8

6
*

CD163

30

CD163

12

CD68

40

0

Time (weeks)

Akata

*

Mouse #2

Mouse #3

Akata

B95-8

Figure 1. Equivalent transformation units of Akata and B95-8 virus demonstrate differential formation of lymphoproliferative diseases in vivo. (A) Survival curve of
Akata (n 5 9, solid line) and B95-8-infected mice (n 5 8, dashed line). P 5 .001, log-rank test. (B-C) Immunohistochemical staining of spleens from EBV-infected mice. (B) The
spleens were stained with hematoxylin and eosin or treated for EBER in situ hybridization. Scale bar, 100 mm. (C) CD68 and CD163 immunohistochemical staining of
the spleen. Scale bar, 100 mm. Cells were counted in 16 ﬁelds from 3 Akata-infected mice and 20 ﬁelds from 3 B95-8-infected mice, respectively. Ratio to total cells were
calculated. *P , .01, Mann-Whitney U test. (D) Production of IL-10 was detected by immunohistochemical staining of spleens collected from EBV-infected mice, following
the manufacturer’s protocol. Rabbit anti-human IL-10 (LS-B7432) was purchased from LifeSpan BioSciences, Inc. (Seattle, WA). Biopsies were collected from 3 Akata- andB95-8-infected mice. Scale bar, 25 mm.

CD1631 macrophages was increased 2- to 3-fold by the injectionof Akata exosomes (Figure 2D). To count the number of CD681
cells and CD1631 cells, 2 mice were analyzed in each group, and3 ﬁelds were counted in each mouse.
To exclude the possibility of contamination of virus particles within
the collected exosomes, the EBV genome was ampliﬁed by PCR.

2554

The B95-8 genome, but not the Akata genome, was detected athigh levels in the mice injected with Akata exosomes after B95-8
infection, indicating that the proliferating cells were infected with
B95-8, but not with Akata (Figure 2E). These results suggest that
exosomes derived from Akata-infected lymphoma caused thesevere LPD in the humanized mouse model, which was associatedwith increased inﬁltration of macrophages in lymphoma tissue.

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0

50

A

Akata
exosomeB95-8
exosome

C

B

B95-8
+ exosome

Akata

Exosome i.v.
B95-8
exosome

50

HE
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Survival rate (%)

100

P = 0.01

Akata
exosome

EBER

0
0

5

10

15

20

25

Time (weeks)

D

B95-8
+ Akata exosome

Akata

*
200

Counts / eld

CD68

Akata
B95-8
+Akata exosome

150
100
50

CD163

0

E

CD68 CD163

Fold change

B95-8 genome
10000

1

Akata

B95-8 B95-8 +Akata-exosome

Akata genome

Fold change

Akata

BART cluster 1

B95-8

BART
cluster 1

Akata

BART cluster 1

B95-8

BART
cluster 1

BART cluster 2

100

1.0
0.5
0

Akata

B95-8

BART cluster 2

B95-8 +Akata-exosome

Figure 2. Exosomes collected from Akata-LCLs accelerate LPD formation. (A) Electron microscope image of exosomes isolated from Akata-LCL (left) and B95-8-LCL culture
medium by ultracentrifugation. Scale bar, 100 nm. (B) Survival curve of B95-8/miRNA-rich exosome-treated mice (n 5 8, solid line) or B95-8/EBV-miRNA-deleted exosomestreated mice (n 5 5, dashed line). P 5 .01, log-rank test. (C) The spleens were stained with hematoxylin and eosin or treated for EBER in situ hybridization. Scale bar, 50 mm.
(D) CD68 and CD163 immunohistochemical staining of the spleen (left). Scale bar, 50 mm. The cells positive for each macrophage marker were counted (right). Two mice
were treated per group, and 3 ﬁelds were counted in each mouse. *P , .01, Student t test. (E, left top) qPCR revealed that the B95-8 virus-speciﬁc region was ampliﬁed in LPD in
B95-8/miRNA-rich exosome-treated mice. (Left bottom) Akata virus-speciﬁc region was not ampliﬁed. (Right) The binding sites of primer pairs used to speciﬁcally amplify theB95-8 virus genome and Akata virus genome are indicated. Indicated primer pair detects B95-8 genome (top) and Akata genome (bottom), respectively. Half of the BART cluster
1 miRNAs, and all BART cluster 2 miRNAs were deleted in the B95-8, but not in Akata virus.

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EBV CODING miRNAs REGULATE MACROPHAGE

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EBER

CD68

Figure 3. Depletion of CD1631macrophages induces
the elimination of EBER1lymphoma cells in humanize
mice model. Clodronate liposome (300 mL/mouse) was
injected into Akata-infected mice (intraperitoneally).
After 5 days, the spleens were collected. EBER was
detected by in situ hybridization (upper). Scale bar,
50 mm. CD68/163 was detected by immunohistochemical
staining. Scale bar, 20 mm. The effects of clodronate liposome in mice (lower). EBER and CD68/CD163 expression was analyzed in each mouse spleen. -, no EBER
or CD68/163-positive cells in 1 microscopic ﬁeld; 1, ,10
positive cells; 11, .10 positive cells. **P , .05, Student
t test.

CD163

PBSliposome

Clodronateliposome

CD68+
Macrophage

CD163+
Macrophage

EBER
++

+

+

++

+

++

-

+

+

++

+

-

-

-

+

+

-

-

+

-

-

-

+

-

-

-

+

++

-

-

+

++

+

+

CD1631 macrophage depletion induces theelimination of EBER1 lymphoma cells in
the humanized mouse model
A large number of inﬁltrated macrophages in LPDs suggest an
essential role for macrophages in EBV-related lymphoma. Accumulating evidence has revealed the critical roles of macrophages for the tumor microenvironment.22,23
To determine the speciﬁc functions of macrophages in the tumor, the macrophages in the Akata-infected mice were depleted
by clodronate liposomes, which speciﬁcally eliminated themacrophages. Surprisingly, a single injection of clodronate liposomes into mice with LPD eliminated CD1631 macrophages
as well as the EBER1 lymphoma cells (Figure 3). Notably, although CD681 macrophages were not completely cleared,
EBER1 lymphoma cells completely disappeared in some of themice. Three and 6 mice were treated with phosphate-bufferedsaline or clodronate liposomes, respectively. This may reﬂect
the function of CD1631 macrophages in human patients. These
results suggest that macrophages are essential for the development of EBV1 lymphoma, and especially suggest that CD1631
macrophages may act as key bystander cells.

Incorporation of exosomes into monocytes in an“eat me” signal-dependent manner
Exosomes derived from Akata-LCL increased the inﬁltration ofmacrophages, especially CD1631 macrophages. The phenotype
of macrophages can be differently regulated by various cytokines,
such as interferon-g and IL-4, which are produced from T helper 1
and T helper 2 cells, respectively.24,25 Here, to investigate whether the

2556

**

-

macrophage phenotype could be regulated directly or indirectly by
the lymphoma-derived exosomes, human peripheral blood mononuclear cells (PBMCs) were treated with exosomes in vitro.
Similar to Figure 2, exosomes were isolated from the culture supernatant of Akata- and B95-8-LCL by ultracentrifugation. Fractionation of isolated exosomes by iodixanol gradient centrifugation26
detected CD63, an exosome marker, from fractions 3 thorough6 (Figure 4A), indicating that exosomes were successfully isolated
from the culture supernatant. Because CD63 is highly glycosylated,27
the different sizes of CD63 between Akata and B95-8 exosomes
suggest the existence of a different glycosylation machinery in Akataand B95-8-LCLs. To monitor the transfer of the exosomes secreted
from the lymphoma cells to immune cells, we labeled the exosomescollected from Akata- and B95-8-LCL with a red ﬂuorescent lipiddye (PKH26). Labeled exosomes were captured on magnetic beads
and conﬁrmed by confocal microscopy (supplemental Figure 2D).
PBMCs were treated with the PKH26-labeled Akata and B95-8
exosomes for 48 hours. Flow cytometry analysis showed that
almost all the monocytes incorporated both exosomes(Figure 4B), and that incorporation was dependent on the doseof the exosomes and accumulated with time (supplemental
Figure 3). In contrast, incorporation of exosomes by lymphocytes
was observed only when treated with a high dose of Akata
exosomes for 6 hours, not with B95-8 exosomes (supplemental
Figure 3). Exosome1 lymphocytes were not observed 24 and48 hours after treatment with both Akata and B95-8 exosomes.
These results suggest that EBV1 lymphoma-derived exosomes
could transiently affect lymphocytes or attach to lymphocytes

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Clodronate

A

Anti-CD63 : Akata-exosome

Anti-CD63 : B95-8-exosome

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

75

75

50

50

37

37

C
Lymphocyte

Monocyte

Monocyte

Monocyte

Exosome Exosome +Akataexosome

0
0 102

B95-8exosome

103

104

105

100

101

102

103

104

Exosome positive

Exosome positive

w/o exosome

w/o exosome

exosomeexosomeexosome + MFG-E8 4 g/ml

exosome + Annexin 0.2 M

exosome + MFG-E8 8 g/ml

exosome + Annexin 0.4 M

Exosome positive
Figure 4. “Eat me” signal mediates exosome uptake by monocytes/macrophages. (A) Exosomes were collected from culture supernatant of Akata- and B95-8-LCLs byultracentrifugation and further fractionated by iodixanol gradient centrifugation. CD63 expression in each exosome was detected by western blotting under nonreducing conditions.
(B) PBMCs were treated with 2.5 mg/mL Akata and B95-8 exosomes for 48 hours. Flow cytometric analysis revealed strong incorporation of exosomes into monocytes, but not into
lymphocytes. (C) Each dose of recombinant MFG-E8 and annexin V was added to the PBMCs and cultured for 4 days. Incorporation of PKH-labeled exosomes by monocytes was
detected as described in Figure 4B. The inhibition of excess of MFG-E8 or annexin V on exosome uptake was repeated 5 times, using PBMCs derived from 2 individuals.

in high concentration, whereas substantial effects are expectedon monocytes. The results indicate that exosomes are secreted
from EBV-infected B cells and are then mainly incorporated into
monocytes/macrophages, suggesting that lymphoma-derived
exosomes could directly affect the macrophage phenotype.
Recognition of phosphatidylserine (PS), also known as the “eatme” signal, is a key step for the uptake of exosomes.16 The
uptake of PKH-stained exosomes by monocytes was partially
blocked by the excess amounts of PS-binding molecules, humanrecombinant milk fat globule epidermal growth factor VIII
(MFG-E8) and annexin V, in a dose-dependent manner (Figure 4C,
left and right, respectively). These results indicate that EBV1
lymphoma-derived exosomes are incorporated into monocytes
in an eat me signal-dependent manner.

Treatment with exosomes shifts the phenotype ofmonocytes to the immune regulatory phenotype
via BART miRNA delivery
To elucidate the direct effects of EBV1 lymphoma-derived
exosomes on monocytes/macrophages, we investigated theeffects of exosome uptake on monocytes phenotype in vitro.
Akata exosomes showed enhanced CD69 expression in theCD141 monocytes compared with B95-8 exosomes (Figure 5A).
This result suggests the regulatory role of Akata exosomes ininﬂammatory responses of macrophages. Importantly, EBV 1lymphoma-derived exosomes deliver noncoding RNAs, such as
BART miRNAs and EBER, and can affect immune regulation.10,16
We compared the expression level of EBER1 between Akata andB95-8 exosomes by reverse transcription quantitative polymerase
chain reaction (RT-qPCR). The expression of EBER1 was slightly
higher in B95-8 exosomes (about 1.6-fold) than in Akata exosomes, but was not signiﬁcantly different (supplemental Figure 4).
Although several differences in the genomes have been reported between Akata and B95-8,28 1 of the characteristic differences is a lack of the 12-kb BART locus in the B95-8 genome,
where a number of BART miRNAs are encoded. Therefore, we
focused on the effects of exosome-mediated delivery of BART
miRNAs on macrophage phenotype.
Next, we examined whether similar effects were observed bytreatment with exosomes collected from different cell lines.

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B

A
Akata
exosome
5

CD69

10

Q1
1.42%

Q2
78.7%

5

10

10

4

10

3

10

Q1
0.183%

Q2
35.3%

5

10

4

2

Q1
0.22

Q2
3.70

Q4
93.4

Q3
2.71

4

10

3

10

3

10

2

10

0

Non-treated
PBMC

B95-8
exosome

10
Q4
8.29%

Q3
11.6%

0

Q4
4.47%

0

Q3
60.1%

D

B
1000

Mono / PBMC (%)

50

Arbitrary unit

800
600
400
200

40
30
20
10
0

0
Daudi exosome

Poor

X50-7 exosome

C

Rich

EBV miRNAs

E
*

102

Q2
7.37%

5

10

5

10

4

10

10

3

10

3

2

2

4

Q4
47.9%

10
0

Q3
44.2%

Q4
27.9%

X50-7 exosome
5

10

Q1
0.253%

Q3
11.7%

5

10

4

Q1
7.40%

EBV miRNAs
poor

101

10

EBV miRNAs
rich

0

10-1

TNF-

IL-10

F

+ control

Q2
69.7%

10

IL-10

Q2
9.32%

20

4

10

3

10

3

10

2

10
0

Q2
58.1%

2

Q4
28.4%

10
0

Q3
1.63%

Q4
51.7%

Q3
31.6%

Relative mRNA

CD69

10
0

10

Q1
2.35%

Relative mRNA

Q1
0.544%*+ BART miRNAs

Daudi exosome

15
10

0

CD14

*

5

*
０
-

0
+

0.2
+

0.4
+

Annexin (M)
exosomeFigure 5. Treatment with exosomes shifts the phenotype of monocytes to the immune regulatory phenotype via BART miRNAs delivery. (A) PBMCs were cultured with
PKH26-labeled Akata-LCL and B95-8-LCL exosomes. CD14 and CD69 expression was detected by ﬂow cytometry. (B) Exosomes were harvested from Daudi and X50-7 culture
supernatant. BART miRNA expression level in exosomes was measured by qPCR. Each column indicates each BART miRNA. (C) PBMCs were cultured with each type of exosome.
CD14 and CD69 expression was detected by ﬂow cytometry (left). Exogenous BART miRNAs were introduced into Daudi cells, and exosomes were harvested from cells
transduced with control or BART miRNA-expressing vector. CD69 expression was upregulated on monocytes (right top) by exosomes collected from BART miRNA-transduced
cells as compared with the control (right bottom). (D) Monocyte ratios in PBMCs were analyzed by ﬂow cytometry. (E) Total RNA from PBMCs was collected. Each mRNA
expression level was determined by qPCR. Fold-induction was calculated as the ratio of EBV miRNA-rich value to EBV miRNA-poor value. (F) mRNA expression level of IL-10 was
detected by qPCR. The relative amount of IL-10 mRNA was standardized by the absolute amount of that the samples without exosome and annexin. Statistical signiﬁcancewas calculated by comparing that on 0 mg/mL of annexin. The triplicated value were analyzed twice. *P , .05, Student t test.

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CD14

These results suggest that exosome-mediated delivery of BART
miRNAs could contribute to the phenotypic changes of primarymonocytes. In particular, induction of TNF-a and IL-10 expression suggests that BART miRNAs are key molecules to induce theimmune regulatory phenotype.

BART miRNAs dramatically change gene
expression in THP-1 cells
To explore how BART miRNAs affect the phenotypic changes inmonocytes, we established an inducible BART miRNA expression system (Tet-Off system) in THP-1, human monocytic leukemia cells (BART/THP-1; Figure 6A).
Conditional induction of BART miRNAs using doxycycline (Dox)caused cell proliferation and TNF-a upregulation in a dosedependent manner (Figure 6B-C). These results were consistent
with those obtained in PBMCs (Figure 5D-E). Microarray analysis
was performed to elucidate the comprehensive changes of gene
expression. According to microarray data, about 400 genes wereupregulated and about 100 genes were downregulated by morethan 2-fold in BART miRNA-expressing cells (BART) relative to both
cells treated with Dox (Control) and the cells treated with empty
vector (Empty) (Figure 6D). Among the upregulated genes, arginase
1 (ARG1), a tumor-associated macrophage marker, was prominently
upregulated by BART miRNAs (supplemental Figure 5A). In addition, the expression levels of several molecules involved in maturation and trafﬁcking of lysosomes, such as ATP6V1B1 and RILP,31,32
were upregulated by BART miRNAs, 1 of the characteristic featuresof the M2-like macrophage.33 HLA-DR, DM, and CIITA, which is atrans-activator of HLA-class II expression, were downregulated,suggesting the inhibitory role of BART miRNAs on acquired immune responses (supplemental Figure 5B). Among them, upregulation of ARG1 and RILP and downregulation of CIITA were also
validated by RT-qPCR (Figure 6E).
In silico prediction identiﬁed MEF2C and CD1c as targeted genes
by several BART miRNAs (Figure 6F; supplemental Figure 5C),
which are a transcription factor involved in the development ofvarious cell lineage and regulation of apoptosis of macrophageand a surface molecule involved in presentation of lipid antigen,respectively.34-38 Downregulation of MEF2C and CD1c by BART
miRNAs was validated using a 39-UTR luciferase reporter assay in
HEK293T cells (Figure 6F; supplemental Figure 5C).
To investigate the role of these genes in the macrophage
phenotype, we performed a knockdown experiment using smallinterfering (si)RNA. THP-1 cells were transduced with siRNAs for
48 hours and subsequently stimulated with lipopolysaccharide(LPS). LPS-induced expression of TNF-a and IL-10 was examinedby RT-qPCR at early (4 h) and later (20 h) points. Decreased
expression of MEF2C (20% to approximately 50%, data notshown) led to the enhanced expression of IL-10 (Figure 6G),
consistent with the data in Figure 5E. Expression of TNF-a was
not affected by MEF2C, suggesting that other targeted molecules are involved in the regulation of TNF-a. The collective
results suggest that BART miRNAs could regulate gene expression, which involves in immune regulation in THP-1 cells.

Amount of tumor-produced BART miRNAs
correlates with clinical outcomes in EBV1 DLBCLof elderly patients
Finally, to investigate the signiﬁcance of BART miRNA expression in EBV-induced lymphomagenesis, BART13, a BART miRNA
that is abundantly expressed in L591 cells derived from patientswith EBV1 lymphoma, was analyzed in 13 EBV1 DLBCL biopsies
obtained from elderly patients by ﬂuorescent in situ hybridization (Figure 7A). This result was also obtained by RT-qPCR in the
representative cases (supplemental Figure 6).
The index of BART131 area/number of EBER1 cells (BART/EBER),
indicating the amount of BART13 production in EBER1 lymphoma
cells, signiﬁcantly differed between the 2 groups (Figure 7B). The
number of EBER1 cells did not signiﬁcantly differ between thegroups (Figure 7C). In contrast, the proportion of the BART131 area
relative to the negative area was higher in samples of group 2
patients (Figure 7D). Surprisingly, patients with low BART/EBER
indices (,10; group 1) had higher survival rates compared withthose with high BART/EBER indices (.10; group 2; Figure 7E).
Overall, our results demonstrated that the amount of BART miRNA
produced by EBV1 lymphoma cells differed between the 2 groups,
suggesting that BART miRNAs play critical roles in tumorigenesis.
Furthermore, in EBV1 DLBCL biopsy samples, BART13 wasdetected in EBER1 lymphoma cells with large nuclei, and also ina few macrophage cells with small nuclei and large volume ofcytoplasm (Figure 7F, upper). In addition, expression of theBART2 miRNA was detected in morphologically macrophages
with small nuclei and large cytoplasm, as well as in Hodgkin/Reed-Sternberg cells with large nuclei in EBV1 Hodgkin’s lymphoma biopsy (Figure 7F, lower).
These results suggest that BART miRNAs derived from EBV1
tumor cells are likely to be transferred from lymphoma cells to
non-EBV-infected macrophages in EBV-related lymphomas.

Discussion
We demonstrated that EBV1 lymphoma cells secrete exosomes
to support tumor cell survival through the regulation of inﬂammatory

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Daudi, Burkitt lymphoma cells, and the LCL X50-7 display different
expression levels of BART miRNAs.29,30 Exosomes were collectedfrom the culture supernatant of these cells, and the expression
levels of BART miRNAs in each exosome were compared. BART
miRNAs were highly expressed in X50-7-derived exosomes compared with Daudi-derived exosomes, reﬂecting the differencein BART miRNA levels between 2 cell lines (Figure 5B). As
compared with Daudi-derived exosomes (BART miRNA-poor),
treatment with X50-7-derived exosomes (BART miRNA-rich)upregulated CD69 expression in CD141 monocytes (Figure 5C,left), similar to the results depicted in Figure 5A. In addition,
treatment of PBMCs with BART miRNA-recovered exosomes,
collected from Daudi cells exogenously introduced with BART
miRNA-expressing vector, resulted in enhanced CD69 expression in CD141 monocytes as compared with that in the control(Figure 5C, right panel). Furthermore, the survival rate of monocytes was increased (Figure 5D), and the expression of tumor
necrosis factor-a (TNF-a) and IL-10 was consistently upregulated(Figure 5E) in cells treated with BART miRNA-rich X50-7-derived
exosomes. Treatment with an overdose of annexin V, which binds
to PS to block the eat me signal (Figure 4C) also inhibited IL-10
expression in monocytes (Figure 5F).

Cluster 1

150
100

Parent

50

BART
5p

3p

6-

p

6-

p

-5

17

16

17

-3

5p

3p

5-

15

5-

5p

3p

1-

5p

1-

3p

4-

4-

3-

3-

Cluster 2

200
150
100
50

19

-3
p
19
-5
p
20
-3
p
20
-5
p
13
-3
p
13
-5
p
14
-3
p
14
-5
p

p

p

12

-5

11

p

11

-3

p

-5

10

10

-3

22

5p

3p

9-

5p

B

9-

3p

5p

8-

18

8-

p
-5

3p

p
-3

18

7-

p
-5

7-

p
-3
21

21

0

D
Day 2

Day 0
control*

15

Relative TNF- mRNA

Relative cell proliferation

C
20

BART

10
*
5
0

0
2
4
Doxycycline (g/ml)

4

Upregulated genes

**

3

339

**

2
1
0

413

1028

Downregulated genes
0

0

2

4
741

Dox (g/ml)

BART miRNAs expression

107

529

BART miRNAs
BART vs Control
BART vs Empty

E

1.5
1
0.5
Dox +

Dox -

5

*

Relative expression

2

0

CIITA

ARG1

*

Relative expression

Relative expression

RILP
2.5

4
3
2
1
0

Dox +

1.5
*

1
0.5

Dox -

0

BART miRNAs

BART miRNAs

Dox +

Dox -

BART miRNAs

F

G
IL-10

*

*

BART11-5p
WT

1

Mutant
MEF2C

ut
.

T

W

m

Ct
rl

0

5’

TAG
Stop 14238

MEF2C 3’-UTR

4261 4373

3’

Relative expression

Arbitrary unit2

2.5

Scramble2

siMEF2C

p = 0.06

*

1.5
1
0.5
0

0

4

20

LPS stimulation (h)Figure 6. BART miRNAs alter gene expression in THP-1 cells. (A) BART miRNAs were overexpressed in THP-1 cells with the Tet-Off system. (B) BART miRNA expression was
regulated by doxycycline. After 7 to 9 days of culture, the number of cells was counted. Cell proliferation is expressed as the fold-change relative to the cell number at day 0.
*P , .05, using Student t test, compared with control values of each doxycycline concentration. (C) Total RNA was collected from BART/THP-1 cells at the indicated timeand doxycycline concentration. TNF-a mRNA expression level was measured by qPCR. **P , .01, using Student t test, compared with day 0. (D) Microarray data revealed that2560

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Copy number

5p

0
3p

Copy numberAresponses in macrophages both in vivo and in vitro (Figure 8). In
detail, we found that: exosomes are an important factor for theformation of EBV1 lymphoma in a humanized mouse model comparing the Akata and B95-8 strains; exosomes collected fromlymphoma cells could regulate the activity of macrophages andinduce the immune regulatory phenotype in vitro characterized
by the enhanced expression of TNF-a, IL-10, and ARG1, whichwere partly regulated by BART miRNAs; and the amount oflymphoma-produced BART miRNAs correlates with clinical outcomes in EBV1 DLBCL in elderly patients.

CD163 1 macrophages, which are thought to be M2-likemacrophages, were completely eliminated in all 6 mice byclodronate liposomes, whereas CD681 macrophages were not(Figure 3). One possible explanation for this result is that M2
macrophages, which support tumor growth, are more sensitive
to clodronate than M1 macrophages, which attack and eliminate
tumors.39,40 Accordingly, the selective elimination of CD1631
macrophages may alter the tumor microenvironment, making it
more difﬁcult for EBER1 tumor cells to survive than that caused
when both CD1631 and CD681 macrophages are eliminated byclodronate liposomes.
In vitro studies demonstrated that exosomes were mainly incorporated into monocytes/macrophages. Incorporation of exosomes by monocytes increased in dose- and time-dependent
manners, whereas that by lymphocytes was transiently detected inonly high-dose treatment, suggesting that the activity of monocytes could be mainly regulated by tumor-derived exosomes, butthat of lymphocytes might possibly still be regulated in the local
environment in vivo. We evaluated the overlapping mechanism of
the engulfment of apoptotic cells and intake of the tumorderived exosomes by monocytes/macrophages. To maintainhomeostasis, phagocytes engulf dead cells expressing the eatme signal. Considering previous results indicating that PS is
exposed by both apoptotic cells and exosomes,16,41-43 and on thebasis of our results, the mechanism of macrophage-speciﬁc uptake of tumor-derived exosomes can be considered to overlap
with that of engulfment of apoptotic cells by macrophages
through the eat me signal. However, the apoptotic cells engulfed
by monocytes/macrophages should enter the lysosomal pathway, whereas the exosome should not. Hence, an exosome-In this study, we focused on the delivery of BART miRNAs
by exosomes. Pegtel et al reported that BART miRNAs inthe exosome can be transferred into monocyte-derived dendritic cells.10 Similarly, we detected BART miRNAs in monocytes
treated with lymphoma-derived exosomes by next-generation
sequencing and RT-qPCR (supplemental Figure 7). Importantly,
B95-8 has a deletion in the BART region and lacks the expressionof the majority of BART miRNAs, which is thought as the mostsigniﬁcant difference between the Akata and B95-8 strains.
Exosomes collected from EBV1 lymphoma cells have also beenreported to carry EBER and drive antiviral responses in dendritic cells.16 Thus, we compared the expression level of EBER1
between exosomes collected from Akata- and B95-8-LCL by
RT-qPCR. Although the expression level of EBER1 was not signiﬁcantly different between exosomes collected from Akata- andB95-8-LCL (supplemental Figure 4B), its general inﬂammatory
function could work in the phenotype caused by the exosomes
derived from Akata-infected cells.
In addition to BART miRNAs, the Akata and B95-8-strains might
differentially induce the expression of EBV-coding genes in theinfected cells.28 Therefore, different gene expression in tumor cells
might be reﬂected in exosomes secreted from Akata- andB95-8-infected tumor cells. Although the expression of LMP-1
and EBNA3B, which is reportedly involved in the transformationand immortalization of B cells,3,4,44 were not signiﬁcantly different
between Akata- and B95-8-LCL (supplemental Figure 4A,C), we
could not exclude the possibility that the different expression ofthese genes and components in exosomes affects tumorigenesis.
Treatment with exosomes containing high levels of BART miRNAs
enhanced survival and activation in monocytes/macrophages. In
particular, the enhanced expression of TNF-a and IL-10 was acharacteristic feature of the immune regulatory phenotype. Furthermore, inducible expression of BART miRNAs in THP-1 also
showed similar results, supporting the idea that BART miRNAs
could regulate inﬂammatory responses in macrophages andinduce the immune regulatory phenotype. Although treating
monocytes with exosomes in vitro for 6 hours induced the production of TNF-a, the production of IL-10 was not induced, regardless of elevated mRNA level (data not shown; Figure 5). This
discrepancy could be caused by the different kinetics of translation between TNF-a and IL-10. Production of IL-10 in vivo waselevated in the spleens of Akata-infected mice compared with
B95-8-infected mice (Figure 1D), suggesting that the long-time
exposure to exosomes produces IL-10 in macrophages.
In silico prediction and 39-untranslated region (UTR) luciferase
assay determined MEF2C as a target gene of BART miRNAs,
which is a transcription factor involved in the developmentof various cell lineages and apoptosis of macrophages.34-36

Figure 6 (continued) about 400 genes were upregulated and 100 genes were downregulated by more than 2-fold in BART/THP-1 cells relative to both control cells treated withdoxycycline (control) and cells treated with empty vector (Empty). (E) Microarray results were validated by RT-qPCR. Expression of ARG1 and RILP were upregulated by inductionof BART miRNAs. Expression of CIITA was downregulated by induction of BART miRNAs. Expression levels were normalized to GAPDH. Averages from 3 independent
experiments were shown. *P , .05, Student t test. (F) 39-UTR luciferase assay was performed with HEK293T cells (left). Either the empty vector or BART miRNA overexpression
vector was cotransfected along with the psiCHECKTM-2 vector, a luciferase reporter vector. Ctrl, psiCHECKTM-2 vector; WT, inserted wild-type 39-UTR sequence of target genes;mut., including mutated target sites. *P , .05, Student’s t-test. Position of the predicted target sequences (miRANDA) of BART miRNAs in the 39-UTR of human MEF2C mRNAs(right). (G) Downregulation of MEF2C by siRNA treatment enhanced LPS-stimulated expression of IL-10. Expression level was normalized to GAPDH. Three siRNAs were mixed tominimize off-target effects. An average of 3 independent experiments was shown. *P , .05, Student t test.

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Since Valadi et al reported that exosomes contain abundantmiRNAs and transfer them from cell to cell,12 several studies haverevealed the physiological signiﬁcances and roles of exosomes inthe development of diseases, especially in the tumor development and metastasis.11-15 Although it has been already reported
that EBV1 lymphoma cells secrete exosomes, their roles in thedevelopment of lymphoma remained to be elucidated.10,16 In
this study, we demonstrate the signiﬁcant role of tumor-derived
exosomes for the development of EBV1 lymphoma, using a humanized mouse model.

speciﬁc mechanism for incorporation after the eat me signal
pathway may exist.

A

EBER

E

BART13

100

Survival rate (%)

Patient #7(Group 1)

Patient #6(Group 2)

Group1(BART / EBER < 10)8060

P = 0.0004

Group 2(BART / EBER > 10)

40
20

0

20

40

60

Time (months)

B

C
10

102

101

Group 1

Group 2101

P = 0.0589% of BART13-positive area

*

103

<100

D
-1

Number of EBER
positive cells (/m2)

Index (BART13 / EBER)

10

4

-2

10

10-3

10-4

10-5

Group 1

Group 2

*

10

0

10-1

10-2

10-3

Group 1

Group 2F
Tumor cells

Macrophage

Negative staining

HRS

Macrophage

Negative staining

EBV+ DLBCL(BART13)

EBV+ HL(BART2)

Figure 7. Amounts of tumor-produced BART miRNAs in EBV1DLBCL in elderly patients. (A) EBV-encoded noncoding RNA (EBER) served as a highly sensitive marker todetect EBV infection. EBER and BART13 were detected from DLBCL biopsy samples by ﬂuorescence in situ hybridization. Representative results are shown. The deﬁnitionof group 1 (n 5 6) and 2 (n 5 7) is indicated in B. (B) Comparison of the index of BART131 area/number of EBER1 cells. Group 1, BART/EBER ,10; gGroup 2, BART/EBER .10.
*P , .05. (C-D) The numbers of EBER1 cells and BART131 cell areas were automatically counted. Comparison of the number of EBER1 cells (C) and BART131 cell areas (D) in thetissue area on a slide. *P , .05. (E) Survival ratio of patients with DLBCL. *P 5 .0004, log-rank test. (F) BART miRNAs were detected from EBV1 DLBCL (top) and EBV1
Hodgkin’s lymphoma (bottom) biopsy samples by in situ hybridization. (Left) Tumor cells; (middle) macrophages; (right) negative staining (using scramble miRNAs for theprobe). (Top) BART-13 was detected; (bottom) BART-2. Representative BART miRNA signals (blue dots) are indicated by arrows. Scale bar, 10 mm.

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Downloaded from https://ashpublications.org/blood/article-pdf/131/23/2552/1468595/blood794529.pdf by TOKYO MEDICAL UNIVERSITY user on 08 April 20200Figure 8. Scheme of EBV1B-cell lymphoma microenvironment establishment. (1) EBV-infected cells release exosomes containing EBV-miRNAs, which areincorporated into macrophages. (2) Lymphoma-derived
exosomes alter gene expression and convert the macrophages into “tumor associated macrophages.” (3)Accumulation of BART miRNAs and upregulation of tumorsupporting molecules, TNF-a, IL-10, and ARG1, enhancethe development of EBV1 B-cell lymphoma.

Exosomes1
EBV infected
tumor cell2
Phosphatidylserine recognition
Macrophage

3

Mf

BART miRNAs

TNF- IL-10 ARG1 HRS

Inflammatory
niche

Downregulation of MEF2C by siRNA enhanced the LPSstimulated IL-10 expression in THP-1, indicating the regulatoryrole in inﬂammatory responses. In contrast, expression of TNF-aand ARG1 was not affected by downregulation of MEF2C.
Production of ARG1 in vivo was detected only in 1 of 3 Akatainfected mice and no B95-8-infected mice (supplemental Figure 8), suggesting that other molecules were involved in theexpression of these genes. Because exosomes can transfer not
only miRNAs but also various biomolecules, such as proteins andlipids,45 it is possible that these molecules might synergisticallyinduce the macrophage phenotype with BART miRNAs. Although downregulation of MEF2C was insufﬁcient in monocytes treated with exosomes for 48 hours (data not shown),
elevated production of IL-10 in Akata-infected mice suggeststhat longer exposure to exosomes is needed for the induction of
IL-10-producing macrophages in vivo (Figure 1D).
The roles of BART miRNAs in tumorigenesis are complicated. It
has been reported that BART miRNAs suppress apoptosis by
inhibiting Bim, a pro-apoptotic protein, and LMP1.46,47 In addition, an in vivo xenograft model of EBV-driven carcinogenesis
using nasopharyngeal carcinoma cells demonstrated that the
BART miRNAs potentiate tumor growth and developmentin epithelial cells.48 In contrast, Lin et al reported that BART
miRNAs suppress B-cell tumorigenesis by inhibiting BZLF1.49 We
think these opposite functions of BART miRNAs reﬂect the useof EBV strain M81. M81 was isolated from a patient with nasopharyngeal carcinoma and has a strong tropism to epithelial
cells,50 which basically differs from strains Akata and B95-8.
Furthermore, M81 contains the mutation in BZLF1, whichmakes the infected cells constitutively active in replication

instead of formation of latent infection. Once Akata and B95-8
infect the cells, they establish a markedly more latent infectionin these cells.51 Consistently, production of BZLF1 was detected
in neither the spleens of Akata- nor B95-8-infected mice in ourmodel (data not shown), suggesting that the different tumorigenic activity between the 2 strains was not related to levels
of BZLF1.
Analysis of BART miRNAs expression in EBV1 DLBCL of elderly
patients indicated that the potency of the production of BART
miRNAs by EBV1 tumor cells was signiﬁcantly correlated withclinical outcomes (Figure 7A-E). Although the differences in the
biology and pathogenesis between the 2 groups should befurther investigated, the production of BART miRNAs by tumor
cells may be useful for the diagnosis of the 2 clinical groupsof EBV1 DLBCL in elderly patients. Recently, the expression of
EBV-coding miRNAs in several EBV-related lymphomas wasinvestigated by next-generation sequencing.52 The expression
proﬁle was quite different among the types of lymphoma. EBV1DLBCL of the elderly signiﬁcantly expressed BART 13-3p, whichis consistent with our results of in situ hybridization and RT-qPCR,
using lymphoma sections (Figure 7A-D; supplemental Figure 6).
It is expected that viral miRNAs could be easily distinguishedfrom endogenous miRNAs,53 which may lead to fewer adverse
effects of the therapy. Furthermore, it is reported that some
BART miRNAs share seed sequence homology among themselves,54 suggesting that targeting the seed sequences of BART
miRNAs may be more efﬁcient. However, at present, further
clinical information is required; for example, which miRNAs arestrongly expressed in lymphoma tissue, and the generality ofblood® 7 JUNE 2018 | VOLUME 131, NUMBER 23

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Macrophage (TAM)their expression among patients. Further studies are warranted
to determine the efﬁcient and effective strategy to target BART
miRNAs for the therapy of EBV1 lymphomas.

Methods
EBV

For virus titration, cord blood lymphocytes were plated at adensity of 2 3 105 cells per well in 6-well plates and then inoculated with serial 10-fold dilutions of the virus preparation.
The number of wells containing proliferating lymphocytes wascounted 6 weeks after infection, and the titer of the virus in
50% transforming dose was determined using the Reed-Muench
method (supplemental Figure 1).

Humanized mice
NOG mice were purchased from the Central Institute for Experimental Animals (Kanagawa, Japan). The detailed method of
humanization of NOG mice has been described previously.17
In brief, 1 3 105 CD341 cord blood cells were administeredintravenously. After about 3 months, peripheral blood was collected from the mice, and human CD21 expression was checked
for humanization. All experiments were approved by the Institutional Review Board of Tokai University. The animals received
humane care as required by the institutional guidelines for animalcare and treatment in experimental investigations.

Mouse experiments
Akata, B95-8, EBV miRNA-rich Akata exosomes, and EBV-miRNAdeleted B95-8 exosomes were injected intravenously into thehematopoietically humanized NOG mice. When the animal
weight reached the determined value, the mice were killed, andhistological analysis was performed on the obtained spleens.
Clodronate liposome injection trial was performed with EBV-infected
mice. Mice were intraperitoneally injected with clodronate- or
phosphate buffered saline-liposomes (ClodronateLiposomes.org,
Amsterdam, The Netherlands).

EBV viral load determination by qPCR
Total DNA was extracted from the whole blood of EBV-infected
mice with the DNeasy Blood and Tissue kit (QIAGEN, Hilden,
Germany), according to the manufacturer’s instructions. EBV DNA
loads were determined by the qPCR assay. The PCR was performed with the THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka,
Japan), following the manufacturer’s protocol. The primers used
for detection of B95-8 were 59-ATCGACGTATCGCTGGAAAC-39
and 59-AGTCCTGATCGTCCTCCTC-39. The primers used for
detection of Akata were 59-ACACCAAGATCACCACCCTC-39 and
59-TTAGGGTGCCACATCCTGTTC-39.

BART miRNA expression analysis
RNAs were isolated from cells or exosomes using Sepasol-RNA I
Super G (Nacalai Tesque, Kyoto, Japan). Reverse transcription
was performed with the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). qPCR was

2564

Cells
Akata and Daudi cells from EBV1 patients with Burkitt lymphoma, B95-8 cells from EBV-transformed marmoset lymphocytes, THP-1 cells from a patient with acute monocytic leukemia,and LCLs (Akata-LCL, B95-8-LCL, and X50-7) from an EBVtransformed umbilical cord blood B lymphoblast line were
cultured in RPMI 1640 medium (Nacalai Tesque) supplemented
with 10% (vol/vol) fetal bovine serum (FBS), 100 U/mL penicillin,and 100 mg/mL streptomycin (Life Technologies, Carlsbad, CA)
in 50-mL ﬂasks.

Lentiviral transfer
High-titer lentiviral supernatant was observed after cotransfection of a miRNA expression vector and pCAG-KGP1R for gag
protein, pCAG-4RTR2 for Rev/tat, and pCMV-VSVG viral packaging construct into 293T cells using ExtremeGENE (Roche
Diagnostic, Basel, Switzerland). The cells were plated at a concentration of 4 to 5 3 105/mL in a 24-well plate, and spin-infectedwith the desired lentiviral supernatant for 2 hours.

Exosome isolation
Akata-LCL, B95-8-LCL, Daudi, and X50-7 were incubated in
RPMI-1640 medium supplemented with 10% FBS (vol/vol), andcell supernatants were harvested. Exosomes were collectedfrom the supernatants by ultracentrifugation at 110 000 3 g for
70 minutes.21 Collected exosomes were labeled with PKH26
dye (SIGMA-ALDRICH, Saint Louis, MO), following the manufacturer’s protocol.

Exosome fractionation
In some experiments, exosomes were separated by density
gradient centrifugation after ultracentrifugation, as reported
previously.26 Brieﬂy, 10%, 20%, and 30% Iodixanol solutions
were prepared by mixing Optiprep (Axis-Shield, Oslo, Norway)
with buffer containing 0.25 M sucrose, 10 mM Tris at pH 8.0, and
1 mM EDTA, ﬁnal pH set to 7.4. Exosome pellets were resuspended in 3 mL of 30% Iodixanol solution. Subsequently, 1.3 mLof 20% and 1.2 mL of 10% Iodixanol solutions were carefully
layered on top of the suspension.

Constructs
Genomic DNA from L591, derived from an EBV1 patient with
Hodgkin’s lymphoma, was extracted using the DNeasy Tissue
extraction kit (QIAGEN). The genomic sequences of thesegments in BART cluster 1 and 2 were ampliﬁed by genomic
PCR, using Pfx polymerase (Invitrogen, Carlsbad, CA). The
oligonucleotide sequences used for PCR were Bartcluster1NotI-F, 59gsstgcggccgcattgctcaggccaaagtt39; Bartcluster1-BamH1R, 59gaatggatcctgaaacccaagtttccttgc39; Bartcluster2-NotI-F,
59aattgcggccgctgccattattcccttga39; and Bartcluster2-NotI-R,
59aattgcggccgcattaacgggggaaggaa39.
The PCR products were puriﬁed with a PCR puriﬁcation kit(QIAGEN) and sequenced. The puriﬁed genomic PCR productsof BART cluster 1 and cluster 2 were serially cloned into themammalian lentiviral expression vector, pCS2-Ires-GFP.

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Akata and B95-8 strains were prepared as described previously.55 Virus production by EBV-infected Akata cells was
stimulated by the brief treatment with anti-IgG antibody (Dako,
Carpinteria, CA), and the culture ﬂuid was used as the inoculum
after ﬁltration through a 0.45-mm membrane ﬁlter.56

performed with TaqMan Fast Universal PCR Master Mix, using
StepOnePlus Real-Time PCR System (Applied Biosystems). The
sequences of primers and probes were described previously.10
RNA isolation, reverse transcription, and qPCR were performed
following each manufacturer’s protocol.

For the Tet-off inducible system, we used the Lenti-X Tet-Off
Advanced Inducible Expression System (Clontech, Mountain
View, CA) and the associated products.

collected from BART/THP-1 cells at the time and doxycycline
concentration indicated in Figure 6.

39-UTR luciferase assay

Flow cytometry analysis of
exosome-treated PBMCs
The supernatants of EBV-infected cells were harvested. Exosomes were isolated from these supernatants and added to
PBMCs. CD14 and CD69 expressions on the cell surface weredetected by ﬂow cytometry. Paciﬁc Blue anti-human CD14
antibody (clone: HCD14, Biolegend, San Diego, CA) and PE/Cy7
anti-human CD69 antibody (clone: FN50, Biolegend) were used.

Total RNAs of PBMCs or THP-1 cells were isolated using
Sepasol-RNA I Super G. RT-PCR was performed with the HighCapacity Reverse Transcription Kit (Applied Biosystems), andqPCR was performed with the THUNDERBIRD SYBR qPCR Mix,following each manufacturer’s protocol. The primers were as
follows:
IL-10 59-CGGCGCTGTCATCGATTT-39
59-GAGTCGCCACCCTGATGTCT-39
TNF-a 59-TCTGGCCCAGGCAGTCAGATCAT-39
59-CGGCGGTTCAGCCACTGGAG-39
ARG1 59-GGAAACTTGCATGGACAACC-39
59-TCCACGTCTCTCAAGCCAAT-39
RILP 59-CAAGATGTTAGGGACACCAGAG-39
59-CAGCTTTACCCCGATACCATAG-39
CIITA 59-AGCTGAAGTCCTTGGAAACC-39
59-CGTCGCAGATGCAGTTATTG-39
MEF2C 59-TGTAACACATCGACCTCCAAG-39
59-TGTTCAAGTTACCAGGTGAGAC-39
EBER1 59-AGCACCTACGCTGCCCTAGA-39
5-AAAACATGCGGACCACCAGC-39
EBNA3B 59-CCACGCTGTCTATGATTCCA-39
59-CGATGTTCAGGTTTTGCTCA-39
GAPDH 59-CTGCACCACCAACTGCTTAG-39
59-TTCAGCTCAGGGATGACCTTG-39

siRNA transfection
siRNA was transfected by Neon electroporation system (Invitrogen) following the manufacturer’s protocol. In brief, 1 3 105
THP-1 cells were resuspended in 10 mL buffer containing 5 mM
siRNA. Electroporation was performed under the condition with
1300 V, 10 milliseconds of pulse width and 3 pulses. Predesigned siRNA against human MEF2C was purchased from Bioneer
Corporation (Daejeon, Korea). To minimize the off-target effects,
3 different siRNAs were mixed and introduced into the cells
simultaneously.

Patients and specimens
The Institutional Review Board of Tokai University, School of
Medicine, approved this study, and all human samples werehandled accordingly. The biopsy samples of patients with DLBCLand the patient prognosis information were provided by Sato,
Kashiwagi, Matsui, Okamoto, and Nakamura.

In situ hybridization of BART miRNAs

Western blotting
Protein concentration in exosomes was measured by absorbance at 280 nm. Proteins were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred onto
polyvinylidene ﬂuoride membranes. The membranes were exposed to mouse anti-LMP1 antibody (ab78113, clone: CS 1-4;
Abcam, Cambridge, UK) or mouse anti-CD63 antibody (sc-5275,
clone: MX-49. 129. 5; Santa Cruz Biotechnology, Santa Cruz,
CA). The protein signals were detected using a ChemiDoc Touch
System (BIO-RAD, Hercules, CA).

THP-1 cell functional assay
BART clusters 1 and 2 were overexpressed in THP-1 cells with
the Tet-Off system (BART/THP-1). BART miRNA expression was
regulated by doxycycline. Ctrl/THP-1 and BART/THP-1 cells werenormally cultured with 10% FBS RPMI medium with 1 mg/mL
doxycycline. For proliferation assay, cells were precultured with
4 mg/mL doxycycline and cultured with 0, 2, or 4 mg/mL doxycycline
in 0.1% FBS RPMI medium. After 7 to 9 days of culture, thenumber of cells was counted. Cell proliferation is expressed as
fold change relative to the cell number on day 0. Total RNA was

In situ hybridization was performed as described previously.57
We purchased the following 59 digoxigenin-labeled locked
nucleic acid probes (miRCURY LNA Detection probe, Exiqon,
Demmark): ebv-miR-BART2-5p, 59-(digoxigenin)GCAAGGGCGAATGCAGAAAATA-39; ebv-miR-BART13, 59-(DIG)TCAGC
CGTCCCTGGCAAGTTACA-39. Slides were photographed using a BZ-X700 All-in-1 ﬂuorescence microscope (Keyence, Osaka,
Japan). The threshold values were determined by our observations. Each positive signal decision was then made automatically,using Keyence software.

DNA microarray
BART/THP-1 cells and ctrl/THP-1 cells (with empty vector) were
cultured without doxycycline for 4 days, and total RNA washarvested. Reverse-transcribed samples were hybridized to Whole
Human Genome DNA microarray 4 3 44K (Agilent Technologies,
Santa Clara, CA) and analyzed according to the manufacturer’s
protocol.

Statistics
Statistical signiﬁcance was determined using Student t testor Mann-Whitney U-test. P , .05 was considered statisticallysigniﬁcant. Survival curves were analyzed with GraphPad Prism
software (GraphPad Software, Inc., La Jolla, CA), using the logrank test.

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Quantitative PCR

39-UTR luciferase assay was performed in HEK293T cells. Either
the empty vector or the BART miRNA overexpression vector was
cotransfected, along with the psiCHECK-2 vector (Promega,
Madison, WI), using X-tremeGENE (Roche). The wild-type
39-UTR sequence of each gene was inserted into the vector. For
the mutation experiment, we introduced a 3-base change in thewild-type sequence, predicted as a BART miRNA target site by
MiRanda, using the Site-Directed Mutagenesis kit (New England
Biolabs, Ipswich, MA). The Dual-Luciferase Reporter Assay System(Promega) was used for detection, following the manufacturer’s
protocol. Renilla luciferase activity was calibrated with ﬁreﬂy luciferase activity.

Study approval
All animal experiments were approved by the Institutional Review
Board of Tokai University. The animals received humane care, as
required by the institutional guidelines for animal care andtreatment in experimental investigations. The Institutional Review
Board of Tokai University, School of Medicine, approved thisstudy, and all human samples were handled accordingly. The
participants were identiﬁed by number.

Acknowledgments

This study was supported by Research and Study Program of Tokai
University Educational System General Research Organization(H. Higuchi); Tokai Scholarship Award (N.Y.); and Precursory Research for
Embryonic Science and Technology, AMED-PRIME, and the Research
Program on Hepatitis from Japan Agency for Medical Research andDevelopment (16fk0210114h0001; A.K.).

Authorship
Contribution: H. Higuchi, N.Y., K.-I.I., T.Y., R.K., J.O., M. Kakizaki, K.F., J.L.,
K.Y., K.O., A.S., M.T., N.K., S.M.A., A.A., T. Kitamura, T. Kashiwagi, T.M.,

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ORCID proﬁles: K.-I.I., 0000-0002-1662-4833; T.M., 0000-0002-25367584.
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Footnotes
Submitted 5 July 2017; accepted 5 April 2018. Prepublished online as
Blood First Edition paper, 22 April 2018; DOI 10.1182/blood-2017-07794529.
*H.H., N.Y., and K.-I.I. contributed equally to this study.
The online version of this article contains a data supplement.
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ONCOLOGY REPORTS 40: 1621-1631, 2018

Overexpression of miR‑221 and miR‑222 in the cancer stroma
is associated with malignant potential in colorectal cancer
MICHIHISA IIDA1, SHOICHI HAZAMA1,2, RYOUICHI TSUNEDOMI1, HIRONORI TANAKA1,HIROKO TAKENOUCHI1, SHINSUKE KANEKIYO1, YUKIO TOKUMITSU1, SHINOBU TOMOCHIKA1,YOSHIHIRO TOKUHISA1, KAZUHIKO SAKAMOTO1, NOBUAKI SUZUKI1, SHIGERU TAKEDA1,TOMIO UENO3, SHIGERU YAMAMOTO1, SHIGEFUMI YOSHINO1,4, KOJI FUJITA5,MASAHIKO KURODA5 and HIROAKI NAGANO1
1Department of Gastroenterological, Breast and Endocrine Surgery;
Department of Translational Research and Developmental Therapeutics against Cancer,
Yamaguchi University School of Medicine, Ube, Yamaguchi 755-8505; 3Department of Digestive Surgery,
Kawasaki Medical School, Kurashiki, Okayama 701-0192; 4Oncology Center, Yamaguchi University Hospital, Ube,
Yamaguchi 755-8505; 5Department of Molecular Pathology, Tokyo Medical University, Tokyo 160-8402, Japan
2

Received January 28, 2018; Accepted June 18, 2018
DOI: 10.3892/or.2018.6575
Abstract. The cancer stroma is important in cancer development, however, whether the aberrant expression of microRNAs(miRNAs) in the cancer stroma is associated with cancer
progression remains to be fully elucidated. The aim of thepresent study was to identify the miRNAs associated with liver
metastasis in the cancer stroma of human colorectal cancer(CRC). Using laser capture microdissection, cancer stroma
was obtained from the primary lesion of six patients with CRC
with liver metastasis (CRCwLM) and six patients with CRC
without liver metastasis (CRCwoLM), and miRNA microarray
analysis was performed. Candidate miRNA expression status
in the stroma was validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis in 40 CRC
cases (wLM, n=20; woLM, n=20), and the association between

Correspondence to: Professor Hiroaki Nagano, Department of
Gastroenterological, Breast and Endocrine Surgery, Yamaguchi
University School of Medicine, 1-1-1 Minami-Kogushi, Ube,
Yamaguchi 755-8505, Japan
E-mail: hnagano@yamaguchi-u.ac.jp
Abbreviations: CRC, colorectal cancer; CRCwLM, colorectal
cancer with liver metastasis; CRCwoLM, colorectal cancer
without liver metastasis; miRNA, microRNA; RT-qPCR, reverse
transcription-quantitative polymerase chain reaction; FFPE,
formalin-fixed paraffin-embedded; SSC, saline-sodium citrate;
SDS, sodium dodecyl sulfate; SD, standard deviation; ISH, in situ
hybridization; LNA-ISH, locked nucleic acid-in situ hybridization;
DIG, digoxigenin-UTP; CAF, cancer associated fibroblast; NF,
normal fibroblast
Key words: colorectal cancer, stromal tissue, tumor microenvironment, microRNA, liver metastasis, microarray analysis

miRNA expression and clinicopathological factors wasassessed in 101 advanced CRC samples. The localization ofcandidate miRNAs in CRCs was analyzed using in situ hybridization analysis (ISH). The microarray analysis identified six
miRNAs with expression differing between the CRCwLM and
CRCwoLM cancer stroma. Validation using RT-qPCR analysisof the stroma showed that the expression levels of miR-221
and miR‑222 in the cancer stroma were significantly higher in
CRCwLM than in CRCwoLM. The RT-qPCR analysis of 101
CRC samples showed that a high expression level of miR-221
or miR-222 in the cancer stroma was associated with liver
metastasis, distant metastasis, and shorter overall survival rateof patients with CRC (P<0.05). Increased levels of miR-221
and miR‑222 were observed in cancer cells and in fibroblasts
in the stromal tissue in the ISH analysis. The results suggested
that the overexpression of miR-221 and miR-222 in the cancer
stroma is associated with the metastatic activity and malignant
potential in patients with CRC.
Introduction
Colorectal cancer (CRC) is the third most commonly
diagnosed cancer in men and the second in women, with
~1.4 million cases and a mortality rate of 693,900 in 2012 (1).
In patients with CRC, ~25% present with metastases at thetime of diagnosis (2), and ~50% of patients with CRC develop
liver metastases (3,4). Metastatic CRC is associated with aparticularly poor prognosis. Despite advances in treatment
over previous decades (5), the 5-year-survival rate for patientswith metastatic CRC remains <10% (6). Extensive investigation of the underlying molecular networks of metastasis isimportant for the development of effective targeted therapy forpatients with metastatic CRC.
Although the overarching focus of cancer research in thelast four decades has been on the malignant cancer cell, it
has become well established that stromal cells in the tumor

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IIDA et al: STROMAL miRNA AND COLORECTAL CANCER AGGRESSIVENESS

microenvironment are important in tumor progression (7,8).
A variety of stromal cells in the tumor microenvironment arerecruited to tumors, and crosstalk between cancer cells andstromal cells is critical for tumor progression and the development of metastases (9). Secreted proteins, including cytokines,
chemokines and growth factors, have been considered tooccupy the main role in this crosstalk. However, tumor-derived
exosomes, which contain various proteins and RNAs, havealso been shown to be involved in this crosstalk (10). In tumor
microenvironments, extracellular microRNAs (miRNAs),
including miRNAs in exosomes, have been suggested to influence tumor progression via bidirectional tumor-to-stromaland stromal-to-tumor communication (11). Therefore, miRNA
expression analysis of stromal cells is important for elucidation of the role of miRNAs in cancer progression in the tumor
microenvironment. Although there have been a number ofreports of comprehensive miRNA expression analysis ofstromal cells (12,13), all have involved the analyses of thedifferences in miRNA expression between cancer stromal cells
and normal stromal cells. In order to understand how stromal
miRNAs are involved in cancer metastasis, it is necessary tocompare the miRNA profile of the cancer stroma in cancer
with metastasis to that of the cancer stroma in cancer without
metastasis. Comprehensive miRNA expression analysis
comparing cancer stroma in cancer with metastasis with thatin cancers without metastasis is lacking. The purpose of thepresent study was to identify the miRNAs whose expression in
the CRC stroma is involved in metastatic ability.
Materials and methods
Patients and tissue samples. The specimens of 113 patientswith primary CRC, who had undergone surgical resection in
Yamaguchi University Medical Hospital (Yamaguchi, Japan)
between January 2008 and December 2013, were used in thepresent study. All patients had undergone resection of the
primary tumor, and among the 31 patients with distant metastases, three patients had undergone hepatectomy. None of thepatients had received preoperative treatments, for example,
chemotherapy and/or radiation.
For screening in the miRNA array analysis, the frozen
specimens of primary CRC from 12 patients were used. For
RT‑qPCR analysis, the formalin‑fixed paraffin embedded tissue(FFPE) specimens of 101 cases of primary CRC were used. Of
these 101 CRC FFPE samples, 40 samples (20 CRCwLM and
20 CRCwoLM) were used for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) validation of thearray analysis (Fig. 1). Survival analysis was performed for all
101 of the cases from which samples were used for RT-qPCR
analysis. All samples were obtained with informed consent
from the patients. The study protocol was approved by theInstitutional Review Board for the Use of Human Subjects at
the Yamaguchi University School of Medicine (H17-83).
Tissue preparation: Laser capture microdissection and RNA
extraction. For the miRNA array analysis, frozen cancer tissue
sections were immediately cut into 5-mm cubes and embedded
in Tissue-Tek OCT compound medium (Sakura Finetech, Co.,
Ltd., Tokyo, Japan) following resection. The sections then were
fixed in liquid nitrogen and stored at ‑80˚C. Frozen specimens

were cut into 10-µm-thick slices using a cryostat, and these
sections were mounted onto foil-coated glass membrane slides(Leica Microsystems GmbH, Wezlar, Germany). The tissue
sections were stained with toluidine blue prior to air drying.
The FFPE specimens were cut into 10-µm-thick sections
using a microtome, and these sections were mounted onto
foil-coated glass membrane slides. The tissue sections were
fixed in 70% ethanol for 30 sec and stained with hematoxylin
and eosin prior to dehydration (5 min each in 70, 95 and100% ethanol). The stained frozen or FFPE sections weremicrodissected using an LMD7000 Laser Microdissection
system (Leica Microsystems GmbH). The cancer cells andcancer stromal tissues were visualized under a bright‑fieldmicroscope (magnification, x100) and selectively separated
by activation of the laser. From each slide, 1x107-2x107 µm 2
epithelial or stromal cells were captured.
RNA extraction and miRNA microarray. Total RNA wasextracted from dissected tissue using the miRNeasy FFPE
kit (Qiagen GmbH, Hilden, Germany) according to the
manufacturer's protocol. The extracted total RNA (250 ng)
was labeled with Cy5 using the 3D-Gene miRNA labelingkit (Toray Industries, Inc., Kanagawa, Japan). The labeled
RNAs were hybridized onto 3D-Gene Human miRNA Oligo
chips containing 2,555 miRNAs (Human_miRNA_Ver20;
Toray Industries, Inc.). The annotation and oligonucleotide
sequences of the probes conformed to the miRBase miRNAdatabase (http://microrna.sanger.ac.uk/sequences/). Following
stringent washes (1st wash: 0.5X saline-sodium citrate(SSC)/0.1% sodium dodecyl sulfate (SDS) solution; 2nd wash:
0.2X SSC/0.1%SDS solution; 3rd wash: 0.05X SSC solution),fluorescent signals were scanned with the 3D‑Gene Scanner(Toray Industries, Inc.) and analyzed using 3D-Gene Extraction
software (Ver 2.0.0.7; Toray Industries, Inc.). The raw data ofeach spot were normalized by substitution with a mean intensity of the background signal determined by all blank spots'
signal intensities of 95% confidence intervals. Measurementsof spots with signal intensities differing from the backgroundsignal intensity by greater than two standard deviations (SDs)were considered to be valid. The relative expression level of agiven miRNA was calculated by comparing the signal intensities of the valid spots throughout the microarray experiments.
The normalized data were globally normalized per array, suchthat the median of the signal intensity was adjusted to 100.
RT‑qPCR analysis. miR-221, miR-222, miR-659, miR-4470,
miR‑4669, miR‑5703 and RNU6B (internal control)‑specific
cDNA synthesis was performed using 10 ng of total RNA
and TaqMan miRNA primers (Applied Biosystems; Thermo
Fisher Scientific, Inc., Waltham, MA, USA). RT-qPCR
analyses were performed using the following TaqMan miRNA
assays: miR-221-3p (ID000524), miR-222-3p (ID002276),
miR-659 (ID001514), miR-4470 (ID464583_mat), miR-4669(ID464125_mat), miR5703 (ID472811_mat) and RNU6B(ID001093) with specific primers (Applied Biosystems;
Thermo Fisher Scientific, Inc.) as previously described (14).
In brief, cDNA was synthesized from total RNA (10 ng) ina 7.5-µl reaction volume (RT primer 1.5 µl, total RNA lysate
2.5 µl, RT mix 3.5 µl) using the TaqMan® MicroRNA Reverse
Transcription kit. The reactions were performed at 16˚C for-264-

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1623

Table Ⅰ. Clinicopathological data for the 12 cases of CRC analyzed in the miRNA microarray.
Features
Case

CRC without liver metastasis-----------------------------------------------------------------------------------1
2
3
4
5
6

CRC with liver metastasis------------------------------------------------------------------------------------7
8
9
10
11
12

Age (years)
Sex
Size (mm)Depth
Histological type
Lymph node metastasis
Liver metastasis
Stage

87
F
90
T3
tu 2
(‑)
(‑)
IIA

48
F
30
T3
tu 2
(+)
(+)
IV

69
F
75
T3
tu 2
(‑)
(‑)
IIA

61
M
92
T4
tu 2
(‑)
(‑)
IIB

75
F
70
T4
tu 2
(‑)
(‑)
IIB

83
M
50
T2
tu 1
(‑)
(‑)
I

80
M
40
T4
tu 2
(‑)
(‑)
IIB

65
M
60
T3
tu 2
(+)
(+)
IV

66
F
40
T3
tu 2
(+)
(+)
IV

64
M
120
T3
tu 2
(+)
(+)
IV

53
M
80
T4
tu 2
(+)
(+)
IV

69
F
28
T4
tu 2
(‑)
(+)
IV

F, female; M, male; T3, tumor invades through the muscularis propria into the pericolorectal tissues; T4, tumor invades the visceral peritoneumor invades or adheres to adjacent organ or structure; T2, tumor invades muscularis propria; tu 1, well-differentiated tubular adenocarcinoma;
tu 2, moderately differentiated tubular adenocarcinoma.

30 min and then at 42˚C for 30 min, and were inactivated
at 85˚C for 5 min. RT‑qPCR analysis was performed using
the LightCycler ®480 System II (Roche Diagnostics, Tokyo,
Japan). The reactions were performed for 10 min at 95˚C, andthen for 55 cycles with denaturation for 15 sec at 95˚C andannealing/extension for 60 sec at 60˚C. The relative expressionof miRNA to RNU6B RNA was calculated using the 2-ΔΔCq
method (15). The CRC samples were categorized into miR-221
and miR-222 weak and strong expression groups according to
the expression levels of miR-221 and miR-222 as determinedby RT-qPCR analysis, using RNU6B as the control. Highexpression was defined as an expression level above the medianvalue of the 101 CRC samples for each miRNA.
miRNA in situ hybridization (ISH). Of the 101 CRC FFPE
samples, 20 samples, (CRCwLM, n=12; CRCwoLM, n=8) wereused for ISH analysis. The locked nucleic acid (LNA)-ISH
system was used to investigate the localization of miR-221
and miR-222. LNA-ISH was performed according to the
manufacturer's protocol and relevant LNA-ISH literature (16).
Briefly, the FFPE advanced colon cancer tissues were cut into
5‑µm‑thick sections and deparaffinized. Following treatment
with proteinase K and post‑fixation with 4% paraformaldehyde (Wako Pure Chemical Industries, Osaka, Japan), theslides were hybridized with the 5' (digoxigenin-UTP)
DIG-labeled miRCURY LNA™ detection probe, miR-221,
and miR-222 (18115-01 and 38499-01; Qiagen GmbH), for
8 h at 52˚C using the Ventana Discovery Ultra instrument(Ventana Medical Systems, Inc. Tucson, AZ, USA). The
digoxigenin was detected with a polyclonal anti-DIG antibody
and an alkaline phosphatase-conjugated secondary antibody(Ventana Medical Systems, Inc.), using nitro blue tetrazolium
and 5-bromo-4-chloro-3-indolyl phosphate as the substrate.
The LNA U6 snRNA probe was used as a positive control forevery specimen. The 5' DIG-labeled miRCURY LNA™ detection probe for human mature miR-221-3p (5'-GAA AGCCAGCAG ACA ATG TAG CT-3'), miR-222-3p (5'-ACC CAG TAG
CCAGAT GTAGCT-3'), U6-positive control, and scrambled

negative control were all purchased from Exiqon A/S (Vedbaek,
Denmark). Signals from the tumor cells and stromal cells wereclassified as negative (‑) or positive (+), respectively.
Statistical analysis. All calculations were performed using the
SPSS software package version 19.0 (SPSS, Inc., Chicago, IL,
USA). Differences between groups were estimated using theMann-Whitney U test. Categorical variables were comparedusing Fisher's exact test. Patient outcome survival curves were
calculated using the Kaplan-Meier method. Factors shown to beof prognostic significance in univariate models were evaluatedin a multivariate Cox proportional hazard model. To validate
the association between survival rates and expression of thecandidate miRNAs, the following approach was used to divide
the patients into two groups according to relative expression
levels of candidate miRNAs. For miRNAs, the median valuesof the expression data were used as the cut-off. Survival was
defined as the interval from the date of diagnosis until the dateof CRC-associated mortality, date of mortality from othercause, or the end of follow-up (June 30, 2017), whichever camefirst. Patients lost to follow‑up were censored at the date of
the final follow‑up contact. All statistical analyses assumed atwo‑sided alternative with a 5% level of significance.
Results
miRNA array analysis of primary CRCwLM or CRCwoLM.
An overview of the experimental design and selection of candidate miRNAs is shown in Fig. 1. To investigate the miRNAs
associated with liver metastasis in cancer stromal tissue,
array‑based miRNA profiling was performed. The stromal
tissue of six cases of primary CRCwoLM and that of six casesof primary CRCwLM were profiled on the Toray 3D‑Gene
Human miRNA Oligo chips ver. 19. The clinicopathological
data of the patients are summarized in Table I. miRNAs withintensity levels higher than the negative control +2 SD wereselected in all cases. This filtering resulted in the identificationof 1,024 miRNAs among a total of 2,555. The Fischer ratio

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IIDA et al: STROMAL miRNA AND COLORECTAL CANCER AGGRESSIVENESS

Figure 1. Flow chart of identification of miR‑221 and miR‑222 as candidate miRNAs. 3D‑Gene Human miRNA Oligo chips were used for screening candidate
miRNAs, and RT-qPCR analysis was used for validation. Samples from six cases of CRCwoLM and six cases of CRCwLM were used for screening, and
20 CRCwoLM samples and 20 CRCwLM samples were used for RT-qPCR validation of the array analysis. Patients in the screening set and validation set
were independent groups; samples in the screening set were frozen sections and validation samples were formalin‑fixed paraffin‑embedded. miRNA/miR,
microRNA; CRC, colorectal cancer; CRCwLM, CRC with liver metastasis; CRCwoLM, CRC without liver metastasis; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2. Validation RT‑qPCR of the six miRNAs identified in the comprehensive array screen. Box and whisker plots of RT‑qPCR analysis of the expression
level in the cancer stroma of CRC with or without liver metastasis (n=20 each) of the four downregulated miRNAs (miR-659, -4470, -4669 and -5703) andthe two upregulated miRNAs (miR-221 and miR-222) in CRC with liver metastasis, compared with CRC without liver metastasis in the microarray screen.
The line within the box indicates the median value, which was used as the cut-off for high or low miRNA expression. miRNA/miR, microRNA; N.S., notsignificant; CRC, colorectal cancer; RT‑qPCR, reverse transcription‑quantitative polymerase chain reaction.

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1625

Table Ⅱ. Dysregulated miRNAs in cancer stromal tissue on comparison of colorectal cancer with and without liver metastasis.
miRNA
hsa-miR-4698
hsa-miR-6862-5p
hsa-miR-491-5p
hsa-miR-7855-5p
hsa-miR-602
hsa-miR-4726-5p
hsa-miR-6845-5p
hsa-miR-371b-5p
hsa-miR-6790-3p
hsa-miR-4533
hsa-miR-663a
hsa-miR-1237-5p
hsa-miR-6742-5p
hsa-miR-4470
hsa-miR-4750-3p
hsa-miR-665
hsa-miR-221-3p
hsa-miR-1238-5p
hsa-miR-659-3p
hsa-miR-4669
hsa-miR-4513
hsa-miR-3180-3p
hsa-miR-3185
hsa-miR-222-3p
hsa-miR-4257
hsa-miR-5196-5p
hsa-miR-5703

Signal intensity in cancer
stroma without liver metastasis

Signal intensity in cancer stroma
with liver metastasis

FoldchangeRegulation

Fischerratio

84.385343.2356621.6555
45.1489
79.6585
282.4948
690.9057
141.0927
209.2740
59.9515
3,729.6112
9,092.0858
91.7006
71.7076
182.9980
622.2152
172.4139
326.3151
109.3211123.4231
333.8607
657.2006
444.7393
86.9045
1,156.5877
154.3040
68.1390

59.945423.2163358.3432
23.2163
43.7653203.2291
480.6378
84.1496140.516832.36442,507.9348
6,981.4697
53.091133.4636
149.9362
380.7198
384.7967
238.1282
51.429958.7635
248.4870
471.0584
294.6611
193.2362
1,437.9049
111.5328
33.4572

0.71040.53700.57640.51420.54940.71940.69570.59640.67140.53980.67240.76790.57900.46670.81930.61192.23180.72970.47040.47610.74430.71680.66252.22351.24320.72280.4910

Down
Down
Down
Down
Down
Down
Down
Down
Down
Down
Down
Down
Down
Down
Down
Down
Up
Down
Down
Down
Down
Down
Down
Up
Up
Down
Down

8.68707.77987.29406.51066.04675.93145.66115.61095.37705.28425.23375.01645.00124.9666
4.9492
4.94484.83594.79284.71224.56894.53414.51944.51794.47534.3621
4.31754.2515miR, microRNA.

was used to evaluate the potential of the selected miRNAs todiscriminate between CRCwLM and CRCwoLM (17). The
1,024 miRNAs were ranked in order of decreasing Fischer
ratio. The 27 miRNAs that were significantly dysregulated incancer stromal tissues, comparing between CRCwoLM and
CRCwLM, are listed in Table II (Fischer ratio >4). For theselection of candidate miRNAs among these 27 miRNAs, topermit comparison between CRCwLM and CRCwoLM, a foldchange of 2.0 or 0.5 was used as the cut-off. Using this cut-off
led to the identification of four miRNAs (miR‑659, ‑4470,-4669 and -5703) that were downregulated and two miRNAs(miR-221 and miR-222) that were upregulated in CRCwLM
compared with CRCwoLM.
Validation of the miRNA array profiling by RT‑qPCR analysis.
To confirm the microarray findings, RT‑qPCR analysis wasused to measure the expression level of these six miRNAs,
described as above, in the stromal tissues of 20 cases CRCwLM
and 20 cases of CRCwoLM. The expression levels of miR-221
and miR‑222 in the cancer stromal tissues were significantly

higher in CRCwLM than in CRCwoLM (P=0.003 and
P= 0.02, respectively), whereas no significant difference wasfound in the expression of the four downregulated miRNAs(miR-659, -4470, -4669 and -5703) between CRCwLM and
CRCwoLM (Fig. 2).
Association of the expression of miR‑221 and miR‑222 in cancer
stromal tissues with clinicopathological factors. To evaluate
whether the expression of miR-221 or miR-222 was associated with clinicopathological factors and prognosis, RT-qPCR
analysis was used to analyze the expression of miR-221 andmiR-222 in 101 CRC FFPE sample sets of cancer stromal
tissues and corresponding cancer cells; RNU6B was used as
the control. The clinicopathological analysis showed that thegroup with a high expression of miR-221 in cancer stromal
tissues, that is, cases with specimens in which the expressionof miR-221 was above the median value, had more advanced
venous invasion (P=0.023), liver metastasis (P=0.028),
and distant metastasis (P=0.003) than the low-expression
group (Table III). The group with a high expression of

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Table III. Expression of miR-221 in the cancer stroma and its association with clinicopathological factors.

Clinicopathological factor
Age (years)
Sex
Male
FemaleDegree of differentiation
Well/moderate
Poor
Tumor size (mm)
<50
≥50
Depth
T2/T3
T4
Lymph node metastasis
AbsentPresent
Lymphatic invasion
AbsentPresent
Venous invasion
AbsentPresent
Liver metastasis
AbsentPresent
Distant metastasis
AbsentPresent
Stage
I/II
III/IV
Recurrence within 3 years
AbsentPresent

miR221 expression in stroma-------------------------------------------------------High (n=50)
Low (n=51)68.3±11.7

67.8±11.6

23
27

26
25

44
6

47
4

25
25

31
20

25
25

33
18

16
34

21
30

5
45

3
48

8
42

18
33

33
17

43
8

29
21

43
8

14
36
High (n=30)
20
10

20
31
Low (n=42)
35
7P-value
P=0.851
P=0.356
P=0.358
P=0.187
P=0.098
P=0.227
P=0.346
P=0.023
P=0.028
P=0.003
P=0.163
P=0.087

miR221 expression in cancer cells---------------------------------------------------------------High (n=50)
Low (n=51)
P-value67.4±12.2

69.0±11.0

25
25

27
24

46
4

45
6

29
21

27
24

25
25

33
18

16
34

21
30

3
47

5
46

9
41

17
34

35
15

41
10

32
18

40
11

15
35
High (n=33)
25
8

19
32
Low (n=39)30
9

P=0.394
P=0.767
P=0.383
P=0.378
P=0.098
P=0.227
P=0.369
P=0.062
P=0.164
P=0.083
P=0.288
P=0.563

T2, tumor invades muscularis propria; T3, tumor invades through the muscularis propria into the pericolorectal tissues; T4, tumor invades thevisceral peritoneum or invades or adheres to adjacent organ or structure; miR, microRNA.

miR-222 in cancer stromal tissues had more advanced depthof tumor invasion (P=0.045), liver metastasis (P=0.009),
and distant metastasis (P=0.003) than the low-expression
group (Table IV). By contrast, unlike in the stromal tissue, theclinicopathological factors and the expression of miR-221 andmiR-222 in cancer cells only showed a tendency to be associated with distant metastasis (Tables III and IV).
Association of the expression of miR‑221 and miR‑222
and survival rates. The overall survival rate of the patients

according to the miR-221 and miR-222 expression status of thecancer stroma are shown in Fig. 3A and B. The survival analysis
showed that the group with a high expression of miR-221 incancer stromal tissues had significantly shorter overall survival
rates than the group with a low expression (P=0.039). Similarly,
the group with a high expression of miR-222 in cancer stromal
tissues had significantly shorter overall survival rate than thegroup with a low expression (P=0.02). However, there was no
association between the overall survival rate and the expression
level of miR-221 or miR-222 in cancer cells (Fig. 3C and D).

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Table IV. Expression level of miR-222 in the cancer stroma and its association with clinicopathological factors.

Clinicopathological factor
Age (years)
Sex
Male
FemaleDegree of differentiation
Well/moderate
Poor
Tumor size (mm)
<50
≥50
Depth
T2/T3
T4
Lymph node metastasis
AbsentPresent
Lymphatic invasion
AbsentPresent
Venous invasion
AbsentPresent
Liver metastasis
AbsentPresent
Distant metastasis
AbsentPresent
Stage
I/II
III/IV
Recurrence within 3 years
AbsentPresent

miR222 expression in stroma-------------------------------------------------------High (n=50)
Low (n=51)
68.7±11.0

67.4±12.2

18
32

31
20

46
4

45
6

23
27

33
18

24
26

34
17

21
29

16
35

2
48

6
45

15
36

11
39

32
18

44
7

29
21

43
8

19
31
High (n=30)
22
8

15
36
Low (n=42)
33
9P-value
P=0.562
P=0.617
P=0.383
P=0.073
P=0.045
P=0.184
P=0.141
P=0.267
P=0.009
P=0.003
P=0.241
P=0.404

miR222 expression in cancer cells------------------------------------------------------------High (n=50)
Low (n=51)
68.8±10.4

67.3±12.7

22
28

27
24

44
6

47
4

26
24

30
21

26
24

32
19

16
34

21
30

4
46

4
47

12
38

14
37

35
15

41
10

32
18

40
11

15
35
High (n=33)
27
6

19
32
Low (n=39)
28
11

P-value
P=0.494
P=0.207
P=0.358
P=0.312P=0.187
P=0.227
P=0.631
P=0.433
P=0.164
P=0.083
P=0.288
P=0.237

T2, tumor invades muscularis propria; T3, tumor invades through the muscularis propria into the pericolorectal tissues; T4, tumor invades thevisceral peritoneum or invades or adheres to adjacent organ or structure; miR, microRNA.

In addition, a Cox proportional hazard regression model wasused to determine whether the expression of miR-221 andmiR-222 in the cancer stroma was an independent risk factor
for overall survival rate (Table V). The univariate analysis
revealed that high levels of stromal miR-221 (P=0.038), highlevels of stromal miR-222 (P=0.024), higher pathological T
stage (T4; P=0.001), and lymph node metastasis (P=0.007)
were significantly associated with poor overall survival rate.
The subsequent multivariate analysis confirmed these results
and showed that the stromal expression of miR-222 [hazard

ratio (HR)=2.280, 95% confidence interval (CI)=1.097‑4.742,
P=0.027), higher pathological T stage (T4; HR=2.078, 95% CI,
1.043-4.142, P=0.038), and lymph node metastasis (HR=3.679,
95% CI, 1.497-9.043, P=0.005) were independent markers forpoor overall survival rates in patients with CRC (Table V).
Localization of miR‑221 and miR‑222 in colon cancer. To
investigate the localization of miR-221 and miR-222 in cancer
tissue, ISH was performed. The negative control groups areshown in Fig. 4A and B. In CRC with liver metastasis, miR-221

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Figure 3. Kaplan-Meier analyses of overall survival according to the expression level of miR-221 and miR-222 in advanced colorectal cancer. Overall survivalof groups is shown for cases with high or low expression of (A) miR-221 in cancer stromal tissues; (B) miR-222 in cancer stromal tissues; (C) miR-221 in cancer
cells; and (D) miR-222 in cancer cells. miR, microRNA.

Table V. Univariate and multivariate Cox proportional hazard models of overall survival rates.

VariablemiR-221 (stroma)
Low, vs. highmiR-222 (stroma)
Low, vs. highmiR-221 (cancer cell)
Low, vs. highmiR-222 (cancer cell)
Low, vs. highAge (years)<60, vs. ≥60
SexMale, vs. female
Tumor size (mm)<50, vs. ≥50
Depth
T2/T3, vs. T4
Lymph node metastasis
Absent, vs. present

Univariate analysis---------------------------------------------------------------------------HR
P-value
95% CI
2.021

P=0.038

1.023-3.991

2.222

P=0.024

1.110-4.444

1.699

P=0.1210.869-3.322

1.506

P=0.220.780-2.938

1.406

P=0.395

0.641-3.086

1.644

P=0.1420.847-3.192

1.036

P=0.9160.537-1.999

2.977

P=0.001

3.369

P=0.007

Multivariate analysis----------------------------------------------------------------------------HR
P-value
95% CI

2.280

P=0.027

1.097-4.742

1.543-5.746

2.078

P=0.038

1.043-4.142

1.400-8.106

3.679

P=0.005

1.497-9.043

HR, hazard ratio; CI, confidence interval; T2, tumor invades muscularis propria; T3, tumor invades through the muscularis propria into thepericolorectal tissues; T4, tumor invades the visceral peritoneum or invades or adheres to adjacent organ or structure; miR, microRNA.

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miR‑221‑positive fibroblasts. The miR‑221 or miR‑222 signal
was observed in <10% of non-cancerous tissues (Fig. 4I and J).
Discussion

Figure 4. Locked nucleic acid-in situ hybridization detection of miR-221 andmiR-222 in primary CRC with or without liver metastasis. Primary CRC
with liver metastasis, without liver metastasis, and normal mucosa wereanalyzed. Positive staining is blue-violet in color. (A) Negative control for
miR-221; (B) negative control for miR-222. (C) miR-221 and (D) miR-222
were expressed at high levels in cancer cells and stromal cells in CRC withliver metastasis (magnification, x200). At x400 magnification (E) miR‑221
and (F) miR-222, black arrowheads indicate positive expression in cancer
cells and black arrows indicate positive expression in fibroblast‑like cells.
Expression levels of (G) miR-221 and (H) miR-222 were low in cancer cells
and stromal cells in primary CRC without liver metastasis. (I) miR-221 innormal mucosa; (J) miR-222 in normal mucosa. CRC, colorectal cancer;
miR, microRNA.

and miR‑222 were localized in the cytoplasm of fibroblasts
in stromal tissue and cancer cells (Fig. 4C-F). By contrast, inprimary CRC without liver metastasis, there was low expression of miR-221 and miR-222 in cancer cells and stromalcells (Fig. 4G and H). In terms of fibroblasts. 9/12 (75%)
CRCwLM and 2/8 (25%) CRCwoLM cases (P= 0.04)
had miR-222-positive fibroblasts. By contrast, 7/12 (58%)
CRCwLM and 3/8 (37.5%) CRCwoLM cases (P=0.36) had

The dysregulation of miRNA expression is frequently detected
in CRC and has been associated with increased metastatic
potential and poor clinical outcome, suggesting the importance of miRNAs in cancer progression (18,19).
In the present study, it was shown that the overexpression of miR-221 and miR-222 in the cancer stromal tissue
of primary tumors was associated with malignant potential
in patients with CRC. To date, several studies have profiled
the expression of miRNAs involved in cancer metastasis in
CRC, however, those studies were performed primarily on
cancer cells or cancer tissue (20,21). In the present study,
the stromal miRNA expression profile of CRCwLM wascompared with that of CRCwoLM using microdissection.
Using miRNA array analysis, six miRNAs were identified
that were differentially expressed between the two patientgroups. Following reassessment of these six miRNAs using
RT-qPCR analysis, differential expression between CRCwLM
and CRCwoLM was confirmed for miR‑221 and miR‑222.
To the best of our knowledge, this is the first comprehensive
miRNA array analysis performed to identify miRNAs in the
CRC stroma whose expression is associated with metastatic
ability. A small number of previous studies have reported
differential miRNA expression profiles between the cancer
stroma and normal stroma. Nishida et al reported oncogenic
miRNAs, including miR-21, miR-221, the miR-17-92a cluster,and the miR-106b-25 cluster, which were upregulated in thecancer stroma compared with the normal stroma. In addition,
the upregulation of miR-25 and miR-92a in stromal tissues
has been associated with a variety of clinicopathologicalfactors (12). Bullock et al observed distinct patterns of stromal
miRNA expression in CRC and paired normal colonic tissue,
and reported that stromal levels of miR-21 and miR-556
predicted short disease-free survival and overall survival rates
in stage II disease (13). Furthermore, Bullock et al reported
the stromal upregulation of miR-214 and downregulation of
miR-192, mir-194, miR-200a, and miR-215 in Dukes C CRC
compared with Dukes A CRC among 95 miRNAs analyzed
using RT-qPCR analysis. However, the results from thisparticular study were limited as the study only compared fiveDukes C samples with five Dukes A samples using RT‑qPCR
analysis. This low number of samples analyzed may have been
insufficient to detect novel stromal miRNAs that are associated with the malignant potential of CRC.
miR-221 and miR-222 are homologous miRNAs, which
are encoded on the X chromosome as part of a locus
designated the miR-221-222 cluster (22). This cluster has
been found to be overexpressed in various types of humancancer, including CRC (23). In previous years, there havebeen reports on the association between the metastatic
potential of CRC and the expression of miR-221 or mir-222.
However, previous studies have analyzed the expression of
miR-221 or mir-222 in cancer cells or cancer tissues, or in
the blood. To the best of our knowledge, previous studies
have not reported that the expression of miR-221 or mir-222
in the cancer stroma was associated with the development

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IIDA et al: STROMAL miRNA AND COLORECTAL CANCER AGGRESSIVENESS

of CRC. It has been shown that miR-221 and miR-222
promote oncogenesis by downregulating the expressionof tumor suppressors, including phosphatase and tensin
homolog, reversion-inducing-cysteine-rich protein withkazal motifs, p53 upregulated modulator of apoptosis, andp27 (24-28). Qin and Lui reported that the overexpressionof miR-221 enhances CRC cell migration and invasionin vitro and metastasis in vivo (26). Furthermore, miR-221
and miR-222 have the same seed sequence. The fact thattwo oncogenic miRNAs with the same seed sequence wereidentified in a comprehensive analysis is of interest, as thesemiRNAs share the same targets and may work coordinately.
To evaluate the clinicopathologic relevance of the identified miRNAs, the present study analyzed the expression of
miR-221and miR-222 in 101 advanced CRC samples. A highexpression level of miR-221 or miR-222 in patients with CRC
was significantly associated with liver metastasis, distantmetastasis, and shorter overall survival rate. Furthermore,
it was found that the association between the malignant
potential of CRC and the level of miR-221 or miR-222 in thecancer stroma was more marked than that in the cancer cells.
Using ISH for miRNA measurement, Uozaki et al reported
that the level of miR-21 in cancer cells was not associatedwith clinicopathological factors, however, the stromal level ofmiR-21 was associated with several factors in gastric cancer,
including tumor stage, size, and nodal metastasis (29). These
results are similar to the results of the present study, and the
combination of these results suggests that the overexpressionof certain oncogenic miRNAs, including miR-21, miR-221
and miR-222, in the cancer stroma may be important forcancer development.
To detect the localization of miR-221 and miR-222
in CRC, ISH was performed in 20 CRC samples. In thisanalysis, miR-221 and miR-222 were upregulated in thecancer cells and stromal cells, particularly in fibroblasts, inmetastatic CRC. Furthermore, the expression of miR-221
and miR‑222 in fibroblasts tended to be higher in fibroblasts
surrounding cancer cells strongly positive for miR-221
and miR-222. A report by Kosaka et al suggested that, intumor microenvironments, extracellular miRNAs may influence tumor progression via bidirectional tumor-to-stroma andstroma-to-tumor communication (11). These results suggestthat the crosstalk of miR-221 and miR-222 between cancer
cells and stromal cells occurs in the cancer microenvironment. The precise mechanisms underlying the upregulationof miR-221 and miR-222 in stromal cells with metastatic
CRC remain to be elucidated. One possible mechanism isthat the crosstalk of miR-221 and miR-222 between cancer
cells and stromal cells is involved in cancer progression. A
previous comprehensive array analysis showed that miR-221
is upregulated in cancer-associated fibroblasts (CAFs)
compared with that in normal fibroblasts (NFs) in breast
cancer (30). Furthermore, Shimoda et al reported that theknockdown of issue inhibitor of metalloproteinase (TIMP)
family genes resulted in acquisition of the properties of
CAFs by NFs (31). TIMP2 and TIMP3, which belong to the
TIMP family, have been reported as being target genes ofmiR-221 and miR-222 (32,33). These results indicate thatmiR-221 and miR-222 from tumor-derived exosomes mad
modify the cellular phenotype of fibroblasts to that of CAFs.

This change in phenotype can promote the invasion andmetastatic potential of the tumor cells.
A limitation of the present study is that no in vivo transfection was performed, therefore, it was not possible to assess
whether overexpression of miR-221 or miR-222 in the cancer
stroma facilitates metastasis in vivo. In order to confirm thisdetailed mechanism, it is necessary to transfect miR-221
and/or miR-222 into the cancer stroma rather than into cancer
cells. However, it is difficult to selectively transfect the cancer
stroma only with specific miRNAs, therefore, no such experiments were performed in the present study. Further functional
studies are required in order to clarify the role of stromalmiR-221/222 expression.
Acknowledgements
The authors would like to thank Mr. Yasutake Yano and
Mr. Naoya Touyama from the Yamaguchi University Schoolof Medicine for their technical support.
Funding
The present study was performed as a research program of
the Project for Development of Innovative Research on Cancer
Therapeutics (P-DIRECT; grant no. 11039020). This study
was funded by the Japan Agency for Medical Research andDevelopment (AMED; grant no. 15cm0106085h0005), and was
supported in part by a grant for Leading Advanced Projects for
Medical Innovation (LEAP; grant no. 16am0001006h0003)
from AMED.
Availability of data and materials
All data generated or analyzed during the present study areincluded within.
Authors' contributions
MI, SH, and HN designed the study; MI, HTakenouchi,
SK, YuT, STo, YoT and SYo collected the data; MI, SH, RT,
HTanaka, HTakenouchi, KF, and MK performed the experiments. MI, SH, KS, NS, STa, TU, and SYa performed thestatistical analysis; MI, SH, and HN wrote the manuscript. All
authors read and approved the final manuscript and agree to beaccountable for all aspects of the research in ensuring that theaccuracy or integrity of any part of the work are appropriately
investigated and resolved.
Ethics approval and consent to participate
Written informed consent was obtained from each patientat the time of enrollment. The study was performed inaccordance with the Declaration of Helsinki on experiments
involving human subjects. The study protocol was approvedby the Institutional Ethics Review Boards of Yamaguchi
University.
Patient consent for publication
Written informed consent was obtained from all patients.

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Competing interests
The authors state that they have no competing interests.
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Expression level of microRNA-200c is associated with cellmorphology in vitro and histological differentiation throughregulation of ZEB1/2 and E-cadherin in gastric carcinoma
ATSuShI KuRATA1, MASATOShI YAMAdA1, ShIN-IChIRO OhNO1, ShIGERu INOuE2,
hIROTSuGu hAShIMOTO3, KOjI FujITA1, MASAKATSu TAKANAShI1 and MASAhIKO KuROdA1
departments of 1Molecular Pathology and 2Preventive Medicine and Public health, Tokyo Medical university,
Tokyo 160-8402; 3department of diagnostic Pathology, NTT Medical Center Tokyo, Tokyo 141-8625, japan
Received june 27, 2017; Accepted October 30, 2017
dOI: 10.3892/or.2017.6093
Abstract. Scirrhous type gastric cancer is characterized bydiffuse infiltration of poorly differentiated adenocarcinoma
cells and poor prognosis. Although association of poorly
differentiated histology with reduction in E-cadherin expression, as well as association of microRNA (miR)-200c with
E-cadherin through regulation of ZEB1/2, has been reported,
participation of miR-200c in gastric carcinogenesis is not
fully understood. We used 6 cell lines originating from gastriccancers, and investigated levels of miR-200c along with its
target mRNAs ZEB1/2 and E-cadherin by qRT-PCR. ZEB1 andE-cadherin protein expression was also assessed via western
blotting. Furthermore, we investigated the expression levels of
miR-200c by in situ hybridization, along with the expressionof ZEB1 and E-cadherin by immunohistochemistry, in 97
gastric adenocarcinoma tissues. Inverse correlation between
miR-200c and ZEB1 levels were obtained by qRT-PCR in celllines (P<0.05). Cell lines with low miR-200c and high ZEB1
exhibited low E-cadherin expression in both qRT-PCR andwestern blotting, and exhibited spindle-shaped morphology,
in contrast to round cell morphology in those cell lines withhigh miR-200c levels. Inverse correlations were also obtained
between miR-200c and ZEB1 as well as between ZEB1 andE-cadherin levels in tissue samples (P<0.001). Cancer tissues
with low miR-200c, high ZEB1, and low E-cadherin expression were associated with poorly differentiated histology,
in contrast to tubular form in cancers with high miR-200c
expression levels (P<0.001). Our data revealed that downregulation of miR-200c primarily regulated cell morphology by

Correspondence to: dr Atsushi Kurata, department of MolecularPathology, Tokyo Medical university, 6-1-1, Shinjuku, Shinjuku-ku,
Tokyo 160-8402, japan
E-mail: akurata@tokyo-med.ac.jp

Key words: gastric adenocarcinoma, microRNA-200c, E-cadherin,
epithelial-mesenchymal transition, cell morphology

downregulation of E-cadherin through upregulation of ZEB1,
leading to poorly differentiated histology in gastric cancer.
Introduction
Gastric cancer is the fourth most common cancer and thesecond leading cause of cancer-related deaths in the world (1).
More than 980,000 new gastric cancer cases are diagnosed
annually, and the disease causes approximately 730,000
deaths per year, with the highest incidence rates in Eastern
Asia (1). Scirrhous cancer, which accounts for approximately 10% of all gastric cancers, is characterized by diffuse
infiltration of poorly differentiated adenocarcinoma cellswith extensive stromal fibrosis, leading to frequent peritoneal
dissemination and extremely poor prognosis (2,3). Molecularanalyses have revealed that reduction in the expression of
the cell-cell adhesion molecule, E-cadherin, was involvedin the development and infiltrative growth of scirrhous typegastric carcinomas (4,5). Reduction in E-cadherin expression
observed in poorly differentiated carcinomas of the lungs andendometrium (6,7), promotes detachment of cells from eachother and increases cell motility; thus, leading to epithelialmesenchymal transition (EMT) (8).
On the other hand, epigenetic regulation including therole of microRNAs (miRNAs, miRs) in carcinogenesis has
recently been intensively studied. miRNAs are endogenous
18-24-nucleotide (nt) single-stranded RNA molecules that act
as post-transcriptional regulators of gene expression (9). They
stabilize target mRNA transcripts through post-transcriptional
gene silencing via either inhibition of the translation process orcleavage of target mRNAs (10,11). miRNAs sharing the sameseed sequence are grouped into families and are theorized totarget overlapping sets of genes (12). miRNAs play diverseroles in numerous cellular processes; in particular, theirexpression is altered during tumorigenesis, and they can act as
oncogenes or tumor suppressors (13).
Recent studies have shown the importance of certain
miRNAs in modulating EMT, and the miR-200 family has
been described to be a key regulator of this process (7,8,14).
The miR-200 family consists of 5 members: miR-200a,
miR-200b and miR-429 which are located on chromosome

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KuRATA et al: miR-200c IN GASTRIC CANCER

1p36 while miR-200c and miR-429 are located on chromosome 12p13 (14). The miR-200 family suppresses zinc-finger
E-box binding homeobox (ZEB) 1 and ZEB2, which in turninhibit the expression of E-cadherin; thus, the miR-200
family maintains the epithelial phenotype through upregulation of E-cadherin (7,8,14). Among the miR-200 family, lossof miR-200c has been reported to be pivotal to inducing
aggressive, invasive and chemoresistant phenotypes in several
cancers (15-17). Although downregulation of miR-200a andmiR-200b have been reported to be associated with poor
prognosis or EBV-association in gastric cancers (18,19), little
is known about miR-200c in this context. Recent reports
demonstrated that miR-200c may affect E-cadherin expression
through regulation of ZEB1/2 expression (20,21); however, theinfluences of miR-200c on cell morphology and its association
with histological type have so far not been reported.
In the present study, we investigated the expressionof miR-200c, as well as corresponding targets at both themRNA and protein levels, in several gastric carcinoma
cell lines. In addition, we examined the expression level ofmiR-200c and respective target proteins in gastric carcinoma
tissues and adjacent non-tumor tissues. Our results revealed
that the expression level of miR-200c was associated withthe morphology of gastric carcinoma cell lines, as well as
histological differentiation in cancer tissues, along with theupregulation or downregulation of ZEB 1/2 and E-cadherin.
Materials and methods
Cell culture and RNA preparation. Six human gastriccarcinoma cell lines, h-111-TC, hGC-27, Kato-III, MKN-1,
MKN-45 and Nu-GC-4 were purchased from Riken Cell Bank
(Tsukuba, japan) and used in the present study. According to
the description by the Riken Cell Bank and literature (22-27),
these cell lines originated from 1 unknown origin (h-111-TC)
and 5 metastatic carcinoma, as well as 2 tubular (well differentiated or adenosquamous) carcinoma (h-111-TC and MKN-1)
and 4 poorly differentiated carcinoma including signet ringcell type. hGC-27 and MKN-1 cell lines were maintained indulbecco's modified Eagle's medium (dMEM; Gibco-BRL,
Rockville, Md, uSA), whereas Nu-GC-4, Kato-III, MKN-45
and h-111-TC cell lines were maintained in RPMI-1640(Gibco-BRL), supplemented with 10% fetal calf serum (FCS),
penicillin, and streptomycin, at 37˚C in a humidified atmosphere of 5% CO2. When they reached 80-90% confluence,the cells were washed with phosphate-buffered saline (PBS)
and homogenized immediately in Isogen reagent (Nippon
Gene, Osaka, japan). Total RNA was extracted according to
the manufacturer's instructions.
Quantitative real-time reverse transcription polymerase
chain reaction (qRT-PCR) analysis. The RNA samples weresuspended in 20 µl of nuclease-free water, and miRNA-200c
levels were quantified using TaqMan MicroRNA Assay technology (Applied Biosystems; Life Technologies, Carlsbad, CA,uSA), as previously described (28). miRNA-200c levels werenormalized against levels of RNu6B. The hsa-miR-200c
oligonucleotide was a synthetic double-stranded 23-nt RNA,
5'-uAAuACuGCCGGGuAAuGAuGGA-3' and 5'-CGuCu
uACCCAGCAGuGuuuGG-3' purchased from BONAC

Corp. (Fukuoka, japan). To assess the mRNA levels of ZEB1,
ZEB2 and E-cadherin, reverse-transcription reactions were
performed using M-MLV reverse transcriptase (Invitrogen,
Carlsbad, CA, uSA). Quantitative PCR (FastStart universal
SYBR-Green Master; Roche, Basel, Switzerland) reactions
were run on an Mx3005P thermocycler (Stratagene, La jolla,CA, uSA) and analyzed using MxPro QPCR software, version
4.01 (Stratagene, Agilent Technologies, Santa Clara, CA,
uSA). The level of gene expression relative to glyceraldehyde
3-phosphate dehydrogenase (GAPdh) was determined. The
primer sequences were as follows: ZEB1 forward, 5'-TGTCACCATGAAACCATTGC-3' and reverse, 5'-AGGTAAAGTG
CGCTTCCTCA-3'; ZEB2 forward, 5'-GGGCATTCAGTGA
CCTGACA-3' and reverse, 5'-GCATTGTTCCCATAGAGT
TC-3'; E-cadherin forward, 5'-TCCTCGGATTCTCTGCT
CTC-3' and reverse, 5'-CTCTGACCTTTTGCCAGGAG-3';
GAPdh forward, 5'-ATGGGGAAGGTGAAGGTCG-3' andreverse, 5'-GGGTCATTGATGGCAACAATATC-3'. The
experiments were performed 3 times, with the average levels
and standard deviations from the mean being obtained.
Correlations between average levels of miR-200c, ZEB1,
ZEB2 and E-cadherin in all cell lines were estimated by
Spearman's rank correlation using the SPSS software package(ver17.0; SPSS japan Inc., Tokyo, japan). The results wereconsidered significant if the P<0.05.
Western blot analysis. Cell lines were lysed in RIPA buffer.
After boiling with sodium dodecyl sulfate loading buffer, equalamounts (10 µg) of the proteins were electrophoresed on 10%
sodium dodecyl sulfate-polyacrylamide gels (SdS-PAGE) andtransferred to Immobilon membranes (Millipore Inc., Bedford,
MA, uSA) by semidry blotting. Five percent milk powder
dissolved in PBS buffer containing 0.1% Tween-20 was used toblock non-specific binding. Then, using standard techniques,
the membranes were probed with the following antibodies:anti-E-cadherin antibody (mouse monoclonal antibody tohuman E-cadherin; clone: hECd-1; dilution: 1:200; Takara,
Tokyo, japan), anti-ZEB1 antibody (rabbit monoclonal antibody to ZEB1; clone: d80d3; dilution: 1:100; Cell Signaling
Technology, danvers, MA, uSA), and anti-β-actin antibody(mouse monoclonal antibody to β -actin; clone: C4; dilution: 1:1,000; Santa Cruz Biotechnology, dallas, TX, uSA).
Antibody-antigen complexes were detected using Immobilon
Western chemiluminescent horseradish peroxidase substrate(Millipore), and signals were recorded on a LAS-3000 mini
system (Fujifilm, Tokyo, japan).
Human tissue specimens and tissue microarrays. human tissue
specimens were obtained from 97 patients who underwent
gastric cancer surgery at Tokyo Medical university hospital
between 2003 and 2010. The Tokyo Medical university
institutional review board approved the present study, andall patients provided written informed consent. The patient
cohort included 74 males and 23 females, ranging in age from40 to 92 years (average 66.3 years). Other clinicopathologicalcharacteristics are listed in Table I. The tumors were cut into
~5-mm sections after fixation in 10% formalin and embedded
in paraffin. From these paraffin blocks, tissue microarrays(TMAs) were constructed, each consisting of 12 specimensof 6-mm diameter on one slide. As previously described (29),-275-

ONCOLOGY REPORTS 39: 91-100, 2018

Table I. Clinical and histopathological features of 97 patients.
Characteristics
Mean age (years)
Sex (male/female)
Lymph nodes metastasis (+/-)
Tumor diameter (mm)depth (M/SM/MP/SS/SE/SI)
Vessel infiltration (+/-)
histology of tubular formation (+/-)

data
66.3±11.9
74/23
33/64
47.8±28.2
25/26/8/25/9/4
50/47
67/30

M, mucosa; SM, submucosa; MP, muscularis propria; SS, subserosa;
SE, serosa exposed; SI, serosa infiltrating (neighboring organ ororgans involved).

TMAs allow the relocation of multiple tissue samples fromconventional histologic paraffin blocks so that tissues frommultiple patients can be analyzed on the same slide.
In situ hybridization. In situ hybridization (ISh) was
performed on TMAs containing 3-µm consecutive sectionsof formalin-fixed, paraffin-embedded (FFPE) samples.
Probes utilized were based on 5' digoxigenin (dIG)-labeled
ISh locked nucleic acid (LNA) technology (miRCuRY-LNA
detection probe; Exiqon A/S, Vedbaek, denmark). Slides
were prepared using the Ventana hX System BenchMark(Ventana Medical Systems, Inc., Tucson, AZ, uSA). We
used the following 5' dIG-labeled LNA probes for ISh:hsa-miR-200c, 5'-TCCATCATTACCCGGCAGTATTA-3';
and non-target negative control sequence, 5'-GTGTAACACGTCTATACGCCCA-3'.
Immunohistochemistry. Immunohistochemistry (IhC) was
performed on TMAs containing 4-µm consecutive sectionsof FFPE gastric tissue samples. After deparaffinization andrehydration, endogenous peroxidases were blocked by incubation in 0.3% hydrogen peroxide solution for 20 min. To
expose antigens, the sections were autoclaved in EdTA buffer(ph 9.0) for 10 min and cooled for 30 min. After rinsing
in 0.05 M Tris-buffered saline containing 0.1% Tween-20(ph 7.6), the sections were incubated with an affinity-purified
anti-ZEB1 antibody (clone: d80d3; dilution: 1:200; Cell
Signaling Technology) and anti-E-cadherin antibody (clone:
hECd-1; dilution: 1:1,000; Takara) for 3 h at RT. Samples
were then washed three times in PBS and incubated withdako secondary antibody for 15 min at RT. horseradish
peroxidase-labeled polymer conjugated to a mixture of goatanti-mouse and anti-rabbit immunoglobulin antibodies (Code:K5007; prediluted; dako, Glostrup, denmark) was used as
the secondary antibody. 3,3'-diaminobenzidine tetrachloride(dAB) was used for color development and sections werecounterstained with hematoxylin.
Evaluation of tissue images. ISh- or IhC-stained slides
were examined by light microscopy using an Olympus BX50
microscope. Three different fields (x200) in each tissue
were randomly selected. digital images of ISh and IhC

93

were captured by the NY-d5000 super system (Microscope
Network; Nikon, Tokyo, japan) and were printed by a truecolor printer (IPSiO SP C420; Ricoh, Tokyo, japan). Positive
ISh results caused the cytoplasm to appear blue, whereas positive IhC results appeared brown. Nuclear immunostaining wasassessed for ZEB1, while cytomembranous immunostaining
was assessed for E-cadherin. Images were interpreted semiquantitatively by assessing the extent and intensity of staining
on the entirety of each tissue section present on the slides,
as previously described (30). Briefly, the total percentage of
positively-stained tumor cells was first determined. Then, thepercentage of weakly-, moderately- and strongly-stained cells
was determined, so that the sum of these categories equatedwith the overall percentage of positivity. A final staining score
was then calculated as the sum of 1 x percentage of weak, 2 x
percentage of moderate, and 3 x percentage of strong staining(maximum score=300).
Statistical analyses of data from human tissue studies.
Prior to statistical analyses, clinicopathlogical data as wellas tissue image results were dichotomized into two groupsof ‘high’ and ‘low’ levels, according to the median values.
Thus, the final score for staining of miR-200c was used todefine high-level expression (final score ≥201) and low-level
expression (final score <201). Likewise, the final score forstaining of ZEB1 was used to define high-level expression(final score ≥1) and low-level expression (final score <1), as
well as that of E-cadherin used to define high-level expression (final score ≥111) and low-level expression (final score<111). Tumor diameter was dichotomized as either highlevel (diameter ≥40 mm) or low-level (diameter <40 mm).
high-age (≥65 years old) and low-age (<65 years old)groups were established according to convention, as wereadvanced (depth ≥mp) and early (depth <mp) cancer groups.
histologically, both a well/moderately differentiated tubular
adenocarcinoma group and a poorly differentiated adenocarcinoma group were established. Then, statistical analyses
including Chi-square test and multivariate analysis using
logistic regression models were performed using the SPSS
software package (ver17.0; SPSS). Results were consideredsignificant if the P<0.05.
Results
Quantitative analysis of miR-200c and target mRNA
expression in gastric carcinoma cell lines. We investigatedmiR-200c expression in 6 gastric carcinoma cell lines by
qRT-PCR analysis. As shown in Fig. 1A, the expression levelof miR-200c descended in the following order (from high tolow): Kato-III > MKN-45 > Nu-GC-4 > h-111-TC > hGC-27> MKN-1. Of note, the latter 2 cell lines hGC-27 and MKN-1
expressed very low levels of miR-200c. We also investigatedthe expression of particular target mRNAs, namely ZEB1and ZEB2, along with E-cadherin, in the same cell lines. The
expression level of ZEB1 descended in the following order(from high to low): hGC-27 > MKN-1 > Nu-GC-4 > h-111-TC> MKN-45 > Kato-III (Fig. 1B). Of note, the 2 cell lines inwhich miR-200c expression level was very low exhibited thegreatest expression level of ZEB1. Similarly, the expression
level of ZEB2 descended in the following order (from high to

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KuRATA et al: miR-200c IN GASTRIC CANCER

Figure 1. The level of miR-200c and respective target mRNAs in gastric carcinoma cell lines. (A) Gastric carcinoma cell lines were arranged according to theexpression level of miR-200c. The expression levels of (B) ZEB1, (C) ZEB2 and (d) E-cadherin expression in gastric carcinoma cell lines arranged according
to the level of miR-200c expression.

low): hGC-27 > h-111-TC > Nu-GC-4 > MKN-45 > MKN-1> Kato-III (Fig. 1C). here, one of the 2 cell lines in which
miR-200c expression level was very low exhibited the greatest
expression level of ZEB2. On the other hand, the expression
level of E-cadherin descended in the following order (fromhigh to low): h-111-TC > MKN-45 > Nu-GC-4 > Kato-III >MKN-1 > hGC-27 (Fig. 1d). Of note, the 2 cell lines in which
the expression level of miR-200c was very low exhibited thelowest expression level of E-cadherin. As shown in Table II,
an inverse correlation was observed between miR-200c and
ZEB1 (P<0.05; by Spearman's rank correlation).
ZEB1 and E-cadherin protein expression in gastric carcinoma cell lines. The expression of ZEB1 and E-cadherin
proteins was examined in the 6 human gastric carcinoma celllines mentioned thus far by western blot analysis. As shown in
Fig. 2, expression of ZEB1 was only detected in hGC-27 andMKN-1 cells, which exhibited the lowest expression level ofmiR-200c determined by qRT-PCR analysis. Conversely, theexpression of E-cadherin was not detected in these 2 cell lines;

Figure 2. Expression of ZEB1, E-cadherin and β -actin proteins in gastriccarcinoma cell lines, presented by western blot analysis performed on 10 µgof the cell extract.

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Table II. Correlations between the average expression levels of miR-200c, ZEB1, ZEB2 and E-cadherin in all cell lines estimatedby Spearman's rank correlation.
ZEB1-----------------------------------------------------------------Correlation coefficient
P-valuemiR-200c
ZEB1
ZEB2-0.8860.019

ZEB2--------------------------------------------------------------Correlation coefficient
P-value-0.429
0.600

0.397
0.208

E-cadherin-----------------------------------------------------------------Correlation coefficient
P-value
0.429-0.600
0.029

0.397
0.208
0.957

Bold indicates P<0.05.

Figure 3. Six gastric carcinoma cell lines arranged according to expression the level of miR-200c. This order is compared with the origin (primary cancer ormetastatic cancer), differentiation state (tubular or poorly differentiated), and shape (round/intermediate/spindle) of the cell lines.

instead, E-cadherin expression was detected in Nu-GC-4 andh-111-TC cell lines, although very weak expression was also
detected in Kato-III cells.
Interpretation of divergent expression levels of miR-200c,
ZEB1/2, and E-cadherin in different gastric carcinoma celllines. The results of qRT-PCR analyses and western blot
analysis were in agreement. Cell lines with a high expression
level of miR-200c exhibited low ZEB1/2 and high E-cadherin
expression, whereas those with a low expression level ofmiR-200c exhibited high ZEB1/2 and low E-cadherin expression. Therefore, we investigated characteristics that illustrated
differences between the group of cell lines with high miR-200c
expression and those with low miR-200c expression. As shownin Fig. 3, we first compared the tumor origin (primary or metastatic tumor) of the cell lines. however, the tumor origin did
not clearly correspond to the levels of miR-200c expression, as
cell lines derived from metastatic tumors exhibited both low
and high miR-200c expression levels. Secondly, we compared

the histological differentiation state of the tumors from which
the cell lines were derived was compared. Although cell lines
from poorly differentiated tumors exhibited high miR-200c
expression, the results were inconclusive as one of the celllines with low miR-200c expression (hGC-27) was originallyfrom a poorly differentiated tumor. Thirdly, we revealed thatthe morphology of the cell lines was well correlated with theexpression level of miR-200c. Cell lines with high miR-200c
expression (Kato-III, MKN-45 and Nu-GC-4) were roundshaped, those with low miR-200c expression (hGC-27 andMKN-1) were spindle-shaped, and those with intermediate
miR-200c expression (h-111-TC) exhibited a phenotype which
was a mid-way between round- and spindle-shaped (polygonalor short spindle-shaped).
miR-200c expression patterns in gastric adenocarcinoma andadjacent non-tumor tissues. In human tissue samples, positivemiR-200c expression, as determined by ISh, was denoted bycytoplasmic granular blue staining. Most of the normal tubules

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KuRATA et al: miR-200c IN GASTRIC CANCER

Figure 5. upregulation/downregulation of miR-200c affects cell morphology
and histology.

protein expression was denoted by nuclear staining. Overall
positivity of ZEB1 was relatively low in normal tubules andgastric carcinomas. ZEB1 was almost negative in tubular
adenocarcinomas; however, positive expression of ZEB1,
although generally weak, was sporadically observed in poorly
differentiated adenocarcinomas. The average staining score
for ZEB1 was 0.84±2.93 in tubular adenocarcinomas, while itwas 5.8±8.6 in poorly differentiated adenocarcinomas. Positive
E-cadherin protein expression was indicated by cytomembranous staining. Normal tubules and tubular adenocarcinomas
were strongly stained, while poorly differentiated adenocarcinomas were weakly stained (Fig. 4). The average staining score
for E-cadherin was 146±52 in tubular adenocarcinomas, while
it was 39±38.6 in poorly differentiated adenocarcinomas.
Figure 4. Representative photomicrographs including in situ hybridizationand immunohistochemistry. (A and B) Staining with h&E and staining for (Cand d) miR-200c, (E and F) ZEB1 and (G and h) E-cadherin in (A, C, E andG) tubular adenocarcinoma and (B, d, F and h) poorly differentiated adenocarcinoma. Original magnification, x200. (A) Left side are normal glands,
right side is carcinoma. (B) Left side is carcinoma, right side is normal
foveolar epithelium. (C) Cytoplasmic granular staining in tubular adenocarcinoma was stronger than that found in normal glands. (d) Cytoplasmic
granular staining in poorly differentiated adenocarcinoma was weaker thanthat observed in normal epithelium. (E) Nuclear immunostaining was negative in tubular adenocarcinoma. (F) Nuclear immunostaining was weak but
positive in poorly differentiated adenocarcinoma. (G) Cytomembranous
immunostaining in tubular adenocarcinoma was as strong as that found innormal glands. (h) Cytomembranous immunostaining in poorly differentiated adenocarcinoma was weaker than that observed in normal epithelium.

stained positive for miR-200c, whereas stromal tissue was notstained. As shown in Fig. 4, miR-200c expression patterns ingastric adenocarcinoma differed substantially between tubular
adenocarcinoma and poorly differentiated adenocarcinoma. In
tubular adenocarcinoma, miR-200c expression was observedwith an intensity level either stronger than or the same asnormal tubules. Conversely, in poorly differentiated adenocarcinoma, miR-200c expression was either not identified orobserved with weaker staining patterns than normal tubules.
The average staining score for miR-200c was 265±81.6 intubular adenocarcinomas, while it was 104±75.2 in poorly
differentiated adenocarcinomas.
ZEB1 and E-cadherin expression patterns in gastric adenocarcinoma and adjacent non-tumor tissues. Positive ZEB1Interpretation of statistical analyses of human tissue studies.
use of the Chi-square test revealed a correlation between
miR-200c and E-cadherin expression levels (P<0.001) and an
inverse correlation between miR-200c and ZEB1 expression
levels (P<0.001) as well as between ZEB1 and E-cadherin
expression levels (P<0.001). We further compared theexpression levels of miR-200c, ZEB1 and E-cadherin withclinicopathological factors (Table III). high expression levelsof miR-200c were correlated with older age (≥65 years old)
(P= 0.002) and tubular histology (P<0.001). high expression
levels of ZEB1 were correlated with poorly differentiatedhistology (P<0.001) and advanced stage (P= 0.040). highexpression levels of E-cadherin were correlated with older age(≥65 years old) (P=0.039) and tubular histology (P<0.001). In
the multivariate analysis (Table IV), among the clinicopathological factors significantly correlated with the expression
levels of miR-200c, ZEB1 and E-cadherin, there was a significant association between the expression level of ZEB1 andadvanced stage (P= 0.031), as well as a significant correlation
between the expression level of ZEB1 and histology (P<0.001).
Discussion
In the present study, we revealed inverse correlations between
miR-200c and ZEB1 in both cell lines and tissues, as well as
inverse correlations between ZEB1 and E-cadherin in tissues,in gastric carcinoma. These well known correlations which
are associated with EMT (8) have been demonstrated bothin vitro and in vivo in several cancers including non-small celllung cancer and pancreatic cancer (15,16) and recently also in-279-

ONCOLOGY REPORTS 39: 91-100, 201897

Table III. Correlations of the expression levels of miR-200c, ZEB1 and E-cadherin with the clinicopathological factors as determined by Chi-squared test.
miR-200c--------------------------------------------------------highLow
P-value
Sex
Male
FemaleAge (years old)≥65
<65
LN metastasis
Positive
Negative

histology
TubularPoor differentiation
Tumor size (mm)
≥40
<40
Stage
Advanced
Early

Vessel invasion
Positive
Negative

37
9

37
14

35
11

22
29

13
33

20
31

45
1

22
29

19
27

28
23

19
27

27
24

20
26

30
21

ZEB1
-------------------------------------------------------highLow
P-value

0.474
0.002

0.289
<0.001

0.224

0.310

0.157

18
8

56
15

11
15

46
25

9
17

24
47

9
17

58
13

15
11

32
39

17
9

29
42

16
10

34
37

0.419

0.063

1.000
<0.001

0.360
0.040

0.259

E-cadherin-----------------------------------------------------highLow
P-value
35
12

39
11

33
14

24
26

15
32

18
32

45
2

22
28

20
27

27
23

23
24

23
2

25
22

25
25

0.812
0.039

0.830
<0.001

0.312

0.840

0.840

LN, lymph node. Bold indicates P<0.05.

Table IV. Multivariate analyses of correlations between the clinicopathological factors and positive expression of miR-200c,
ZEB1 and E-cadherin.

Clinicopathological factors

miR-200c-----------------------------------------------OR (95% CI)
P-value

ZEB1-----------------------------------------------------------OR (95% CI)
P-valueE-cadherin-----------------------------------------------OR (95% CI)
P-valueSexmale vs. female

1.822(0.505-6.568)

0.359

0.294
(0.074-1.170)

0.082

0.438
(0.124-1.553)

0.201

LN metastasis
Positive vs. negative

1.038(0.249-4.320)

0.959

0.347
(0.090-1.342)

0.125

0.458
(0.111-1.888)

0.280

Age (years old)
≥65 vs. <65

histology
Tubular vs. poor differentiation
Tumor size (mm)≥40 vs. <40

Stage
Advanced vs. early

Vessel invasion
Positive vs. negative

2.742(0.916-8.202)

0.071

0.023
(0.003-0.182)<0.0011.073(0.258-4.473)

0.923

1.087
(0.337-3.509)

0.889

0.484
(0.105-2.240)

0.354

0.505
(0.153-1.662)

0.261

7.556
(2.471-23.109)<0.0015.522(1.166-26.148)

0.031

0.809
(0.242-2.703)

0.730

0.998
(0.227-4.378)

0.998

LN, lymph node; OR, odds ratio; CI, confidence interval. Bold indicates P<0.05.

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1.085
(0.370-3.184)

0.882

0.025
(0.005-0.137)<0.001

2.197(0.558-8.653)

0.260

0.533
(0.172-1.652)

0.276

2.182
(0.489-9.732)

0.306

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KuRATA et al: miR-200c IN GASTRIC CANCER

gastric cancer (21,31). however, this study is the first to havedemonstrated that this phenomenon was associated with themorphology of cell lines and histological differentiation, thelatter as visualized by ISh, in gastric carcinoma.
qRT-PCR analysis of 6 cell lines in the present study
revealed that the expression level of miR-200c descended
in the following order: Kato-III > MKN-45 > Nu-GC-4> h-111-TC > hGC-27 > MKN-1. The expression level of
ZEB1 or ZEB2 corresponded to the reverse order, and that ofE-cadherin corresponded to this order. Indeed, significantly
inverse correlations between miR-200c and ZEB1 wereobtained by Spearman's rank correlation. Our results are inline with a previous study in which E-cadherin gene expression was present in MKN-45 cells but absent in hGC-27
cells (32), along with a recent study which revealed that
Kato-III, MKN-45 and Nu-GC-4 cells had high expressionof E-cadherin and low expression of ZEB1, while MKN-1
cells had low expression of E-cadherin and high expression of
ZEB1 (33). however, these authors did not mention any differences in cell morphology in their studies. In the present study,
western blot analysis coincided with qRT-PCR data, albeit
E-cadherin expression in Kato-III and MKN-45 cells wasalmost absent contrary to positive expression as determined
by qRT-PCR analysis. This result was compatible with anotherstudy that revealed that E-cadherin protein expression wasundetectable by western blot analysis, while it was detected
at the dNA and mRNA level by southern and northern blot
analysis, respectively, in Kato-III and MKN-45 cells (4). They
mentioned that the possibility of post-translational modification or minor abnormalities in antibody-recognition sites may
be involved in this discrepancy.
We searched for key differences in the characteristics
between cell lines displaying divergent expression levels of
miR-200c which also corresponded to differences in theexpression levels of ZEB1/2 and E-cadherin. Although histological differences were conspicuous in the tissue studies
mentioned below, cell line groupings based on the expression of miR-200c and associated targets did not correspondto histological differentiation of the derived tumors. Other
authors have also demonstrated that E-cadherin was ‘unexpectedly’ detected in cell lines that originated from poorly
differentiated gastric carcinomas (26). This may be due tothe fact that cell lines have generally been established fromtumors exhibiting heterogenous histology. Indeed, it has beenreported that histological difference in original tumors did not
influence the growth rate of various cell lines in gastric carcinomas (25). Instead, we established that cell lines with highmiR-200c expression (Kato-III, MKN-45 and Nu-GC-4),
low miR-200c expression (hGC-27 and MKN-1), and intermediate miR-200c expression (h-111-TC) exhibited round,spindle, and intermediate, respectively, cell morphologies.
Although studies concerning cell morphology in association
with expression of miR-200 family members are scant, one
research group demonstrated that expression of miR-200c
was associated with round cell morphology whereas that of
miR-200a was associated with spindle cell morphology inmelanoma cell lines (34). The phenomenon where E-cadherin(+) cells are round and E-cadherin (-) cells are spindle in shapehas been reported in esophageal cancer cells and, recently,
in colon cancer cells (35,36). Furthermore, it was formerly

reported that cell morphology of E-cadherin-deficient celllines was generally altered from the fibroblastic cell type tothe epithelial cell type by transfection with complementarydNA that codes for E-cadherin (37). Therefore, the influenceof miR-200c and E-cadherin on cell morphology may be auniversal phenomenon.
In the tissue samples, miR-200c expression, as visualized
by ISh, was strongly expressed in tubular adenocarcinomas but
weakly expressed in poorly differentiated adenocarcinomas.
This result was in agreement with a recent study in which low
expression levels of each member of the miR-200 family wereassociated with histological grade in gastric carcinoma tissues,
as assessed by qRT-PCR analysis (31). Analysis of non-small
cell lung cancer tissue also revealed that low miR-200c
expression levels were associated with poorly differentiatedhistology (15). Assessment of endometrial carcinosarcoma
tissues revealed that members of the miR-200 family were
downregulated in the mesenchymal part of the tumor (38).
Therefore, it is likely that the expression of miR-200c is associated with epithelial histology of tumors in general.
IhC analysis revealed that tubular adenocarcinomas were
negative for ZEB1 and positive for E-cadherin, while poorly
differentiated adenocarcinomas were sporadically positive for
ZEB1 and negative for E-cadherin. Notably, it has long been
pointed out that the level of E-cadherin is significantly lower
in poorly differentiated adenocarcinomas compared to differentiated adnocarcinomas and non-neoplastic mucosa of thestomach (4,32). The reason for this phenomenon was proposed
to be inactivation of the E-cadherin gene due to either a classic
two-hit mechanism or hypermethylation (32). In the present
study, we add EMT mediated by miR-200c as a possible causal
mechanism, since miR-200c and ZEB1 expression levels, aswell as ZEB1 and E-cadherin expression levels, were inversely
correlated (P<0.001). Other authors have also demonstrated
an inverse correlation between ZEB1 and E-cadherin mRNA
expression in gastric cancer tissue (33). Association of poorly
differentiated histology with high expression of ZEB1 and low
expression of E-cadherin, as determined by IhC, has also been
demonstrated in endometrioid carcinoma and non-small celllung cancers (6,7).
In addition to histology, older age was also correlated withhigh expression levels of miR-200c and E-cadherin as assessedby Chi-squared test. however, in terms of multivariate analyses, age was not correlated with miR-200c and E-cadherin
expression, although histology remained correlated. These
results coincide with a well-known fact that poorly differentiated scirrhous gastric carcinoma is more frequent in younger
patients (2,3). Conversely, a high expression level of ZEB1 wascorrelated with advanced stage by multivariate analyses. As
other authors have also reported, gastric cancer patients withhigh ZEB1 mRNA expression displayed significantly poorer
survival than those with low expression (33), ZEB1 may be
independently associated with advanced stage.
Concerning miR-200c expression in tumors in general,a meta-analysis revealed that low expression of miR-200c inprimary cancer tissue from early-stage patients was significantly associated with poor survival (39). Association of low
miR-200c expression with high propensity to metastasis, alongwith highly invasive/aggressive ability, has been demonstrated
in several cancers including non-small cell lung cancer and-281-

ONCOLOGY REPORTS 39: 91-100, 2018

pancreatic cancer (15,16). Recent studies have also demonstrated that migration/invasion abilities of gastric cancer cells
were promoted by decreased expression of miR-200c, while
they were inhibited by ZEB1 siRNA (21,33). In addition, thepresent study demonstrated an association of miR-200c withcell morphology and histological differentiation of the tumor.
Since it has been proposed that the ability of miR-200c toregulate morphological plasticity may be important in relation to dissemination capacity in melanoma (34), we postulatethat downregulation of miR-200c primarily regulates cellmorphology by downregulation of E-cadherin through upregulation of ZEB1/2, with this morphological change causing
poorly differentiated histology (Fig. 5), producing enhanced
migration/invasion abilities, and finally leading to scirrhous
stromal reaction and poor prognosis (2,3).
In summary, using cell culture models and histologicalassessment of gastric carcinoma, we demonstrated an inverse
correlation in the expression level between miR-200c and
ZEB1 in vitro and in vivo, as well as between ZEB1 andE-cadherin in vivo. Low miR-200c expression was associatedwith spindle cell morphology in vitro and poorly differentiated histology in vivo; thus, this factor may be involved in the
carcinogenesis of scirrhous gastric carcinoma.
Acknowledgements
The present study was supported by Grants-in-Aid for scientific research (C) (grant no. 24590431).
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International Journal of Gynecological Pathology
38:377–385, Lippincott Williams & Wilkins, Baltimore
Copyright © 2018 by the International Society of Gynecological Pathologists

Original Article

Fascin as a Useful Marker for Identifying Neural Components
in Immature Teratomas of Human Ovary and Those Derived
From Murine Embryonic Stem Cells
Ryunosuke Umehara, M.S., Atsushi Kurata, M.D., Masakatsu Takanashi, Ph.D.,
Hirotsugu Hashimoto, M.D., Koji Fujita, C.T., Toshitaka Nagao, M.D.,
and Masahiko Kuroda, M.D.

Summary: Immature teratoma of the human ovary is a rare disease, and its diagnosis andgrading are currently based on histologic evaluation of the presence and amount ofimmature neural components in the tumor. Despite the importance of tumor grading,immature neural components especially without rosette formation are difﬁcult to identify,
partly because useful biomarkers for them are not yet available. Toward this goal, weinvestigated 16 immature teratomas from human ovaries as well as 10 of those derived frommurine embryonic stem cells transplanted into immunodeﬁcient mice. Immunohistochemistry was performed for cytokeratin, glial ﬁbrillary acidic protein, S100, and fascin. It wasdemonstrated that glial ﬁbrillary acidic protein and S100 expression was not observed in theimmature neural components of immature teratomas derived from both human ovary andembryonic stem cells, although their expression was detected in mature neural tissues. In
contrast, fascin immunopositivity was clearly found in both mature and immature neuralcomponents regardless of rosette formation in immature teratomas derived from bothhuman ovary and embryonic stem cells. Assessment of immature neural components by
fascin immunostaining yielded the same or slightly increased quantity than quantiﬁcation
based on hematoxylin and eosin staining. These results suggest that fascin immunostaining isuseful as a biomarker in correctly diagnosing and grading human immature teratomas.
Further, fascin immunostaining may contribute to the development of regenerative medicine
through accurate assessment of the maturation status of pluripotent stem cell-derived tumorstransplanted into immunodeﬁcient mice. Key Words: Immature teratoma—Immature neuralcomponents—Immunohistochemistry—Fascin—Regenerative medicine.

Teratoma is a subtype of germ cell tumors, traditionallydeﬁned as a tumor composed of elements from at least 2of the 3 germ cell layers, namely the ectoderm, mesoderm,and endoderm (1,2). However, teratoma is currently
regarded as a tumor that forms embryonic, fetal, and/or
adult-type somatic tissue, which includes monodermal
teratoma such as pure struma ovarii (3). Immature
teratoma is a malignant form of teratoma (4), which
consists of not only mature but also immature tissues, thelatter containing most commonly immature neuralcomponents (1,2). Immature teratoma represents 1% of
all teratomas, and 20% to 35% of malignant germ celltumors of the ovary (1,2,5).
Currently, immature teratomas are histologicallydiagnosed and graded (from grades 1 to 3) based on
the presence and amount of immature neural components

From the Tokyo Medical University (R.U.); Departments ofMolecular Pathology (A.K., M.T., K.F., M.K.); Anatomic
Pathology (T.N.), Tokyo Medical University; and Department of
Diagnostic Pathology, NTT Medical Center Tokyo (H.H.), Tokyo,
Japan.
The authors declare no conﬂict of interest.
Address correspondence and reprint requests to Atsushi Kurata,
MD, Department of Molecular Pathology, Tokyo Medical
University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.
E-mail: akurata@tokyo-med.ac.jp.

377

DOI: 10.1097/PGP.0000000000000528

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in the tumor (2,3). This grading is clinically very importantbecause it correlates with the prognosis and inﬂuences
choice of chemotherapy (2,6). Nevertheless, grading
reproducibility between pathologists is not sufﬁcientenough, partly because immature neural components ofdiffuse (sarcomatoid) pattern without rosette formation
are ambiguous and useful biomarkers for them are not yet
available (7).
Therefore, in the present study, we investigated for
useful immunohistochemical markers that could
identify immature neural components including thosewith diffuse pattern in immature teratoma tissues of
the human ovary. In addition to the well-known
neural markers, glial ﬁbrillary acidic protein (GFAP)and S100, we also examined fascin since its expression
in the nervous system from the embryonic through fetal
to postnatal stages has been reported (8). Moreover,the same immunohistochemical study was applied toimmature teratomas originating from murine embryonicstem (ES) cells transplanted into immunodeﬁcient mice.
Here, we demonstrate that fascin immunolabelingwas detected in immature neural components with orwithout rosette formation in both human ovarian andmurine ES cell-originated immature teratomas.

TABLE 1. Histologic grade, age at diagnosis, history ofgravidity and parity, maximum diameter of the tumor, andstage of the tumor investigated in 16 patients with ovarian
immature teratoma

Histologic
grade
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3

Age atdiagnosis(yr old)

History ofgravidity
and parity(G/P)

Maximum
diameter of
the tumor
(cm)

Stage
of the
tumor

35
22
31
26
37
20
20
25
24
23
18
32
33
33
22
30

nr/0
0/0
nr/0
0/0
0/0
0/0
1/0
nr/0
0/0
nr/0
nr/0
0/0
0/0
0/0
0/0
0/0

13
4.8
3.5
3.5
16
15
15
12.6
14.5
17
20
17.3
14
15
10
22

Ic
Ia
Ic
Ia
Ic
Ia
Ia
Ia
Ia
Ic
Ia
IIb
Ic
Ic
Ic
Ia

nr indicates not recorded.

formalin solution for 24 h and embedded in parafﬁn. We
reviewed each specimen, and 1 slide for each case wasselected that included dense immature neural components.

MATERIALS AND METHODS

Derivation, Culture, and Transplantation of Murine ES
Cells, and Sampling of Resulting Tumors
ES cells derived from C57BL/6J mice were establishedusing standard methods (11), with some modiﬁcations in
the duration of culture, as in our previous study (12).
Brieﬂy, blastocysts collected at 3.5 days postcoitus (dpc)
were cultured on 96-well tissue culture plates with amouse embryonic ﬁbroblast feeder layer in knockout
serum replacement (KSR) medium, which consisted ofhigh-glucose DMEM (Gibco, Frederick, MD) supplemented with 15% KSR (Gibco), 2 mM glutamine(Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM
nonessential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Nakalai Tesque, Kyoto, Japan), and 1000 U/mL
leukemia inhibitory factor (Merck Millipore, Darmstadt,
Germany). After the emergence of primary ES cells, thecells were passaged 3 more times in KSR medium on 10
cm culture plates with a mouse embryonic ﬁbroblast
feeder layer. When they reached 70% to 80% conﬂuence,
they were trypsinized, washed with phosphate bufferedsaline (PBS, pH 7.4), resuspended in 1 mL PBS, andinjected subcutaneously (4×106 ES cells in a total volumeof 100 μL) into the back and left femur of ﬁve 10-wk-old
RAG2−/−male mice (Sankyo Labo Service, Tokyo,
Japan). The mice were housed in cages (5 animals/cage)and provided access to water and food (FR-1; Funabashi

Patients and Tissues
The protocol of the present study was approved by theCommittees of Human Study of Tokyo Medical University and NTT Medical Center Tokyo. A total of 16
specimens of surgically resected immature teratoma of theovary from 16 patients were identiﬁed and retrieved fromthe databases of these 2 institutions between 2003 and2015. These specimens were partly identical to those in ourprevious study in which vascular smooth muscle cellmaturation and Ki-67 index were assessed in teratoma
tissues (9). As shown in Table 1, the age range of theselected patients was 18 to 37 yr old (mean, 26.9 ± 5.84 yrold). The maximum diameter of these tumors was from
3.5 to 22 cm (mean, 13.3 ± 5.27 cm). All patients were
nulliparous, and their teratomas were limited to stage I
with 1 exception. The diagnoses of these 16 specimens had
been performed by at least 2 experienced pathologists. The
grading was based on the criteria of Thurlbeck and Scully(10), with modiﬁcations by Norris et al (4), as follows:
grade 1, 1 or less low magniﬁcation (40×) ﬁeld showing
immature neural components per slide; grade 2, 1 to 3
ﬁelds per slide; and grade 3, 3 or more ﬁelds per slide. Our
study specimens consisted of 5, 6, and 5 cases of grades 1,
2, and 3 immature teratomas, respectively. The surgicallyresected specimens had previously been ﬁxed in 10%
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FASCIN AS A MARKER FOR NEURAL COMPONENTS
Farm, Chiba, Japan) ad libitum. The animal rooms weremaintained in a temperature-controlled (21–25°C) andlight-controlled (12L:12D cycle, lights on at 08:00 h)
environment. All the mice were bred and maintained
under speciﬁc pathogen-free conditions. All animal careand procedures that were performed in this study wereapproved by the Animal Research Committee of Tokyo
Medical University, and conformed to its guidelines foranimal experiments. Four weeks after the injection,
tumors of ∼1.5 cm in the longest diameter were detected.
Then, the mice were sacriﬁced, and the tumors were
removed, ﬁxed in 10% formalin overnight at room
temperature, and embedded in parafﬁn. Staining of
histologic sections was performed with hematoxylin andeosin (HE).
Immunohistochemistry
Immunohistochemistry was performed on 4 μm thick,parafﬁn-embedded sections of surgically resected humanspecimens and tumors sampled from the mice using thepolymer method according to standard techniques. The
monoclonal antibodies to cytokeratin (mouse, clone
AE1/AE3, working dilution 1:400; Dako, Glostrup,
Denmark) and fascin (mouse, clone 55k-2, working
dilution 1:400; Dako) as well as polyclonal antibodies to
S100 (rabbit, working dilution 1:2000; Dako) and
GFAP (rabbit, working dilution 1:2000; Dako) wereused. Deparafﬁnized and hydrated sections were treatedwith 0.3% hydrogen peroxide in methanol for 15 min toblock endogenous peroxidase activity. For immunostaining of cytokeratin and fascin, sections were autoclaved in 10 mmol/L sodium citrate buffer (pH 6.0) at
121°C for 10 min to expose antigens and then cooled for
30 min. After rinsing in 0.1 mol/L PBS, the sections wereincubated with afﬁnity-puriﬁed primary antibodies atroom temperature for 1.5 h. Thereafter, they wereincubated with Envision (+) Dual Link peroxidase(Dako) at room temperature for 30 min. The peroxidase
reaction was visualized using 0.02% 3,3’-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide in
0.1 mol/L PBS (pH 7.4). Finally, nuclear counterstaining
was performed with Mayer’s hematoxylin.
Assessment
Light microscopy performed with a Carl Zeiss HAL
100 instrument and a W-PI 10×/23 ocular lens was used
to analyze the tissues. All the slides were analyzed
microscopically by 2 independent investigators (R.U. andA.K.). The histologic analyses included evaluation ofdifferentiation (eg, keratinizing squamous epithelium,
immature neural components with rosette formation,379etc.) of each tissue area, correspondence of immunopositive cells to tissue differentiation, and presence orabsence of necrosis. In particular, the number of nests ofimmature neural components per slide was independently
assessed by HE staining and fascin immunostaining.
RESULTSHistology and Immunohistochemistry of Human
Ovarian Immature Teratomas
The human ovarian immature teratomas consistedof variable histologic features including mature andimmature components of the 3 germ cell layers, andwere sporadically accompanied by necrosis. In theectoderm, immature and mature neural components
and choroid plexus as well as mature keratinizing
squamous epithelium with skin appendages including
sebaceous glands, hair follicles, and sweat glands wereobserved. In the mesoderm, mature and immature
cartilage tissues, immature bone tissue, mature andfocally immature adipose tissues, and mature smoothmuscle tissue were identiﬁed. In the endoderm,ciliated and mucous columnar epithelium along withpartial salivary glands were present.
Immunohistochemically, mature keratinizing squamous epithelium, skin appendages, columnar epithelium, and salivary glands were positively stainedfor cytokeratin, whereas choroid plexus was focally
positive. Mature neural tissue and focally immaturechondrocytes were immunopositive for GFAP. S100
expression was found in mature neural tissue, choroid
plexus, and mature and immature chondrocytes.
Immature neural components were negative for both
GFAP and S100. In contrast, fascin immunostaining
was robustly observed in all areas of both mature andimmature neural components. In particular, allimmature neural components regardless of presenceof rosette formation were clearly positive for fascin.
Fascin expression was further demonstrated in thebasal layer of squamous epithelium as well as in thesebaceous glands and hair follicles, endothelium,
choroid plexus, and immature chondrocytes (Fig. 1).
The number of nests of immature neural components per slide was assessed ﬁrst by HE staining. As
shown in Figure 2A, the average number of thesenests in immature teratomas that were originallydiagnosed as grades 1, 2, and 3 were 1.2 ± 0.4,
2.23 ± 0.47, and 5.4 ± 1.62, respectively. Thus, thesenests increased as the grade advanced, in accordancewith the criteria. Quantiﬁcation of nests of immatureneural components based on fascin immunostaining
revealed that the number was the same or slightly
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FIG. 1. Human ovarian immature teratomas with squamous and columnar epithelium along with mature neural components (lower left) (A–E,
same fields), choroid plexus, and immature cartilage (F–J, same fields), and mature and immature neural components (K–O, same fields) as
shown by hematoxylin and eosin staining (A, F, K) as well as immunohistochemistry for cytokeratin (B, G, L), glial fibrillary acidic protein(GFAP) (C, H, M), S100 (D, I, N), and fascin (E, J, O) (200×). Cytokeratin immunostaining is positive in squamous and columnar epithelium
along with choroid plexus. GFAP immunopositivity is found in mature neural components. S100 expression is observed in choroid plexus,
immature chondrocytes, and mature neural components. Fascin immunopositivity is detected in the basal layer of squamous epithelium,
endothelium, choroid plexus, immature chondrocytes, and mature and immature neural components.
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FASCIN AS A MARKER FOR NEURAL COMPONENTS

381

C
A

8
7
6
5

D

4
3
2
1
0

Grade 1(n=5)

Grade 2(n=6)

Grade 3
(n=5)

E

B 14

12

fascin immunostaining

10

F
8

6

4

n=3

n=3

G

2

n=3
0

0

2

4

6

8

10

12

HE

FIG. 2. Quantification of nests of immature neural components in human ovarian immature teratomas. (A) Average number with SD of
immature neural component nests per slide based on hematoxylin and eosin (HE) staining according to the different grades. (B) Comparison of
the number of immature neural component nests per slide based on HE staining and fascin immunostaining. (C–G, same fields) Representative
images of subtle immature neural components of diffuse pattern (upper right and lower right) along with mature neural components (left) andcolumnar epithelium (center) as shown by HE staining (C) as well as immunohistochemistry for cytokeratin (D), glial fibrillary acidic protein(E), S100 (F), and fascin (G) (200×).

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increased as compared with that based on HE
staining. On average, the former yielded 1.56 ± 0.66
more immature neural components than the latter(Fig. 2B), because fascin expression could even be
detected in subtle foci of immature neural components
of diffuse pattern (Figs. 2C–G). However, as the grades
were based on the number of low magniﬁcation (40×)
ﬁelds containing immature neural components, the small
increase in the amount of immature components as
detected by fascin immunostaining did not affect theoriginal diagnoses except for 1 specimen, which was
upgraded from the original grades 2 to 3. In thisassessment, the fact that the columnar epithelium
was negative for fascin, but positive for cytokeratin,
was helpful for correctly identifying immature neuralcomponents that occasionally form gland-like structures.
However, choroid plexus is also immunopositive for
fascin, thus, it should be excluded as a fascin+ gland-likestructure.

accuracy of the clinical grading of this type of tumor.
We immunohistochemically assessed human ovarian
teratomas as well as those derived from murine ES cells
using a panel of antibodies, including cytokeratin,
GFAP, S100, and fascin. We demonstrate here for theﬁrst time that fascin immunopositivity was detected in
not only mature but also immature neural components
regardless of the presence of rosette formation, although
expression of the well-known neural markers, GFAP,and S100, was limited to mature ones. The importanceof histologic observation based on HE staining cannot
be overemphasized, but combined utilization of fascin
with GFAP and S100 as a means of subtracting themature neural components from all the neural components may be useful to identify immature foci.
The immunohistochemical results of cytokeratin,
GFAP, and S100 in the present study were generally
the same as previously reported. Cytokeratin immunopositivity was found in epithelial tissue and focallyin choroid plexus, as previously observed in humansacrococcygeal immature teratomas (13). S100 expression was observed in mature and immaturechondrocytes while that of GFAP was demonstrated
focally and only in immature chondrocytes, whichwere generally in accordance with previously documented results in immature teratomas of the ovary(14). The present study also demonstrated thatmature, but not immature, neural tissues were positive
for both GFAP and S100 immunostaining. Similar
results have also been described in immature teratomas of the sacrococcygeal region (13) and central
nervous system (15), in which immature neural tissues
were focally immunopositive for GFAP and S100 incontrast to diffuse positivity in mature neural tissues.
Other studies have also reported that GFAP expression in nerve tissue is low in immature teratomas, buthigh in mature teratomas originating from various
regions including ovaries (16). Taken together, GFAPand S100 immunostainings are not considered usefulin identifying immature neural components.
It is noteworthy that fascin immunopositivity was
detected in all neural components in the present study.
Fascin is a 55 kDa actin-binding protein that regulates
ﬁlaments necessary for formation of ﬁlopodia in cell
migration. In normal adult human tissues, fascin
expression is characteristic of neurons and dendritic
cells that have striking large ﬁlopodia and are highly
motile (17,18), in addition to endothelium and skinbasal layer (8). Fascin expression in various epithelial
neoplasms has been reported to be associated withinvasion of tumor cells and clinically aggressive
manifestations, as we previously described in thymic

Histology and Immunohistochemistry of Immature
Teratomas Derived from ES Cells
The histologic features of the tumors that formed after
transplantation of murine ES cells into immunodeﬁcient
mice were similar to those of human ovarian immature
teratomas, but immature neural components and necrosis
were more abundant; thus, all 10 tumors exhibited
histology corresponding to grade 3 immature teratomas.
The components of these tumors were: immature andmature neural components along with mature keratinizing squamous epithelium without skin appendages in theectoderm, immature cartilage, and striated muscle in themesoderm, and ciliated and mucous columnar epithelium
along with partial salivary glands in the endoderm.
The immunohistochemical results of the murine ES
cell-derived tumors were also similar to those of humanovarian immature teratomas. Cytokeratin staining was
detected in mature keratinizing squamous epithelium aswell as in columnar epithelium; GFAP expression wasobserved only in mature neural tissue; S100 immunopositivity was found in immature chondrocytes andfocally mature neural tissue; and fascin expression was
detected in all mature and immature neural components,
basal layer of the squamous epithelium, endothelium,
and occasionally immature chondrocytes (Fig. 3).
DISCUSSION
In the present study, we investigated useful markers
for immature neural components in immature teratoma in order to improve the reproducibility and
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FASCIN AS A MARKER FOR NEURAL COMPONENTS

383

FIG. 3. Murine embryonic stem cell-derived immature teratoma (A–E, same fields) that contains columnar epithelium, immature neuralcomponents (center to lower half), mature neural components encircled by striated muscle (upper left), and immature cartilage (center) as
visualized by hematoxylin and eosin staining (A) as well as immunohistochemistry for cytokeratin (B), glial fibrillary acidic protein (GFAP)(C), S100 (D), and fascin (E) (200×). Cytokeratin immunostaining is positive in columnar epithelium. GFAP expression is found in matureneural components. S100 immunopositivity is observed in immature chondrocytes. Fascin expression is detected in mature and immature neuralcomponents along with focally immature chondrocytes.

neoplasms (19). In human embryo and fetus, fascin
was widely expressed in the nervous system including
neural tubes along with other organs such as

chondroblasts (8). In accordance with these previous
descriptions, fascin immunopositivity was detected
in all mature and immature neural components
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regardless of the presence of rosette formation, in
addition to the basal layer of squamous epitheliumand skin appendages, endothelium, choroid plexus,
and immature chondrocytes in the present study. In
particular, fascin immunostaining was helpful in
identifying immature neural components without
rosette formation namely of diffuse (sarcomatoid)
pattern, which have confounded pathologists owing totheir obscure features (6,7). Indeed, quantiﬁcation ofnests of immature neural components based on fascin
immunostaining revealed that the number of nests was
the same or slightly increased as compared withquantiﬁcation based on HE staining. This was most
likely due to detection of fascin immunopositivity evenin subtle immature neural components of diffuse
pattern, which could also be identiﬁed by their highcellularity (7).
Several immunohistochemical markers have been
proposed to be speciﬁc for immature neural components in immature teratomas of organs mainly otherthan ovaries. OCT4 expression was described inprimitive neuroectodermal components in immature
teratoma of the central nervous system (20), but
neuroepithelial rosettes in ovarian immature teratomas have been reported to be negative for OCT4 (21).
Glypican 3 expression may also be useful, but theneuroepithelium was positive for glypican 3 in only 2of 16 cases of testicular immature teratomas (22).
SOX2 may be a relatively reliable marker as well; its
expression was found in the neuroepithelium of thecentral nervous system, although not all of the cells in
the neuroepithelial tubules expressed SOX2 in immature teratoma (23). Other investigators have reported
that the neuroepithelium was positive for SOX2
staining in 11 of 14 cases of testicular immature
teratomas (24). Furthermore, columnar epithelium
and embryonal carcinoma were also shown to bepositive for SOX2 (23,24). Thus, immunohistochemistry for a combination of fascin with these markers
especially SOX2 as well as assessment of vascularsmooth muscle cell maturation stage and Ki-67 index
as described in our previous study may further
facilitate accurate diagnosis and grading of immature
teratomas (9).
The signiﬁcance of the present study is not limited
to routine diagnostic tools in the pathology departments. The pluripotent status of ES cells and induced
pluripotent stem cells is partly veriﬁed by formation of
immature teratomas after subcutaneous transplantation into immunodeﬁcient mice (25,26). Histologic
features of murine ES cell-derived immature teratomas have been reported to consist of immature nerve

tissues, keratinizing epithelium, ciliated and mucous
epithelium, muscular tissues, and cartilage (26), as in
the present study. The histologic characteristics ofhuman pluripotent stem cell-derived tumors xenografted into immunodeﬁcient mice have been shownto contain similar components (27). The assessment ofderivatives of the 3 germ layers in the tumors formed
after injection of pluripotent stem cells into immunocompromised mice is called the “teratoma assay”;
however, the problem with this assay is poor
methodology of quantiﬁcation of differentiation
partly due to lack of experienced pathologists (28).
Quantiﬁcation of immature neural components by
fascin immunostaining may contribute to moreaccurate performance of the “teratoma assay.”
Further, in order to develop safe pluripotent stem
cell-based treatments, this tumorigenicity hurdle must
be overcome. One of the strategies to cope with thisrisk is terminal differentiation of pluripotent stem cells(25). Therefore, assessment of the tumor by fascin
immunostaining may serve to monitor and verify
terminal differentiation of pluripotent stem cellderived tumor, thus, leading to development of safer
regenerative medicine in the future.
In conclusion, we have conducted an immunohistochemical study of neural markers on immature
teratoma tissues derived from human ovary andmurine ES cells. Fascin expression was universallyobserved in mature and immature neural components
in both human and murine tissues. These results maylead to the improvement of diagnostic tools anddevelopment of safer treatment modalities.
Acknowledgments: Rag2−/− mouse was kindly gifted by
Dr Katsuko Sudo, Tokyo Medical University. Further, theauthors thank medical students in Tokyo Medical University, Ayumu Kocha, Koji Tanaka, Chihiro Tani, Amika
Nakazawa, Kengo Maehara, and Shun Mochida for theirtechnical assistance.

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Laboratory Investigation (2019) 99:1275–1286
https://doi.org/10.1038/s41374-019-0247-4

ARTICLE

Therapeutic potential of PLK1 inhibition in triple-negative breast
cancerAi Ueda1 Keiki Oikawa2 Koji Fujita2 Akio Ishikawa2 Eiichi Sato3 Takashi Ishikawa1 Masahiko Kuroda2
Kohsuke Kanekura2
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Received: 10 September 2018 / Revised: 15 February 2019 / Accepted: 19 February 2019 / Published online: 17 April 2019
© United States & Canadian Academy of Pathology 2019

Abstract
Triple negative breast cancer (TNBC) is responsible for signiﬁcant number of breast cancer-associated deaths because oflacking of successful molecular-targeted therapy. To explore a therapeutic target for TNBC, we performed a siRNAmediated knockdown screening and identiﬁed Polo-like kinase 1 (PLK1) as a potential therapeutic target for TNBC.
Knockdown of PLK1 as well as a small compound inhibitor for PLK1, BI-2536, induced G2/M arrest and created polyploidcell population, shown by increased DNA content and nuclear size. Inhibition of PLK1 eventually triggered apoptosis in
multiple TNBC cell lines. In addition, we conﬁrmed that PLK1 was signiﬁcantly overexpressed in the tissues from TNBC
patients compared with the tissues of normal mammary glands and benign breast tumors. Our data indicated that PLK1 playsa pivotal role in the regulation of mitosis of TNBC cells. Although future in vivo studies are warranted, targeting PLK1 by aselective inhibitor such as BI-2536 can be an attractive molecular-targeted therapy for TNBC.

Introduction
Breast cancer is the leading cause of cancer-associateddeaths in women worldwide, affecting more than 1 million
new cases and half a million deaths every year. Clinicopathological characterization categorizes the disease into
ﬁve classical subtypes, luminal A, luminal B (HER2
negative), luminal B (HER2 positive), HER2 positive, andtriple negative breast cancer (TNBC). TNBC, deﬁned by alack of immunohistochemical expression of estrogen
receptor (ER), progesterone receptor (PgR) and HER2

* Masahiko Kuroda
kuroda@tokyo-med.ac.jp* Kohsuke Kanekura
kanekura@tokyo-med.ac.jp
1

Department of Breast Oncology and Surgery, Tokyo Medical
University Hospital, 6-7-1 Nishishinjuku, Shinjuku-ku,
Tokyo 160-0023, Japan2

Department of Molecular Pathology, Tokyo Medical University,
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan3

Department of Anatomic Pathology, Tokyo Medical University
Hospital, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023,
Japan

receptor, accounts for approximately 15% of all breast
cancers [1, 2], and is commonly observed in premenopausal
African-American women [3]. In Asian population, TNBC
accounts for approximately 10% of all breast cancer [4].
TNBC is a highly aggressive type of breast cancer and oftencauses metastasis in early stage. However, because TNBC
does not express hormone receptors or HER2, the patientswith TNBC do not beneﬁt from hormonal therapy or antiHER2 therapy. It is also well-known that TNBC is sensitive
to the conventional chemotherapy at ﬁrst, but it has higher
recurrence rate and thus results in poor prognosis [5, 6].
Although the recent report which re-analyzed the dataset ofgene expression proﬁles of TNBC enabled subcategorization of the TNBC into 6 subtypes [7], the moleculartargeted therapy for TNBC, highly effective in other typesof breast cancer, still remains to be investigated.
Genome-wide association studies have shown that
defects in DNA repairing machineries contribute to thepathogenesis of breast cancer [8]. When severe genomic
DNA damages including double-strand breaks (DSBs) andsingle-strand breaks (SSBs) occur, intrinsic DNA damage
repairing pathways cooperatively ﬁx the damage to suppress undesirable mutations. In higher eukaryotes, DSBs are
ﬁxed by two major DNA repairing mechanisms, thehomologous recombination (HR) and the non-homologous
end joining (NHEJ) pathways. BRCA1 is a key modulator

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A. Ueda et al.

protein required for efﬁcient repair of DSB through HR.
Following DSB, serine/threonine kinases Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia andRad3-Related (ATR) are activated and they phosphorylate
BRCA1 [9]. Polo-like knase-1 (PLK1) also phosphorylates
BRCA1 and mediates recruitment of BRCA1 to DSBs,
resulting in promotion of HR [10]. BRCA2 protein is also
involved in the HR. The implications of these DNA
repairing machineries in breast cancer has been shown byrobust association of mutations in BRCA1 and BRCA2
genes with hereditary breast cancer patients. In the patientswith mutations of BRCA1/2 genes, the non-functional DNA
repairing system due to lack of BRCA1/2 fails to ﬁx cancerous mutations and thus they cause breast cancer.
On the other hand, the major chemotherapy used fortreatment of TNBC, including anthracyclines and platinumcompounds, also utilizes DNA damages to inhibit tumor
growth. These chemotherapeutic compounds induce severe
damage in genomic DNA. If the degree of DNA damage isoverwhelming to the repairing machinery, the accumulationof DNA damage is monitored by cell cycle checkpoints,
such as G1/S checkpoint and the G2/M checkpoint, resulting in inhibition of DNA replication and impairment ofsegregation of chromosome at mitosis, respectively. If further irreversible DNA damages accumulate, they trigger
apoptosis. Therefore, the molecule indispensable in the
DNA repairing pathways may be a strong candidate of a
new drug target for TNBC.
In this study, we screened 24 genes involved in the DNA
repair using the siRNA-mediated cell viability assay andfound that knockdown of PLK1 triggered apoptosis in
multiple TNBC cell lines. We also conﬁrmed that both
knockdown of PLK1 as well as a small compound inhibitor
for PLK1, BI-2536, induced G2/M arrest and createdpolyploid cell population, and eventually induced apoptosis.
In addition, overexpression of PLK1 in TNBC tissues was
conﬁrmed by immunohistochemical analysis. Our data
shown here indicate that PLK1 regulates mitosis of TNBC
cells the function of PLK1 is indispensable for survival of
TNBC in vitro. Although further in vivo studies andsophisticated clinical trials are necessary, inhibition of
PLK1 should be considered as a probable option ofmolecular-targeted therapy for TNBC.

materials and methods
Cell lines and reagents
TNBC cell lines, MDA-MB-231, Hs578T, MDA-MB-436,
MDA-MB-468 and HCC1937 cell lines were obtained fromthe American Type Culture Collection (ATCC) (Manassas,
VA, USA) in December 2015. MDA-MB-453 cell line wasobtained from RIKEN BRC Cell Bank (Tsukuba, Ibaraki,
Japan) in July 2015. HCC38 and HCC1806 cell lines weregenerous gifts from Department of Gastroenterologicalsurgery at Yokohama City University Hospital in July 2015.
HCC38, HCC1806 and HCC1937 cells were cultured in
RPMI-1640 medium (Gibco, Waltham, MA) supplemented
with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin at 37 °C in 5% CO2. Hs578T was cultured in DMEM supplemented with 0.01 mg/ml bovine
insulin (Sigma, St. Louis, MO), 10% FBS and antibiotics at
37 °C in 5% CO2. MDA-MB-231, MDA-MB-436, MDAMB-453 and MDA-MB-468 were cultured in Leibovitz’s L15 medium (Gibco) supplemented with 10% FBS, 1%
GlutaMAX (Gibco) and antibiotics at 37 °C in 100% room
air. BI-2536 was purchased from Selleck chemicals(Houston, TX).

siRNA library
The RNAi library was purchased from Dharmacon(Lafayette, CO, USA). The targeted genes were shown in
Table 1. The siRNA for each gene contains four different
siRNA to obtain better knockdown efﬁciency.
Table 1 List of genes targeted in our siRNA library
No.

Name

NCBI Accession number

1

MDC1

NM_014641.2

2ATM

NM_000051.3

3

ATR

NM_001184.3

4

RNF168

NM_152617.3

5

RNF8

NM_003958.3

6

RAP80

NM_001199297.2

7

HSP90

NM_001017963.2

8

BRCA1

NM_007294.3

9

53BP1

NM_001141979.2

10

RIF1

NM_001177663.1

11

Rev7

NM_001127325.1

12

Ku70

NM_001288976.1

13

Ku80

NM_021141.3

14

BARD1

NM_000465.3

15

BRCA2

NM_000059.3

16

RAD51

NM_001164269.1

17

PALB2

NM_024675.3

18

BACH1

NM_001186.3

19

RAD18

NM_020165.3

20

PLK1

NM_005030.5

21

CTIP

NM_002894.2

22

MRE11

NM_001330347.1

23

FAM175

NM_001345962.1

24

RPA1

NM_001355120.1-294-

Therapeutic potential of PLK1 inhibition in triple-negative breast cancer

siRNA screening
MDA-MB-436, MDA-MB-468 and HCC1806 were seeded(8000 cells/well) in a 96-well plate. The cells were transfected with 10 nM of each siRNA using Lipofectamine
RNAi MAX (Invitrogen, Carlsbad, CA). Forty-eight hours
after transfection, the cell viability was assessed by Celltiter glo 2.0 (Promega, Fitchburg, WI) following the manufacturer’s instruction.

1277

Tesque, Kyoto, Japan), the membrane was incubatedovernight with anti-GAPDH antibody (Cell Signaling
Technology, Danvers, MA) or anti-PLK1 mouse monoclonal antibody (Abcam) at 4 °C. After incubation withhorseradish peroxidase-labeled secondary antibodies, protein expressions were detected with ChemiDoc imaging
system (Biorad, Hercules, CA) using Amersham ECL
Select Western Blotting Detection Reagents (GE Healthcare, Chicago, IL). The images were acquired with ImageLab Software (Biorad).

Immunohistochemistry (IHC)
Immunocytochemistry
All samples were obtained from patients treated at Tokyo
Medical University Hospital (Tokyo, Japan) under anapproved Institutional Review Board protocol (TS20180071). Samples were ﬁxed in 10% neutral buffered formalinfollowed by embedding in parafﬁn. Sections were deparafﬁnized and rehydrated to distilled water before staining.
Endogenous peroxidase activity was quenched by incubation with 0.5% H2O2/methanol for 10 min, and heat-induced
epitope retrieval was performed. Immunohistochemistry
was done with anti-PLK1 antibody (Abcam, Cambridge,MA) and visualized using the EnVision DAB kit (DAKO,Glostrup, Denmark) according to the manufacturer’s
instructions.

Scoring of IHC staining
All samples (TNBCs, normal mammary glands, benign
tumors, luminal A breast cancer, luminal B breast cancerand HER-2 positive breast cancer, N = 5 for each group)
were observed with the BZ-9000 Fluorescence Microscope(KEYENCE, Osaka, Japan). To evaluate the intensity andproportion of cells positive for PLK1, we applied a semiquantitative scale of the Quickscore method [11]. First, thestaining intensity (0 = negative, 1 = weak, 2 = intermediate, 3 = strong) and the proportion of the cells
immune-positive (1 = 0–4%, 2 = 5–19%, 3 = 20–39%, 4 =40–59%, 5 = 60–79%, 6 = 80–100%) were scored, theneach score was multiplied to obtain the Quickscore. Either
nucleus or cytoplasmic signal above background wascounted as positive.

Western blot analysis
MDA-MB-231, MDA-MB-436, MDA-MB-468, HCC38
and HCC1937 were grown in 100 mm cell culture dishes
and lysed in M-PER mammalian protein extraction reagent(Thermo Fisher Scientiﬁc) containing phosphatase inhibitors and protease inhibitors (Roche Diagnostics, Basel,
Switzerland). Each sample containing 30 μg of proteins wasapplied to SDS-PAGE and transferred to PVDF membranes. After blocking in Blocking One solution (Nacalai

For staining of endogenous PLK1, MDA-MB-436 cells
were ﬁxed with 4% paraformaldehyde-PBS for 15 min andpermeabilized with 0.5% Triton X-100 for 5 min at room
temperature. After blocking with Blocking One solution(Nacalai Tesque) for 1 h, the cells were incubated forovernight with anti-PLK1 mouse monoclonal antibody(Abcam) at 4 °C. The cells were washed in PBS twice andincubated with Alexa-Fluor 488-conjugated goat antimouse IgG (Abcam) for 1 h. Nuclei were counterstainedwith 4′,6′-diamidino-2-phenylinodole (DAPI). For stainingof phosphorylated histone H2AX (γ-H2AX), MDA-MB231 cells, treated with 10 μM of bleomycin or 10 nM of BI2536 for 6 h, were immunostained with anti-γ-H2AX antibody (Cell signaling). For imaging of spindle assembly,
MDA-MB-231 cells and MDA-MB-436 cells expressing
GFP-tubulin were treated with DMSO or 10 nM of BI-2536
for 6 h or 24 h.

Cell viability assay
To examine the effect of siRNA-mediated knockdown on
the cell viability, TNBC cell lines including HCC1806,
HCC1937, Hs578T, MDA-MB-231, MDA-MB-436,
MDA-MB-453, and MDA-MB-468, seeded (5000 or
8,000 cells/well) in 96-well plates, were transfected with
10 nM of negative control siRNA or siRNA against PLK1
using Lipofectamine RNAiMAX (Invitrogen). To examine
the effect of BI-2536, a selective inhibitor for PLK1,
HCC1806, HCC1937, Hs578T, MDA-MB-231, MDA-MB436, MDA-MB-453, and MDA-MB-468 were incubatedwith DMSO or BI-2536 (from 0.25 to 100 nM) for 48 h.
Cell viability was assessed by Calcein-AM solution(Dojindo, Tokyo, Japan) or Cell-titer glo (Promega). The
viability was measured with the microplate reader inﬁnite
F200 PRO (TECAN, Männedorf, Switzerland).

Cell cycle analysis
MDA-MB-436 cells and MDA-MB-231 cells were seeded(1 × 105 cells/well) in 6-well plates and transfected with

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Fig. 1 a A simple scheme of functions of genes screened in this study.
b The cell viability of MDA-MB-436 cells transfected with the siRNAlibrary. c The cell viability of HCC1806 cells transfected with thesiRNA library. d The cell viability of MDA-MB-468 cells transfectedwith the siRNA library

10 nM of negative control siRNA or siRNA against PLK1.
Forty-eight h after transfection, the cells were collected,
washed with PBS containing 2% FBS and then resuspended in 70% ice-cold ethanol to ﬁx and permeabilize
the cells. The ﬁxed cells were washed in PBS and resuspended in PBS containing 100 μg/ml RNase A and20 μg/ml propidium iodide. After incubation for 30 min atroom temperature, samples were analyzed by ﬂuorescenceactivated cell sorter (FACS). All experiments were repeatedthree times independently. FlowJo software (TreeStar Inc.,
Ashland, OR) was used for data analysis.

Transmission electron microscopy

Apoptosis analysis using Annexin V-7AAD staining
MDA-MB-436 cells and MDA-MB-231 cells were seeded(1 × 105 cells/well) in 6-well plates and treated with 10 nM BI2536 for 24 h. The cells were collected, washed twice with 2%
FBS-PBS and re-suspended in a binding buffer. Then cells
were incubated with PE-Annexin V (BD Pharmingen, Franklin
Lakes, NJ) and AAD (BD Pharmingen) for 15 min at room
temperature in the dark. Samples were analyzed by FACS. All
experiments were repeated ﬁve times independently.

MDA-MB-231 cells treated with or without 10 nM of
BI-2536 were ﬁxed in 4% paraformaldehyde/2.5%
glutaraldehyde-PBS for 1 h. After wash with PBS, thesamples were treated with 1.5% osmium tetroxide-PBS andembedded in the epoxy resin. The embedded samples werethen ultrathin sectioned and imaged by LEM-1200EX
transmission electron microscopy (JEOL, Tokyo, Japan).

Results
siRNA library screening identiﬁed PLK1 as atherapeutic target of TNBC cell lines
To identify the gene involved in the DNA damage repair
and indispensable for survival of TNBC cell lines, wesearched literatures and chose 24 genes related to the DNA
damage repair and create a library of siRNAs (Fig. 1a andTable 1). The siRNA screening was performed in 3 different
TNBC cell lines, MDA-MB-436 (a mutation in BRCA1),-296-

Therapeutic potential of PLK1 inhibition in triple-negative breast cancer

1279

Fig. 2 a Immunoblot analysis of
PLK1 and GAPDH in TNBC
cell lines. b Immunoblot
analysis of PLK1 and GAPDHin TNBC lines transfected with
control siRNA (si-control) or
siRNA targeting PLK1
(siPLK1). c
Immunocytochemical analysis
of PLK1 in MDA-MB-436 cells.
The most right panel shows apseudocolor image of PLK1. A
white arrow indicatesaccumulation of PLK1 in thespindle. d The cell viability of
TNBC lines transfected with sicontrol or siRNA targeting
PLK1. Data were mean ± S.D.
from 3 independent
experiments. *p < 0.05, **p <0.01, ***p < 0.001

HCC1806 (mutations in both BRCA1/2) and MDA-MB468 (wild type BRCA1). The cell viability assay showedthat knockdown of PLK1 decreased number of viable cells
in all three TNBC lines examined (Fig. 1b–d).

Inhibition of PLK1 decreases the viability of various
TNBC cell lines
It has been shown that TNBC can be subclassiﬁed into
6 subtypes by gene expression proﬁles (BL1; basal-like 1,
BL2; basal-like 2, IM; immunomodulatory, M; mesenchymal-like, MSL; mesenchymal stem-like, LAR; luminal
androgen receptor) [7]. Therefore, we next examined if thecytotoxic phenotype by inhibition of PLK1 was speciﬁc to
the cell lines tested in the screening above, or it is commonly shared by various type of TNBC lines. To conﬁrm
this, we ﬁrst performed the immunoblot analysis of PLK1 invarious TNBC cells lines including MDA-MB-231, MDAMB-436, MDA-MB-468, HCC38 and HCC1937 and conﬁrmed that all TNBC line endogenously expressed highlevel of PLK1 (Fig. 2a). We also conﬁrmed that transfectionof siRNA targeting PLK1 effectively decreased theexpression of endogenous PLK1 in TNBC cell lines(Fig. 2b). The immunocytochemical analysis revealed thatthe expression of PLK1 was highly enhanced in the mitotic
cells and it localized to kinetochores and spindles asreported previously (Fig. 2c) [12]. Next, we examined
whether inhibition of PLK1 induces cell death in different
types of TNBC. We tried seven TNBC lines with the different gene expression proﬁles and conﬁrmed that all TNBC
lines were vulnerable to depletion of PLK1 and the cell
viability was decreased by transfection with siRNA targeting PLK1 (Fig. 2d), suggesting that PLK1 is essential forsurvival of TNBC cells.

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BI-2536, a selective inhibitor for PLK1, triggered
apoptosis in TNBC lines
PLK1 is reported to be involved in mitosis and also highly
expressed in cancer tissues [13–20], therefore its selective
inhibitor could be a potential anti-cancer drug. The highthroughput screen identiﬁed BI-2536 as a small molecule
inhibitor for PLK1, and it has been shown that BI-2536
prevents tumor growth in vitro and in vivo [21]. To test theeffect of pharmacological inhibition of PLK1 on TNBC
cells, we treated MDA-MB-231 cells (wild type BRCA1)
and MDA-MB-436 cells (a mutation in BRCA1) with different dose of BI-2536 and performed the cell viability
assays (Fig. 3a–d). The cell viability was decreased in adose-dependent fashion in both cell lines regardless of
BRCA genotype. Flowcytometric analyses with Annexin-V
and 7-aminoactinomycin D (7-AAD) staining indicated thattreatment with BI-2536 triggered apoptotic cell death in
MDA-MB-231 cells and MDA-MB-436 cells shown by
increase of number of cells with both early apoptotic phase(Annexin-V positive, 7-AAD negative) and late apoptotic
phase (Annexin-V positive, 7-AAD positive) (Fig. 3e–h).
Electron microscopic images showed that treatment with
BI-2536 caused fragmentation of the nucleus in MDA-MB231 cells, also indicating that inhibition of PLK1 triggers
apoptosis in TNBC (Fig. 3i). We further examined theeffect of BI-2536 on a variety of TNBC lines of different
subtypes and conﬁrmed that BI-2536 induced cell death in
all TNBC lines examined (Fig. 3j).

Inhibition of PLK1 increases polyploidy and size ofthe nucleus in TNBC cells
Because it has been reported that PLK1 drives transition of
G2/M and its role to establish the bipolar spindle is indispensable [22], inhibition of PLK1 results in a fatal error ofmitosis and increases number of polyploid cells [23, 24]. To
investigate whether inhibition of PLK1 increases polyploidcells in TNBCs, we demonstrated the propidium iodide (PI)staining to quantify the DNA content in TNBC cells. For thisend, we tested the effect of depletion of PLK1 in both MDAMB-231 cells (wild type BRCA1) and MDA-MB-436 cells(a mutation in BRCA1), and siRNA-mediated knockdown of
PLK1 increased the rate of cells harboring 4n DNA comparedwith the cells transfected with si-control regardless of BRCA
genotype (Fig. 4a–d). Treatment with BI-2536 also drasticallyincreased the rate of tetraploid cells in TNBC lines(Fig. 4e–h). It has been shown that accumulation of DNA
damage induces G2/M arrest and thus many anti-cancer
genotoxic compounds induce G2/M arrest as a result of DNA
damage [25]. To examine whether the inhibition of cell cycle
by BI-2536 is dependent on DNA damage, we compared BI2536 with a traditional genotoxic drug, bleomycin. When wetreated the TNBC cells with BI-2536 or bleomycin, both BI2536 and bleomycin increased the cell population with 4n
DNA content, but the effect was much more drastic with BI2536 (Fig. 4f, h). We also evaluated the damage of DNA bystaining with anti-phosphorylated histone 2AX (γ-H2AX)
antibody, a marker for DSB. After 6 h incubation, bleomycincaused severe DNA damages, shown by increased staining
with γ-H2AX antibody whereas BI-2536 did not, suggestingthat bleomycin increased 4n population via inducing DSB
followed by arrest at G2/M checkpoint but BI-2536 increased
4n polyploid cells independent of DNA damage (Fig. 4i).
Because PLK1 is known to contribute to bipolar spindle
formation, we next examined whether inhibition of PLK1
disrupts spindle assembly, resulting in incomplete segregationof chromosome. To visualize bipolar spindle, we overexpressed GFP-fused tubulin in MDA-MB-231 cells and
MDA-MB-436 cells. When treated with BI-2536, abnormalmonopolar spindle was formed and separation of chromosome was inhibited (Fig. 4J). The increased DNA content
might result in the larger nuclear size and thus we next
measured the size of nucleus in BI-2536 treated MDAMB231 cells. As Fig. 4k, l show, the treated cells have larger
nuclear size compared with control cells, also suggesting theincrement of DNA content.

PLK1 is overexpressed in TNBC
To investigate the clinical relevance of the PLK1 to TNBC,
we assessed the expression of PLK1 in samples of normal
mammary glands (n = 5), benign mammary tumors (n = 5),
luminal A breast cancer (n = 5), luminal B breast cancer(n = 5), HER-2 positive breast cancer (n = 5) and TNBC
patients (n = 5). The immunohistochemical analyses
showed that PLK1 was hardly observed in the benign
tumors or in normal mammary glands, on the other hand,
PLK1 was highly expressed in cancer tissues, especially in
TNBC (Fig. 5a, d). The high magniﬁcation image of TNBC
tissue showed that especially cancer cells at mitotic stages
with condensed chromosomes had intense signals of PLK1
(Fig. 5d). We also immunohistochemically analyzed thebreast cancer tissues with anti-Ki67 antibody because PLK1
was highly expressed in cells at mitotic phases thus wehypothesized that the expression of PLK1 might correlatewith the Ki67, a cell proliferation marker. The immunohistochemical analysis with anti-Ki67 antibody showed thatthe breast cancer tissues, especially TNBC was immunopositive with anti-Ki67 antibody (Fig. 5e).
To evaluate PLK1 expression level in detail, we semiquantiﬁed PLK1 staining by the Quickscore [11], obtainedby multiplying the rate of positive cells and intensity ofsignals (Please see the material and method section.). The
mean of the Quickscore of PLK1 expression was 5.4 for
TNBC, 4.8 for Luminal B, 3.2 for HER-2, 2.4 for Luminal-298-

Therapeutic potential of PLK1 inhibition in triple-negative breast cancer

1281

Fig. 3 a Dose-response curve of
BI-2536 on MDA-MB-231
cells. Y-axis indicates relativecell viability and X-axis showsconcentration of BI-2536 (nM).
Statistical signiﬁcance wascalculated by one-way ANOVA
followed by Dunnett’s test. *p <0.05, **p < 0.01, ***p < 0.001.
b Microscopic images of MDAMB-231 cells treated withDMSO or 5 nM of BI-2536.
c Dose-response curve of BI2536 on MDA-MB-436 cells. Yaxis indicates relative cell
viability and X-axis showsconcentration of BI-2536 (nM).
Statistical signiﬁcance wascalculated by one-way ANOVA
followed by Dunnett’s test. *p <0.05, **p < 0.01, ***p < 0.001.
d Microscopic images of MDAMB-436 cells treated withDMSO or 5 nM of BI-2536.
e, f Flowcytometric analysis of
MDA-MB-231 cells treated withDMSO or 10 nM BI-2536,
stained with PE-Annexin V and
7-AAD. g, h Flowcytometricanalysis of MDA-MB-436 cells
treated with DMSO or 10 nM
BI-2536, stained with PEAnnexin V and 7-AAD.
i Transmission electron
microscopic images of MDAMB-231 cells treated with orwithout 10 nM BI-2536. The
arrows show fragmented
nucleus. Scale bars show 2 μM.
j Effects of BI-2536 on thesurvival of TNBC lines. Data
were mean ± S.D. from 3
independent experiments.
Statistical signiﬁcance wascalculated by Student t test. *p <0.05, **p < 0.01, ***p < 0.001

A, 0.6 for benign breast tumors and 0 for normal mammary
glands (Fig. 5f) and the Quickscore of TNBC was signiﬁcantly higher than those of normal mammary glands andbenign tumors. There was no signiﬁcant difference in the
Quickscore between the normal mammary glands andbenign tumors. We also quantiﬁed the Ki67-psotive cells in

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A. Ueda et al.

the breast cancer tissues, and TNBC tended to have higher
positivity of Ki67 (Fig. 5g). We next investigated whether
the Quickscore of PLK1 and the Ki67-positivity correlates.

As expected, there was a good correlation between them(r = 0.633), and it might be a part of reasons why PLK1
was highly expressed in TNBC tissues (Fig. 5c, f).

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1283

Fig. 4 a, b Flowcytometric analysis to quantify the DNA content in
MDA-MB-231 cells transfected with control siRNA (si-control) or
siRNA targeting PLK1 (siPLK1). Statistical signiﬁcance was calculated by Student t test. c, d Flowcytometric analysis to quantify the
DNA content in MDA-MB-436 cells transfected with control siRNA(si-control) or siRNA targeting PLK1 (siPLK1). Statistical signiﬁcancewas calculated by Student t test. e, f Flowcytometric analysis toquantify the DNA content in MDA-MB-231 cells treated with DMSO,
10 μM of Bleomycin or 10 nM of BI-2536. Statistical signiﬁcancewas calculated by one-way ANOVA followed by Dunnett’s test.
g, h Flowcytometric analysis to quantify the DNA content in MDAMB-436 cells treated with DMSO, 10 μM of Bleomycin or 10 nM of
BI-2536. Statistical signiﬁcance was calculated by one-way ANOVA
followed by Dunnett’s test. i Immunocytochemistry of MDA-MB-231
cells treated with 10 μM of bleomycin or 10 nM of BI-2536 for 6 h,
stained with anti- γ-H2AX antibody. Arrows show γ-H2AX positive
cells. j Confocal imaging of bipolar spindle formation of MDA-MB231 cells and MDA-MB-436 cells expressing GFP-tubulin, treatedwith DMSO or 10 nM of BI-2536 for 6 h (MDA-MB-436) or for 24 h
(MDA-MB-231). The nucleus was visualized with DAPI. k Microscopic images of MDA-MB-231 cells treated with DMSO or 10 nM
BI-2536. The nuclei were visualized with DAPI. Scale bars show
20 μm. l A box plot showing the size of nuclei of MDA-MB-231 cells
treated with DMSO or 10 nM BI-2536. The size of nuclei was determined by measuring major axis of each nucleus. N = 23 for eachgroup. Statistical signiﬁcance was calculated by Student t test. All data
were mean ± S.D. from at least 3 independent experiments. *p < 0.05,**p < 0.01, ***p < 0.001

targeted therapy for TNBC yet and thus TNBC has thepoorest outcome among breast cancer. The RNAi library
screening is a powerful tool to identify potential therapeutictargets for diseases with unmet medical needs [28–30]. In
this study, we performed siRNA-mediated screening of 24
genes, involving in the DNA repair and BRCA1 signaling,
to identify the gene whose expression is indispensable forsurvival of TNBC and identiﬁed PLK1 as such a genebecause knockdown of PLK1 triggered cell death in thescreened TNBC lines, MDA-MB-436 (a mutation in
BRCA1), HCC1806 (mutations in both BRCA1/2) and
MDA-MB-468 (wild type BRCA1). Although we examined
three lines with different BRCA features for the initial
screen, no gene other than PLK1 was essential for survivalof TNBC when depleted by siRNA. We also examined theeffect of siRNA against PLK1 and BI-2536 on the viabilityof TNBC lines with various genetic signatures because
inhibition of PLK1 might be effective in the speciﬁc type of
TNBC cells [31, 29, 32], but BI-2536 was effective for all
the TNBC lines examined. The absence of BRCA function
results in failure of HR and thus the repair of undesirable
mutations in genomic DNA is dependent on the NHEJ
pathway or poly (ADP ribose) polymerase (PARP)-dependent SSB-repairing pathway. Actually, PARP inhibitors
such as olaparib and talazoparib are in clinical trials for
BRCA-negative TNBC [33, 34]. However, these compounds are supposed to be ineffective for BRCA-positive
TNBCs which accounts for more than 50% of TNBC. As
we demonstrated here, inhibition of PLK1 efﬁciently
inhibited growth of all TNBC lines examined regardless of
BRCA1-genotype, suggesting that inhibition of PLK1 may
be a broader therapeutic for TNBC than inhibition of
PARP1. In addition, the expression of PLK1 was correlatedwith cellular proliferation, implying that inhibition of PLK1
may be especially effective for the aggressive type of
TNBC. Further in vivo experiments using TNBC animalmodels will help to show the superiority of activity andbroader applicability of PLK1 inhibition.
PLK family members are known to be involved in mitosis
and cytoskeletal reorganization in normal and malignant
cells [35]. PLKs family consists of 5 members, PLK1, −2,−3, −4 and −5, and PLK1 is the most investigated memberof the PLK family which plays an important role in mitosis,
especially in G2/M phase [36]. Most importantly, PLK1
drives mitotic events including centrosome disjunction,
activation of cyclin and cyclin-dependent kinases, spindle
assembly and chromosome separation [37, 38]. PLK1 is also
known to phosphorylate BRCA1 to facilitate the recruitment
to the DSB site [10]. Clinically, PLK1 is highly overexpressed in many types of cancer including glioma, headand neck cancer, ovarian cancer, prostate cancer, non-small
cell lung cancer and breast cancer [13–20]. Consistent withthese previous studies, the immunohistochemical analyses

Discussion
Breast cancer is one of the biggest threats for women healthworldwide. Recent advances in development of moleculartargeting therapies including anti-HER2 therapy and hormonal therapy have drastically improved the outcome andquality-of-life of patients with breast cancer. However,
TNBC, diagnosed by a lack of hormone receptors andHER2 receptor, is still enigmatic breast cancer with worst
prognosis due to its high recurrence rate after conventional
chemotherapy and deﬁciency of molecular-targeting therapy [5]. To identify a therapeutic target for TNBC, we
performed siRNA-mediated screening and identiﬁed PLK1
as a possible therapeutic target for TNBC. Depletion of
PLK1 by siRNA-mediated knockdown or BI-2536, a
selective inhibitor for PLK1, decreased viability of various
TNBC lines through inhibition of mitosis. Furthermore,immunohistochemical analyses showed that PLK1 washighly expressed in the tissues from TNBC patients compared with benign breast tumors or normal mammary gland,
proposing PLK1 as a possible therapeutic target for TNBC.
TNBC is a heterogeneous cancer and has a wide varietyof genetic mutations. A recent study classiﬁed the TNBC
into 6 subtypes by the gene expression proﬁle [7]. To ﬁnd a
new therapeutic approach to improve prognosis of TNBC, aseries of signal transduction pathways such as the Notch
signaling pathway, Wnt/β-catenin pathway and Hedgehog
pathway, have been pre-clinically and clinically studied[26, 27]. However, still there is no validated molecular-

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A. Ueda et al.

Fig. 5 a Immunohistochemistry of PLK1 in normal mammary gland.
b Immunohistochemistry of PLK1 in benign tumors. c Immunohistochemistry of PLK1 in luminal A, luminal B, HER-2 positive or
TNBC tissues (# shows each patient). d The high magniﬁcation imageof TNBC tissue immunostained with anti-PLK1 antibody. Arrows
show condensed chromosomes. e Immunohistochemistry of Ki67 inluminal A, luminal B, HER-2 positive or TNBC tissues. f A box plotof semi-quantiﬁed Quickscore of PLK1 in normal mammary glands,

benign tumor (three ﬁbroadenoma cases, one benign plyllodes caseand one intraductal papilloma case), luminal A, luminal B, HER-2
positive and TNBC. g A box plot of quantiﬁed Ki67-positive cells in
the luminal A, luminal B, HER-2 positive and TNBC tissues. h The
correlation of the Quickscore of PLK1 and rate of Ki67-positive cells.
Statistical signiﬁcance was calculated by one-way ANOVA followed
by Tukey’s test. *p < 0.05, **p < 0.01. N = 5

we performed here also indicated that PLK1 is highly
overexpressed in breast cancer, especially in TNBC, but not
in benign tumors or normal mammary gland. In breast

cancer, overexpression of PLK1 is reported to correlate withpoor prognosis [39]. It is also shown that expression level of
PLK1 is particularly higher in TNBC than other subtypes-302-

Therapeutic potential of PLK1 inhibition in triple-negative breast cancer

1285(Fig. 5c, f) [40, 41], and higher tumor grade of TNBC
(poorer differentiation and higher genome instability,
resulting in aggressive tumor growth) correlates with PLK1
expression [42]. Therefore, PLK1-targeting therapy in
TNBC is rational and worth being considered.
BI-2536 is a potent and selective PLK1 inhibitor(IC50 = 0.83 nM), and its robust anti-tumor activity has
been conﬁrmed in vitro and in vivo [21]. When given
intravenously once or twice per week, BI-2536 is welltolerated and inhibits growth of diverse xenograft solid
tumor models such as colon cancer, pancreas cancer, nonsmall cell lung cancer, prostate cancer and osteosarcoma[21, 43, 44]. These favorable preclinical data prompted
clinical trials of BI-2536 for several types of cancer
including breast cancer [45]. Although phase I trials showedthat BI-2536 has acceptable safety proﬁles, its effectivenesswas not validated so far in phase II trials yet, thus further
sophistication of the design or modiﬁcation of protocol may
be required [45–48]. The immunohistochemical analysis
showed that PLK1 was highly expressed in the TNBC cells
at mitotic phase and the expression level was correlatedwith the rate of Ki67-positivity (Fig. 5d, h), suggesting thatthe criteria based on the results of Ki67 immunostaining,which are routinely performed for diagnosis of breast cancer, may help to choose appropriate TNBC patient group fortreatment with PLK1 inhibitors. For further improvement ofclinical outcome by inhibition of PLK1 may be achieved bydifferent types of PLK1 inhibitors. BI-2536 was identiﬁed
as a ATP- competing compound for PLK family proteins[21], and other types of PLK1 inhibitors have been underclinically investigated [49].
In summary, we identiﬁed PLK1 as a therapeutic target
for TNBC through siRNA-mediated screen and demonstrated that inhibition of PLK1 by siRNA-mediatedknockdown or a chemical inhibitor promoted cell cycle
arrest and apoptosis in multiple TNBC lines. We also
conﬁrmed that PLK1 was highly overexpressed in the
TNBC tissue. These our in vitro data will be further
strengthened by a future study using TNBC animal model.
The lack of druggable target is the problem for poor prognosis of TNBC, and the effectiveness and unique action
proﬁle of PLK1 inhibition, in addition to relatively speciﬁc
expression to TNBC tissue, suggest that PLK1 is a promising molecular target for TNBC.

Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science andTechnology of Japan and Project for Development of Innovative
Research on Cancer Therapeutics.

Acknowledgements We appreciate the members of department of
Molecular Pathology, Tokyo Medical University and department ofBreast Oncology and Surgery, Tokyo Medical University Hospital for
their supports and helpful suggestions.
Funding This work was partly supported by the grants from the JSPS
KAKENHI Grant number (17K16520 to A. U., 16H06247 to K. K.,
17H04067 to M. Kuroda). This work was also supported in part by
Japan Agency for Medical Research and Development (AMED),

Compliance with ethical standards
Conﬂict of interest The authors declare that they have no conﬂict ofinterest.
Consent for publication We obtained the written forms of informed
consent from all the patients regarding the use of their clinical data,
following the ethical guideline of Tokyo Medical University. The datafrom patients were analyzed anonymously.
Publisher’s note: Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional afﬁliations.

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Laboratory Investigation
https://doi.org/10.1038/s41374-020-0409-4

ARTICLEmiR-27a ameliorates chemoresistance of breast cancer cellsby disruption of reactive oxygen species homeostasis
and impairment of autophagy
Shinobu Ueda1 Masakatsu Takanashi1 Katsuko Sudo2 Kohsuke Kanekura1 Masahiko Kuroda1
●

●

●

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1234567890();,:

1234567890();,:

Received: 25 November 2019 / Accepted: 28 January 2020
© The Author(s), under exclusive licence to United States and Canadian Academy of Pathology 2020

Abstract
In patients with breast cancer, primary chemotherapy often fails due to survival of chemoresistant breast cancer stem cells(BCSCs) which results in recurrence and metastasis of the tumor. However, the factors determining the chemoresistance of
BCSCs have remained to be investigated. Here, we proﬁled a series of differentially expressed microRNAs (miRNAs)
between parental adherent breast cancer cells and BCSC-mimicking mammosphere-derived cancer cells, and identiﬁed hsamiR-27a as a negative regulator for survival and chemoresistance of BCSCs. In the mammosphere, we found that theexpression of hsa-miR-27a was downregulated, and ectopic overexpression of hsa-miR-27a reduced both number and size of
mammospheres. In addition, overexpression of hsa-miR-27a sensitized breast cancer cells to anticancer drugs bydownregulation of genes essential for detoxiﬁcation of reactive oxygen species (ROS) and impairment of autophagy.
Therefore, enhancing the hsa-miR-27a signaling pathway can be a potential therapeutic modality for breast cancer.

Introduction
Breast cancer is a leading cause of cancer-associated deaths
for women world-wide. Although recent improvements ofregimens for the conventional chemotherapy for breast cancer
gradually reduce the rate of relapse, a signiﬁcant proportion ofpatients suffer from unexpected relapse and metastasis evenafter years or decades of periods with no detectable tumors[1, 2]. Current hypotheses suggest that these recurrence andmetastases arise from residual disseminated tumor cells,
which disseminate from primary lesions, undergoing an
extended period of dormancy in the stroma of metastasizedorgans [3–9]. Importantly, these dormant tumor cells seem tohave a special drug resistance machinery which protects them

Supplementary information The online version of this article (https://doi.org/10.1038/s41374-020-0409-4) contains supplementarymaterial, which is available to authorized users.
* Masahiko Kuroda
kuroda@tokyo-med.ac.jp
1

Department of Molecular Pathology, Tokyo Medical University,
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan2

Preclinical Research Center, Tokyo Medical University,
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan

from chemotherapy, and accumulating evidences suggest thatthe dormant disseminated breast cancer cells should beequated with breast cancer stem cells (BCSCs), characterized
by self-renewal and differentiation into multiple cell types ofbreast cancer [10].
The molecular mechanism by which BCSCs tolerate
cytotoxic chemotherapy still remains enigmatic; however,their ability to maintain intracellular reactive oxygen species(ROS) levels lower than their matured progeny cells may be
implicated in the survival [11]. Intracellular ROS plays pivotal
roles in cellular signaling and homeostasis including inﬂammation, apoptosis, DNA damage, and posttranslational modiﬁcation thereby its level is strictly regulated by a complexed
antioxidative defense system including the Keap1-Nrf2
pathway [12–14]. Under normal condition, ROS is produced as a byproduct of biochemical reactions, such as oxidative phosphorylation at mitochondria and oxidative foldingof proteins in endoplasmic reticulum. Once excessive amountof ROS is produced, cytosolic Keap1 is inactivated by oxidation of its cysteine residues and releases a transcription
factor Nrf2. After translocation into the nucleus, Nrf2 induces
expression of a series of antioxidant genes including glutathione (GSH) reductase [15–17] which produces endogenous antioxidant, reduced GSH.
Another possible mechanism explaining the special chemoresistance of BCSCs is higher level of basal autophagic-305-

S. Ueda et al.

ﬂux [3, 18, 19]. Autophagy is a cellular process which
degrades organelles and proteins in response to various stimuli, such as nutrient starvation, and autophagy augments
supply of amino acids to BCSCs which survive for years indormancy. Importantly, not only playing roles in supplying
ingredients, but autophagy also regulates cell death in bothpromoting and inhibiting fashions [18]. In breast cancer,
autophagy has been reported to confer protection against
chemotherapy. Adriamycin-resistant MCF-7/ADM cells
maintain a high basal level of autophagy and inhibition of
autophagy counteracts their resistance to the drug [19].
Moreover, survival of dormant CSCs in secondary sites isdependent on autophagy and autophagy helps their subsequent growth as macrometastases [3, 20, 21].
The master regulator gene(s) which confers these diverse
phenotypes to BCSCs remains unidentiﬁed. Although conventional oncogenes, such as KRAS and EGFR, play roles inboth BCSCs and their progeny cells, their activation cannot
fully explain the complexed phenotypes [22]. Recently, evidences showing implication of miRNAs in oncogenesis havebeen emerged [23]. miRNAs are short 20–22 nucleotide RNA
molecules that are negative regulators of gene expression in avariety of eukaryotic organisms [24], and pro-oncogenic“onco-miR” and antioncogenic “anti-onco-miR” can modulates multiple genes by interacting 3′UTR of target genes[23, 25, 26], thus miRNA might explain the multiple featuresof BCSCs [27, 28].
In this study, we performed miRNA proﬁling between
BCSC-mimicking mammosphere and their parental cancer
cell lines, and identiﬁed hsa-miR-27a was speciﬁcallydownregulated in the BCSC model. Overexpression of hsamiR-27a attenuated formation and survival of mammosphere,
and ameliorated drug resistance to chemotherapeutic compounds through downregulation of ROS-detoxifying genes.
Consistently, inhibition of hsa-miR-27a augmented both basal
and induced autophagic ﬂux in breast cancer cells which
confers protection against chemotherapy. Although further
in vivo studies are necessary, enhancement of hsa-miR-27a
signaling could be an attractive modality for treatment ofrelapsed/metastasized breast cancer.

Materials and methods
Cell Lines
Breast cancer cell lines were obtained from the American
Type Culture Collection (Manassas, VA, USA). MCF-7
cells were cultured in RPMI1640 medium (Life Technology, Carlsbad, CA, USA) supplemented with 10% fetal
bovine serum (FBS) (Cell Culture Technologies, Canada)
and penicillin/streptomycin (Sigma, St. Louis, MO, USA).
MDA-MB-231 cells were cultured in Leibovitz’s L-15

Medium (Sigma) supplemented with 10% FBS, penicillin/
streptomycin, L-glutamine (Sigma) and 2 g/L of NaHCO3(Wako Pure Chemical Industries, Osaka, Japan). All cells
were grown at 37 °C in a humidiﬁed atmosphere containing
5% CO2.

miRNA microarray analysis
Total RNA (100 ng/sample) was isolated using ISOGEN(Nippon Gene, Tokyo, Japan), and then labeled andhybridized using the Human microRNA (miRNA) Microarray kit (Agilent Technologies, Santa Clara, CA, USA)according to the version 1.5 of the “Protocol for use with
Agilent miRNA microarrays.” Hybridized signals weredetected on a G2505B DNA microarray scanner (Agilent
Technologies), and all scanned images were analyzed using
the Agilent Feature Extraction software (v9.5.3.1).

miRNA transfection
The sequences of miRNA mimics used in this study were as
follows: hsa-miR-27a sense (5′-AGGGCUUAGCUGCUUGUGAGCA-3′) and antisense (5′-UUCACAGUGGCUAAGUUCCGC-3′); hsa-let-7a-1 sense (5′-UGAGGUAGUAGGUUGUAUAGUU-3′) and antisense (5′-CUAUACAAUCUACUGUCUUUC-3′); nontargeted control miRNA sense(5′-AUCCGCGCGAUAGCACGUAUU-3′) and antisense(5′-UACGUACUAUCGCGCGGAUUU-3′). The miRNA
mimics were transfected with HiPerFect reagent (ﬁnalconcentration, 100 nM; Qiagen, Hilden, Germany).

Quantitative real-time PCR assays
MiRNAs were quantiﬁed using TaqMan® miRNA Assays(Thermo Fischer Scientiﬁc Inc., USA) with some modiﬁcations [29]. Brieﬂy, 10 ng of total RNA was reverse
transcribed (RT) using the TaqMan® miRNA RT Kit. For
amplifying miRNA speciﬁc stem-loop, TaqMan® primers(hsa-miR-27a, 000408; hsa-miR-29b, 000413; hsa-miR101, 002253; hsa-miR-125a-5p, 002198; hsa-miR-193a-3p,
002250; RNU6B, 001093; Applied Biosystems) were used.
Quantitative RT-PCR using FastStart Universal Probe
Master Mix (Roche Diagnostics) was used for quantiﬁcationof mRNA. The sequences of mRNA primers and probes
were listed in Supplementary Table 1. Data were analyzed
using the Light Cycler 96 Software Ver.1.1 (Roche Diagnostics GmbH). The comparative Ct method was used for
relative quantitation of samples.

Cell viability
The breast cancer cells were treated with paclitaxel (Pac)or doxorubicin (DXR) at indicated concentration for 24 h.

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miR-27a ameliorates chemoresistance of breast cancer cells by disruption of reactive oxygen species. . .

The cell viability after the treatment was determined using
Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan).

Measurement of ROS

considered statistically signiﬁcant and was denoted as follows: *P < 0.05; **P < 0.01; ***P < 0.001.

Results

Breast cancer cells were incubated with 10 μM 2′,7′-dichloroﬂuorescein diacetate (DCFH-DA, Sigma) for 15 min at
37 °C, washed with phosphate-buffered saline (PBS) with 2%
FBS, and subjected to ﬂow cytometric analysis on BD FACS
Aria III (BD Biosciences, San Jose, CA, USA).

FACS analysis
MCF-7 and MDA-MB-231 cells transfected with control
miRNA, let-7a-1, or hsa-miR-27a. After 2 days of transfection, cells were dissociated by exposure to 0.25%
trypsin/EDTA. Cells were incubated with anti-humanCD44v9 antibody (3 μg/ml in 0.2% BSA in PBS, LKGM001, Cosmo Bio, Japan) for 30 min on ice, washedwith PBS with 2% FBS. Cells were incubated withphycoerythrin-conjugated antibody to rat IgG2a (2 μg/ml
in 0.2% BSA in PBS, 558067, BD Pharmingen) for
30 min on ice, washed with PBS with 2% FBS. FACS was
performed with BD FACS Aria III (BD Biosciences, San
Jose, CA, USA). Aldehyde dehydrogenase (ALDH)
activity was determined with ALDEFLUOR assay(STEMCELL Technologies, Canada).

GSH assay
Intracellular levels of GSH were determined using the GSHGlo Glutathione Assay kit (Promega), as described previously [30].

Western blot analysis and autophagy assay
Breast cancer cells, lysed with TNE buffer containing
1 mM of PMSF (Sigma), were subjected to SDS-PAGE
followed by western blot (WB) analyses. The primary
antibodies used for WB were as follows: p62 (1:1000,
ab207305, Abcam), beta-actin (1:5000, A1978, Sigma),and Autophagy watch kit (8486. MBL, Japan) for LC3
and tubulin. The signals were visualized with HRPconjugated anti-mouse IgG secondary antibody (1:50,000, NA9310, GE Healthcare) or HRP-conjugatedanti-rabbit IgG secondary antibody (1:50,000, NA9340,
GE Healthcare), and Amersham ECL select detection
regent (GE Healthcare).

Statistical analysis
Data were analyzed using the unpaired Student’s t test,
and were presented as means ± SD. P value < 0.05 was

Hsa-miR-27a expression is downregulated in
mammospheres, and hsa-miR-27a overexpression
suppresses BCSC properties
The current CSC hypothesis is based on the existence offewer number of stem cell-like cells which can initiate selfrenewal and also differentiate into multiple types of cells
associated with the cancer in the special environment
called niche [31]. To dissect the molecular characteristics
of BCSCs using cultured cancer cell lines, we need amodel which mimic the niche for BCSCs [32]. Mammosphere, a sphere-like structure consisting of threedimensionally cultured breast cancer cells is thought tomimic the environment of niche and promote BCSC-likephenotypes [33]. First, we successfully established the
mammosphere models from MCF-7, HCC-70, and HCC1954 cell lines. Using these models, we comprehensively
proﬁled miRNAs which differentially expressed between
parental adherent cancer cells and mammosphere cells
using the miRNA array (Fig. 1a). The results indicated thatmiR-125a-5p expression was higher in the mammospheres
than in their parental adherent breast cancer cells, whereas
the expressions of miR-101, miR-193a-3p, miR-27a, andmiR-29b were lower in the mammospheres than in theirparental adherent breast cancer cells (Fig. 1a). The results
were further validated by qRT-PCR with Taqman probes(Fig. 1b). Among them, we were especially interested inmiR-27a because miR-29b, miR-101, and miR-193a-3p
are already established anti-oncomiRs and the miR-125a5p can work as both pro-oncogenic and anti-oncogenic[34, 35], and although miR-27a is reported to be oncogenic
for breast cancer, its expression is downregulated by formation of mammosphere.
To investigate the role of hsa-miR-27a in maintenance of
the BCSC properties, we examined the role of hsa-miR-27ain the mammosphere harboring more BCSC-like phenotypes. The established cancer stem cell markers for BCSC is
the ratio of CD44+/CD24− [33, 36], and MDA-MB-231 cellline has been shown to have high CD44+/CD24− ratio,suggesting that MDA-MB-231 may have similar characteristics with BCSCs. MDA-MB-231 cells, transfectedwith hsa-miR-27a, formed 0.36-fold fewer number ofmammospheres than control cells transfected without themiRNA mimic (Fig. 1c, d). In addition, the size of mammospheres derived from MDA-MB-231 cells transfectedwith hsa-miR-27a was smaller (0.43-fold) than the mammospheres from control cells (Fig. 1d). Next, we examined-307-

S. Ueda et al.

Fig. 1 Hsa-miR-27a negatively regulates the formation of mammospheres. a Relative miRNA expression levels in adherent parental
breast cancer cell lines and corresponding mammospheres, as determined using miRNA arrays. b Validation of miRNA expression levelsin the parental breast cancer cell lines and corresponding mammospheres, determined by quantitative RT-PCR. The expression level of
RNU6B was used as an internal control; n = 3. c Microscopic imagesof mammospheres established from MDA-MB-231 cells, in the presence or absence of introduction of hsa-miR-27a. The arrowheads
show the mammospheres. The scale bar shows 200 μm. d The efﬁciency and size of mammospheres established from MDA-MB-231
cells, in the presence or absence of introduction of hsa-miR-27a; n = 3.
Data are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001(Student’s t test).

whether the introduction of hsa-miR-27a affects the
ALDH activity, which is an established marker for BCSC[37], in breast cancer cells using ALDEFLUOR, a ﬂuorescent substrate for ALDH. The rates of ALDEFLUORpositive populations in the MCF-7 and MDA-MB-231 cells
transfected with hsa-miR-27a were decreased comparedwith their parental control cells (MCF-7: hsa-miR-27a
4.47% vs. control 6.65%; MDA-MB-231: hsa-miR-27a

26.6% vs. control 41.6%) (Fig. 2a). Consistently, the MDAMB-231 cells transfected with antisense-miR-27a (as-miR27a), an antagonistic miRNA mimic against miR-27a,
contained a higher proportion of ALDEFLUOR-positive
cells than the cells transfected with nontargeting control
cells (Fig. 2b).
The BCSCs population expressing variant of CD44
(CD44v) is known to aggressively disseminate and-308-

miR-27a ameliorates chemoresistance of breast cancer cells by disruption of reactive oxygen species. . .

Fig. 2 Expression of hsa-miR-27a attenuates expressions of BCSC
markers. a Flow cytometry of MDA-MB-231 cells transfected with
miR-27a or control, stained with ALDEFLUOR dye. The ALDHspeciﬁc inhibitor (DEAB) was used to determine the background
ﬂuorescent signals and deﬁne the ALDEFLUOR-positive population.
SSC side scatter. b Flow cytometry of MDA-MB-231 cells transfectedwith antagonizing as-miR-27a or as-control, stained with ALDEFLUOR dye. DEAB was used to determine the background ﬂuorescentsignals and deﬁne the ALDEFLUOR-positive population. SSC side
scatter. c Flow cytometric analysis of MCF-7 cells transfected with
control miRNA, let-7a-1 or hsa-miR-27a, immunostained with anaggressive type of breast cancer marker anti-human CD44v9 antibody.

metastasize [38]. CD44v has been reported to contributeto ROS defense by promoting the synthesis of GSH
through stabilization of xCT, a subunit of glutamatecystine transporter [30], and importantly, 3′UTR of
CD44 harbors two partially complementary seedsequence to hsa-miR-27a (miRDB: mirdb.org). To
investigate if hsa-miR-27a could suppress the expressionof CD44v, we introduced hsa-miR-27a into MCF-7 cells
and MDA-MB-231 cells, and measured the rates of cells
positive for CD44v. As expected, the overexpression of
hsa-miR-27a inhibited the expression of CD44v in both
MCF-7 cells and MDA-MB-231 cells (Fig. 2c). Theseﬁndings indicate that hsa-miR-27a regulates the key
features of BCSCs.

Attenuation of chemoresistance of breast cancer
cells by miR-27a
Even after vigorous chemotherapy, BCSCs still survive andeventually promote relapse and metastasis in breast cancer
patients [39]. If the chemoresistant feature of BCSCs can be
mitigated, the outcome could be drastically improved.
Therefore, we next investigated whether miR-27a attenuates
the chemoresistant capacity of breast cancer cells. We
introduced hsa-miR-27a into MCF-7 and MDA-MB-231
cells, and then challenged them with anticancer drugs, DXR
and Pac. The microscopic images showed that hsa-miR-27a
increased dead cell population and decreased the number ofattached live cells after treatment with 100 nM of DXR-309-

S. Ueda et al.
Fig. 3 hsa-miR-27a sensitizes
breast cancer cells toconventional
chemotherapeutic agents.
a Typical microscopic images of
MCF-7 cells and MDA-MB-231
cells transfected with or without
hsa-miR-27a. b Viability ofbreast cancer cell linestransfected with or without hsamiR-27a, incubated with DXRor Pac at indicatedconcentrations for 3 days. The
error bars show ± SD from three
independent experiments. *P <0.05; **P < 0.01; ***P < 0.001(Student’s t test).

(Fig. 3a). We also measured the viability of the cells treatedwith DXR (10 or 100 nM), or Pac (10 or 20 nM), andconﬁrmed that introduction of hsa-miR-27a sensitizedMCF-7 and MDA-MB-231 cells to these chemotherapeutic
compounds and decreased their viability (Fig. 3b). These
data indicate that hsa-miR-27a negatively regulates chemoresistance of breast cancer cells.

Hsa-miR-27a impairs ROS defense by targeting CTH,
xCT, and Nrf2
To dissect the molecular mechanism underlying the miR27a-mediated chemosensitization, we next sought to ﬁndmolecular targets of miR-27a. Each miRNA can interfere
multiple genes by interaction with 3′UTR of mRNAs.
We used the bioinformatic prediction tool, miRDB andlisted potential target genes of hsa-miR-27a. Among them,

we focused on a group of genes related to cellular response
to ROS including cystathionine gamma-lyase (CTH),xCT cystine/glutamate transporter (SLC7A11), and Nrf2
(NFE2L2) (Fig. 4a), because activation of these genes
promote chemoresistance [40]. The 3′UTRs of these genes
are partially complementary to hsa-miR-27a-3p (Fig. 4b).
When we introduced hsa-miR-27a mimic to MCF-7, we
conﬁrmed that hsa-miR-27a signiﬁcantly downregulated theexpression levels of CTH, SLC7A11, and NFE2L2 (Fig. 4c).
In MDA-MB-231 cells, although we observed similar tendency to MCF-7 cells, hsa-miR-27a signiﬁcantly suppressed only SLC7A11 mRNA expression (Fig. 4c).
We next sought to determine whether the expression of
hsa-miR-27a is functionally associated with ROS defense inbreast cancer cells. To address this question, we measured
the intracellular ROS levels in MCF-7 and MDA-MB-231
cells transfected with hsa-miR-27a by staining with the-310-miR-27a ameliorates chemoresistance of breast cancer cells by disruption of reactive oxygen species. . .

Fig. 4 hsa-miR-27a disturbs ROS homeostasis by targeting GSH
synthesis and ROS defense-related genes. a Functional network ofcandidate genes targeted by hsa-miR-27a, determined by STRING(string.org). b Sequences of hsa-miR-27a and its binding sites in the 3′
UTRs of SLC7A11, CTH, and NFE2L2 mRNAs. c Expression of hsamiR-27a target genes, determined by RT-qPCR. Data were normalizedagainst the corresponding level of β-actin mRNA. The error bars show

± SD from three independent experiments. d Basal and enhanced ROS
levels in the MCF-7 and MDA-MB-231 cells, incubated with orwithout 0.1 mM H2O2 for 20 min, 3 days after transfection with
miRNA mimics. Cellular ROS levels were determined by DCFH-DA.
e Intracellular GSH/GSSG ratio 2 days after transfection with miRNA
mimics. n = 3. Data are shown as mean ± SD. *P < 0.05; **P < 0.01;***P < 0.001 (Student’s t test).

ﬂuorescent ROS probe DCFH-DA. Because these potential
target genes are involved in detoxiﬁcation of ROS in cells
and especially Nrf2 is the master regulator of cytosolic

response to ROS, downregulation of these genes would
promote higher ROS levels in cells. As expected, hsa-miR27a-overexpressing cells exhibited higher DCFH-DA-311-

S. Ueda et al.

Fig. 5 Inhibition of hsa-miR-27a expression induces autophagy.
a Expression of LC3-II 48 and 72 h after transfection with as-hsa-miR27a. (**P < 0.01; data are expressed relative to the corresponding

value for vehicle-treated cells and are shown as means ± SD from
quadruplicate experiments.) b Expression of p62 at 48 and 72 h by ashsa-miR-27a post transfection.

staining than control cells (Fig. 4d) under normal culture
condition. To further examine the effect of hsa-miR-27a on
defense against ROS, we burdened the cells with 0.1 mMhydrogen peroxide (H2O2). After exposure to H2O2, hsamiR-27a-overexpressing MCF-7 cells and MDA-MB-231
cells exhibited higher DCFH-DA staining than control cells(Fig. 4d).
Cytosolic redox state is maintained as reduced condition
by abundant existence of GSH [41, 42]. The methionine
cycle is the major source for cysteine, a precursor of GSH
in the trans-sulfuration pathway and cysteine availability israte-limiting for GSH synthesis [43]. In the trans-sulfuration pathway, cystathionine γ-lyase (CTH/CSE) converts
cystathionine into cysteine. Cystine is taken up by cells
primarily through xCT, a heterodimeric protein consistingof a transporter subunit and a regulatory subunit (4F2hc,
also known as CD98, encoded by the SLC3A2) [44]. In
cells, cystine is reduced to cysteine and used for synthesis
of GSH. Therefore, if hsa-miR-27a targets CTH and xCT(SLC7A11), production of GSH will be attenuated. To
conﬁrm this, we measured intracellular GSH level. As
expected, transfection of hsa-miR-27a signiﬁcantly lowered the cytosolic GSH in MCF-7 cells compared with
control cells (Fig. 4e). We also observed similar tendency
in MDA-MB-231 but it was not statistically signiﬁcant,probably due to poor downregulation of CTH in MDAMB-231 cells (Fig. 4c). Overall, these results suggest that
expression of hsa-miR-27a increases intracellular ROS in
MCF-7 cells by attenuating Nrf2 signaling and GSH
synthesis pathway.

Inhibition of the expression of miR-27a induces
autophagy
Another big problem of BCSCs to be conquered is that they
can survive for years or decades in dormancy after chemotherapy. Still little is known about the mechanisms
responsible for it; however, several studies have suggested
that autophagy is implicated in this process, and autophagy
supplies nutrition required for the long nap [45].
To determine whether miR-27a regulates autophagic
activity of breast cancer cells, we measured autophagy ﬂux
in the breast cancer cells and examined whether it isaffected by transient transfection of antagonizing as-hsamiR-27a. Once autophagic signaling is initiated, LC3, amammalian homolog of ATG8 is lipidated and converted to
LC3-II which associates with the autophagosome membrane. The amount of LC3-II correlates with the number of
autophagosomes, thus it is used for measurement of activityof autophagy. When we antagonized endogenous hsa-miR27a by as-hsa-miR-27a, which mimicked the condition of
BCSCs, LC3-II was increased in MCF-7 cells at 72 h after
as-hsa-miR-27a transfection (Fig. 5a). p62 is autophagic
substrate and eventually delivered to lysosomes for degradation [46]. Hence, we measured the protein levels of p62.
The expression of p62 was increased in MCF-7 and MDAMB-231 cells at 48 h, and gradually decreased by 72 h
(Fig. 5b).
Together, these data suggested that hsa-miR-27a negatively regulates autophagic ﬂux and BCSCs with lower
expression of hsa-miR-27a may beneﬁt from nutrition-312-miR-27a ameliorates chemoresistance of breast cancer cells by disruption of reactive oxygen species. . .

supply from enhanced autophagy which is essential fordormancy for decades.

Discussion
Breast cancer is one of the most common types of cancer
among women throughout the world [47]. Accumulating
evidences suggest that metastatic tumor cells have alreadydisseminated at an early stage of breast cancer [6, 48, 49].
These disseminated cells at the micrometastatic sites harborfeatures of CSCs, dormancy and chemoresistance [5, 6].
Therefore, for further improvement of the prognosis for breast
cancer patients, we need to target BCSCs; however, currently
there is no available therapy for BCSCs. To identify a therapeutic target for BCSCs, we comprehensibly proﬁled miRNAs differentially expressed between mammosphere and theparental cancer cells, and found that hsa-miR-27a is themaster negative regulator of BCSC phenotypes. In mammosphere hsa-miR-27a was downregulated. The overexpressionof hsa-miR-27a suppressed the mammosphere formation anddownregulated expression of BCSC markers. Furthermore,
overexpression of hsa-miR-27a chemosensitized breast cancer
cells via attenuation of ROS-defense system and autophagy.
Therefore, hsa-miR-27a is a possible therapeutic target
for BCSCs.
BCSCs, characterized by dormancy and chemoresistance, play roles in relapse and metastasis of breast cancer.
The dormant stage without detectable mitosis is one ofnotable phenotypes to distinguish BCSCs and breast cancer
cells. When patients undergo chemotherapy, actively proliferating cells are killed by chemotherapeutic reagents but
the dormant cells are chemoresistant and survive for years.
To date, biomarkers including CD44+/CD24− ratio and
ALDH activity are established as markers for BCSCs[36, 37], and the expression of CD44v is shown to be
involved in the more aggressive metastatic phenotype [30].
As we demonstrated above, hsa-miR-27a suppressed
expression of all of these markers and activity of ALDH,
suggesting that hsa-miR-27a is a master negative regulatorof stemness of breast cancer. Previously, hsa-miR-27a isreported as an oncomir and its expression is high in breast
cancer cells, promoting cancer cell proliferation, and inhibition of hsa-miR-27a decreases the proliferation [50, 51].
The downregulation of a tumor-suppressive transcription
factor FOXO by hsa-miR-27a is implicated in this phenotype, and inactivation of FOXO results in rapid turnover ofcell cycles and increased number of cancer cells [50].
Therefore, hsa-miR-27a promotes cell proliferation in breast
cancer cells. In this study, however, we identiﬁed novelroles of hsa-miR-27a in maintenance of features of BCSCs
and hsa-miR-27a inhibits proliferation of breast cancer cells
resulting in enhanced chemosensitivity and imbalance ofcytosolic ROS. Which cellular component(s) determines
these two controversial biological effects of hsa-miR-27a
remains to be investigated, but we speculated that microenvironment in mammosphere culture converts the breast
cancer cells to be more static state, and slowed cell cycle
progression contribute to the discrepancy.
Detoxiﬁcation of ROS is a big issue for cancer cell tosurvive the chemotherapy. DXR and Pac, which are widelyused chemotherapeutic compounds for breast cancer, produce ROS to induce apoptosis in breast cancer cells[52, 53], and to deal with it, breast cancer cells need tosense the aberrant ROS and neutralize the ROS. Importantly, BCSCs harbor highly active antioxidative machineries and their enhanced production of GSH contributes tothe survival under chemotherapy [54]. When we introduced
hsa-miR-27a mimic to breast cancer cells, it downregulated
not only Nrf2 but also CTH and xCT, both are essential for
GSH production, and thus increased intracellular ROS leveland susceptibility of breast cancer cells to DXR and Pac.
The cancer cells with disturbed antioxidative response arealso prone to DNA damages by radiation and thereby targeting hsa-miR-27a signaling will enhance the effect ofboth chemotherapy and radiotherapy.
Another determinant of survival of BCSCs is autophagy.
After long dormant period, metastatic BCSCs cause relapse
and it is a major cause of death of patients with breast
cancer. Pharmacologic or genetic inhibition of autophagy
signiﬁcantly decreases cell survival and metastasis of breast
cancer in animal models and in vivo preclinical models of
dormancy [45]. The Inhibition of autophagy in BCSCs
results in accumulation of ROS and apoptosis [45]. Therefore, maintenance of autophagic ﬂux is critical for survivalof BCSCs. In addition, the enhanced autophagy promotes
chemoresistant in MCF-7 cells [19], and basal andstarvation-induced autophagy ﬂux is higher in the ALDH+
population derived from mammospheres than the bulk
population [46], implying the roles of autophagy not only inthe maintenance of dormancy but acquiring chemoresistance. In this study, we demonstrated that hsa-miR-27a
negatively regulates autophagy ﬂux in MCF-7 and MDAMB-231 cells, and thus hsa-miR-27a inhibits phenotypes of
BCSCs via multiple mechanisms. Why BCSCs have higher
autophagic ﬂux remains unknown. Intracellular ROS is anestablished trigger for autophagy but the ROS level in
BCSCs is maintained lower than their progeny thus it
cannot explain it, and further studies are expected.
In summary, we identiﬁed hsa-miR-27a as the master
negative regulator of BCSC phenotypes through impairing
ROS defense and autophagy. These our in vitro data will befurther strengthened by a future study using animal models.
These novel insights give us a deeper understanding of therole of miRNA in maintaining BCSC features, and providing us novel molecular targets for breast cancer.

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S. Ueda et al.

Data availability
All datasets analyzed during the present study are available
from the corresponding author on reasonable request.
Funding This research was supported by Grants-in-Aid for ScientiﬁcResearch (17K10564 and 17H04067), a Grant-in-Aid for Exploratory
Research from Japan Society for the Promotion of Science (JSPS), andthe Strategic Research Foundation Grant-aided Project for Private
Universities from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (MEXT).
Author contributions SU designed the study; SU, MT, and KS performed the wet-lab experiments; SU did data analysis and statisticalanalysis; SU, MT, and KK performed interpretation of data; SU, MT,
KK, and MK drafted the manuscript.

Compliance with ethical standards
Conﬂict of interest The authors declare that they have no conﬂict ofinterest.
Publisher’s note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional afﬁliations.

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-315-Modern Rheumatology

ISSN: 1439-7595 (Print) 1439-7609 (Online) Journal homepage: https://www.tandfonline.com/loi/imor20

Difference in immunohistochemical characteristics
between Takayasu arteritis and giant cell arteritis:
It may be better to distinguish them in the sameage
Atsushi Kurata, Akira Saito, Hirotsugu Hashimoto, Koji Fujita, Shinichiro Ohno, Hiroshi Kamma, Toshitaka Nagao, Shigeto Kobayashi, Akira
Yamashina & Masahiko Kuroda
To cite this article: Atsushi Kurata, Akira Saito, Hirotsugu Hashimoto, Koji Fujita, Shin-ichiro
Ohno, Hiroshi Kamma, Toshitaka Nagao, Shigeto Kobayashi, Akira Yamashina & Masahiko Kuroda(2019): Difference in immunohistochemical characteristics between Takayasu arteritis and giant
cell arteritis: It may be better to distinguish them in the same age, Modern Rheumatology, DOI:
10.1080/14397595.2019.1570999
To link to this article: https://doi.org/10.1080/14397595.2019.1570999

Accepted author version posted online: 21
Jan 2019.
Published online: 18 Feb 2019.
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MODERN RHEUMATOLOGY
https://doi.org/10.1080/14397595.2019.1570999

ORIGINAL ARTICLE

Difference in immunohistochemical characteristics between Takayasu arteritis
and giant cell arteritis: It may be better to distinguish them in the same age
Atsushi Kurataa, Akira Saitoa, Hirotsugu Hashimotob, Koji Fujitaa, Shin-ichiro Ohnoa, Hiroshi Kammac,
Toshitaka Nagaod, Shigeto Kobayashie, Akira Yamashinaf and Masahiko KurodaaaDepartment of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; bDepartment of Diagnostic Pathology, NTT Medical CenterTokyo, Tokyo, Japan; cDepartment of Pathology, Kyorin University School of Medicine, Tokyo, Japan; dDepartment of Anatomic Pathology,
Tokyo Medical University, Tokyo, Japan; eDepartment of Rheumatology, Juntendo Koshigaya Hospital, Saitama, Japan; fDepartment ofMedical Education Promotion Center, Tokyo Medical University, Tokyo, Japan
ABSTRACT

Objectives: This study aimed to compare Takayasu arteritis (TAK) with giant cell arteritis (GCA)
through immunohistochemistry principally of inflammatory cells; these two disorders may be on the
spectrum within a single disease state.
Methods: Nine TAK and 5 GCA surgically resected vessel specimens were selected. TAK specimen
was divided into each three acute-, chronic-, and healed-phase samples based on intimal andadventitial thickening. Immunohistochemical analysis was performed of smooth muscle cells andinflammatory cells including lymphocytes, plasma cells, macrophages, and dendritic cells, where
three healed-phase TAK specimens were excluded due to paucity of inflammation.
Immunopositive cells per three different fields in intima, media, and adventitia were counted ineach specimen, and their numbers in these three layers along with total 3 layers were compared
between the two disorders.
Results: Intimal smooth muscle maturity estimated by ratio of h-Caldesmonþ cells to a-SMAþ cells significantly increased in chronic- and healed- over acute-phase increases in TAK. Mann-Whitney tests
demonstrated significantly more adventitial lymphoplasmacytic infiltration and less intimal fascinþ
dendritic cells, as well as overall more CD8þ T-cells, more CD20þ B-cells and lower CD4/8 ratio in TAK
than in GCA.
Conclusion: Different inflammatory involvement is suggested in the pathogenesis of TAK and GCA.

Introduction
Takayasu arteritis (TAK) is a non-specific inflammatory
disease of unknown etiology that causes stenosis, occlusion,or dilatation of the aorta and its immediate branches alongwith pulmonary arterial involvement. Signs and symptoms
differ widely by location and size of the affected vessels.
TAK is particularly prevalent among young Asianfemales [1,2].
On the other hand, giant cell arteritis (GCA) mainly
involves the temporal artery, is most prevalent in aged
Caucasians, and complication with polymyalgia rheumatica(PMR) is seen in 30–50% of cases [3]. However, it has
recently been claimed that TAK and GCA may be phenotypes within the spectrum of a single disorder, on the
grounds that (1) TAK may occur in aged subjects, (2) GCA
may involve aorta, (3) signs and symptoms in these two disorders broadly overlap [4], (4) arteriographic lesions of
TAK and GCA are similar [5], and (5) histology of TAK
and GCA may be indistinguishable [6]. The background to
this claim may reflect the paucity of immunohistochemical
studies comparing TAK with GCA. The present study was,CONTACT Atsushi Kurata
160-8402, Japanakurata@tokyo-med.ac.jp

ARTICLE HISTORY

Received 24 April 2018
Accepted 22 October 2018
KEYWORDS

Giant cell arteritis;
immunohistochemistry;
lymphocytes; smoothmuscle cells;
Takayasu arteritis

therefore, designed to assess similarities or differences in
immunohistochemical characteristics between TAK and
GCA specimens.

Material and methods
Patients and tissues
The present study protocol was approved by theCommittee of Human Studies of Tokyo Medical
University, Kyorin University School of Medicine inTokyo, and NTT Medical Center Tokyo. Following
informed patients’ consent, the tissues were used for the
diagnostic work-up and transferred to the archival files atthese hospitals, according to the data and ethical protection rules of the Medical Faculty. Surgically resected vessel
specimens from four TAK and five GCA patients wereidentified and retrieved from the database of Tokyo
Medical University 2007–2016. Additionally, five TAK
specimens were obtained from Kyorin University Schoolof Medicine and NTT Medical Center Tokyo and includedin the study. These nine TAK and five GCA specimens

Department of Molecular Pathology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo

 2019 Japan College of Rheumatology

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A. KURATA ET AL.

Table 1. Clinical summary of subjects and involved region.
Diagnosis
TAK
TAK
TAK
TAK

Age
80
49
76
27

Sex
F
M
M
F

Affected vasculature
Ascending aorta
Thoracic descending aorta
Aortic arch
Aortic arch and brachiocephalicarteryAortic arch
Celiac to hepatic artery

TAK
TAK

51
40

M
M

TAK
TAK

45
25

F
F

TAK

58

F

GCA
GCA
GCA
GCA

77
79
63
76

M
F
F
F

Ascending aorta
Aortic arch, brachiocephalic artery,and bilateral common carotid andsubclavian artery
Aortic arch, brachiocephalic artery,and bilateral common
carotid artery
Bilateral temporal artery
Bilateral temporal artery
Right temporal artery
Right temporal artery

GCA

71

F

Bilateral temporal artery

Glucocorticoids
before operation
Not doneNot doneNot donePSL 5–30 mg/D
for 4Y6M
Not donePSL 3–10 mg/D
for 3MPSL 5 mg/D for 17Y
PSL 10 mg/D
for 2M14D

Thickness ratioa
0.30
0.34
0.69
1.03

TAK stage
acute
acute
acute
chronic

1.18
1.66

chronic
chronic

Ascending aorta
Ascending aorta

2.20
2.47

healedhealedNot done

Ascending aorta

4.06

healedNot doneNot doneNot done
PSL 1000 mg/D
for 3D
Not done

Temporal artery
Temporal artery
Temporal artery
Temporal artery

Resected site
Ascending aorta
Thoracic descending aorta
Ascending aorta
Ascending aorta and brachiocephalicarteryThoracic descending aorta
Hepatic artery

Temporal artery

PSL: prednisolone.
a
Ratio of average thickness between intima and adventitia to that of media, measured based on Elastica van Gieson staining.

Figure 1. Representative pulse wave analysis and ultrasound examination. Pulse wave analysis of the four extremities shows lower blood pressure in upper thanlower extremities (a). Ultrasound examination of left carotid artery visualizes homogeneous and moderately echogenic circumferential thickening of the arterial
wall, the ‘Macaroni sign’ (b), which is also illustrated as a schema (c).

were clinically diagnosed based on the diagnostic criteriaof the Japanese Circulation Society for TAK [7], and theclassification criteria of the American College of
Rheumatology for GCA [8], because the Japanese
Circulation Society has not provided new diagnostic criteria for GCA. As shown in Table 1, the age range of TAK
patients was 25–80 years old (mean 50.1 ± 18.0 years old),while that of GCA patients was 63–79 years old (mean73.2 ± 5.7 years old). Female predominance was seen inboth disorders (5/9 in TAK and 4/5 in GCA). Strict diagnostic tools including pulse wave analysis of the four
extremities and imaging were used especially in aged TAK
subjects (Figure 1). Specimens were resected from aorta
and its immediate branches in TAK except for one casefrom hepatic artery, whereas in GCA, all specimens were
resected from temporal artery (Table 1). Glucocorticoids
treatment had been performed before the resection in fourcases in TAK and one case in GCA.

Immunohistochemistry
Surgically resected specimens had previously been fixed in
10% formalin solution for 24 h and embedded in paraffin.
Immunohistochemical analysis, along with HE andElastica van Gieson re-staining, was performed on 4 lmthick, paraffin-embedded sections of these specimens. The
polymer method was used for immunohistochemistry
according to standard techniques using the antibodies
listed in Table 2. Deparaffinized and hydrated sections
were treated with 0.3% hydrogen peroxide in methanol for
15 min to block endogenous peroxidase activity. Sections
for immunostaining of h-Caldesmon, CD3, CD8, CD79a,
CD68, and fascin were autoclaved in 10 mmol/L sodium
citrate buffer (pH 6.0) at 121  C for 10 min; sections for
immunostaining of CD4 and CD138 were autoclaved in
Tris-EDTA buffer (pH 9.0) at 121  C for 10 min to expose
antigens, and then cooled for 30 min. After rinsing in-318-

MODERN RHEUMATOLOGY

3

Table 2. Antibodies used in immunohistochemistry.
Antibody to

Type

Clone

a-SMA
h-Caldesmon
CD3
CD4
CD8
CD20
CD79a
CD138
CD68
S100
Fascin

M
M
M
M
M
M
M
M
M
R
M

1A4
h-CD
PS1
4B11
1A5
L26
HM57
MI15
PG-M1
Polyclonal
55K-2

Cell types identified

SourceDilution

Antigen retrieval

Pretreatment

SMAs
Differentiated SMAs
T-cells
helper/inducer T-cells
Cytotoxic/suppressor T cells
B-cells
B-cells
Plasma cells
Macrophages
S100a, S100b
Mature dendritic cells, endothelial cells

Dako, Glostrup, Denmark
Dako
Nichirei, Tokyo, Japan
Novocastra, Newcastle, UK
Novocastra
Dako
Dako
Dako
Dako
Dako
Dako

�100
�100
�1
�50
�100
�200
�200
�200
�50
�2000
�200

–
pH 6 CBpH 6 CBpH 8 EDTA
pH 6 CB–pH 6 CBpH 8 EDTA
pH 6 CB–pH 6 CB–
Autoclave
Autoclave
Autoclave
Autoclave–
Autoclave
Autoclave
Autoclave–
Autoclave

pH6 CB: pH6 citrate buffer solution; pH8 EDTA: pH8 EDTA buffer.

0.1 mol/L phosphate-buffered saline (PBS, pH 7.4), sections were incubated with affinity-purified primary antibodies at room temperature (RT) for 1.5 h. Thereafter,
they were incubated with Envision (þ) Dual Link peroxidase (Dako, Glostrup, Denmark) at RT for 30 min. The peroxidase reaction was visualized using 0.02% 3,30 diaminobenzidine tetrahydrochloride and 0.01% hydrogen
peroxide in 0.1 mol/L PBS. Finally, nuclear counterstaining
was performed with Mayer’s hematoxylin.
Assessment and statistical analyses
All slides including those used for HE staining, Elastica van
Gieson staining, and immunohistochemistry, were captured
using a whole-slide scanner (Nanozoomer, Hamamatsu
Photonics, Hamamatsu City, Japan), and viewed with theassociated proprietary viewing software. We first histologically reviewed all TAK specimens with Elastica van Gieson
staining, and thicknesses of intima, media, and adventitia
were measured in at least three regions (Figure 2). Mean
thickness of intima, media, and adventitia were then calculated. The ratio of average thickness between intima andadventitia to that of media was calculated, and specimens
with ratio <1 were designated acute phase, >1 and <2
chronic phase, and >2 healed phase, since evolution of
TAK is divided into these three phases and eventually progresses to exuberant adventitial and intimal fibrous thickening [9]. Each of the nine specimens was thus divided into
three parts representing acute, chronic, and healed phases(Table 1). This method was not applied to GCA specimens,
since adventitial thickening in proportion to disease progression is not common in GCA.
In assessing immunohistochemical results for smoothmuscle cells, three different squares, each 0.13 � 0.13 mm insize (an area of 0.169 mm2), were randomly chosen from theintima; numbers of positive cells per square for immunostaining of a-SMA and h-Caldesmon were counted in thesame fields in nine TAK and five GCA specimens. The average number of positive cells as well as the average ratio of
h-Caldesomonþ cells to a-SMAþ cells was calculated ineach specimen.
Concerning counting of inflammatory cells immunostained by the nine other antibodies, three TAK specimens
in healed phase were excluded, due to the virtual absenceof inflammatory cells in these samples. For counting of

inflammatory cells in the remaining six TAK and five GCA
specimens, three different squares, 0.13 � 0.13 mm in size
(an area of 0.169 mm2), representing the greatest inflammatory infiltrates were chosen from the intima, media, andadventitia, and the numbers of positive cells were counted.
Average positive cell numbers in the three squares werethen calculated in the intima, media, and adventitia ineach specimen. All counting was performed in the samesquares for inflammatory cells immunostained by the different antibodies. In the intima, sites within, or in thevicinity of an atheromatous plaque were avoided, to
exclude an influence of atheroma. In fascin immunohistochemistry, endothelial cells were excluded from counting,since this antibody also immunostains endothelium. The
ratio of CD4þ cells to CD8þ cells was also calculated.
Total average numbers in intima, media, and adventitia
were also calculated in each specimen for each immunohistochemical analysis. Differences in calculated average numbers between the six TAK and five GCA specimens were
statistically analyzed in the three different layers and as
total average numbers from three layers, using
Mann–Whitney tests. The level of significance was set
at p < .05.

Results
Histology and immunohistochemistry of smoothmuscle cells
The ratio of average thickness of intima to media was
135%, and adventitia to media was 174% in the nine TAK
specimens. The ratios of average thickness between intima
and adventitia to that of media were 0.30, 0.34, 0.69, 1.03,
1.18, 1.66, 2.20, 2.47, and 4.06, from which the first threecases were designated acute phase, the middle three cases
chronic phase, and the final three cases healed phase,according to the assessment described in the material andmethod section (Table 1).
Average ratio of h-Caldesmonþ cells to a-SMAþ cells in
the intima in TAK specimens was 30.8 ± 4.7% in acutephase, 55.0 ± 11.1 in chronic-phase, and 56.3 ± 5.3 in healedphase (Figure 3). Thus, while this ratio increased from theacute through chronic to the healed phase, it was almost thesame in the chronic and healed phases. The difference inthis ratio between acute versus chronic and healed phase

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A. KURATA ET AL.

Figure 2. Acute and healed phases of Takayasu arteritis shown by Elastica van Gieson staining and immunostaining of smooth muscle cells. Staining of acute (a)and healed (b) phase supplemented with length of intima (upper left), media, and adventitia indicated by different colored bars (�40). Immunostaining in smoothmuscle cells of the same field as (a) (c and e) and as (b) (d and f). Not only medial but also intimal smooth muscle cells are diffusely immunopositive for a-SMA (cand d). Although medial smooth muscle cells are diffusely immunopositive for h-Caldesmon, intimal smooth muscle cells are rarely immunopositive in the acutephase (e), while they are diffusely immunopositive in the healed phase (f). Insets show high power field of red-colored squares (�200).

(n ¼ 3 versus 6) was statistically significant byMann–Whitney test (p ¼ .02). Additionally, average ratio of
h-Caldesmonþ cells to a-SMAþ cells in the intima in GCA
specimens was 51.4 ± 21.3%.
Histology and general observation of
immunohistochemistry in inflammatory cells
Destruction of the media in TAK progressed from the acutethrough chronic to the healed stage, as paralleled by intimal
and adventitial fibrous thickening. Destruction of the media
started not only from the adventitial side but also from theintimal side (Figure 2(a)), and destruction from the intimalside was demonstrated to the same degree as that from theadventitial side in four (including three acute phase) out ofnine cases. Multinucleated giant cells appeared in only three
specimens in the acute phase of TAK, showing phagocytosisof medial elastic fibers. In GCA, multinucleated giant cells
appeared in four out of five specimens, with features ofphagocytosis of internal elastic lamina. In the remaining
specimen (63 years old female patient), calcification ofinternal elastic lamina was observed. All these giant cells
were immunohistochemically positive for CD68 inboth disorders.
Inflammatory cell infiltration was observed to variable
degrees in both TAK and GCA (Figures 4 and 5). Cells
tended to infiltrate abundantly in the adventitia anddecrease through media to intima in TAK, but infiltrateconsiderably also in the intima in GCA. The greatest inflammatory infiltrates in the adventitia of TAK tended to occur
around vasa vasorum of the aorta. In summary, while in-320-

MODERN RHEUMATOLOGY

Figure 3. Ratio of h-Caldesomonþ cells to a-SMAþ cells in the intima of TAK
specimens in acute phase (n ¼ 3) and chronic plus healed phase (n ¼ 6) as well
as GCA specimens (n ¼ 5) as visualized by average (x), median (–), interquartile
range (box), and min–max (I). Asterisk indicates p < .05 by Mann–Whitney test.

both disorders T-cell infiltrate was enriched, macrophages
moderately infiltrated, and dendritic cells were relatively lessevident, B-cells, and plasma cells were enriched in
TAK only.
Interrelationship between clinical data and immunohistochemical results was not evident except that glucocorticoids
treatment was not performed before the resection in all thethree patients with acute phase TAK.

5

(3–26), 7.3 (0.67–17), 5 (0.67–7.7), 5.7 (1–6.7), 18 (3.3–23),
4.7 (2.3–7.3), and 13 (8–16), respectively. CD4/8 ratio was
2.9 (0.88–7.1) in TAK and 5.6 (4.3–12) in GCA, which wassignificantly higher in GCA than in TAK. No other significant differences between the two disorders were observed in
the media, although prominent infiltration of B-cells andplasma cells was noted in a limited number of
TAK specimens.
In the intima, median values (min–max) of CD3þ,
CD4þ, CD8þ, CD20þ, CD79aþ, CD138þ, CD68þ, S100þ,and fascinþ cells per 0.169 mm2 were 18 (2.3–42), 14(1.3–21), 5 (0.33–9), 0 (0–5), 1 (0–7.7), 0.67 (0–33), 4.2(0.33–34), 0.5 (0–8.7), and 0.17 (0–8), respectively, in TAK;values were 30 (7.3–66), 16 (6–38), 4 (0–10), 2.3 (0–19), 4(0–17), 4.3 (0.33–12), 8.3 (7–10), 2.7 (1–6.7), and 8.7(3.3–11), respectively, in GCA. CD4/8 ratio was 4.2(1.3–7.9) in TAK and 6 (3.7–20) in GCA. Significantly
greater numbers of intimal fascinþ dendritic cells wereobserved in GCA than in TAK.
Regarding total average numbers from the three layers,
for CD3þ, CD4þ, CD8þ, CD20þ, CD79aþ, CD138þ,
CD68þ, S100þ, and fascinþ cells, median value (min–max)
per 0.169 mm2 were 78 (63–102), 52 (38–76), 19 (11–35), 43(13–95), 22 (16–60), 20 (2.3–44), 9.2 (4.8–19), 1.3(0.78–7.1), and 3.2 (1.1–15), respectively, in TAK; values
were 65 (17–83), 41 (13–63), 4.7 (2.3–19), 8.6 (3.9–32), 5.7(1–20), 5.3 (1.9–7.3), 12 (4.2–14), 4.6 (2.4–5.6), and 9(8.7–12), respectively, in GCA. CD4/8 ratio was 2.7 (2.4–7)
in TAK and 6.3 (3.5–12) in GCA. Thus, significantly more
CD8þ T-cells were found in TAK than in GCA, reflecting
the significantly higher CD4/8 ratio in GCA than in TAK.
Further, CD20þ B-cells were significantly more abundant in
TAK than in GCA.

Statistical analyses of immunohistochemistry ininflammatory cells

Discussion

Inflammatory cell infiltrates in the three individual layers
and total three layers for both disorders are compared in
Figure 6. In the adventitia, median values (min-max) ofCD3þ, CD4þ, CD8þ, CD20þ, CD79aþ, CD138þ, CD68þ,
S100þ, and fascinþ cells per 0.169 mm2 were 166 (120–233),
109 (81–182), 47 (20–91), 129 (33–265), 61 (19–105), 22(5.7–29), 4.3 (2–19), 1.7 (1.3–5.3), and 6.2 (1.3–19), respectively, in TAK; corresponding numbers in GCA were 66(12–96), 58 (11–81), 7.7 (3–20), 16 (4–70), 8 (0.3–34), 3.3(0.3–10), 4.6 (2.3–14), 4.7 (2–8), and 8.3 (6–15), respectively.
Significantly more CD3þ, CD4þ, and CD8þ T-cells; CD20þ
and CD79aþ B-cells; and CD138þ plasma cells wereobserved in TAK than in GCA. CD4/8 ratio was 3.1(1.2–5.8) in TAK and 6.9 (2.5–13) in GCA, which was significantly higher in GCA than in TAK.
In the media, median values (min–max) of CD3þ, CD4þ,
CD8þ, CD20þ, CD79aþ, CD138þ, CD68þ, S100þ, and fascinþ cells per 0.169 mm2 were 53 (14–115), 24 (6–76), 9.7(6.3–29), 3.6 (0–56), 15 (0–73), 28 (0.7–70), 15 (7.3–29), 1.8(1–7.7), and 3.6 (1.3–19), respectively, in TAK; corresponding numbers in GCA were 68 (18–129), 43 (14–101), 4.7

In the present study, we assessed TAK and GCA specimens
mainly through use of immunohistochemistry, with a viewto elucidation of similarities or differences between thesetwo disorders. To the best of our knowledge, such immunohistochemical comparisons have not been performed todate, except for one study which compared necrotizing aortitis in patients younger than 65 years suggestive of TAK
with non-necrotizing aortitis suggestive of GCA [10].
However, in that study, grouping did not conform to clinical criteria, cases suggestive of TAK exhibited no sex predominance, and inflammatory cells were assessed only inthe media and adventitia. In the present study we comparedcases of clinically diagnosed TAK with GCA and compared
three layers including intima. Furthermore, we used antibodies against h-Caldesmon for smooth muscle cells, CD79afor B-cells, CD138 for plasma cells, and fascin for dendritic
cells for the first time. Several novel features were found in
TAK compared with GCA, including greater adventitial
lymphoplasmacytic infiltration and smaller numbers of
intimal dendritic cells along with overall more
B-cells,
more CD8þ cells, and a lower CD4/8 ratio.

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Figure 4. Distribution of inflammatory cells shown by Elastica van Gieson and HE staining along with T-cell immunostaining in TAK and GCA. Elastica van Gieson
staining reveals adventitia to media of TAK (a), media to intima of TAK (b) and all three layers of GCA (c) (�200). The same fields as (a) (d, g, j, and m), (b) (e, h, k,
and n) and (c) (f, i, l, and o) are shown. Yellow dotted lines indicate borderline between adventitia (lt.) and media (rt.) in (d), between media (lt.) and intima (rt.) in(e), and between adventitia (lt.), media (center), and intima (rt.) in (f). Immunostaining for CD3 (g, h, and i), CD4 (j, k, and l), and CD8 (m, n, and o) shows variableinfiltration of T-cells. Numbers of CD4þ and CD8þ T-cells are almost the same in TAK (j versus m, and k versus n), but CD4þ T-cells outnumber CD8þ T-cells in GCA(l versus o).

The time course of TAK is assumed to progress fromacute through chronic to healed phase, and eventually progress to exuberant adventitial and intimal fibrous thickening[9]. Therefore, in the present study, we simply assessed
ratios of average thickness between intima and adventitia tothat of media, and designated specimens with ratios <1, >1
and <2, and >2 as acute, chronic, and healed phases,
respectively. Although this method has not been broadly
supported, we assume it to be appropriate, because giant
cells that have been reported to be characteristic of the early

granulomatous phase [2] appeared in only three specimens
with the acute phase; furthermore, inflammatory cells almost
disappeared in three specimens of the healed phase. In addition, the average ratio of h-Caldesmonþ cells to a-SMAþcells in the intima was significantly increased in the chronicand healed phases over that seen in the acute phase. Thisratio reflects maturity of vascular smooth muscle as we haverecently reported [11]. As we previously investigated thisratio in association with plaque stability in coronary andcarotid arteries [12,13], intimal smooth muscle may also be-322-

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7

Figure 5. Distribution of inflammatory cells shown by immunostaining for B-cells, plasma cells, macrophages, and dendritic cells in TAK and GCA. The same fields
as (a) in Figure 4 (a, d, g, and j), (b) in Figure 4 (b, e, h, and k) and (c) in Figure 4 (c, f, i, and l) are shown. Immunostaining for CD79a (a, b, and c), CD138 (d, e,
and f), CD68 (g, h, and i), and fascin (j, k, and l) shows variable infiltration of B-cells, plasma cells, macrophages, and dendritic cells, respectively. CD79aþ B-cells
and CD138þ plasma cells are rich in TAK (a, b, d, e) but scant in GCA (c, f). Giant cells are positive for CD68 in both disease states (g, i). Fascinþ dendritic cells are
predominantly found in the adventitia in TAK (j) but are rich in the intima in GCA (l).

divergent in TAK and mature over time. This ratio was also
divergent in GCA specimens, suggestive of similar timephase samples to those of TAK; however, statistical analysis
was not performed due to small sample size.
Medial destruction in TAK progressed from acutethrough chronic to healed stage, as paralleled by intimal andadventitial fibrous thickening. Destruction of media started
from not only the adventitial side but also the intimal side,
as shown by the same degree of medial destruction fromboth sides in four (including three acute phase) out of ninespecimens. This result appears to conflict with a previous
report documenting that inflammation commences from theadventitia and progresses to the intima in TAK [9].
However, our results also support greater adventitial thanintimal lymphocytic infiltration in TAK as mentioned
below. Furthermore, mean thickness ratio of adventitia tomedia (174%) was greater than that of intima to media(135%) in all nine TAK specimens, reflecting a more prominent cicatrix after inflammation in the adventitial side.

Multinucleated giant cells that appeared in three acute-phase
TAK specimens were all immunohistochemically positive for
CD68, suggesting derivation from macrophages, as has beenreported in giant cells in GCA [14,15], in contrast to the
hypothesis of smooth muscle-cell origin [2]. The features ofphagocytosis and calcification of internal elastic lamina seenin GCA were absent in TAK, but elastophagocytosis of
media was found. Therefore, even if initial elastophagocytosis were to start from the intimal side, subsequent inflammation is likely to persist mainly on the adventitial sidein TAK.
Concerning comparison of the distribution of inflammatory cells in TAK and GCA, CD3þ, CD4þ, and CD8þ Tcells; CD20þ and CD79aþ B-cells; and CD138þ plasma cells
were significantly more abundant in the adventitia in TAK
than in GCA. Although B-cells and plasma cells were obviously more abundant in TAK than in GCA also in the
media, their numbers did not show a significant difference,
presumably because of the relatively large standard

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Figure 6. Average numbers of inflammatory cell infiltrates in three individual layers and total average of three layers as visualized by average (x), median (–), interquartile range (box), and min–max (I). Asterisk indicates p < .05 by Mann–Whitney test.

deviation. In fact, prominent infiltration of B-cells andplasma cells in the media was noted in a limited number of
TAK specimens. Our results agree with those of a previous
study which reported that inflammatory cells, especially

CD20þ B-cells, were more abundant in the adventitia than
the intima in TAK; however, no giant cells were found intheir 10 specimens of TAK, suggesting the presence of only
chronic and healed phases in their specimens [16]. Other-324-

MODERN RHEUMATOLOGY

authors compared necrotizing aortitis in elderly patients(suggestive of TAK) with non-necrotizing aortitis (suggestiveof GCA) and demonstrated that medial T-cells were significantly more abundant in the latter, although no significant
difference was obtained in the adventitia [10], suggesting a
relatively greater extent of media-dominant (compared withadventitia-dominant) inflammation in GCA than in TAK.
S100þ dendritic cells were found in all three layers inboth TAK and GCA, but their numbers were generally
smaller than those of lymphocytes in the present study. This
result is in accordance with previously reported studies in
TAK and GCA [14,16]. Furthermore, fascinþ dendritic cells
were significantly more abundant in the intima in GCA
than in TAK. As we have previously reported in granulomatous disorders, fascin is a specific marker only for maturedendritic cells that are capable of antigen presentation [17].
Therefore, our present results indicate that antigen presentation in the intima is relatively lower in TAK compared with
GCA. This may support the notion that commencement ofinflammation by antigen-presentation predominantly occurs
in the intimal side (intima or media-dominant inflammation) in GCA, in contrast to TAK where inflammatory infiltrate is dominant in adventitia.
Regarding average numbers of inflammatory cells in thethree layers as a whole, CD20þ B-cells were significantly
more abundant in TAK than in GCA. In addition, althougha significant difference was only obtained in the adventitia,
CD79aþ B-cells along with CD138þ plasma cell infiltration
was more extensive in TAK than in GCA, as mentioned earlier. Transcription of CD79 protein can be identified in the
majority of B cells, similarly to CD20, but CD79 positivity
continues throughout B cell differentiation into the plasma
cell stage [18]. Therefore, these results may reflect abundant
plasmacytic infiltration in TAK. Indeed, characteristic plasmacytic infiltration in TAK has long been pointed out [2].
Furthermore, the role of B cells in formation of ectopic
lymphoid organs in the adventitia has recently been highlighted in active TAK specimens [19]. In addition, there
were significantly more CD8þ T-cells overall in TAK thanin GCA, reflecting a significantly higher overall CD4/8 ratio
in GCA than in TAK. Taken together, inflammatory cells in
TAK seem to be divergent including B-cells, plasma cells,and CD8þ T-cells, in contrast to GCA, which has beenreported to contain an infiltrate of mainly CD4þ T-cells andmacrophages [14,20,21].
Hypersensitivity pneumonitis and sarcoidosis, which arecommon in granulomatous disorders in the same organ, are
two different diseases, partly reflected by the significant difference in CD4/8 ratio in infiltrating T-cells [22]. Likewise,
we postulate that TAK and GCA are different disorders,since TAK involves adventitia-dominant divergent inflammatory infiltration, as opposed to GCA which is associatedwith intima-dominant, relatively uniform inflammatory
infiltration. Other authors have also recently highlighted thedifferences in these two disorders from the clinical standpoint [23,24].
Several limitations of the present study should be
acknowledged. First, the relatively small sample size may

9have reduced statistical power. For example, although infiltration of B-cells and plasma cells in the media is presumably characteristic of TAK as previously reported [2], thesefindings were not significantly different between TAK and
GCA. Second, out of 9 TAK patients, six were aged >40
and four were aged >50 at onset (Table 1), despite the factthat age restriction (�40 years) was included in the six criteria (adherence to three of these is required) of theAmerican College of Rheumatology in 1990 [25] and thatusual occurrence before the age of 50 years was stressed at
the Chapel Hill Consensus Conference 2012 [26]. However,recent studies by other authors included approximately 30%
of TAK patients aged >40 at onset [24,27], and these maintained that the age restriction (�40 years) may not be essential for diagnosis because clinical characteristics of earlyand late-onset TAK were comparable [24]. Third, theresected artery differed between the two disorders: mainly
aorta in TAK but exclusively temporal arteries in GCA.
Although involvement of large vessels including aorta has
been reported in 15–25% of GCA patients [3,28], description of their histology is limited to single case reports andto our knowledge, no immunohistochemical findings wereattached to these reports [29,30]. Further study is necessary
to confirm if inflammatory cell infiltrates in large vessels
compare with those in temporal artery in GCA.
In conclusion, we have conducted an immunohistochemical study of TAK and GCA specimens. Both disorders werecommon in granulomatous vasculitis with infiltration of
giant cells; however, significantly more adventitial lymphocytes and plasmacytes along with lower numbers of intimal
fascinþ dendritic cells, as well as overall more CD8þ T-cells,
more CD20þ B-cells, and a lower CD4/8 ratio were demonstrated in TAK than in GCA. These results suggest that the
inflammatory process in TAK may be different from that in
GCA; thus, the two disorders are likely to constitute twodifferent disease states.

Conflict of interest
None.

Funding
This research was supported by Grants-in-Aid for scientific research(C) [grant number 16K08675].

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open

Generation of mouse model of
TGFBI-R124C corneal dystrophy
using CRISPR/Cas9-mediatedhomology-directed repair
Kohdai Kitamoto1,9, Yukako Taketani1,2,9, Wataru fujii3, Aya Inamochi1, Tetsuya Toyono1,
Takashi Miyai1, Satoru Yamagami4, Masahiko Kuroda5, Tomohiko Usui1,6* & Yasuo Ouchi7,8*Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types ofcorneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C
mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic
mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODNmediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin
and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stromathat stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C
mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near
the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp)
was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the
mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial
wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of
TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help
delineate the pathogenic mechanisms of human corneal dystrophy.
Corneal dystrophy is a hereditary disease that causes corneal opacity; 22 types of corneal dystrophy are currently
classified according to the International Committee for Classification of Corneal Dystrophies (IC3D)01. Amongthese, transforming growth factor-beta-induced (TGFBI) corneal dystrophies occur most frequently in East Asian
populations2. TGFBI corneal dystrophies are caused by a point mutation, or insertion or deletion of some frames,
in TGFBI, which is located on chromosome 5q311,2. Various mutations in TGFBI cause corneal opacities withdifferent phenotypes, such as granular corneal dystrophy (GCD), lattice corneal dystrophy (LCD), Reis-Bücklers
corneal dystrophy (RBCD), and Thiel-Behnke corneal dystrophy (TBCD)3. There are genotype-phenotype correlations in TGFBI corneal dystrophy; for example, the R124H mutation causes GCD type2 (GCD2), and the
R124C mutation causes LCD type1 (LCD1)2. Even though we know that the corneal opacities are caused by accumulated TGFBI protein (TGFBIp)4, the mechanism or pathophysiology is not yet clearly understood5.
Animal models help elucidate the pathophysiology of corneal dystrophies. Previously, Bustamante et al. useda lentiviral vector to generate knock-in mice overexpressing the human R555W mutation, which causes GCD
type1 (GCD1)6. However, R555W mutant mice did not show any corneal phenotype according to this report6.
Tgfbi-knockout mice also failed to show corneal abnormalities even after systemic depletion of Tgfbi expression7. Recently, Yamazoe et al. established an R124H mutant transgenic mouse model showing corneal opacities8.
However, these mice harbour an artificial human TGFBI mutant gene cassette and do not reflect the complexity
1

Department of Ophthalmology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan. 2Schepens Eye
Research Institute, Boston, MA, USA. 3Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo,
Japan. 4Department of Ophthalmology, Nihon University, Tokyo, Japan. 5Department of Molecular Pathology, Tokyo
Medical University, Tokyo, Japan. 6Department of Ophthalmology, International University of Health and Welfare,Tokyo, Japan. 7Department of Regenerative Medicine, School of Medicine, Chiba University, Chiba, Japan. 8Gene
Expression Laboratory, Salk Institute for Biological Studies, Ja Jolla, CA, USA. 9These authors contributed equally:
Kohdai Kitamoto and Yukako Taketani. *email: tomohiko-tky@umin.ac.jp; o-yasuo@chiba-u.jp
Scientific RepoRtS |(2020) 10:2000 | https://doi.org/10.1038/s41598-020-58876-w

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www.nature.com/scientificreports/of the human disease. Therefore, it is imperative to develop physiologically relevant animal models to decipher
the mechanisms driving TGFBI corneal dystrophy.
The popularity of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9) gene-editing technology has increased dramatically because it enables rapid and precise genetic
manipulation of various cell types9–11. Moreover, CRISPR/Cas9 is a powerful one-step strategy for establishingknockout animal models because it allows single-nucleotide conversion via single-stranded donor oligodeoxynucleotide (ssODN) template-mediated homology-directed repair (HDR)12,13. Single-nucleotide polymorphism(SNP)-based animal models of human disease are a useful and physiologically relevant approach that reflects
point mutations or substitutions in the human genome.
To understand the effect of TGFBI mutation in corneal dystrophy, we generated mutant mice with the Tgfbi
R124C mutation, which is the representative mutation causing LCD1, using the CRISPR/Cas9 approach. We
then examined how the expression of this phenotype affected corneal integrity and wound healing in this mousemodel.

Results

Generation of mouse model of TGFBI-R124C corneal dystrophy using ssODN-mediated basepair substitution introduced via CRISPR/Cas9. We used CRISPR/Cas9-induced HDR-mediated nucle-

otide conversion technology to develop a clinically relevant mouse model of TGFBI corneal dystrophy14. We
targeted the arginine residue at position 124 of TGFBI; this amino-acid residue is important in the developmentof TGFBI corneal dystrophy. To generate Tgfbi R124C knock-in mice without inducing off-target cleavage of
CRISPR/Cas9, we took advantage of Cas9 nickase (Cas9D10A), a mutated Cas9 that makes single-strand breaks,
which allows us to generate region targeted knock-in mice without off-target mutations13,15,16. Two single-guide
RNAs (sgRNAs) were designed to target exon4 of Tgfbi (Fig. 1a). To convert arginine 124 into cysteine, we
designed a 107 nt ssODN containing the BsrGI restriction enzyme site (Fig. 1b). The BsrGI restriction enzymesite enables easy genotyping of TGFBI-R124C mice by PCR-RFLP analysis, without affecting the amino-acidsequence of the Tgfbi R124C protein. A mixture of Cas9D10A mRNA, sgRNAs, and ssODN was injected into
zygotes, as reported previously13. Then, two-cell embryos were transferred into pseudopregnant recipient female
mice. The genotype of the resultant pups was examined by PCR-RFLP analysis. We PCR amplified 475 bp fragment around the target site and digested the PCR product with BsrGI restriction enzyme. As the PCR productincluding a cleavage site for BsrGI, PCR-RFLP digestion using BsrGI produced an undigested 475 bp fragment
in wild-type mice, and fragments of 253 and 222 bp in homozygotic TGFBI-R124C mice (Fig. 1c). The genotype of the pups was further examined using DNA sequencing, and 5 in 19 pups had the TGFBI-R124C mutant
allele. DNA sequencing of TgfbiR124C/R124C mice confirmed that the mutation had been successfully induced(Fig. 1d). One of the mutant mice was used for breeding, and the descendants were used for the experiments.
Both TgfbiR124C/R124C and TgfbiR124C/wt mice were viable and fertile, showing normal body weight and life span.

TGFBI-R124C mice develop corneal opacity with high frequency.

We used a stereomicroscope to analyse the effects of the TGFBI-R124C mutation on the development of corneal opacity in mice. In
TGFBI-R124C homozygous mice, corneal opacity was first observed at 12 weeks of age and tended to graduallyworsen until 24 weeks of age. While wild-type littermates (n = 9) did not show any substantial changes in thecorneas (Fig. 2a), all corneal opacity was observed in the central cornea of both TGFBI-R124C homozygous andheterozygous mice (Fig. 2b,c). In TGFBI-R124C homozygous mice, corneal opacity was observed in 51 of 71 mice
(71.8%) at 20 weeks of age and in 57 mice (80.3%) at 40 weeks of age (Fig. 2d). On the other hand, corneal opacity
was not observed in any TGFBI-R124C heterozygous mice below 24 weeks of age, and only 1 of the11 mice (9.1%)
developed bilateral corneal opacity until 40 weeks of age (Fig. 2d). Over 71% of the diseased TGFBI-R124C
homozygous mice showed bilateral corneal opacity at 12 weeks of age, and the percentage gradually increased to
91% at 40 weeks of age, whereas 5 mice developed unilateral corneal opacity. In summary, total number of the eye
showed corneal opacity at 40 weeks of age are 105 (73.9%) and 2 (9.1%) in the TGFBI-R124C homozygous andheterozygous mice, respectively (Table 1).

Abnormal deposits in the subepithelial stroma of TGFBI-R124C mice.

We used histological analysis to evaluate corneal opacity in TGFBI-R124C mice. As demonstrated in Fig. 3a,b, several parts of the subepithelial stroma showed degeneration and fibrosis. No inflammatory cell infiltration was observed. These areas werepositive for Masson’s trichrome staining, which indicates an increase in collagen fibres (Fig. 3c,d). All corneal
tissues were negative for Congo-red staining (Fig. 3e,f). Transmission electron microscopy (TEM) imaging of
the corneas showed pathognomonic, irregular, and amorphous deposits in the subepithelial stroma (Fig. 3g,h).

TGFBIp deposits in the subepithelial stroma of TGFBI-R124C mice. To assess whether Tgfbi muta-

tions result in deposits of TGFBIp in the corneas of TGFBI-R124C mice, we analyzed the expression levels of the
Tgfbi gene and the TGFBIp protein. First, we performed immunohistochemical analysis to determine the localization of TGFBIp. In the corneas of wild-type mice, TGFBIp immunoreactivity was mainly observed in the corneal epithelium (Fig. 4a). In the corneas of TGFBI-R124C homozygous mice, TGFBIp expression was observedin all the corneal layers (Fig. 4a), whereas no significant staining was observed in control sections.
Expression of Tgfbi mRNA in the corneas of wild-type mice and homozygous mutant mice was examined via
real-time reverse transcription-polymerase chain reaction (RT-PCR). Corneal expression of Tgfbi mRNA did
not significantly differ between wild-type and TGFBI-R124C mice (Fig. 4b; p = 0.31). Conversely, expressionof TGFBIp in the cornea of homozygous mice was significantly increased compared with that of wild-type mice
(Fig. 4c,d; p = 0.030, supplementary file).

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Figure 1. Generation of TGFBI-R124C mouse model via offset-nicking by CRISPR/Cas9 system. (a,b)
Schematic representations of targeting strategy for the generation of TGFBI-R124C mice. Two sgRNAs
were designed and in vitro-transcribed to target exon 4 in Tgfbi. The red arrowhead indicates the expectedcleavage site for sgRNAs (a). Schematic of sgRNAs and ssODN donor sequences designed to target Tgfbi
for HDR-mediated knock-in via CRISPR/Cas9 (b). The target nucleotide sequence used to induce TGFBIR124C mutation is indicated in red. The red arrowhead indicates the expected nicking sites for sgRNAs. Tgfbi
sgRNA#1 and Tgfbi sgRNA#2 are marked in blue. Protospacer adjacent motifs (PAMs) are highlighted in pink.
(c) PCR-RFLP genotyping of TGFBI-R124C mutant mice. The image shows an undigested 475 bp fragment inwild-type mice; fragments of 253, 222 and an undigested fragment of 475 bp in heterozygous TGFBI-R124C
mice; and fragments of 253 and 222 bp in homozygous TGFBI-R124C. (d) Representative sequencing results
used to validate the mutation in Tgfbi of TGFBI-R124C homozygous mutant mice. In TGFBI-R124C mice, abase substitution (CGC → TGT) is observed.

TGFBI-R124C mutation affects corneal wound healing. Most patients with R124C LCD present with
impaired corneal wound healing17; therefore, we examined whether the TGFBI-R124C mutation affected woundhealing in TGFBI-R124C homozygous mice. The areas bearing corneal epithelial defects showed no significant
differences in wild-type and TGFBI-R124C mice at 24 hours after induction of corneal abrasion. However, at
48 hours after induction of corneal abrasion, the area bearing an epithelial defect in TGFBI-R124C mice waslarger than that in wild-type mice (Fig. 5a,b; p = 0.011).

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Figure 2. Corneal opacity in TGFBI-R124C mice. (a–c) The representative pictures of wild-type at 28 weeks
of age mouse and TGFBI-R124C mice eyes at 32 weeks of age. Corneal opacity is not observed in wild-typemice (a). Corneal opacity is observed in the centre of the cornea in heterozygous (b) and homozygous mice (c).
Dashed line represents corneal opacity. (d) Frequency of corneal opacity by genotype. In TgfbiR124C/R124C mice,corneal opacity occurred in 78.9% at 24 weeks of age. Then the frequency of corneal opacity gradually increased
to 80.3% of mice at 40 weeks of age. Over 91% of the diseased TGFBI-R124C homozygous mice showed bilateralcorneal opacity at 40 weeks of age. In eleven TgfbiR124C/WT mice (Het), bilateral corneal opacity was observed
one mouse (9.1%) before 24 weeks of age. No corneal opacity was observed in wild-type mice (Wt).

Genotypes
TgfbiWT/WT
Total number of eyes analyzed n = 18

TgfbiR124C/WT TgfbiR124C/R124C
n = 22n = 142Age (weeks)

n

%

n

%

n

%

≦20

0

0

0

0

87

61.3

≦40

0

0

2

9.1

105

73.9

Table 1. Summary of the corneal opacity observed in the TGFBI-R124C mutant mice eye in this study.

Discussion

The mechanisms driving the formation of corneal opacity in TGFBI corneal dystrophy are largely unknown5.
Establishing clinically relevant mouse models of TGFBI corneal dystrophy would be very helpful to understandthe mechanisms or pathophysiology underlying corneal opacity. Two animal models of TGFBI corneal dystrophy
have been established via introduction of human mutant TGFBI genes into mice6,8. However, these models weregenerated by artificial induction of human genetic mutations, and the frequency of corneal opacity observed wasinsufficient to explain the patient phenotype. Therefore, these models are inadequate for studying the pathogenesis of TGFBI corneal dystrophy.
In this study, we established a mouse model of TGFBI corneal dystrophy using HDR-mediated base substitution introduced via CRISPR/Cas9. To the best of our knowledge, this study is the first to show that a base substitution introduced via CRISPR/Cas9 into Tgfbi can induce the development of corneal dystrophy.
In TgfbiR124C/R124C mice, more than 70% of eyes developed corneal opacity by 40 weeks of age (Table 1). The
frequency of corneal opacity in our mutant mice was nearly four times greater than that in previously establishedmouse models8. On the other hand, no corneal opacity was observed in TgfbiR124C/WT mice up to 20 weeks of age,and the frequency of corneal opacity by 40 weeks was lower than that in TgfbiR124C/R124C mice. In cases of TGFBI
corneal opacity, homozygous patients have earlier onset than heterozygous patients18; our finding is consistent
with that. Among the mutant mice produced by Yamazoe et al., 8% of heterozygous mutant mice were foundto have corneal opacity by 40 weeks of age8; the same result was obtained for the TGFBI-R124C mice that westudied.
Although corneal opacity was mostly bilateral in the TgfbiR124C/R124C mice, some mice developed unilateralcorneal opacity (Fig. 2d). Among the TGFBI-R124C mice with unilateral corneal opacity, some mice developed
bilateral corneal opacity. In cases of TGFBI corneal dystrophy, some cases with unilateral corneal opacity developed corneal opacity in the other eye several years later19. It is possible that even in the mice that developed unilateral corneal opacity during our study, bilateral corneal opacity could occur later.
In patients with TGFBI corneal dystrophy (such as lattice and granular dystrophy), deposits and opacities
occur in the centre of the cornea3,20. Consistent with the patterns observed in human corneal dystrophy, we

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Figure 3. Microscopic examination of the cornea in wild-type and TGFBI-R124C mutant mice. Corneal
sections of TGFBI-R124C a homozygous mouse at 22 weeks of age and a wild-type mouse at 24 weeks ofage. (a,b) H&E staining of corneal tissue shows eosinophilic areas of subepithelial stroma (arrowhead) andno inflammatory cell infiltrate in TGFBI-R124C homozygotes. (c,d) Masson trichrome staining showed
faint positive staining in the corneal tissue of homozygotic TGFBI-R124C mice (arrowhead). (e,f) Congored staining of corneal tissue was negative for both homozygotic TGFBI-R124C and wild-type mice. (g,h)
Transmission electron microscopy (TEM) images of corneal tissues. TEM revealed irregular and amorphousdeposits in the subepithelial stroma (red arrowhead). Scale bar: 50 µm in (a–f), 2 nm in (g,h).
detected a well-defined mass in the central cornea of the TGFBI-R124C mice (Fig. 2b,c). Corneal deposits in
TGFBI-R124C homozygous mice appeared more frequently and developed earlier than in TGFBI-R124C heterozygous mice. Human patients bearing TGFBI-R124H homozygous mutations present with more severe corneal
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Figure 4. Expression levels of Tgfbi mRNA and protein in the cornea of wild-type and TGFBI-R124C mutant
mice. (a) Immunohistochemical analysis of TGFBI expression in the cornea of wild-type and homozygotic
TGFBI-R124C mice. TGFBI expression was detected in all the corneal layers of homozygotic TGFBI-R124C
mice, with particularly strong expression around deposit sites (red arrowhead). No detectable staining wasobserved in the control sections. Scale bar = 50 µm. (b) Real-time RT-PCR analysis of Tgfbi mRNA levelsin the corneas of wild-type and TGFBI-R124C mutant mice. The expression level of Tgfbi was calculatedusing the ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected to normalise
the gene expression. No significant difference was observed between wild-type and TGFBI-R124C mice(p = 0.31). (c)Western blot analysis of Tgfbi protein levels in the corneas of wild-type and TGFBI-R124C
mutant mice. d. Comparison of Tgfbi protein expression levels in the corneas of wild-type (n = 4) and TGFBIR124C homozygotic mice (n = 4). Expression levels of Tgfbi protein were normalised to Gapdh expression. A
significant difference was observed between wild-type and TGFBI-R124C mice (p = 0.030).

opacity than patients harbouring heterozygous mutations18,21. Taken together, our mice model presented very
similar symptoms to human corneal dystrophy.
Consistent with patterns of TGFBI corneal degeneration in humans2, the deposits in TGFBI-R124C homozygous mice showed eosinophilic staining (Fig. 3b). The deposits caused by the TGFBI-R124C mutation consist ofamyloids3, which are positively stained by Congo-red22. However, positive Congo-red staining was not observedin TGFBI-R124C mice (Fig. 3f). Conversely, the deposits were stained faintly positive by Masson trichrome staining, revealing the presence of hyaline substance (Fig. 3d). These discrepancies may result from differences indegradation of TGFBIp between humans and mice. Corneal opacity manifests as LCD when arginine-124 is
replaced with cysteine, as GCD1 when it is replaced with leucine, and as GCD2 when it is replaced with histidine.
Korvatska et al. speculated that these phenotypic variations in the degradation process are due to this mutation23.
There are differences in the fragment size of keratoepithelin in the cornea, for each of these diseases23.
Conversely, some cases of TGFBI-R124C mutation do not result in amyloid deposits24,25. The pathway of
TGFBIp degradation is still unclear. Nonetheless, the absence of amyloid deposits in our study may be because
TGFBIp in the TGFBI-R124C mice was processed similarly to how it is processed in GCD1 in humans. In
humans, GCD1 is caused by the R124L mutation and does not involve amyloid deposits.
The observation periods that we used maybe another reason why we did not observe amyloid deposit formation in TGFBI-R124C mice. In mouse models of amyloid-associated diseases, the formation of amyloid plaques
occurs via accumulation of mutant protein and requires a certain length of time. For example, Tg2576 mice,
which are used in the well-known model of Alzheimer’s disease, are generated by overexpressing a mutant formof amyloid precursor protein26; however, 11–13 months are required to develop sufficient deposits of amyloid

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Figure 5. Corneal wound healing in wild-type and TGFBI-R124C mutant mice. (a) Representative images ofcorneal epithelial fluorescein staining at 0, 24, 48, and 72 h, after 2 mm long corneal debridement were induced
in wild-type (n = 11) and TGFBI-R124C mutant (n = 13) mice. Dashed line represent stained area. (b) Bar
graph showing the corneal epithelial healing rates in wild-type and TGFBI-R124C homozygotic mice after
induction of 2 mm long corneal debridement. At 48 hours after induction of corneal abrasion, the area bearing
an epithelial defect in TGFBI-R124C mice was larger than that in wild-type mice (p = 0.011).
plaque26,27. Because we did not use artificial overexpression of mutant genes to establish our mouse model of
TGFBI corneal dystrophy, our mice likely required more time to form amyloid plaques. Furthermore, because thelifespan of mice is shorter than that of humans, it is possible that there was not sufficient time to form amyloid
plaques in our mice.
Under TEM, hyaline in GCD1 or GCD2 appears as unstructured club-shaped or trapezoidal deposits3. In
TGFBI-R124C mice, hyaline appeared as an unstructured amorphous deposit, unlike the hyaline observed inhuman TGFBI corneal dystrophy. Although the reason for this discrepancy is unclear, deposits in R124H transgenic mice also presented an amorphous appearance, similar to that in our TGFBI-R124C mice8. Different mechanisms may drive hyaline formation in human and mouse corneas.
Corneal deposits in TGFBI corneal dystrophy may be caused by abnormal accumulation of TGFBIp resulting from mutated TGFBI4. Our results support this notion because sites with aggregations of TGFBIp also
showed eosinophilic deposits in our study (Fig. 4a). TGFBIp was expressed at higher levels in the corneas of
TGFBI-R124C mice than in those of wild-type mice (Fig. 4d). Nevertheless, the expression levels of Tgfbi mRNA
did not differ significantly between wild-type mice and TGFBI-R124C mice (Fig. 4b). Therefore, accumulationsof TGFBIp in the corneas of TGFBI-R124C mice may be caused by abnormal TGFBIp degradation and notenhanced the production of Tgfbi. Choi et al. previously reported that TGFBIp is usually digested via autophagy,
but mutant TGFBIp inhibits the fusion of autophagosomes and lysosomes, which results in TGFBIp accumulation28. These mechanisms will be examined further in our future studies of TGFBI-R124C mice.
Patients with TGFBI corneal dystrophy often present with recurrent erosion of the corneal epithelium29. In
LCD involving R124C mutations, regeneration of corneal epithelium after corneal erosion or phototherapeutic
keratectomy requires an extended length of time17,30. Our results indicate that regeneration of the corneal epithelium after induction of corneal abrasion required more time in TGFBI-R124C mice than in wild-type mice. The
domain containing R124 in fascilin1-1 (FAS1-1) is used for binding to integrin2. TGFBIp mediates cell adhesion
and spreading via integrin31. A study using keratocytes has shown that TGFBI is involved in the proliferation,adhesion, and migration of corneal epithelium32. Mutated TGFBIp may have influenced the repair of epithelial
wounds in our mice. Also, we assume that mutant TGFBIp affected the basement membrane function of epithelial
cells because TGFBIp aggregated near the basement membrane in our mouse model. Taken together, our findings suggest some insights into the kinds of mechanisms underlying delayed wound healing in human corneal
dystrophy.
In this study, we established the TGFBI-R124C mutant mouse model. The mice showed corneal opacity similar to that observed in human TGFBI corneal dystrophy. The TGFBI-R124C mutant mice might not be reliable
as a model of LCD1, given that amyloid deposition was not detected in these mice. Nonetheless, our findings
reveal that the corneal opacity was caused by the accumulation of mutant TGFBIp, similar to that observed in
TGFBI corneal dystrophy in humans. Epithelial wound healing was delayed in TGFBI-R124C mice comparedwith wild-type mice. This delayed wound healing in mutant mice was similar to that observed in human patientswith R124C LCD. Characterising corneal dystrophy via our TGFBI-R124C mouse model will help to uncover themechanisms driving the pathogenesis of human TGFBI corneal dystrophies.
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Material and Methods

Ethics statement. All animal care and experiments involving animals were conducted in full compliancewith the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The study was approved by
the Animal Research Committee of The University of Tokyo.
SgRNA design and plasmid construction
We first designed the sgRNAs at the following loci;sgRNA target1: 5′-CAGGCCTCAGCTTTTCTGTGCGG [Chr13: 56625270 to 56625292 (GRCm38.p4)]
sgRNA target2: 5′-GAGGCCTGAGATGGAGGGACCCGG [Chr13: 56625285 to 56625308 (GRCm38.p4)].
To construct the plasmid encoding sgRNA target#1 and sgRNA target#2 for Tgfbi, the respective oligonucleotides corresponding to the protospacer of each sgRNAs were phosphorylated using T4PNK (Takara Bio Inc.,
Shiga, Japan), annealed, and cloned into a pGEM-T-Easy-T3-BB-sgRNA plasmid via the BbsI restriction enzyme
site, as described previously33. The oligonucleotide sequences are as follows:
Tgfbi-sgRNA#1-Forward: AAGGCAGGCCTCAGCTTTTCTGTG
Tgfbi-sgRNA#1-Reverse: AAACCACAGAAAAGCTGAGGCCTG
Tgfbi-sgRNA#2-Forward: AAGGGGAGGCCTGAGATGGAGGGACC
Tgfbi-sgRNA#2-Reverse: AAACGGTCCCTCCATCTCAGGCCTCC
In-vitro transcription of sgRNAs and Cas9 nickase mRNA
SgRNAs and Cas9-nickase (Cas9D10A) mRNA were prepared as described previously13. Briefly, the constructed
sgRNA plasmids and pCAG-T3-hCasD10A-pA (Addgene#51638)13 plasmid were linearised using the DraI and
SphI, restriction enzymes, respectively. SgRNAs and Cas9D10A mRNA were transcribed in vitro using T3-RNA
polymerase (Promega, CA), according to the manufacturer’s protocol. The template DNAs were digested with 1
U of TURBO DNase (Thermo Fisher Scientific, MA) at 37 °C for 30 min. Then the RNA transcripts were extractedwith phenol: chloroform precipitated with absolute ethanol with ammonium acetate, washed, dried, and resuspended in RNase-free water. The RNA solutions were stored at −80 °C until use.

Establishment of TGFBI-R124C mouse model. In the 124th amino acid of mouse Tgfbi exon 4, arginine(CGC) was replaced with cysteine (TGT) by offset-nicking of CRISPR/Cas9 system13 with using the two sgRNAs.
The ssODN was designed as follows:
5 ′- TATG AG AC CATG GG AG TT GT GG GATC GACC AC CACACAGC TG TATACAGATG TACAGAAAAGCTGAGGCCTGAGATGG AGGGACCCGGAAGCTTCACCATCTTTGC-3′
Mutant mice were generated as reported previously13. Briefly, 100 ng/μl Cas9D10A mRNA, 10 ng/μl of eachsgRNAs, and 200 ng/μl ssODN were injected into the zygotes of C57BL/6NCr mice. The two-cell embryos weretransferred into the oviductal ampullae of 8-week-old female mice from the Institute of Cancer Research (ICR)
mated with vasectomized ICR males.
After birth, genomic DNA was extracted from the tail tip using Nucleospin Kit (TaKaRa Bio Inc., Shiga, Japan).
The obtained sample was amplified via the following primers using a KOD FX kit (Toyobo Co., Ltd., Osaka, Japan):
R124C-Forward: 5′-CATCTGACTCCTGCGGTTCC-3′
R124C-Reverse: 5′-CTTTCACTTTCCTTGGGGCTG-3′
PCR products were then sequenced to ensure that the mutation had been introduced.

RFLP assay. RFLP assay was performed to assess the genotype of mice used in this study. DNA was extracted

from the mice, and the mutation site was amplified via PCR as described in the previous section. The obtained
PCR products were treated with the restriction enzyme BsrGI (New England Biolab, Ipswich, MA) and electrophoresed on a 2% agarose gel.

Determination of corneal opacity. Eighteen eyes from 9 wild-type mice, 22 eyes from 11 heterozygous mice,and 142 eyes from 71 homozygous mice were examined using a slit lamp. The examination was performed undergeneral anaesthesia induced via intramuscular injection of a xylazine (5 mg/ml) and ketamine (50 mg/ml) mixture.
Examinations were conducted every month from the age of 12 weeks to 40 weeks. Mice were divided into those withbilateral or unilateral corneal opacity. The age of the mice and the presence of corneal opacity was recorded.
Histology. Eyes were removed from 22-week-old mice under general anaesthesia induced as mentioned previously. The eyes were fixed in 4% paraformaldehyde, after which the corneal buttons were excised, subjected tostandard processing, embedded in paraffin, and sectioned at 5–7 μm. These sections were then deparaffinised
using xylene, dehydrated using graded ethanol, and stained using H&E, Congo-red to detect the presence ofamyloids and Masson’s trichrome to detect hyaline, as per the manufacturers’ instructions.
The deparaffinised sections were also evaluated using immunohistochemistry. The sections were washed with
PBS, blocked with 5% skim milk for 30 min at room temperature, and incubated with a rabbit anti-TGFBI antibody (Proteintech Group Inc., Rosemont, IL;#10188-1-AP, 1:1000) for 60 min at room temperature. Adjacentsections were processed without primary antibody used as negative controls. Peroxidase-labelled anti-rabbit polyclonal antibody (Nichirei Biosciences Inc., Tokyo, Japan, # 714341) was used as the secondary antibody and thesections were incubated for 30 min at room temperature. Immunoreactivity was visualized using the Dako Liquid
DAB + Substrate Chromogen System (Agilent Technologies, Inc., Santa Clara, CA, # K3467).
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Transmission electron microscopy. Enucleated eyeballs were fixed using 4% paraformaldehyde and 2.5%

glutaraldehyde and then embedded in epoxy resin. Ultrathin sections were double-stained with 2.5% gadolinium
acetate and lead citrate and examined using an electron microscope (JEM1200EX II, JEOL Ltd., Tokyo, Japan).

Real-time reverse transcription PCR.

Total RNA, obtained from mouse corneas, was processed using
ISOGEN (Nippon Gene, Toyama, Japan). cDNA was obtained using PrimeScript RT Master Mix (TaKaRa BioInc.). Real-time RT-PCR was performed using SYBR Premix Ex Taq II (Tli RNaseH Plus, TaKaRa Bio Inc.).
Thermal cycling conditions for RT-PCR were as follows: 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The expression level of the target gene was calculated using the ΔΔCT method. Glyceraldehyde 3-phosphate dehydrogenase(GAPDH) was selected to normalize the gene expression. Primers used for mouse Tgfbi and Gapdh are shown
below.
Tgfbi-mRNA-Forward: 5′-AGAGGAAGATCTGCGGCAAG-3′
Tgfbi-mRNA-Reverse: 5′-TCTCGGCAGGGATCTTCTCA-3′
Gapdh-mRNA-Forward: 5′-CACATTGGGGGTAGGAACAC-3′
Gapdh-mRNA-Reverse: 5′-AACTTTGGCATTGTGGAAGG-3′

Western blot analysis.

Corneas were ground in liquid nitrogen and then homogenised in 90 μl radioimmunoprecipitation assay (RIPA) buffer on ice. Homogenised samples were agitated for 2 h and centrifuged for
15 min at 4 °C at 14,000 × g. Then, 10 μg protein was loaded per lane, separated on a 10% acrylamide gel (Bio-Rad,
Richmond, CA) via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred topolyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo System (Bio-Rad). Membranes were
blocked using Blocking One solution (Nacalai Tesque, Inc., Kyoto, Japan) and incubated with a rabbit anti-TGFBI
antibody (1:500, Protein Tech) or mouse anti-GAPDH antibody (1:1000, Abcam, Cambridge, UK, # ab8245)
overnight at 4 °C. After washing with TBS-T, the membranes were incubated with HRP-conjugated secondary
antibody (Goat anti-rabbit IgG, 1:5000,# 32460 or Goat anti-mouse IgG, 1:5000,# 32430) at room temperaturefor 3 h. Immunodetection was performed via chemiluminescence using Super Signal West Femto Maximum
Sensitivity Substrate (Thermo Fisher Scientific, IL) and ImageQuant LAS 4000 mini (Fujifilm, Tokyo, Japan). The
expression levels of TGFBIp were evaluated with respect to that of GAPDH.

Corneal wound healing model. Corneal epithelial debridement was performed using a previously
reported method with several modifications34. A circle of filter paper (diameter 2 mm) was soaked with 70%
EtOH and placed onto the central cornea for 30 s; then, the corneal epithelium was debrided with a #11 scalpel.
The cornea was evaluated using fluorescein staining at 0, 24, 48, and 72 h after debridement. The wound area wascalculated using ImageJ (https://imagej.nih.gov/ij/).
Statistical analysis. Comparisons between groups were performed using the Wilcoxon rank-sum test.
P < 0.05 was considered statistically significant. All statistical analyses were performed using JMP Pro 14 (SASInstitute, Cary, NC).

Data availability

The datasets analyzed during the current study available from the corresponding author on reasonable request.
Received: 18 September 2019; Accepted: 6 December 2019;
Published: xx xx xxxx

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20. Eifrig, D. E., Afshari, N. A., Buchanan, H. W. IV, Bowling, B. L. & Klintworth, G. K. Polymorphic corneal amyloidosis: A disorderdue to a novel mutation in the Transforming Growth Factor β-Induced (BIGH3) gene. Ophthalmology 111, 1108–1114 (2004).
21. Okada, M. et al. Severe corneal dystrophy phenotype caused by homozygous R124H keratoepithelin mutations. Investig. Ophthalmol.
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23. Korvatska, E. et al. Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at
Arg-124 are associated with abnormal turnover of the protein. J. Biol. Chem. 275, 11465–9 (2000).
24. Morishige, N. et al. Unusual Phenotype of an Individual With the R124C Mutation in the TGFBI Gene. Arch Ophthalmol 118,
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Acknowledgements

This work was supported by a grant-in-aid for Scientific Research from the Japanese Ministry of Education,
Culture, Sports, Science and Technology. We would like to thank Azusa Kamikawa, assistant researcher, for her
assistance with the experiments.

Author contributions

K.K. and Y.T. conducted the experiments and prepared all figures, with equal contributions. K.K. wrote the initial
draft of the manuscript. W.F. generated the mutant mice. A.I., T.T. and T.M. supported the experiments. S.Y. and
M.K. contributed to data collection and interpretation and critically reviewed the manuscript. T.U. and Y.O.
designed the study, and wrote the manuscript as corresponding authors. The final version of the manuscript wasapproved by all authors.

Competing interests

The authors declare no competing interests.

Additional information

Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-58876-w.
Correspondence and requests for materials should be addressed to T.U. or Y.O.
Reprints and permissions information is available at www.nature.com/reprints.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps andinstitutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
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© The Author(s) 2020

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REGULAR ARTICLE

Replenishing exosomes from older bone marrow stromal cells with
miR-340 inhibits myeloma-related angiogenesis
Tomohiro Umezu,1,2 Satoshi Imanishi,2 Kenko Azuma,2 Chiaki Kobayashi,1,2 Seiichiro Yoshizawa,1 Kazuma Ohyashiki,1 andJunko H. Ohyashiki2
1

Department of Hematology and 2Department of Molecular Oncology, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan

Key Points• Exosomal miR-340 derived from young
BMSCs inhibited tumor
angiogenesis via the
HGF/c-MET signaling
pathway.
• The anti-angiogenic effect of exosomes fromolder BMSCs was restored by direct transfection of young
BMSC-derived exosomal miRNAs.

The study of bone marrow stromal cells (BMSCs) and the exosomes they secrete is considered
promising for cancer therapy. However, little is known about the effect of donor age on
BMSCs. In the present study, we investigated the therapeutic potential of BMSC exosomes
derived from donors of different ages using an in vivo model of hypoxic bone marrow in
multiple myeloma (MM). We found that donor age was strongly related to senescent changes
in BMSCs. Exosomes derived from young BMSCs signiﬁcantly inhibited MM-induced
angiogenesis in Matrigel plugs. The exosomal microRNA (miRNA) expression proﬁle wasdifferent between young and older BMSCs, despite similarities in the size and quantity of
exosomes. Of note was the observation that the antiangiogenic effect of older BMSCs wasenhanced by direct transfection of miR-340 that was preferentially expressed in exosomes
derived from young BMSCs. We found that miR-340 inhibited angiogenesis via the hepatocyte
growth factor/c-MET (HGF/c-MET) signaling pathway in endothelial cells. Our data provide
new insights into exosome-based cancer therapy by modiﬁcation of BMSC-derived exosomes.

Introduction
The tumor microenvironment has gradually been recognized as a key contributor to cancer progression,angiogenesis, and metastasis1 and to the development of drug resistance. 2 Therefore, future
development of anticancer therapies3 should consider the tumor microenvironment. Bone marrowstromal cells (BMSCs) interact with multiple myeloma (MM) cells in the bone marrow (BM). BMSCs also
create a permissive microenvironment for MM cell growth and survival.4 An increase of angiogenesis is
involved in the progression of MM and the cell-to-cell contact between MM cells, whereas BMSCs
regulate the secretion of key mediators of angiogenesis, such as vascular endothelial growth factor andbasic fibroblast growth factor.5
It is well established that MM is an incurable disease. It commonly occurs in older people with a medianage at diagnosis of approximately 70 years.6,7 A previous study by Andre et al demonstrated that
BMSCs in MM patients have an early senescent profile.8 Furthermore, BMSC aging results in adecrease of their ability to maintain the BM microenvironment, leading to increases in the incidenceof hematological malignancies, anemia, and immune dysfunction.9,10 Recent evidence indicates that
exosome-mediated cell-to-cell communication plays an important role in maintaining the BM
microenvironment.11
Exosomes are 30- to120-nm membrane-bound vesicles secreted naturally by almost all cells and exist in
all body fluids.12 Accumulating evidence has shown that exosomes contain proteins, lipids, DNA,
messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA that can be transferred fromproducer cells to recipient cells, thereby facilitating cell-to-cell communication.13-16 More recent studies
in animal models have indicated that healthy BMSC-derived exosomes are effective for treatment ofSubmitted 28 November 2016; accepted 13 April 2017. DOI 10.1182/
bloodadvances.2016003251.

© 2017 by The American Society of Hematology

The full-text version of this article contains a data supplement.

812

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cardiovascular disease,17 lung injury,18,19 kidney injury,20,21 andliver injury.22 These findings suggest that the contents transferredby healthy BMSC-derived exosomes have a therapeutic potential.
Kordelas et al demonstrated that the clinical symptoms of graftversus-host disease are significantly improved after treatment withhealthy BMSC-derived exosomes.23 In a manner similar to thatof BMSCs, BMSC-derived exosomes have lower antigenicity
than do various cell types used for cell therapy, such as activated T cells.24,25 Although aging of BMSCs has been studied
extensively, the effect of exosomes derived from older BMSCs on
the BM microenvironment remains unclear. Therefore, we hypothesized that the therapeutic potential of healthy BMSC-derived
exosomes depends largely on the donor age, possibly because ofage-specific differences in their contents (eg, mRNA, miRNA, andprotein).
To test this hypothesis, we investigated the donor age–relateddifferences and therapeutic effects of healthy BMSC-derived
exosomes by using a heterotransplant mouse model of chronic
hypoxia-resistant (HR) MM cells.26 HR-MM cells showed continuous growth under persistent hypoxic conditions, and exosomes
derived from HR-MM cells affected angiogenic functions in bothin vitro and in vivo models.26 In the present study, we modified
BMSC-derived exosomes from older healthy donors by directlytransfecting them with miRNA expressed specifically in young
BMSC exosomes. This approach resulted in exosomes derived
from older BMSCs acquiring antiangiogenic properties, suggesting that exosomal miRNA replacement has a therapeuticpotential.

Methods
Cell lines and culture conditions
Human MM cell lines (RPMI8226, KMS-11, and IM-9) werepurchased from the Health Science Research Resource Bank
(Osaka, Japan). Sublines that survived well under long-term hypoxia(HR-MM cell lines; RPMI8226-HR, KMS-11-HR, and IM-9-HR)26
were maintained under hypoxic conditions (1% O2, 5% CO2, and
94% N2) in a humidified, gas-sorted hypoxic incubator (MCO-5M;
Panasonic, Osaka, Japan). Pooled human umbilical vein endothelial
cells (HUVECs) and human BMSCs from young (donor ages: 19
and 20 years old; young BMSCs) and older (donor ages: 68 and 72
years old; older BMSCs) healthy donors were purchased from
Lonza Inc. (Allendale, NJ). BMSCs were harvested by trypsinizationand either passaged for expansion or subjected to analyses(passages 1-5). During days 1 to 5, the number of viable cells wasdetermined using a commercial kit (Cell Counting Kit-8; Dojindo,
Kumamoto, Japan). See the supplemental experimental proceduresfor details.

Measurement of terminal restriction
fragments (TRFs)
Genomic DNA were extracted from each group of BMSCs by using
the Gentra Puregene Cell kit (Qiagen, Hiden, Germany) anddigested by restriction enzymes (Hinf I or Rsa I). Digested DNA
were fractionated by electrophoresis on 0.7% agarose gel and thentransferred to nylon membranes. Hybridization was performed with
the Telo TTAGGG Telomere Length Assay kit (Roche, Mannheim,
Germany) according to the manufacturer’s instructions. Smears ofthe developed films were captured on a VersaDoc 1000 imaging
system (Bio-Rad, Hercules, CA).

Senescence-associated (SA) b-galactosidase(b-gal) staining
BMSCs were fixed in 0.2% glutaraldehyde, washed with phosphatebuffered saline (PBS), and incubated overnight at 37°C (without
CO2) in staining solution (1 mg/mL of X-gal in 40 mM of citric acid/sodium phosphate, pH 6.0, 5 mM of potassium ferrocyanide, 5 mMof potassium ferricyanide, 150 mM of NaCl, and 2 mM of MgCl2).
After incubation, the cells were washed with PBS and imaged for thepresence of blue staining.

Isolation and characterization of exosomes
BMSCs (4 3 104 cells/cm 2 ) were cultured in T-25 flasks (BDBiosciences, San Jose, CA) containing 5 mL of serum-free AIM-V
medium (Invitrogen, Carlsbad, CA). The culture supernatants were
harvested after 48 h of incubation, and the exosome fraction was
purified using Exoquick-TC reagent (System Biosciences, Mountain
View, CA) according to the manufacturer’s instructions. Exosomes
were quantified by nanoparticle tracking analysis (NanoSight LM10;
Malvern, Herrenberg, Germany) and observed under a transmission
electron microscope (JEM-1200EX; JEOL, Tokyo, Japan), as described previously.27 See the supplemental experimental proceduresfor details.

Direct transfection of miRNA mimics into exosomes
miRNA mimics (Ambion; Invitrogen) were labeled with the Label
IT small interfering RNA Tracker Cy3 kit (Mirus, Madison, WI)
according to the manufacturer’s instructions. mirVana miRNA mimics(see supplemental Table 1 for mimic ID) (Applied Biosystems) weretransfected directly into exosomes using Exo-fect (System Biosciences). See the supplemental experimental procedures for details.

miRNA expression profiles
Cellular and exosomal miRNAs were isolated using the miRNeasykit (Qiagen), and miRNA profiling of both cells and exosomes was
performed using the TaqMan low-density miRNA array (Thermo
Fisher Scientific, Carlsbad, CA) according to the manufacturer’s
recommendations. The relative expression of each gene was calculated using the comparative threshold cycle method, as described
previously.28 See the supplemental experimental procedures fordetails.

Tube formation assay
The formation of capillary-like structures was assessed, as described previously.27 HUVECs (2 3 104 cells/well) were seeded on
Matrigel (growth factor reduced, 280 mL/well; BD) in 24-well plates
and treated with exosomes (1.2 3 107 particles) in endothelial basal
medium-2 (480 mL/well; Lonza Inc.), supplemented with or without
endothelial growth medium-2 SingleQuots and 5% fetal bovine
serum for 24 h. BMSC-derived exosomes were titrated to determine
the optimal concentration for tube formation assay (supplemental
Figure 1). The total area of tube formation was quantified as themean pixel density obtained from image analysis of 5 random
microscopic fields using ImageJ software (http://rsb.info.nih.gov/
nih-image/).

In vivo Matrigel plug assay
A mixture of growth factor–reduced Matrigel (200 mL) and 4 3 106
HR-MM cells was admixed with exosomes derived from BMSCs(4 3 107 particles). The Matrigel mixtures were injected subcutaneously

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

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813

(donor age: 19 years)

young BMSCs(donor age: 20 years)

older BMSCs

B

E
10

(donor age: 68 years)

older BMSCs

M
ar
k
yo er
un
g
B
M
ol
SC
de
s
rB
M
SC
s

young BMSCs

Cell number (x105/ml, log)A

young BMSCs
older BMSCs

(kB)
20
1
8

(donor age: 72 years)

0

24

48

72

5

96 120hrs
2Telomere length (mean TRF, kbp)

20
0
s

s

older BMSCs

SC(donor age: 72 years)

*

15

10

5

de
ol

0

yo

G

de

β-gal

β-gal

ol

un

g

rB

B

M

M

SC

SC

s

s

yo

un

g

(donor age: 20 years)

40

SC

young BMSCs

20

*

M

β-gal

β-gal

F
60

rB

(donor age: 68 years)

DM(donor age: 19 years)

older BMSCs

B

young BMSCs

SA-β-gal positive cells (%)

CI
older BMSCs

Nanoparticle concentration (x 108 particle/ml)

young BMSCs

H

3

2

young BMSCs
older BMSCs

1

0
CD63

0

100

200

300

Particle size (nm)CD81

young BMSCs

older BMSCs
Figure 1.

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23 MAY 2017 x VOLUME 1, NUMBER 13

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400

into 8-week-old male nude mice (BALB/c-nu/nu; CLEA Japan, Tokyo,
Japan). All experimental groups included 3 mice. Three weeks after
implantation, the Matrigel plugs were harvested and processed foranalysis. The Matrigel plugs were fixed with 4% paraformaldehyde
and embedded in paraffin. Then, 8-mm-thick paraffin sections
were prepared using a microtome (RM2245; Leica Biosystems,
Heidelberg, Germany). Neovascularization was evaluated byimmunohistochemistry. The total CD31-positive area was quantified as the mean pixel density obtained from image analysis of 6
random microscopic fields by using ImageJ software. See thesupplemental experimental procedures for details on analysis ofcapillary density.

Western blotting
Western blots were probed with antibodies against cMET (rabbit
polyclonal, 1:500; C-28; Santa Cruz Biotechnology, Santa Cruz,
CA), phosphorylated cMET (p-cMET) (rabbit polyclonal, 1:100;
ab5662; Abcam, Cambridge, MA), hepatocyte growth factor (HGF)(rabbit polyclonal, 1:100; ab83760; Abcam), and b-actin (mousemonoclonal, 1:2500; Chemicon, Temecula, CA). CD63 (mousemonoclonal anti-CD63 antibody, 1:100, sc-5275; Santa Cruz
Biotechnology) and CD81 (mouse monoclonal anti-CD81 antibody; 1:100; sc-23962; Santa Cruz Biotechnology) were usedas markers of exosomes. Horseradish peroxidase–conjugatedsecondary antibodies were purchased from GE Healthcare Life
Science (Uppsala, Sweden). See the supplemental experimental
procedures for details.

EthicsAll animal experiments were conducted in compliance with theinstitutional guidelines of the Animal Experimental Center of Tokyo
Medical University/Animal Biosafety Level II Laboratory for Useof Animals. The experimental protocols were approved by theInstitutional Animal Care and Use Committee of Tokyo Medical
University. The use of patient samples was approved by theInstitutional Review Board of Tokyo Medical University (no. 2648,
approved 22 April 2014). Written informed consent was obtained
from all participants before specimen collection, according to the
Declaration of Helsinki.

Statistical analyses
Data are presented as the mean 6 standard deviation (SD).
Comparisons between 2 groups were made with the Student t test.
Multiple group comparisons were made by analysis of variance.
GraphPad Prism (version 5c) for Macintosh (GraphPad Inc., La
Jolla, CA) was used for statistical analyses. Results were considered
statistically significant at P , .05.

Results
Donor age–dependent senescent changes in BMSCs
There were no morphological differences between BMSCs derived
from young or older healthy donors (Figure 1A). The cell proliferation rate was not significantly different between young andolder BMSCs for at least 72 h in culture, although older BMSCs
had a gradual reduction in their proliferative capability (Figure 1B).
The number of SA-b gal-positive cells among older BMSCs was
significantly higher than that among young BMSCs at the same
passage (Figure 1C-D; P , .01). Similarly, TRFs in older BMSCs(11 6 0.99 kbp) were shorter than were those in young BMSCs(14.8 6 1.41 kbp) (Figure 1E-F), suggesting that senescenceassociated changes in BMSCs are dependent on the donor age.

Characterization of exosomes derived from young
and older BMSCs
The size and structure of exosomes appeared to be similar between
young and older BMSCs (Figure 1G). Additionally, the expression ofcommon exosomal markers (CD63 and CD81) was similar between
young and older BMSCs (Figure 1H). The particle size of BMSCderived exosomes was approximately 50 nm. On average, after 48 hof culture, 1.9 3 108/mL exosomes and 2.3 3 108/mL exosomes
were found in the culture supernatants of young BMSCs and older
BMSCs, respectively (Figure 1I).

In vivo model of the MM hypoxic niche
We previously established HR-MM cells derived from MM cell lines(RPMI8226-HR, KMS-11-HR, and IM-9-HR) to evaluate thepotential role of MM cells in the hypoxic BM environment. We foundthat HR-MM cells induce tumor angiogenesis under chronic hypoxic
conditions.26 In this study, we used an in vivo Matrigel plug angiogenesis assay to analyze the angiogenic responses to HR-MM
cells in vivo (supplemental Figure 2). HR-MM cells formed tumors
with increased amounts of CD31-positive endothelial cells in the
Matrigel plug (supplemental Figure 2A,C), whereas parental cells did
not grow (supplemental Figure 2B-C). Among them, RPMI8226-HR
cells induced higher levels of angiogenesis in comparison with other
HR-MM cell lines (supplemental Figure 2A). Therefore, we primarily
used RPMI8226-HR cells in this study.

Antiangiogenic effects of BMSC-derived exosomesfrom young donors
BALB/c nude mice were injected subcutaneously with HR-MM cells(RPMI8226-HR) seeded in Matrigel plugs containing exosomes(4 3 10 7 particles per 200 mL Matrigel) derived from young
BMSCs (young BMSC exosomes) or older BMSCs (older BMSC

Figure 1. Characterization of BMSCs. (A) Morphology of BMSCs derived from 2 young healthy donors (young BMSCs; passage 2; donor ages: 19 and 20 years old) and 2
older healthy donors (older BMSCs; passage 2; donor age: 68 and 72 years) by phase-contrast inverted microscopy. Scale bar, 50 mm. (B) BMSC growth measured after 24, 48,
72, 96, and 120 h in culture. Cell proliferation of young BMSCs (pink line) and older BMSCs (blue) was evaluated. Each value represents the mean 6 SD (n 5 6). (C-D)
Representative images of SA-b-gal staining (arrows indicate positive cells) and quantification of cell senescence. Data represent the mean 6 SD. *P , .01 in comparison with young
BMSCs using a Student t test. Scale bar, 50 mm. (E-F) Telomere lengths in young and older BMSCs determined by Southern blotting of TRFs. Data represent the mean 6 SD.
*P , .05 in comparison with young BMSCs using a Student t test. (G) Transmission electron micrographs of exosomes derived from young and older BMSCs. Scale bar, 50 nm.
(H) Western blots of exosomal lysates (CD63 and CD81 are common exosomal markers) derived from young and older BMSCs. Exosomes were isolated from 5 mL of culture
medium collected from BMSCs (4 3 104 cells/cm2) grown for 48 h. Equal volumes of exosomal lysate (30 mL) were loaded in the lanes of gels. (I) Nanoparticle concentration andsize distribution of exosomes derived from young BMSCs (pink line) and older BMSCs (blue line).

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A

B
RPMI8226-HR+
PBS (control)

RPMI8226-HR
Matrigel plug

RPMI8226-HR+
older BMSC
exosomes

RPMI8226-HR+
young BMSC
exosomes

PBS (control)oryoung BMSC exosomesorolder BMSC exosomes

BALB/c-nu/nuC
Vessel density(relative CD31+ area)

1.5
1.0

*
0.5

0.0
RPMI8226-HR
young BMSC
exosomes
older BMSC
exosomes

CD138 / CD31

CD138 / CD31

DAPI / CD31

DAPI / CD31

DAPI / CD31

**
+

+

+

–

+

–

–

–

+

E

D
1.2

Cell viability (fold of control)

CD138 / CD31

Control

young BMSC
exosomes

older BMSC
exosomes

1.0
0.8
0.6
0.4

F
P = 0.038

0.0
young BMSC
exosomes
older BMSC
exosomes

–

+

–

–

–

+

Relative tube formation(fold of control)

0.2

1.5

P < 0.001

P = 0.007

1.0
0.5

0.0
young BMSC exosomes
older BMSC exosomes

–
–

+
–

–
+

Figure 2. Healthy BMSC exosomes inhibit HR-MM cell-induced angiogenesis in Matrigel plugs. (A) Schematic of the Matrigel plug assay. A mixture of Matrigel and
4 3 106 RPMI8226-HR cells was admixed with exosomes derived from young BMSCs (young BMSC exosomes; 4 3 107 particles per 200 mL Matrigel) or older BMSCs(older BMSC exosomes; 4 3 107 particles per 200 mL Matrigel). (B) After 3 weeks, the Matrigel plugs were harvested and photographed. Paraffin-embedded sections of Matrigel
plugs were stained with hematoxylin and eosin and then subjected to immunostaining for CD138 (brown) and CD31 (red). Cell nuclei were stained with 49,6-diamidino-2phenylindole (DAPI) (blue). Scale bar, 500 mm. (C) Quantification of vessel density in Matrigel plugs by pixel density. Young and older BMSC exosomes inhibited tumor angiogenesis
in Matrigel plugs compared with the control (RPMI8226-HR only) (*P , .01; **P , .001 vs control, Student t test, n 5 6). Values represent the mean 6 SD. (D) Effect of
BMSC exosomes on the viability of HR-MM cells. RPMI8226-HR cells were cultured with young or older BMSC exosomes (2.5 3 107 particles/mL). Cell viability was assayed after

816

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23 MAY 2017 x VOLUME 1, NUMBER 13

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A

B

Young BMSC exosome-specific miRNA mimics were

ol
d

yo
un

(B) Schematic of the modification of older BMSC exosomes.
transfected directly into older BMSC exosomes by Exo-fect.

older BMSCs

exosomes transfected with miR-340 mimics (miR-340
exosomes) and older BMSC exosomes transfected with
miR-365 mimics (miR-365 exosomes) significantly inhibited
HUVEC tube formation in comparison with the control (older

24 upregulated
exosomal miRNAs(C) Quantification of tube-like structures to determine theantiangiogenic effect of modified exosomes. Older BMSC

C
1.5

12 downregulated
exosomal miRNAs

8

-8

ΔCt

Relative tube formation(fold of control)miR) (control vs miR-340 exosomes; *P , .01, Student t test;
the mean 6 SD. Ct, comparative threshold cycle.

directtransfection

young BMSC exosomes-specific miRNA mimics

BMSC exosomes transfected with negative control miR; control**P , .001, control vs miR-365 exosomes). Values represent• Tube formation
Assay (in vitro)• Matrigel plug
assay (in vivo)

older BMSC exosomes

er

g

B

M

exosomes was determined by TaqMan miRNA array analysis.

SC

SC

s

s

Differential miRNA expression of young and older BMSC

M

exosomes derived from young and older BMSCs. (A)B

Figure 3. Differential miRNA expression profiles of

1.0

*
0.5

**

co

nt
ro
l
m miR
iR
m 10
iR a
m -41
iR 1
-4
m 87
iR a
m -1
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m 8-5
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m -34
iR 0
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m 365
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85

0.0

exosomes) (Figure 2A). We observed hyperproliferative CD138positive RPMI8226-HR cells and CD31-positive endothelial cells
attracted into the Matrigel plug (Figure 2B, left). However, older
BMSC exosomes inhibited the proliferation of RPMI8226-HR
cells and angiogenesis in the Matrigel plug (Figure 2B, right) in comparison with the control (RPMI8226-HR 1 PBS) (Figure 2B-C; P , .05).
In addition, the antiangiogenic effect was more evident with young
BMSC exosomes than with older BMSC exosomes (Figure 2B, middle,
and Figure 2C; P , .001).
To test whether BMSC exosomes directly affect the cell viabilityof HR-MM cells, we measured the cell proliferation of RPMI8226HR cells treated with young or older BMSC exosomes (2.5 3 107
particles/mL). Neither older BMSC exosomes nor young BMSC
exosomes affect the survival of RPMI8226-HR cells (Figure2D). We assessed effector caspase activity in RPMI8226-HR
cells treated with BMSC exosomes (2.5 3 10 7 particles/mL).
There was no significant difference in caspase 3/7 activity in
RPMI8226-HR cells treated with and without BMSC exosomes(supplemental Figure 3B). Furthermore, we performed experiments to assess nonapoptotic cell death mechanisms in HR-MM
cells, including autophagic cell death. The expression of p62
protein showed no significant difference between RPMI8226-HR
cells treated with BMSC exosomes and without (supplemental
Figure 3B).

We next performed an endothelial tube formation assay with
HUVECs treated with young or older BMSC exosomes (2.5 3 107
particles/mL). Consistent with the results obtained from the in vivo
Matrigel plug assay, HUVEC tube formation was inhibited to ahigher degree by young BMSC exosomes than they were by older
BMSC exosomes (young BMSC exosomes vs older BMSC exosomes;
P 5 .007) (Figure 2E-F).

Identification of young BMSC exosome-specificmiRNAs
To identify the exosomal miRNAs associated with the antiangiogenic effect of young BMSC exosomes, we first comparedexosomal miRNA expression profiles between young and older
BMSCs (National Center for Biotechnology Information [NCBI],
gene expression omnibus; GSE78865). A subset of 12 miRNAs
was significantly downregulated in older BMSC exosomes. ThesemiRNAs were specifically expressed in young BMSC exosomes(Figure 3A). Subsequently, we replenished the 12 miRNAs in older
BMSC exosomes by direct transfection with miRNA mimics (Figure3B) and investigated whether young BMSC exosome-specific miRNAs
affect HUVEC tube formation. Exosomes transfected with miR-340 or
miR-365 mimics significantly inhibited angiogenesis in comparison
with scrambled control miRNA-containing exosomes (control miR
exosomes) (Figure 3C; P , .01, P , .001).

Figure 2. (continued) 48 h using Cell Counting Kit-8. Values represent the mean 6 SD. (E) The formation of tube-like structures was observed using the cell-permeable
dye Calcein AM (green). Endothelial tube formation of HUVECs treated with young BMSC exosomes, older BMSC exosomes, or the control (without exosomes). Scale bar,
500 mm. (F) Quantification of tube-like structures by pixel density. Young and older BMSC exosomes significantly inhibited HUVEC tube formation in comparison withthe control (control vs young BMSC exosomes; P , .001, control vs older BMSC exosomes; P 5 .038, Student t test). Values represent the mean 6 SD.

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

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817

**

8

vivo. (A) Exosomal miR-340 expression levels in young BMSC
quantified by reverse transcription polymerase chain reaction(young BMSC exosomes vs older BMSC exosomes; *P , .01,

4

young BMSC exosomes vs miR-340 exosomes; **P , .001,*

Student t test). Values represent the mean 6 SD. (B-C) Recipient2

cells were treated with exosomes directly transfected with Cy3miR-340 mimics. Cy3-miR-340 signals were detected in the

ex

SC
M

Cy3-miR-340 / DAPI

Cy3-miR-340 / DAPI
Calcein AM

cytoplasm of RPMI8226-HR cells (B) and HUVECs (C). Nuclear
and cytoplasmic staining were performed with DAPI (blue) andCalcein AM (green), respectively. Scale bar, 25 mm. (D) A mixture

m

SC
M
ol
d

er

B

ex

iR
o.
co -340nt
exrol m o.
iR
ex
o.

0

E

of Matrigel and 4 3 106 RPMI8226-HR cells was admixed with

1.5

older BMSC exosomes directly transfected with miR-340 mimics

Vessel density(relative CD31+ area)(miR-340 exosomes; 4 3 107 particles per 200 mL Matrigel), miR-

RPMI8226-HR RPMI8226-HR RPMI8226-HR+++
control miRmiR-340miR-365exosomes
exosomes
exosomess365 mimics (miR-365 exosomes; 4 3 107 particles per 200 mL
Matrigel), or negative control miR (control miR exosomes). The

1.0

Matrigel mixture was injected subcutaneously into nude mice.
Three weeks after implantation, the Matrigel plugs were harvested.
Paraffin-embedded sections of Matrigel plugs were stained with0.5

hematoxylin and eosin and then subjected to immunostaining for*
0.0
RPMI8226-HR
control miR-exo.
miR-340-exo.
miR-365-exo.
DAPI / CD31

DAPI / CD31

+
+
–
–

+
–
+
–

CD31 (red). Scale bar, 500 mm. (E) Quantification of vessel density
in Matrigel plugs by pixel density. Both mir-340 and miR-365*

exosomes inhibited tumor angiogenesis in Matrigel plugs in

+
–
–
+

comparison with the control (*P , .001 vs control, Student t test,n 5 6). Values represent the mean 6 SD. Exo, exosomes.

DAPI / CD31

Enrichment of older BMSC exosomes with young
BMSC exosome-specific miRNAs inhibits
angiogenesis in vivo
Older BMSC exosomes transfected with miR-340 mimics(miR-340 exosomes) exhibited a 6-fold increase in the expressionof miR-340 in relation to young BMSC exosomes (Figure 4A). We
also visualized with an in vitro model the uptake of exosomal Cy3labeled miR-340 into HR-MM cells (RPMI8226-HR; Figure 4B) and
HUVECs (Figure 4C) via exosomes. Matrigel plug assays were performed
using Cy3-labeled miR-340. We checked the colocalization of Cy3labeled miR-340 and HR cells in the Matrigel plug. Cy3-miR-340
signals were detected in not only the cytoplasm of HR cells(supplemental Figure 4A-B) but also the cytoplasm of endothelial
cells (supplemental Figure 4C-F).
Matrigel plug assays confirmed that older BMSC exosomestransfected with miR-340 mimics (miR-340 exosomes) or miR365 mimics (miR-365 exosomes) significantly inhibited angiogenesis in the Matrigel plug in comparison with scrambled control
miRNA-containing exosomes (control miR exosomes) (Figure 4D-E;
P , .001). We also observed inhibition of the hyperproliferationof HR-MM cells in conjunction with the antiangiogenic effectof older BMSC exosomes transfected with miR-340 andmiR-365 mimics (Figure 4D). These findings suggested that theantiangiogenic effect of older BMSC exosomes was restored
by transfection with young BMSC exosome-specific miRNAs (ie,miR-340 and miR-365). We performed Matrigel plug assays with
KMS-11-HR and IM-9-HR cells and primary myeloma cells of MM
818

exosomes, older BMSC exosomes, and miR-340 exosomes were

HUVECs

6

B
g
yo
un

D

Figure 4. Antiangiogenic effects of modified exosomes inC

B
RPMI8226-HRo.

Relative expression level(mIR-340/ath-miR-159)

A

patients. As in the experiment using RPMI8226-HR cells, miR-340
exosomes significantly inhibited angiogenesis induced by HR-MM
cells (supplemental Figure 5A) or CD1381 cells from a MM
patient with a high tumor burden in the Matrigel plug (supplemental Figure 5B).

Phosphorylated cMET and HGF protein levels vary in
HUVECs cocultured with HR-MM cells
We selected various confirmed targets of miR-340 and miR-365
according to reporter assays or Western blotting using miRTarBase.29 Within this list, we searched for a candidate target of miR340 with a high predicted efficacy as calculated by the context
scores. We focused on the cMET tyrosine kinase receptor thatexerts context-dependent effects on endothelial cells. cMET expression was significantly higher in HUVECs than in MM cells (RPMI8226
and RPMI8226-HR) (Figure 5A). There was no difference in cMET
expression between parental and HR-MM cells (RPMI8226-HR)(Figure 5A-B; P , .01). HGF protein expression was higher in HRMM cells than in parental and endothelial cells (Figure 5A-B;
P , .01). We next investigated changes in the expression of cMET,
phosphorylated cMET (p-cMET), and HGF in HUVECs cultured
with or without MM cells using a noncontact coculture system(Figure 5C). Although there were no significant differences in thelevels of cMET, p-cMET was significantly upregulated in HUVECs
cocultured with HR-MM cells (Figure 5D-E; P , .01, P , .001).
Additionally, HGF expression in HUVECs was induced by coculture
with HR-MM cells (Figure 5D-E; P , .001).

UMEZU et al

23 MAY 2017 x VOLUME 1, NUMBER 13

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C

A
cMET

RPMI8226
or
RPMI8226-HR

D

cMET
p-cMET

HGF

HGF
s
EC

+

−

−

−

+

HUVECs (1% O2)

4
2
s

RP
M

0.5

1
s

0

EC

0

UV

22

2

RP

M

I8

4

H

6-

H

22

R

6

0
I8

5

6

+ c
+R RP ontr
PM MI ol
8
I8 22
22 6
6H
R

0.0

2

M

**

10

**

*

*

3

RP

8

+ co
+R RP ntr
PM MI8 ol
I8 22
22 6
6H
R

4

1.0

+ c
+R RP ontr
PM MI ol
8
I8 22
22 6
6H
R

I8

H

UV

6H
22

I8
RP
M

EC

R

6

0

15

1.5

Relative expression level(HGF/β-actin)

6E

Relative expression level(p-cMET/β-actin)*

Relative expression level(cMET/β-actin)

822

Relative expression level(cMET/β-actin)−

RPMI8226-HR

HUVECsB

Relative expression level(HGF/β-actin)

RPMI8226

UV
H

22

β-actin

RP
M

I8

RP
M

I8

6H

22

R

6

β-actinFigure 5. p-cMET and HGF protein levels vary in HUVECs cocultured with HR-MM cells. (A) Western blots showing the expression of cMET and HGF in MM cells(RPMI8226 and RPMI8226-HR) and HUVECs. (B) The intensities of cMET and HGF bands in panel A were quantified and normalized to the levels of b-actin (*P , .01 vs
RPMI8226, Student t test, n 5 3). (C) Schematic of the nonadherent coculture system of MM cells (blue) and HUVECs (green). MM cells and HUVECs were cocultured separately
using a Transwell filter (polycarbonate membrane insert, 0.45-mm pores). (D) cMET, p-cMET, and HGF levels in HUVECs were measured by Western blotting after coculture
with RPMI8226 or RPMI8226-HR cells under hypoxic conditions (1% O2). (E) The intensities of cMET, p-cMET, and HGF bands in panel D were quantified and normalized to thelevels of b-actin (*P , .01; **P , .001 vs control (HUVECs only), Student t test, n 5 3).

Exosomal miR-340 suppresses tube formation via
inhibition of the cMET signaling pathway

directly transfected with miR-340 mimics; 45% reduction) incomparison with the control (HUVECs only) (Figure 6C).

To determine whether cMET expression was suppressed by theaddition of older BMSC exosomes transfected with miR-340
mimics (miR-340 exosomes), we examined cMET protein levels
in HUVECs treated with miR-340 exosomes (Figure 6A). cMET
protein levels were reduced by approximately 60% in HUVECs
treated with miR-340 exosomes in comparison with control miR
exosomes (Figure 6A). These results indicated that exogenous miR340 interacted with cMET for translational suppression of cMET in
HUVECs via exosomes, whereas inhibition of cMET expression by
miR-340 exosomes did not affect the expression of HGF (Figure 6A).
To confirm direct binding between miR-340 and the cMET 39untranslated region (UTR), we performed a luciferase reporter
assay using a reporter plasmid containing the cMET 39-UTR withthe miR-340-binding site (Figure 6B). A reduction of the luciferase
activity from the pLuc-cMET-39-UTR plasmid was observed in
HUVECs treated with miR-340 exosomes (older BMSC exosomes

In the in vitro assay (Figure 6D), endothelial cell tube formation wasenhanced by treatment with conditioned medium derived from HRMM cells or HGF. Such enhanced tube formation was inhibitedby the addition of miR-340 exosomes (Figure 6E-F; P , .01).
Furthermore, we assessed endothelial cMET activation in vivo inMatrigel plugs by immunohistochemistry. In Matrigel plugs, CD31positive endothelial cells attracted by HR-MM cells expressed
p-cMET (Figure 6G).

Discussion
The advantages of exosome therapy are its stability, biocompatibility, biological barrier permeability, low toxicity, and native
mechanism of cell-to-cell transfer. Exosomes derived from BMSCs
have low inherent immunogenicity because of composition similar
to that of the body’s own cells.30 The use of BMSC exosomes istherefore especially promising for exosome-based cancer therapy.

23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

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819

B

A

hsa-miR-340

3’ UUAGUCAGAGU -AACG-AAAUAUU 5’

Human cMET 3’UTR(1487-1508)

5’ UA - AAGUGUUAUAUUU-UUUAUAAA 3’

cMET
HGF
β-actin

H
+c UV
on EC
tro s
lm
iR

ex
o.
H
+m UV
iR EC
-3 s
40
ex
o.

C

Mutated sensor vector

Sensor vector#

1.2

Relative luciferase activity

Relative luciferase activity

1.2
1.0

*

0.8
0.6
0.4
0.2

1.0
0.8
0.6
0.4
0.2
0.0

0.0
young BMSC exosomes
Control miR exosomes
miR-340 exosomes

–
–
–

+
–
–

–
+
–

–
–
–

–
–
+

HUVECs/Luc/β-gal

D

E

–
+
–

–
–
+

HUVECs/Luc/β-gal

HUVECsHUVECs+control miR exo. +miR-340 exo.

miR-340 exo.

+
–
–

G

Basal medium
Basal medium+
Conditioned med.
or
HGF

Basal medium+
conditioned med.
from HR cells

p-cMET

Basal medium+
HGF

HUVECson Matrigel

Relative tube formation(Fold of control)

F

*

10

CD31

*

8
6
4
2

0
miR-340 exo.
Conditioned med. from HR cells
HGF

–
–
–

+
–
–

–
+
–

+
+
–

–
–
+

+
–
+

DAPI / p-cMET / CD31

Figure 6. Regulation of HGF/cMET signaling in endothelial cells by exosomal miR-340 derived from young BMSCs. (A) Representative Western blots of the expressionof cMET and HGF in HUVECs after treatment with older BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) or negative control miR (control miR exo.). (B) The
hsa-miR-340-binding site spanning nucleotides 1487–1508 (accession no. NM_000245) of the human cMET 39-UTR was predicted to be evolutionarily conserved. The seedsequence of hsa-miR-340 (AAAUAU) is shown in red. (C) Luciferase reporter assay using a reporter plasmid containing the cMET 39-UTR with the miR-340-binding site. The
luciferase reporter vector to assess miR-340-specific activity contained complementary miR-340 sequences in its 39-UTR. Normalized firefly luciferase activity was obtained

820

UMEZU et al

23 MAY 2017 x VOLUME 1, NUMBER 13-345-

There are 2 purposes in our study. One is to clarify the difference in
BMSC and BMSC exosome changes by aging. The other is to findexosomes with therapeutic effects and develop exosome-based
therapy.
Target miRNA-overexpressing cells have generally been used as
exosome-producing cells to obtain target miRNA-enriched exosomes. However, the expression of exosomal factors other than thetarget miRNA also changes using this method, because the biology
of exosome-producing cells is modified by either transfected miRNAor electroporation.31 To overcome this problem, we adopted the“Exo-fect” system, which enables direct transfection of miRNAs into
isolated exosomes. The transfection efficiencies achieved by Exofect were approximately 75% (supplemental Figure 6). As a result,
we found that only 1 exosomal miRNA of young healthy donors
restored the antiangiogenic function of exosomes derived from older
healthy donors.
We initially hypothesized that the function of exosomes is highly
dependent on the BMSC donor age. We found that young BMSC
exosomes and miRNA had significant antiangiogenic effects. In thisstudy, we first investigated the differential gene expression profilesof young BMSCs and older BMSCs (NCBI, gene expression
omnibus; GSE78235) and found that 128 genes were upregulated
and 117 genes were downregulated in older BMSCs in comparison
with young BMSCs (supplemental Figure 7A). By performing gene
ontology (GO) term analysis using the Functional Annotation Tool of
the Database for Annotation, Visualization, and Integrated Discovery (http://david.ncifcrf.gov), we identified major changes between
the functions of young and older BMSCs, such as developmental
processes and cell proliferation and adhesion (supplemental Figure 7B).
Although we did not identify GO terms related to cellular senescence, we found that older BMSCs displayed downregulationof aldehyde dehydrogenase 1, which is involved in self-renewal
and differentiation, indicating that the donor age might affect thedifferentiation potential of BMSCs. Previous studies on age-relatedchanges in BMSCs clearly show that the age of the BMSC donor
directly influences BMSC differentiation, proliferation, and metabolic profiles.32,33 Furthermore, the immunoregulatory potential ofyoung BMSCs is higher than that of older BMSCs.34 Age-related
increases in the expression of apoptotic and senescent genes witha concomitant decrease in Sirt1 gene expression also inhibits stem
cell function to some degree.35 These findings suggest that the ageof BMSC donors should be considered in regard to their therapeutic
efficacy. This interference further suggests that the loss of young
BMSC exosome-specific miRNAs is related to cancer, because thedevelopment of the majority of cancers is considered to be related
to age.

In the present study, we extracted several exosomal miRNAs specificto young BMSCs and identified 2 miRNAs (miR-340 and miR-365)
that exerted an antiangiogenic effect (Figures 3C and 4D). Our insilico analysis showed that miR-365 targeted interleukin-6 (IL-6), andwe conducted a luciferase reporter assay with a reporter vector
containing the 39-UTR of IL-6 with the predicted miR-365 bindingsite (supplemental Figure 8A). Unexpectedly, overexpression of
miR-365 did not decrease the activity of the luciferase reporter(supplemental Figure 8B).
We therefore focused on miR-340, which restored and evenincreased the antiangiogenic effect of older BMSC exosomes. The
first identified target of miR-340 was microphthalmia-associatedtranscription factor, a master regulator of melanocyte development
and melanogenesis in melanoma cells.36 Although several miR-340
target genes have been identified to date, the HGF receptor, cMET,
has been reported as a target of miRNA several times.37 It is known
that the HGF/cMET interaction plays a key role in the regulationof angiogenesis, and high levels of HGF expression and cMET
activation (p-cMET) have been found in BM endothelial cells of MM
patients in comparison with normal endothelial cells of healthy
donors.38 Consequently, miR-340 might act as a negative regulatorof angiogenesis by inhibiting cMET expression. On the basis of aprevious report,37 we assessed direct binding between the cMET
39-UTR and miR-340 by a luciferase reporter assay (Figure 6C).
Furthermore, Moschetta et al demonstrated that resistant R5 cells
and plasma cells from MM patients at relapse with drug resistancehave a high HGF-independent p-cMET content with constitutive
activation of the cMET receptor.39 The expression of cMET andp-cMET did not show a difference between parental and HR-MM cells.
We also did not observe constitutive activation of cMET in CD1381
cells obtained from MM patients in this study. Unfortunately, using
our system, we could not analyze the effect of miR-340 on
overcoming drug resistance by inhibition of cMET expression.
One of the reasons why we replenished miR-340 in older BMSC
exosomes was to use older BMSC exosomes as an experimental
control (miR-340-insufficient exosomes) to demonstrate miR-340
functions. However, the principal reason for modifying exosomes bydirect transfection of miRNA was establishment of novel strategies
for exosome-based therapy. An important issue that should be
addressed is whether miR-340-enriched exosomes have a direct
inhibitory effect on tumor growth. Although we cannot completely
rule out the possibility of either direct growth inhibition of tumor cells
or remaining natural killer cell activity in the nude mouse, miR-340enriched exosomes did not induce HR cell proliferation in vitro(Figure 2D). We believe the inhibition of tumor angiogenesis by
miR-340-enriched exosomes was due to tumor growth suppression

Figure 6. (continued) according to firefly luciferase activity/b-gal activity. Sensor vector: the luciferase activity of HUVECs/Luc/b-gal (HUVECs co-transfected with luciferase
reporter and b-gal control vectors) treated with young BMSC exosomes was significantly reduced in comparison with control cells (HUVECs only, *P , .01, n 5 3). miR-340
exosomes (older BMSC exosomes transfected with miR-340 mimics) also reduced the luciferase activity in HUVECs in comparison with control miR exosomes (older BMSC
exosomes transfected with negative control miR) (#P , .01, Student t test, n 5 3). Using the mutated sensor vector, there was no difference in luciferase activity with or without exosomes.
(D) Schematic of the endothelial cell tube formation assay to confirm the antiangiogenic effect of miR-340 exosomes. HUVECs were cultured on Matrigel in 3 types of medium (basal
medium, basal medium supplemented with conditioned medium from RPMI8226-HR cells, or basal medium supplemented with HGF). (E) Endothelial tube formation in response to older
BMSC exosomes transfected with miR-340 mimics (miR-340 exo.) observed under a bright field by phase-contrast microscopy. Scale bar, 500 mm. (F) Quantification of tube formation asshown in panel E. The conditioned medium from HR-MM (RPMI8226-HR) cells and HGF induced endothelial tube formation in vitro. Induction of tube formation by conditioned medium of
HR-MM cells and HGF was inhibited by older BMSC exosomes transfected with miR-340 mimics (miR-340 exosomes) in comparison with the control (older BMSC exosomes transfectedwith negative control miR; control miR exosomes) (*P , .01, Student t test). Values represent the mean 6 SD. (G) Immunohistochemical staining of CD31 (red) and p-cMET (green) in
Matrigel plugs seeded with RPMI8226-HR cells at 3 weeks after transplantation into nude mice. Scale bar, 200 mm. med., medium.

23 MAY 2017 x VOLUME 1, NUMBER 13

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in the Matrigel plug, although further studies are required to test thishypothesis.
In conclusion, we provide new information on the age-relatedchanges of BMSC-derived exosomes and the inhibitive effectof exosomal miRNA on tumor angiogenesis. The present study
provides evidence of the therapeutic potential of exosomal miRNAs
derived from young BMSCs and offers a foundation for the development of novel strategies for exosome-based therapy via replenishment of exosomal miRNAs.

of Japan (15H04303, 15K06841, and 16K07183); and the Tokyo
Biochemical Research Foundation.

Authorship
Contribution: T.U., K.O., and J.H.O. designed the study and wrote themanuscript; T.U., S.I., K.A., and C.K. performed experiments andanalyzed data; and S.Y. provided patient samples.

Acknowledgments

Conflict of interest disclosure: K.O. received research support
from Celgene KK, Chugai Pharmaceutical KK, and Janssen Pharma
KK. The remaining authors declare no competing financial interests.

This study was supported in part by the Private University Strategic
Research-Based Support Project (S1311016); grants-in-aid from
the Ministry of Education, Culture, Sports, Science, and Technology

Correspondence: Tomohiro Umezu, Tokyo Medical University,
6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan; e-mail:t_umezu@tokyo-med.ac.jp.

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23 MAY 2017 x VOLUME 1, NUMBER 13

EXOSOMAL miR-340 INHIBITS TUMOR ANGIOGENESIS

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Int J Hematol (2017) 105:419–422
DOI 10.1007/s12185-017-2199-z

RAPID COMMUNICATION

Up‑regulated exosomal miRNA‑140‑3p in CML patientswith musculoskeletal pain associated with discontinuationof tyrosine kinase inhibitors
Michiyo Asano1,3 · Tomohiro Umezu1,2 · Seiichiro Katagiri1 · Chiaki Kobayashi1,2 · Tetsuzo Tauchi1 ·Moritaka Gotoh1 · Keiko Ando1 · Seiichi Okabe1 · Junko H. Ohyashiki2 · Kazuma Ohyashiki1

Received: 29 September 2016 / Revised: 7 February 2017 / Accepted: 7 February 2017 / Published online: 14 February 2017
© The Japanese Society of Hematology 2017

Abstract We analyzed the exosomal miRNA fromperipheral blood from CML patients with musculoskeletal pain after stopping tyrosine kinase inhibitors to identify possible factors related to this manifestation. Exosomal miRNA profiling using TaqMan low-density arrayrevealed that exosomal miR-140-3p was significantly
elevated in CML patients showing musculoskeletal pain,
when compared to those without such pain (P = 0.0336)
or healthy individuals (P = 0.0022). All five CML
patients with musculoskeletal pain and increased exosomal miR-140-3p levels sustained deep molecularresponses: four of them achieved symptom relief and asignificant decrease in exosomal miR-140-3p levels wasevident. Because exosomal miR-140-3p is considered
to have an inflammation-associated biological function
in airway smooth muscle cells and targets Myomarker
muscle-specific transmembrane protein, it appears that its
overexpression in circulating exosomal miR-140-3p may
have some role in the mechanism underlying self-limited
musculoskeletal pain.

Keywords Exosomes · miRNA-140-3p · Chronic myeloid
leukemia · Musculoskeletal pain · Discontinuation of TKIs

Tyrosine kinase inhibitors (TKIs), including imatinib, arenow a standard frontline therapy for chronic myeloid leukemia (CML) that yields successful outcomes. Althoughclinical trials for TKI discontinuation are still ongoing,
approximately 60% of CML patients who achieved a deep
molecular response for more than 2 years maintained amajor molecular response after discontinuation of imatinib[1]. However, long-term prognosis and/or adverse events
after TKIs cessation remain unclear. Recent reports showedthat transient musculoskeletal pain occurs in approximately
30% of CML patients after stopping imatinib, without consistent biochemical abnormalities [2–4]. We also reported
on nine CML patients with musculoskeletal pain after TKI
cessation (eight receiving imatinib and one nilotinib), but
none of them showed laboratory abnormalities, including
C-reactive protein, creatinine kinase, and electrolytes [5].

* Kazuma Ohyashikiohyashik@rr.iij4u.or.jp

Keiko Ando
ok1126@yahoo.co.jp

Michiyo Asanooymch061417@yahoo.co.jp

Seiichi Okabe
okabe@tokyo-med.ac.jp

Tomohiro Umezu
t_umezu@tokyo-med.ac.jp
Seiichiro Katagiri
patchsei@yahoo.co.jp
Chiaki Kobayashi
chiaki-k@tokyo-med.ac.jp
Tetsuzo Tauchi
tauchi@tokyo-med.ac.jp

Junko H. Ohyashiki
junko@hh.iij4u.or.jp
1

Department of Hematology, Tokyo Medical University,
160-0023 Tokyo, Japan2

Department of Molecular Oncology, Institute of Medical
Science, Tokyo Medical University, 160-0023 Tokyo, Japan

3

Present Address: Department of Hematology, Erasmus
Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The
Netherlands

Moritaka Gotoh
gotohm@wj8.so-net.ne.jp

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M. Asano et al.

Exosomes are cell-derived vesicle and released from various cells. They contained genetic elements, such as microRNA (miRNA), and transport to different cell types; therefore, exosomes are conceivable to contribute to “cell to cellcommunication”. miRNAs play key roles in the regulationof gene expression, including cell cycle, apoptosis, immune
responses, and muscle development. To ascertain the factors underlying musculoskeletal events after TKI cessation,
we investigated circulating exosomal miRNA in five CML
patients who did not experience musculoskeletal eventsand five patients with musculoskeletal pain after stopping
TKIs, as well as three healthy individuals. Peripheral blood
was obtained approximately 3 months after successful TKI
cessation in CML patients. Exosomes were extracted using
Total Exosome Isolation Reagent (Invitrogen, Carlsbad, CA,USA), and the exosomal miRNA profiling was performed
with a TaqMan Low-Density Array (Thermo Fisher Scientific, Carlsbad, CA, USA), as reported previously [6]. The
relative expression level of each gene was calculated using
the comparative thresholds cycle (Ct) method [6]. Synthetic
spike control (ath-miR-159: Hokkaido System Science,
Hokkaido, Japan) was used as an invariant control for the
exosomal miRNA. This study was approved by the institutional review board of Tokyo Medical University (no. 930
approved 24 June 2008 and no. 3052 approved 9 June 2015).
We compared exosomal miRNA profiles between CML
with musculoskeletal pain and those without musculoskeletal events, in addition to the healthy controls, using
the GeneSpring software (Agilent Technologies, Santa
Clara, CA, USA) (Fig. 1a) (GEO: GSE85903). Three-way
ANOVA, among healthy controls, CML patients with orwithout musculoskeletal pain, revealed exosomal miR140-3p to be the most significant value (P = 0.00778). At test analysis using the R software identified 10 differentially expressed exosomal miRNAs between CML withor without musculoskeletal pain: seven miRNAs were upregulated (miR-107, P = 0.0416; miR-145, P = 0.0499;
miR-140-3p, P = 0.0336; miR-539, P = 0.0394; miR495, P = 00399; miR-299-5p, P = 0.0291; and miR425, P = 0.0475) and three were down-regulated (miR122, P = 0.0033; miR-218, P = 0.0489; and miR-523,
P = 0.0433) in CML with musculoskeletal pain. We thencompared those miRNA expression levels between CML
with musculoskeletal pain and healthy subjects; among
them, miR-140-3p was significantly elevated in only CML
patients with musculoskeletal pain (P = 0.0022) (Fig. 1b).
All five CML patients with musculoskeletal pain andelevated exosomal miR-140-3p sustained deep molecularresponse: four of them released their symptoms during
the courses. We then compared those miRNA expression
levels in each CML patient with and without musculoskeletal pain: a significant reduction of exosomal miR140-3p levels was evident (P = 0.0044, P = 0.0358,

P < 0.001, P = 0.0284, respectively) after releasing musculoskeletal pain (Fig. 1c, d). The remaining one patientsustained musculoskeletal pain with up-regulated exosomal miR-140-3p until now (approximately 400 days
after stopping imatinib mesylate). In the group without
musculoskeletal pain, two CML patients had molecularrelapse (squares in Fig. 1b), while the remaining threemaintained deep molecular response (triangles in Fig. 1b)(median observation period: 55 months after stoppingTKIs): there was no significant difference in exosomal
miR-140-3p level whether they had molecular relapse or
not (P = 0.1141).
Based on the results obtained from exosomal miRNAprofiling, we focused on the up-regulation of miR-140-3p
in the current study. The miRTarBase (http://mirtarbase.
mbc.nctu.edu.tw/) revealed two experimentally validated
microRNA targets of miR-140-3p: CD38 and nuclear receptor-interacting protein 1 (NRIP1; also known as receptorinteracting protein 140, RIP140). NRIP1 is a nuclear protein that specifically interacts with the hormone-dependent
activation domain AF2 of nuclear receptors. A recent study
demonstrated that reduced expression of RIP140 alters theregulation of fatty-acid oxidation in muscle [7]. Although
we did not perform any functional analysis of miR-140-3p,
it is possible that altered fatty-acid oxidation due to inhibition of RIP140 may be linked to musculoskeletal symptoms in CML patients. The protein encoded by CD38 is a
non-lineage-restricted, type II transmembrane glycoprotein that is expressed in airway smooth muscle cells. Judeet al. reported that miR-140-3p directly binds to the CD38
3′-UTR and then indirectly activates the p38 MAPK and
NF-κB pathway in airway smooth muscle cells [8]. miR140-3p inhibits the expression of CCL11, CXCL10, CCL5
and CXCL8, and is therefore considered to exert inflammation-associated biological function of airway smooth muscle[9].
A recent study demonstrated a new target gene of
miR-140-3p, Myomarker in vitro [10]. The Myomarker,
muscle-specific transmembrane protein, is necessary formyoblast fusion. Because Myomarker genetic deletion inmice completely abolishes muscle formation, Myomarker
expression is considered to be crucial for vertebrate muscle regeneration [11]. In the current study, we found
up-regulation of exosomal miR-140-3p, suggesting thatelevated miR-140-3p may have a possibility to inhibit
Myomarker expression and leading to musculoskeletal
events in CML patients. However, there was no report forthis association in human musculoskeletal pain; therefore, further study is required to conclude this possible
relationship. In the current study, the elevated exosomal miR-140-3p in CML patients became the level ofhealthy control, after release of musculoskeletal pain. It
is reported the association between musculoskeletal pain

13

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Up-regulated exosomal miRNA-140-3p in CML patients with musculoskeletal pain associated with…

NC AE−

B

AE+

P = 0.0022

miR-140-3p expression

A

5

0

miR-140-3p expression

miR-140-3p expression

P = 0.008
-5

-10

-15

NC

AE+

P = 0.2057

-10

-15

AE−

NC

AE+

D

P = 0.6343

0

P = 0.0336

-5

-11

C

421

AE+ −

0

STIM_141
-5

-10

-15

0

100

200

300

days

400

500

Fig. 1 a Exosomal miRNA profiling by TaqMan (Thermo Fischer
Science) mRNA array. b A different expression pattern was foundbetween CML patients with (AE+) or without musculoskeletal pain(AE−) and healthy subjects (normal control: NC). Using SDS2.2
and DataAssist software (Thermo Fisher Sciences), we calculated
the expression of miRNA based on their Ct value normalized by theCt value of ath-miR-159. Data were analyzed with the GeneSpring
and R software packages. Bars indicate mean ± standard error. Open
triangles indicate CML patients without musculoskeletal pain andmaintained deep molecular response, and open squares indicate those

who had molecular relapse. c Sequential analysis of exosomal miR140-3p levels in four CML patients with musculoskeletal pain. A
significant reduction of exosomal miR-140-3p levels was noted after
release of musculoskeletal pain (P = 0.008). AE+: samples during
musculoskeletal pain. AE + ⇒−: samples after release of musculoskeletal pain. d Representative case (STIM_141) showing reductionof exosomal miR-140-3p level. X-axis indicates days after imatinib
cessation, and y-axis does exosomal miR-140-3p expression level.
Back bar indicates period of musculoskeletal pain

and inflammatory indicators in some patients with CML
stopping TKIs [3, 4]; nevertheless, we did not find any
positive association.
Although the number of CML patients in this study
is too small to draw firm conclusions, further researchshould investigate whether up-regulation of exosomal
miR-140-3p expression in peripheral blood is correlatedwith musculoskeletal events in CML patients after TKI
cessation.

Author contribution TT, SO, SK, MG, KA, and KO treated thepatients; MA, TU, and CK performed exosomal miRNA profiling
analysis; TU performed statistical analysis; MA, JHO, and KO wrote
the paper; KO supervised the project. All authors contributed to thedata analysis, drafting, and revision of the manuscript.

Acknowledgements This work was supported by JSPS KAKENHI
Grant no. JP15H04303 (K.O.) and the Private University Strategic
Research-Based Support Project (S1311016) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (J.H.O.,
T.U.).

Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.

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International Journal of

Molecular Sciences
ArticleDownregulation of Plasma miR-215 in Chronic
Myeloid Leukemia Patients with Successful
Discontinuation of Imatinib

Kazuma Ohyashiki 1,2, *, Tomohiro Umezu 2 , Seiichiro Katagiri 1 , Chiaki Kobayashi 2 ,Kenko Azuma 3 , Tetsuzo Tauchi 1 , Seiichi Okabe 1 , Yutaka Fukuoka 4 and Junko H. Ohyashiki 3
1
2
3
4

*

Department of Hematology, Tokyo Medical University, Tokyo 160-0023, Japan; patchsei@yahoo.co.jp (S.K.);
tauchi@tokyo-med.ac.jp (T.T.); okabe@tokyo-med.ac.jp (S.O.)
Department of Molecular Science, Tokyo Medical University, Tokyo 160-0023, Japan;t_umezu@tokyo-med.ac.jp (T.U.); chiaki-k@tokyo-med.ac.jp (C.K.)
Department of Molecular Oncology, Institute of Medical Science, Tokyo Medical University, Tokyo 160-0023,
Japan; k_azuma@tokyo-med.ac.jp (K.A.); junko@hh.iij4u.or.jp (J.H.O.)
Department of Electrical Engineering, Kogakuin University, Tokyo 163-8677, Japan;
fukuoka@cc.kogakuin.ac.jp
Correspondence: ohyashik@rr.iij4u.or.jp; Tel.: +81-3-3342-1510; Fax: +81-3-5381-6651

Academic Editors: William Chi-shing Cho and Nalini Santanam
Received: 8 January 2016; Accepted: 8 April 2016; Published: 15 April 2016

Abstract: Approximately 40% of chronic myeloid leukemia (CML) patients who discontinue imatinib(IM) therapy maintain undetectable minimal residual disease (UMRD) for more than one year
(stopping IM (STOP-IM)). To determine a possible biomarker for STOP-IM CML, we examined
plasma miRNA expression in CML patients who were able to discontinue IM. We first screened
candidate miRNAs in unselected STOP-IM patients, who had sustained UMRD after discontinuing
IM for more than six months, in comparison with healthy volunteers, by using a TaqMan low-density
array for plasma or exosomes. Exosomal miR-215 and plasma miR-215 were downregulated in
the STOP-IM group compared to the control, indicating that the biological relevance of the plasmamiR-215 level is equivalent to that of the exosomal level. Next, we performed real-time quantitative
RT-PCR in 20 STOP-IM patients, 32 patients with UMRD on continued IM therapy (IM group) and28 healthy volunteers. The plasma miRNA-215 level was significantly downregulated in the STOP-IM
group (p < 0.0001); we determined the cut-off level and divided the IM group patients into two groups
according to whether the plasma miR-215 was downregulated or not. The IM group patients witha low plasma miR-215 level had a significantly higher total IM intake, compared to the patients withelevated miR-215 levels (p = 0.0229). Functional annotation of miR-215 target genes estimated by
the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic tools
involved cell cycle, mitosis, DNA repair and cell cycle checkpoint. Our study suggests a possible roleof miR-215 in successful IM discontinuation.
Keywords: plasma miR-215; chronic myeloid leukemia; imatinib; discontinuation

1. Introduction
Tyrosine kinase inhibitors (TKIs), including imatinib (IM), are now a standard frontline therapy
for chronic myeloid leukemia (CML) that yields successful outcomes [1]. CML therapy is widely
accepted, and achieving cytogenetic and molecular responses at certain time points after TKI therapy
and switching among TKIs have been proposed as practical guidelines [2]. In addition, clinical trials
for TKI discontinuation are still on-going, with the most famous studies being the STIM study [3] andthe TWISTER study [4]. In the STIM study, approximately 40% of CML patients with a molecularInt. J. Mol. Sci. 2016, 17, 570; doi:10.3390/ijms17040570

www.mdpi.com/journal/ijms-353-

Int. J. Mol. Sci. 2016, 17, 570

2 of 11

remission of at least a two years’ duration safely discontinued IM; they maintained undetermined
minimal residual disease (UMRD) for more than two years, and 60% maintained major molecularresponse (MMR: MR4.0 ) [3]. The STIM study showed that male gender, low Sokal score and IM
duration of >72 months were associated with maintaining treatment-free remission [3]. The TWISTER
study, using a patient-specific primer for BCR-ABL, revealed that some CML patients who maintained
UMRD after stopping IM had BCR-ABL DNA, leading to molecular relapse [4]. Early reduction of
BCR-ABL1 transcription by TKIs induces a deep molecular response [5], and expansion of cytotoxic
natural killer (NK) cells [6] could serve as a predictive marker for future treatment-free remission.
We have reported that extracellular microRNAs (miRNAs), including plasma miRNAs andexosomal miRNAs, are altered in hematopoietic neoplasias [7–9], and some support malignant
progression via the microenvironment [10,11]. miRNAs are noncoding single-stranded RNAs of
21–25 nucleotides that have recently been implicated in the regulation of cellular processes, such as
apoptosis, proliferation, development or differentiation, not only in normal hematopoiesis, but also
in hematological malignancies. The biological and clinical implications of cellular miRNAs are now
being extensively studied. Recently, extracellular miRNAs, also known as secretary miRNAs, havebeen proposed as having multiple functions, including immune-modulation, angiogenesis and cancer
progression [11–13]. Extracellular miRNAs, such as exosomal miRNAs, are extensively studied
in non-hematologic diseases, such as cardiovascular diseases, endocrine disorders or pulmonary
diseases [12]. Unlike solid tumors, the cellular component is easily obtained in the context of leukemia;
however, analysis of the cell-free fraction, including plasma, is worthwhile in cases of complete
remission when neoplastic cells are not present in the peripheral blood. We attempted to identify
circulating miRNAs in CML patients who maintained UMRD after stopping IM (STOP-IM), and we
studied target molecules by using bioinformatics tools.
2. Results
2.1. miRNA Expression Profiling by the TaqMan miRNA Array
To identify candidate plasma miRNAs with altered expression in the STOP-IM group, we screened
miRNA expression using a TaqMan miRNA array on seven unselected CML patients from the STOP-IM
group and seven healthy volunteers. Between these two groups, we observed differential expressionof 69 miRNAs identified by using GeneSpring software (Agilent Technologies, Santa Clara, CA,
USA) (Figure 1; Gene Expression Omnibus (GEO) Accession No. GSE75392). Based on the Wilcoxon
rank sum test of the R statistical software, only two miRNAs, miR-215 (p = 0.006841) and miR-134(p = 0.028805), had greater than a 1.5-fold change in expression.
To determine whether miRNA expression in plasma indeed reflects exosomal miRNA,
we compared the expression profiles by using a TaqMan low-density array. We randomly chose
three patients in the STOP-IM group and three healthy volunteers. Eleven miRNAs in plasma and35 exosomal miRNAs were found to be significantly different between the two groups. Among thesemiRNAs, downregulation of miRNA-215 expression was highly significant in the STOP-IM group(plasma miRNAs, p = 0.00311 (Table S1); exosomal miRNAs, p = 0.00039 (Table S2)); therefore,
we concluded that plasma miR-215 expression mirrors exosomal miRNA expression and focusedon expression of this miRNA for further study.

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3 of 10Int. J. Mol. Sci. 2016, 17, 570

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Int. J. Mol. Sci. 2016, 17, 570

3 of 10Figure 1. miRNAprofiling theby the TaqMan(Thermo(Thermo Fischer
Science,
Waltham,
MA, USA)
FigureFischer
Science,
Waltham,MA,miRNA
USA)miRNA
miRNA
Figure1.1.miRNA
miRNAprofilingprofilingby
by the TaqMan
TaqMan (Thermo Fischer
Science,
Waltham,MA,
USA)
array. A differential expression pattern was found between stopping imatinib (STOP-IM) patients andarray.
patternwas
foundbetweenstopping
imatinib(STOP-IM)
patients
andarray.AAdifferential
differentialexpression
expression
patternwas
foundbetweenstopping
imatinib(STOP-IM)
patients
control subjects. Using Sequence Detection System (SDS, Version 2.4, Thermo Fisher Science, Waltham,control
subjects.
Using
Sequence
Detection
System(SDS,
Version
2.4,
ThermoFisherScience,
Waltham,and control
subjects.
Using
Sequence
Detection
System(SDS,
Version
2.4,
ThermoFisherScience)and
MA, USA) and DataAssist software (Thermo Fisher Science, Waltham, MA, USA), we calculated the
MA,
USA)
and
DataAssist
software(ThermoFisherScience,
Waltham,wecalculated
the
DataAssist
software(ThermoFisherScience),wecalculatedof USA),
miRNAsbased
normalized
by thetheCexpression
t value ofMA,
ath-miR-159.
Data
wereon theirexpressionof miRNAs
basedon theirCt value
analyzed
withGeneSpring
software(Agilent
Technologies,
Santa
Clara,
CA,
USA).
value normalized
by theanalyzed
Ct valuewithof ath-miR-159.
wereexpressionof miRNAsbybased
theirof
Ctath-miR-159.
Ct value normalized
the Con
Data were
GeneSpringData
softwaret value(Agilent Technologies,
Santa
Clara, CA,
USA).Technologies, Santa Clara, CA, USA).
analyzedwith GeneSpring
software(Agilent

2.2. Quantification of Individual miRNA by Real-Time Quantitative Reverse Transcriptase-Polymerase
Chain Reaction

2.2.
QuantitativeReverse
ReverseTranscriptase-Polymerase
Transcriptase-Polymerase
2.2.Quantification
QuantificationofofIndividual
Individual miRNA
miRNA by
by Real-Time
Real-Time Quantitative
Since
the
expressionofmiR-215
was
typically
downregulated
in
both
the
plasma and exosomes
Chain
Reaction
Chain Reactionof the STOP-IM group compared to healthy individuals, we analyzed the plasma miR-215 expression

Since
the
expression
ofbymiR-215
miR-215
was
typically
downregulated
in
the
and
level
inexpression
CML
patientsof
individual
real-timequantitative
RT-PCR. Plasma
samples
were obtained
Since
the
was
typically
downregulated
in bothboththeplasma
plasmaandexosomes
exosomesfrom20
patients
inthe
STOP-IM
group
and
32
of
the
IM
group,
as
well
as
from
28
healthy
volunteers.
theSTOP-IM
STOP-IMgroup
groupcompared
compared toto healthy
healthy individuals,
individuals, weofofthewe analyzed
analyzed thethe plasma
plasma miR-215
miR-215expression
expression
ACML
validation
analysisusing a large
number ofsamples revealed
that expressionof miR-215 waslevelininCML
patients
by individual
individual
real-timequantitative
RT-PCR.
level
patients
byreal-timequantitative
RT-PCR. Plasma
Plasma samples
sampleswere
wereobtained
obtainedsignificantly lower in the STOP-IM group than in the healthy volunteers (p < 0.0001) or in the IMfrom20
patients
inthe
STOP-IM
group
and
32
of
the
IM
group,
as
well
as
from
28
healthy
volunteers.
from 20 patients
inthe
STOP-IM
group
and
32
of
the
IM
group,
as
well
as
from
28
healthy
volunteers.
group (p = 0.0439) (Figure 2A).
validation analysis
analysis using
using aa large
large number
number ofof samples
AAvalidation
samples revealed
revealed thatthat expression
expression ofof miR-215
miR-215was
was
significantly
lower
in
the
STOP-IM
group
than
in
the
healthy
volunteers(p<0.0001)
or
in
the
IM
group
significantly lower in the STOP-IM group than in the healthy volunteers (p < 0.0001) or in the IM(p = 0.0439)(Figure2A). 2A).
group(p = 0.0439)(FigureFigure 2. Expression of miR-215 (A). The cutoff level for miR-215 expression between the STOP-IM
and control groups (B). The area under the curve level was 0.9204. The cutoff value was thus
determined to be 0.6627, which was used in the following analyses of plasma miR-215 expression.
Abbreviations: IM, imatinib mesylate; CI, confidence interval; CML, chronic myeloid leukemia;
STOP-IM, patients with sustained undetectable minimal residual disease (UMRD) for more than six
months after discontinuing IM; N, healthy volunteers; IM, CML patients taking IM who have a
Figure2.2.
Expression
ofmiR-215
miR-215 (A).
(A); The
The cutoff level for miR-215
FigureExpressionofmiR-215 expression
expression between
betweenthe
theSTOP-IM
STOP-IM
molecular
response.

andcontrol
control groups
groups (B).
(B). The
The area under theandthe curve
curve level
level was
was 0.9204.
0.9204. The cutoff
cutoff value
value was
was thus
thus
determinedtotobe
be0.6627,
0.6627,which
which waswas used
used inin thethe following analyses of plasma
determined
plasma miR-215
miR-215 expression.
expression.
Abbreviations: IM,
IM, imatinib
imatinib mesylate;mesylate; CI,
CI, confidence
confidence interval;
Abbreviations:interval; CML,
CML, chronic
chronic myeloid
myeloid leukemia;
leukemia;
STOP-IM,patients
withsustained
undetectable
minimalresidualdisease(UMRD)
more
than
STOP-IM, patients with sustained undetectable minimal residual disease (UMRD) forfor
more
than
six
six months
discontinuingN, healthy
volunteers;
CML
patients
takingIMIMwho
whohavehavea
monthsafterafter
discontinuing
IM; IM;N, healthy
volunteers;
IM,IM,
CML
patients
takinga molecularresponse.
molecular
response.

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Int. J. Mol. Sci. 2016, 17, 570

4 of 112.3. Clinical and Biological Relevance of miR-215 Expression
To estimate a cutoff value for miR-215 expression in plasma for distinguishing between STOP-IM
and healthy control groups, we performed receiver operating characteristics (ROC) curve analysis(Figure 2B). The area under the ROC curve was 0.9204% with 85.00% sensitivity (95% confidence
interval (CI), 62.22%–96.79%) and 85.71% specificity (95% CI, 42.13%–85.71%). The cutoff value wasthus determined to be 0.6627, which was used in subsequent analyses of miR-215 expression. We next
analyzed the clinical relevance of the plasma miR-215 expression level in CML patients who sustained
MR4.0 while on IM (IM group). There were no significant differences in age (p = 0.5903), sex (p = 1),
Sokal score (p = 0.2719) or prior interferon therapy history (p = 0.7026). Notably, the patients witha low plasma miR-215 level had a significantly higher total IM intake, compared to the patients witha high miR-215 level (p = 0.0229), while no significant differences existed with regard to the daily IM
dose (p = 0.395) or duration of IM therapy (p = 0.0871) between the two CML groups with or withouta low expression level of plasma miR-215 (Table 1). These two CML groups also did not significantly
differ in the International Scale at the time of plasma miR-215 examination (p = 0.4126). The durationof UMRD from CML diagnosis (p = 0.3827) and the duration of stable molecular response (maintain
MMR) (p = 0.7548) did not differ between these two groups (Table 1).
Table 1. Clinico-hematologic characteristics of chronic myeloid leukemia (CML) patients under imatinib
therapy with or without down-regulation of plasma miR-215 expression.
Plasma miR-215 Levels (Number of CML
Patients Who Are Taking Imatinib)<0.6627>0.6627n = 22n = 10Age (years)
Sex (male/female)
Sokal score (low/intermediate/high)Prior interferon therapy (yes/no)
Total IM dose (g)Daily IM dose (mg)
Duration IM (months)
UMRD from diagnosis (months)
Duration of UMRD (months)
International scale percentage at the time ofplasma miRNA examination

55.91 ˘ 2.436
18/4
19/3/0
7/15
931.0 ˘ 65.32
377.3 ˘ 11.27
84.05 ˘ 5.654
53.27 ˘ 8.855
41.82 ˘ 5.283

58.50 ˘ 4.634
8/2
7/2/1
4/6
670.5 ˘ 71.67
360.0 ˘ 16.33
65.30 ˘ 9.656
70.80 ˘ 22.24
38.60 ˘ 9.782

0.0026 ˘ 0.0003

0.0031 ˘ 0.0006

p Value
0.59031*
0.2719 *0.7026 *0.02290.395
0.0871
0.38270.75480.4126IM, imatinib mesylate; UMRD, undetectable minimum residual disease; MR, molecular response; MMR, major
molecular response; * determined by the chi-square test.

2.4. Functional Annotation of miRNA-215 Target Genes
The clinical relevance of miR-215b was not evident, so we sought experimentally-validated
target genes of miRNA-215 in miRTarBase or determining the biological significance of miR-215.
More than 100 target genes were extracted by miRTarBase and the target genes for which strong
evidence existed are summarized in Table 2. Six genes, including WNK lysine deficient protein kinase
1 (WNK1), activated leukocyte cell adhesion molecule (ALCAM), retinoblastoma 1 (RB1), protein
tyrosine phosphatase receptor type T (PTPRT), activin A receptor type IIB (ACVR2B) and catenin beta
interacting protein 1 (CTNNBIP1), were experimentally validated as genes with strong evidence forbeing targets (Table 2) [14–23]. To estimate the biological implication of miR-215 target genes, wethen used the DAVID bioinformatic tool for functional annotation of target genes and possible related
pathways (p < 0.0001). Approximately 30% of the target genes were related to cellular metabolic
process, and miR-215 target genes were subsequently determined to be involved in the cell cycle,
cellular stress, mitosis, DNA repair and cell cycle checkpoints (Figure 3 and Table S3).

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5 of 10

Table 2. Top 10 of the predicted target genes for miR-215 that identified by MiRTarBase 5 of 11

Int. J. Mol. Sci. 2016, 17, 570

Validation Methods
Strong EvidenceLess Strong Evidence
ID
Target
Sum ReferencesTable
2. Top 10of the predicted
ReporterWesterntarget genes for miR-215 that identified by MiRTarBase.
qPCR Microarray NGS pSILAC Other
Assay
Blot
Validation Methods
MIRT005583
WNK1
*
*
*
*
4
[14]
StrongEvidence
Less Strong Evidence
MIRT024327
ALCAM
*
*
*
*
4
[15,16]
TargetID
Sum References
ReporterWestern
Blot*qPCR MicroarrayNGS
pSILAC Other
MIRT024341
RB1* Assay**4[15]
MIRT005583 ZEB2
WNK1
*
*
*
*
4 3
[14] [17]MIRT052961
*
*
*
ALCAM
*
*
*
4 3 [15,16] [18]MIRT024327 PTPRTMIRT054778****
MIRT024341
RB1
*
*
*
*
4
[15]
MIRT006340
ACVR2B**MIRT052961
ZEB2
*
*
*
3 2
[17] [19]MIRT007364***
MIRT054778CTNNBIP1
PTPRT
*
*
3 2
[18] [15,20]
ACVR2B**2 2[19] [15,21]
MIRT006340 DTL
MIRT024273**
CTNNBIP1**
2[15,20]
MIRT007364
MIRT024412
GABPB1* **2 [15,21][15,22]
MIRT024273
DTL*
2
MIRT024495* ***
GABPB12 2 [15,22][15,23]
MIRT024412 SYNGR1SYNGR1**
2[15,23]
MIRT024495
Asterisks
indicate
data from validation methods; NGS, next-generation
sequencing; pSILAC,
stableAsterisks indicate data from validation methods; NGS, next-generation sequencing; pSILAC, stable isotope
isotopelabelinglabeling
by amino
in cell culture.
by aminoacid inacidcell culture.

Figure3. A3.functionalannotation
Bioinformatic
tools
v6.7
demonstrated
FigureA functionalannotationanalysis
analysisusing
using DAVIDDAVID Bioinformatic
tools
v6.7
[24][24]
demonstrated
that thatthe thetargetgenes
miR-215were
werehighlyrepresented
in the categories
cellularprocess
metabolictargetgenesforfor miR-215
highlyrepresented
in the categories
of cellularof
metabolic(Gene
Ontology(GO):(GO):44237),44237),
cell cycle
07049),
stimulusto(GO:51716),(GO:mitosisprocess(Gene
Ontologycell(GO:
cycle(GO:cellular
07049),response
cellular to
response
stimulus
51716),(GO:
07067),
DNA
repair(GO:
06281)
and
cell
cycle
checkpoint(GO:
00075).
The
percentages
ofgenes
mitosis (GO: 07067), DNA repair (GO: 06281) and cell cycle checkpoint (GO: 00075). The percentages
that contributed
to representative
categories
are depicted.
of genesthat contributed
to representative
categories
are depicted.

3. Discussion
3. Discussion
During the last decade, emerging evidence has shown that miRNAs are a key molecular tool for
During the last decade, emerging evidence has shown that miRNAs are a key molecular tool forcancer diagnosis and prognosis. A section of tumor tissue or a circulating cell-free fraction, such as
cancerdiagnosis and prognosis. A section of tumor tissue or a circulating cell-free fraction, such asplasma, may be used for miRNA analysis for finding novel diagnostic and prognostic markers [12].
plasma,may
be majority
used for ofmiRNA
findingnovel
diagnostic
and prognostic
markers
In thevast
reportsanalysis
dealing for
with
miRNA
diagnosis,
investigators
identified
tumor [12].
In thevast majority
reports
dealingwithis miRNA
diagnosis, investigators
tumor
cell-derived
miRNA.ofWhen
a cell-freefractionused, biologically-relevant
miRNA is identified
considered tocell-derived
miRNA.
Whenacell-free
fractionisused,biologically-relevant
miRNA
isconsidered
be exosomal miRNA derived from circulating cancer cells. For miRNA analysis in leukemia, using to
be exosomal
fromcirculating
cancercells. For
analysis
in ofleukemia,
using
leukemia miRNAcells mayderived
indeed be
morereliable fordetermining
the miRNA
biologicalrelevancethese cells
than using
However,to findreliablea novel for
prognostic
markerthein patients
withrelevancecomplete remission,
leukemiacells plasma.
may indeed
be moredetermining
biologicalof these cells
suchasSTOP-IMpatients,
we
need
to
use
a
different
type
of
miRNA
analysis.
For
instance,
wethan using plasma. However, to find a novel prognostic marker in patients with complete remission,
recently
showedthat
microRNA-148b,
a
type
of
immunemiRNA
incirculating
peripheral
bloodsuch as STOP-IM patients, we need to use a different type of miRNA analysis. For instance, wemononuclear cells (PBMCs), is downregulated in STOP-IM patients [13], in accordance with increased
recently
showed that microRNA-148b, a type of immune miRNA in circulating peripheral blood
NK cells [6]. This finding indicated the possibility that maintaining UMRD after stopping IM might bemononuclear cells (PBMCs), is downregulated in STOP-IM patients [13], in accordance with
due to immune surveillance. In such cases, the miRNA level may reflect the component of circulating
increased NK cells [6]. This finding indicated the possibility that maintaining UMRD after stopping
immune cells; therefore, miRNA analysis requires careful attention and interpretation in a complete
IM might
be dueto immune surveillance. In such cases, the miRNA level may reflect the component
remissionstate.

of circulating immune cells; therefore, miRNA analysis requires careful attention and interpretation
in a complete remission state.
To further elucidate the possible role of miRNA in STOP-IM patients, we analyzed plasma
miRNA. We and others have reported the clinical relevance of plasma or serum miRNA in hematologic
neoplasia [7–9,25]. Recent technical advances in exosome separation and characterization enabled-357-

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6 of 11

To further elucidate the possible role of miRNA in STOP-IM patients, we analyzed plasma
miRNA. We and others have reported the clinical relevance of plasma or serum miRNA in hematologic
neoplasia [7–9,25]. Recent technical advances in exosome separation and characterization enabled
analyzing exosomal miRNA rather than plasma or serum miRNA [11]. Theoretically, using exosomal
miRNA for analysis would be ideal, but in practical terms, the procedure remains complicated. For thisreason, we compared the results obtained from plasma and exosome. Regardless of the template source,
plasma miR-215 expression was downregulated in STOP-IM patients (Tables S1 and S2). Our CML
patients in the STOP-IM group did not take IM for more than six months before the plasma miRNA
assay, indicating that the downregulation of plasma miR-215 in STOP-IM patients was not due tocurrent IM intake. It is well known that approximately 60% of CML patients who discontinue IM
relapse within 12 months [3–5] and CML stem cells may persist after stopping IM [4]. Since the total
IM intake dose was significantly higher in CML patients with low miR-215 levels in the IM group,
CML stem cells may be more suppressed, although the prognosis in the CML-IM group did not differ
between patients with and without low plasma miR-215 expression. Long-term follow-up of CML-IM
patients may clarify this matter.
In the current study, plasma miR-215 level was significantly low in the STOP-IM group, whencompared to normal subjects, nevertheless, none of these groups took IM for more than six months.
We speculated that the difference of plasma miR-215 level in the IM group (CML patients with UMRD,
but maintaining IM therapy) could predict a subset of CML patients who could stop IM safely, but thecurrent study failed to predict the selection of the candidate correctly. Therefore, we searched miR-215
target genes by miRTarBase or those that may be linked to immunological surveillance or residual
CML stem cells; miR-215 has previously been demonstrated to be dysregulated in several humanmalignancies and to be correlated with tumor progression [19,21,26,27]. Meta-analyses have revealed
that elevated miR-215 expression is correlated with poor prognosis in renal cell carcinomas [28],glioma [29] and pancreatic cancer [30]. In contrast, decreased expression was associated withadvanced disease stage in colorectal cancer [31], and miR-192-5p/215 is a putative tumor suppressor
in non-small cell lung cancer [32]. Taken together, miR-215 may potentially be both an oncomiRand a tumor-suppressive miR, with its actual role depending on the type of cancer and the pathway
involved. In the current study, it was impossible to conduct a functional analysis of miR-215, such asa reporter assay using specimens from patients in a complete remission state. As an alternative, we
utilized bioinformatics tools, such as miRTarBase and DAVID, to identify the miR-215 target gene.
miRTarBase includes information on miRNA–target interactions (MTIs), with the collected MTIs
having been experimentally validated by reporter assay, Western blot, microarray and next-generation
sequencing [33]. Based on miRTarBase, we particularly focused on RB1 and ACVR2, showing strong
evidence in this study.
Deng et al. [32] reported upregulation of miR-215 in gastric cancer tissues, and functional analysis
revealed that inhibition of miR-215 significantly suppressed gastric cancer cell proliferation, possibly
via G1 arrest. In addition, miR-215 was able to target RB1 through its 31 -UTR in gastric cancer
cells [32]. Conversely, Braun et al. [34] reported that p53 induces upregulation of miR-192, miR-194
and miR-215, and miR-192/miR-215 enhanced CDKN1/p21 levels, colony suppression and cell cycle
arrest; therefore, miR-192/miR-215 may suppress carcinogenesis through p21 accumulation and cell
cycle arrest. Senanayake et al. [19] reported that miR-192, miR-215 and miR-194 had significantly lower
expression in nephroblastomas, and they identified the target as activating receptor type 2B (ACVR2B),
indicating that downregulation of miR-215 may affect an important step in the pathogenesis of renal
childhood neoplasms. ACVR2B is known to be associated with human embryonic stem cells [35].
Human embryonic stem cells express receptors for nodal (ACVR1B and ACVR2B) and a co-receptor
for nodal, and they control NANOG expression via SMAD2/SMAD3; therefore, miR-215 targeting
ACVR2B may affect the self-renewal capability of stem cells [36]. Although all of the evidence isprovided by databases, DAVID bioinformatics tools clearly showed that miR-215 targets genes in thepathway involved in cell cycle regulation.

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7 of 11The data presented in the current study suggest that the expression level of plasma miRNAs
in CML patients who maintained a deep molecular response to IM is lower than in normal subjects.
Of the 32 CML patients taking IM at the time of plasma miRNAs examination, 16 patients discontinued
IM after the assay, and five have had a molecular relapse. Unfortunately, we observed no significant
differences in the plasma miR-215 level or in the clinical data between these two IM groups (datanot shown), possibly because of the retrospective study for miRNAs assessment. The plasmamiR-215 level in CML patients who had been maintaining molecular remission without imatinib
was actually downregulated; therefore, further prospective studies may provide new insight into
available biomarkers for therapy-free remission in CML patients.
4. Experimental Section
4.1. Patients and Samples
Fifty-two consecutive patients with CML, who maintained MR4.0 or more by IM therapy,
were enrolled in this study. Samples from unselected 20 patients who had sustained UMRD after
discontinuing IM (STOP-IM group) were collected more than 6 months after discontinuation [13].
The STOP-IM patients maintained the negativity of nested PCR for more than two years before
stopping IM therapy. Thirty-two patients who were receiving IM and maintained less than MR4.0
at the time of plasma collection were categorized as the IM group. Twenty-eight healthy volunteers
served as the control group. CML patients who had not received treatment and those who did
not respond to IM were not included in this study. No significant difference existed in sex orage between the CML groups and healthy volunteers. Molecular response was determined by the
BCR-ABL1 transcript, as reported previously [6,13], and was represented as an International Scale(IS). UMRD was confirmed by the absence of BCR-ABL1 transcript as determined by nested reverse
transcription-polymerase chain reaction (RT-PCR) [13]. This study was approved by the institutional
review board of Tokyo Medical University (No. 930, approved 24 June 2008). Written informed
consent was obtained from all patients prior to specimen collection, in accordance with the Declarationof Helsinki.
4.2. TaqMan Low-Density Array Screening
Total RNA was isolated with the mirVana PARIS kit (Ambion, Austin, TX, USA) fromseven randomly-selected healthy volunteers and seven randomly-selected STOP-IM patients.
Five hundred microliters of plasma were diluted with 500 µL of binding solution. After a 5-min
incubation, 1 µL of 1 nM ath-miR-159 (Hokkaido System Science, Hokkaido, Japan) was added,
followed by vortexing for 30 s and incubation on ice for 10 min. Subsequent phenol extraction andcartridge filtration were performed according to the manufacturer’s instructions. The RT reaction
and pre-amplification step were set up according to the recommendations. miRNAs were reverse
transcribed with Megaplex Prime Pools (Human Pools A v2:1; Thermo Fisher Sciences). The miRNA
expression profiles were determined with a TaqMan miRNA Array Human Card A (Thermo Fisher
Sciences). To compare the results between plasma miRNA expression and exosomal miRNA expression,
we extracted the exosome by Total Exosome Isolation Reagent (Invitrogen, Carlsbad, CA, USA) andthen assessed exosomal miRNA expression in a subset of samples, as described previously [11].
Quantitative RT-PCR was performed on an Applied Biosystem 7900 HT thermocycler according to
the manufacturer’s recommended program [13]. With the use of SDS2.2 software (Invitrogen) and
DataAssist (Thermo Fisher Sciences), the expression of plasma miRNAs was calculated based on cycle
threshold (Ct ) values normalized to those of ath-miR-159, which was spiked in each plasma sample.
Data analysis was done using GeneSpring software (Agilent Technologies). The Benjamin-Hochberg
algorithm was used for estimation of false discovery rates, as we reported previously [13].

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8 of 114.3. Real-Time Quantitative RT-PCR for miR-215
We determined the amounts of the individual miRNA by real-time quantitative RT-PCR witha TaqMan MicroRNA Assay (Thermo Fisher Sciences) and the following miRNA-specific stem-loop
primer: hsa-miR-215 (hsa-miR-215-4373084). Subsequently, quantitative real-time PCR was performed
with an ABI Prism 7000 sequence detection system (Thermo Fisher Sciences). The reaction was initiatedby incubation at 95 ˝ C for 2 min, followed by 50 cycles of 95 ˝ C for 15 s and then 60 ˝ C for 1 min.
All reactions were run in duplicate. Mean (Ct ) values for all miRNAs were quantified with sequence
detection system software (SDS Version 1.02, Thermo Fisher Scientific) [13]. The relative expressionof miR-215 was calculated by using the ∆Ct method as reported previously [8]. The spike control(ath-miR-159) was used as an invariant control for plasma miRNA.
4.4. Statistical and Bioinformatics Analyses
Data were expressed as means ˘ standard deviation (SD). Mann-Whitney U and χ-square tests
were used to determine statistical significance for comparisons between the control and test groups.
Multiple groups were compared by one-way analysis of variance (ANOVA). Statistical analysis
was done using R software and GraphPad Prism software (Version 5c for Macintosh; GraphPad
Software Inc., La Jolla, CA, USA). miRNAs with a ∆Ct of >1.0 or <´1.0 and p-values of >0.05 weredeemed as being differentially expressed.
Following identification of differentially-expressed miRNAs, the predicted target genes for thesealtered miRNAs were subjected to the experimentally-validated miRNA-target interactions database
miRTarBase [37]. Moreover, functional annotation of target genes was determined by the Database forAnnotation, Visualization and Integrated Discovery (DAVID Bioinformatics tools v6.7) [24].
5. Conclusions
In the current study, we found that plasma miR-215 was downregulated in CML patients who
had stopped IM therapy compared to normal subjects. Further studies will reveal the biological andclinical significance of miR-215, including the plasma miR-215 level, and provide further information
for treatment-free remission in CML patients. In conclusion, plasma miRNA analysis, which may
reflect exosomal miRNA, could be a potent prognostic biomarker in hematologic malignancies thathave a current remission status.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/
17/4/570/s1.
Acknowledgments: This work was supported by the Private University Strategic Research Based Support Project(S1311016) and in part by the Grant-in-Aid (15H04303) from MEXT (Ministry of Education, Culture, Sports,
Science and Technology, Tokyo, Japan).
Author Contributions: Kazuma Ohyashiki designed and performed the results. Kazuma Ohyashiki,
Seiichiro Katagiri, Tetsuzo Tauchi and Seiichi Okabe contributed essential reagents or tools. Tomohiro Umezu,
Chiaki Kobayashi and Kenko Azuma performed PCR analysis and bioinformatic analysis. Yutaka Fukuoka
performed bioinformatic analysis. Kazuma Ohyashiki wrote the paper. Junko H. Ohyashiki performed criticalreading. All authors contributed toward data analysis, drafting and revising of the manuscript.
Conflicts of Interest: Kazuma Ohyashiki received research support from Novartis and Bristrol-Meyer Squibb.
The authors report no other conflicts of interest in this work.

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Contents lists available at ScienceDirect

Leukemia Research
journal homepage: www.elsevier.com/locate/leukres

Research paper

Genetic variations of bone marrow mesenchymal stromal cells derived fromacute leukemia and myelodysplastic syndrome by targeted deep sequencing
Kenko Azumaa, Tomohiro Umezub, Satoshi Imanishia, Michiyo Asanob,1, Seiichiro Yoshizawab,⁎
Seiichiro Katagirib, Kazuma Ohyashikib, Junko H. Ohyashikia,ab

Department of Molecular Oncology, Institute of Medical Science, Tokyo Medical University, 160-0023, Japan
Department of Haematology, Tokyo Medical University, 160-0023, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
Bone marrow mesenchymal stromal cells
Targeted deep sequencing
Genetic variants
Myeloid malignancies

Bone marrow mesenchymal stromal cells (MSCs), which support proliferation and diﬀerentiation of hematopoietic stem cells, may play a crucial role in the pathogenesis of myeloid neoplasms. To determine whether MSCs
in myeloid neoplasms harbor distinct somatic mutations that may aﬀect their function, we used a targeted genesequencing panel containing 50 myeloid neoplasm-associated genes with coverage of ≥500. We compared thegenetic alterations between MSCs and bone marrow hematopoietic (BM) cells from patients with acute leukemia(n = 5) or myelodysplastic syndrome (MDS, n = 5). Non-synonymous somatic mutations, such as DNMT3AR882H and FLT3-D835Y, were only detected in BM cells with high allelic frequency. We found several nonsynonymous genetic variants overlapping BM cells and MSCs, including TP53 and ASXL1, partially owing to theheterogenous cell fraction of MSC samples and lineage ﬁdelity. We also found MSC-speciﬁc genetic variants withvery low allelic frequency (7% to 8%), such as NF1-G2114D and NF1-G140. Further studies in large cohorts areneeded to clarify the molecular properties of MSCs including age-related genetic alterations by targeted deep
sequencing.

1. Introduction
The bone marrow mesenchymal stromal cell (MSC) population, themajor component of hematopoietic microenvironment [1,2], is comprised of a mixture of several adherent cell types including ﬁbroblasts,
endothelial cells, macrophages, osteoclasts [3,4], and mesenchymal
stem cells that can diﬀerentiate into adipocytes, astrocytes, cardiomyocytes, chondrocytes, hepatocytes, muscles, neurons, and osteoblasts [5]. The deﬁnition of MSC is therefore somewhat obscure owing
to the complexity of cells in the stromal cell fraction. The hematopoietic
microenvironment is involved in the pathogenesis of acute myeloid
leukemia (AML) as well as myelodysplastic syndrome (MDS) [6,7].
Accordingly, dysfunction of MSCs leading to insuﬃcient stromal support, impaired osteogenic diﬀerentiation activity, and increased IL-6
secretion has been reported in AML and MDS [8–10]. In addition to thefunctional abnormalities of MSCs, a distinct gene expression proﬁle in
MSCs from AML and MDS has also been reported using either microarray or RNA sequencing [11–13].
Several reports demonstrated cytogenetic abnormalities in stromal
cell fractions derived from AML and MDS patients [14–19]. More recent⁎

1report using whole exome sequencing demonstrated that genetic alterations in the stromal compartment in 16 AML patients were nonspeciﬁc [13], while other studies using mouse models suggested nicheinduced oncogenesis [20,21]. Therefore, whether MSCs derived fromhematologic disorders harbor speciﬁc somatic mutations that may playa key role in the pathogenesis of myeloid neoplasms remains unclear. In
practical terms, genetic analysis of MSCs is challenging because it isdiﬃcult to exclude genetic polymorphisms by simultaneous study ofgerminal cells, in addition to lineage ﬁdelity of MSCs. To address thesequestions and to gain more insight into genetic properties of MSCs
derived from patients with acute leukemia and MDS, we analyzed genetic variants in 50 major genes associated with myeloid malignancies
by a targeted deep sequencing.
2. Materials and methods
2.1. PatientsTen consecutive patients with myeloid malignancies (ﬁve withacute leukemia and ﬁve with MDS) were included in this study

Corresponding author.
E-mail address: junko@hh.iij4u.or.jp (J.H. Ohyashiki).
Current address: Department of Hematology, Erasmus Medical Center, Wytemaweg 80 3015CN, Rotterdam, the Netherland.

http://dx.doi.org/10.1016/j.leukres.2017.09.008
Received 30 March 2017; Received in revised form 15 September 2017; Accepted 17 September 2017
Available online 21 September 20170145-2126/ © 2017 Elsevier Ltd. All rights reserved.

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Table 1
Patient characteristics and cytogenetic results.
UPN

Age (years)

GenderDiagnosis

Karyotypes of BM cells

Karyotypes of BMSCs

Acute leukemia
36
74

Male

AML

45,XY,-18[6]/46,XY[27]3755

FemaleAML

Multiple missing chromosomesa [11]/46,XX[7]

38
39

77
61

Male
Male

AML
BAL

46,XY,inv(9)(p12q13)[15]/46,XY[6]46,XY,-4,add(5)(q31),-13, add(14)(q32),+2 ∼ 4mar[19]/46,XY,-4,add(5)(q31),add(14)(q32),-20,+2mar[1]/46,XY,-8,8,+2mar[1]46,XX,inv(16)(p13q22)[4]/47,XX,idem,+22[18]47,XY,+7[2]/46,XY,-15,der(1)t(1;15)(q24;q13)[1]/46,XY[22]46,XX,t(3;12)(q24;q13),t(1;8)(p24;q22)[2]/47,XX,+8[1]/46,XX,-7,+der(7)t(1;7)(q11;p21)[1]/45,XX,-5,6,-12,+2mar[1]/46,XX[21]46,XY[6]46,XY[1]4042FemaleMyelodysplastic syndrome
10
60
Male
18
75
Male

25
13

72
70

Male
Male

35

67

Male

t-MDS/AML
RA (intermediate)
RCMD (very high)

RCMD (intermediate)
RAEB-2(intermediate)
RAEB-2 (high)46, XX[5]46,XY,del(20)(q11)[20]/46,XY[1]41-44,X,-Y,del(5)(q13), del(17)(p12)[16]/-18[14]/t(1;3)(p22;q22),del(7)(p15)[11]/add(2)(q37),-13[10]/-22,+mar[5]/-20[4]/del(11)(q23)[3]46,XY,-7,+mar[21]45,X,-Y[6]/46,XY[16]46,XY[2]46,XY[4]45,XY,del(1)(p12),-15[2]/46,XY[13]No metaphase

46,XY[10]46,XY,del(3)(p21),add(4)(p15)[8]/multiple deletion

UPN, unique patient number; BM, bone marrow; BMSC, bone marrow stromal cell; RA, refractory anemia; RCMD, refractory cytopenias with multilineage dysplasia; RAEB-2, refractory
anemia with excess blasts-2; AML, acute myeloid leukemia; BAL, biphenotypic acute leukemia; MDS, myelodysplastic syndrome; (x), IPSS-R; [n], number of metaphases.
amultiple missing chromosomes: representative karyotype = 42,XX,-5,-7,-8,-10, 43,XX,-6,-15,-17; missing 17 in 6 cells, missing 5 in 4 cells, missing 11 in 3 cells, missing 11b in 3 cells,
missing 19 in 3 cells, missing 6 in 23 cells, and missing 7 in 3 cells.

(Table 1). All patients were treated at Tokyo Medical University, Tokyo,
Japan from April 2011 to September 2015. The patients’ mean age was65.3 years (range 42–77 years). The diagnoses were established according to the 2008 World Health Organization criteria: three patientswere diagnosed with de novo AML, one patients with biphenotypic
acute leukemia (BAL), one patient with therapy-related myelodysplastic
syndrome/acute myeloid leukemia (t-MDS/AML), one patient with refractory anemia (RA), two patients with refractory anemia with excess
blasts (RAEB), and two patients with refractory cytopenia with multilineage dysplasia (RCMD). Cytogenetic analyses of both bone marrow
and MSCs were performed as reported previously [22]. Bone marrow
and MSCs obtained from three patients with non-Hodgkin’s lymphoma(NHL) without bone marrow invasion were also used as non-myeloid
malignancy controls for deep sequencing. Written informed consent
was obtained from all patients. The study was validated by the internal
review boards of Tokyo Medical University and followed both the Declaration of Helsinki and “Guidelines for Genetic Tests and Diagnoses in
Medical Practice” by The Japanese Association of Medical Sciences.

monoclonal antibodies from BD Pharmingen (San Jose, CA, USA). MSCs
diﬀerentiated to adipocytes and osteoblasts after exposure to MSC differentiation media (PromoCell, Heidelberg, Germany) for at least one
week. Adipocyte and osteoblast diﬀerentiation was determined bystaining with Oil Red (Sigma, St. Louis, MO, USA) and Alizarin Red(Wako, Osaka, Japan), respectively (Supplementary Fig. 1).

2.2. Primary MSC culture

2.4. GeneRead DNAseq library preparation

MSCs were obtained by the classical adhesion method with a minor
modiﬁcation [23,24]. Brieﬂy, 0.5–1 mL of freshly obtained bonemarrow aspirates were cultured in an equivalent volume of RPMI1640
medium (Thermo Fisher Scientiﬁc, Waltham, MA, USA) containing 10%of fetal bovine serum (FBS; GE Healthcare UK, Buckinghamshire, England), 1% of penicillin streptomycin (P/S; Thermo Fisher Scientiﬁc),and Dulbecco's Modiﬁed Eagle Medium (DMEM) (Thermo Fisher Scientiﬁc) containing 10% of FBS (GE Healthcare, UK), 1% of P/S, and 1%
of non-essential amino acids (NEAA; Thermo Fisher Scientiﬁc). Primarycultured cells were cultured for 3–5 days, and medium was exchanged
to DMEM (with 10% of FBS, 1% of P/S, 1% of NEAA) for non-hematopoietic expansion, after removing non-adherent cells. Over a period of
1–2 weeks, the adherent cells cultured with DMEM were split bytrypsinization and harvested for cryopreservation with Cell Banker 1(ZENOAQ, Fukushima, Japan) until use.
The cultured MSC population was identiﬁed as CD73+, CD90+,
CD105+, CD34-, CD45- and HLA-DR- by ﬂow cytometry with < 5%
CD34+ and CD45+. Data for all cell surface markers were obtainedusing a BD Accuri C6 (Becton Dickinson, Franklin Lakes, NJ, USA), with

The targeted gene fragments were ampliﬁed from 40 ng of genomic
DNA from each patient using the GeneRead DNAseq Panel PCR Kit V2(QIAGEN) and GeneRead DNAseq Targeted Panel V2 (QIAGEN), resulting in an average amplicon size of 150 bp. Amplicons were puriﬁedusing Agecourt AMP XP Beads (BECKMAN COULTER, Brea, CA, USA).
Following library construction, puriﬁed amplicons were end-repaired(GeneRead DNA Library I Core Kit; QIAGEN) and patient-speciﬁc barcode adapters were ligated (GeneRead Adapter I Set 12-plex; 12 different ones in total; QIAGEN). Constructed libraries were puriﬁed using
the GeneRead Size Selection Kit (QIAGEN) and Agecourt AMP XP Beads(BECKMAN COULTER). Puriﬁed libraries were then ampliﬁed using the
GeneRead DNA I Amp Kit (QIAGEN), and puriﬁed using the QIAquick
PCR Puriﬁcation Kit (QIAGEN), resulting in an average amplicon size of
280 bp. Each library was eventually pooled equimolarly in one tube for
DNA sequencing. The quality of libraries was conﬁrmed by an Agilent
2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) andquantiﬁed using the Qubit 2.0 ﬂuorometer (Thermo Fisher Scientiﬁc)with the Molecular Probes Qubit dsDNA HS Assay Kit (Thermo Fisher
Scientiﬁc).

2.3. Genomic DNA extraction
MSCs were collected for DNA extraction after one passage of culture. Bone marrow mononuclear cells containing hematopoietic cells(hereafter BM cells) were separated using Ficoll-Hypaque gradients asreported previously [25]. Buccal mucosa cells were also collected fromthree patients (from whom informed consent was obtained) by gently
stroking the intraoral cavity three times using sterile foam tipped applicators (GE Healthcare, UK). Genomic DNA was extracted from
BMSCs, BM cells and buccal mucosa cells using a Genta PureGene Cell
Kit (QIAGEN, Hilden, Germany), according to the suppliers’ instruction.

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2.5. Targeted sequencing and data analysis

variants, tumor protein p53 (TP53-R202H) and additional sex combs
like 1 (ASXL1-L1444R), were found in MSCs of UPN37 in addition to
BM cells. Notably, neuroﬁbromin 1 (NF1-G2114D) in UPN36 was the
only MSC-speciﬁc genetic variant, while the VAF was low compared tothose in leukemia cell-speciﬁc genetic variants (Table 2). We also noteda genetic variant of tet methylcytosine dioxygenase 2 (TET2-S1107P)
with low VAF in UPN39 and UPN40; the genetic variant was detected in
MSC and BM cells and not detected in germ line controls.

The Myeloid Neoplasms Panel (QIAGEN), with 50 known or putative mutation gene targets of hematopoietic diseases, was used for DNA
sequencing with MiSeq (Illumina, San Diego, CA, USA). All sequencing
data were mapped and annotated by QIAGEN cloud-based mapping andannotating tool GeneRead Panel Variant Calling. High-probability oncogenic genetic variants were extracted from annotated data based on
the available database ClinVar (https://www.ncbi.nlm.nih.gov/
clinvar/) and COSMIC (http://cancer.sanger.ac.uk/cosmic).
All other non-polymorphous non-synonymous/synonymous andintronic genetic variants were extracted individually by ﬁltering population frequencies (< 1%) with a coverage of ≥500 using the sequencing data analysis tool VariantStudio (Illumina).

3.3. Non-synonymous mutations detected in patients with MDS
Unlike acute leukemia patients, the VAFs in specimens from MDS
were variable, and we did not detect MSC-speciﬁc genetic variants(Table 3). BM cell-speciﬁc genetic variants DNMT3A-G543C and lysine
methyltransferase 2A (KMT2A-R2890P), with VAFs of approximately
50%, were detected in UPN18. In UPN25, genetic variants of SET
binding protein 1 (SETBP1-G870S) and DNMT3A-F354S were detected,
and the VAFs in BM cells (40% and 38%, respectively) were higher inthose in BMSCs (12% and 13%, respectively), indicating heterogeneityof MSCs obtained by the classical adhesion method. We also noted agenetic variant of KRAS proto-oncogene, GTPase (KRAS-T58I), in
UPN25 with low VAF (10%). Mutation of neuroﬁbromin 1 (NF1E1720D) in BM cells, MSCs, and buccal mucosa cells (germ line controls) was found in UPN35.

3. Results
3.1. Limitation of cytogenetic analysis to identify clonal nature of MSCs
We tried to compare the cytogenetic results between BM cells andMSCs, however we could only obtain suﬃcient metaphases in a limited
number of samples (Table 1). Unfortunately, we could not obtain metaphases in one MSC sample from UPN 13, and ﬁve patients showednormal karyotypes in less than 10 metaphases (UPN 38, 39, 40, 10 and18). Although the majority of metaphases were normal karyotype, threepatients (UPN 36, 37, and 25) harbored a clonal cytogenetic change
distinct from those of BM cells. The remaining one patient (UPN 35)
had multiple chromosome deletions in addition to structural changes in
MSCs; however, BM cells showed normal male karyotype. In brief, we
could not conclude the clonal nature of MSCs by conventional cytogenetic analysis, possibly due to the low mitotic activity of MSCs.
Therefore, we next performed mutation analysis.

3.4. Synonymous and intronic mutations in patients with acute leukemia
and MDSWe next assessed the synonymous genetic variants that do not alter
the amino acid sequence of a protein (Table 4). We found genetic
variants with VAF of approximately 50% in structural maintenance ofchromosomes 1A (SMC1A-Q391) in UPN40 and KMT2A-E3639 in
UPN18. In UPN40, SMCA1Z-Q391 was also detected in germ-line controls. We also found genetic variants of JAK1-E186 in three patients(UPN13 and 18) and JAK2-I214 in four patients (UPN10, 13, 18, and39); however, the VAFs were less than 20%. We also noted MSC-speciﬁc
genetic variants of NF1-G140 with low VAF (8%) in UPN40 (Table 4).
To further elucidate MSC-speciﬁc genetic alterations, we summarized the intronic genetic variants in Table 4, although biological signiﬁcance is poorly understood. Similar to the results in synonymous
genetic variants of NF1-E1720D, shared genetic variants were found in
3 genes: SMC3, RAD21 cohesin complex component (RAD21), and NF1
were detected in three patients (UPN35, 39, and 40), with VAF of approximately 0.5. The polycomb protein SUZ12 (SUZ12-T > T/G) was
BMSC-speciﬁc genetic variants with low VAF (Supplementary ﬁle 1).

3.2. Non-synonymous mutations detected in acute leukemia patients
We were able to obtain buccal mucosa cells as a source in 3 of 10
patients, however germ line controls were not available in the remaining patients. We therefore carefully extracted genetic variants to
exclude polymorphisms using two available databases: ClinVar and
COSMIC. We then focused on non-synonymous genetic variants thatalter the amino acid sequence of a protein in acute leukemia patients(Table 2). Genetic variants were mainly compared between MSC andBM cells, since we could not obtain germ-line controls, except for threepatients (UPN35, 39 and 40). In BM cells with acute leukemia, BM cellspeciﬁc genetic variants with a variant allele frequency (VAF) of approximately 50% corresponding heterogeneous mutations were detected in three of the ﬁve patients: DNA methyltrasnferase 3A(DNMT3A-R882H) and MPL proto-oncogene, thrombopoietin receptor(MPL-Ter636H) in UPN36; fms related tyrosine kinase 3 (FLT3-D836H)
in UPN37; and FLT3-D836Y in UPN39. In contrast, two genetic

3.5. Non-synonymous mutations in non-myeloid malignancies
To clarify whether genetic variants are found in non-myeloid malignancies, we used stage I NHL specimens. We performed targeted

Table 2
Non-synonymous genetic variants detected in patients with acute leukemia.
Gene

UPN36
BM

DNMT3A
TP53
ASXL1
FLT3
MPL
NF1
TET2

UPN37
MSC

BM

MSC

R202H (0.50)
L1444R (0.55)
D835H (0.46)

R202H (0.46)
L1444R (0.50)

UPN38

UPN39

BM

BM

MSC

UPN40
MSC

Bu

BM

MSC

S1107P (0.06)

S1107P (0.07)

Bu

R882H (0.42)

D835Y (0.43)

Ter636H (0.45)
G2114D (0.07)
S1107P (0.06)

S1107P (0.07)

UPN, unique patient number; BM, bone marrow mononuclear cells; MSC, bone marrow stromal cells; Bu, buccal mucosa cells; mutated amino acid position is described in “Nonsynonymous”; Variant allele frequency (VAF) in this table is shown as raw data in the parenthesis. The VAF is the proportion of reads at a site which contain the variant allele. If you have
500 reads total, and 100 of them have the variant, then its frequency is 100/500 = 0.2.

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Table 3
Non-synonymous genetic variants detected in patients with myelodysplastic syndrome.
Gene

UPN10
BM

UPN13
MSC

BM

UPN18
MSC

SETBP1
DNMT3A
KMT2A
FLT3
NF1
KRAS
TET2

UPN25

BM

MSC

G543C (0.38)
R2890P (0.39)
P336L (0.50)

UPN35

BM

MSC

G870S (0.40)
F354S (0.38)

G870S (0.12)
F354S (0.13)

BM

MSC

Bu

E1720D (0.52)

E1720D (0.50)

E1720D (0.49)

P336L (0.54)
T58I (0.10)
S1107P (0.06)

UPN, unique patient number; BM, bone marrow mononuclear cells; MSC, bone marrow stromal cells, Bu, buccal mucosa cells; mutated amino acid position was described in “Nonsynonymous”; Variant allele frequency (VAF) in this table is shown as raw data in the parenthesis. The VAF is the proportion of reads at a site which contain the variant allele. If you have
500 reads total, and 100 of them have the variant, then its frequency is 100/500 = 0.2.

deep sequencing using the same panel of 50 genes related to myeloid
malignancy. We did not identify any non-synonymous mutations, either
leukemia cell-speciﬁc or overlapping in BM cells and MSCs with highallelic frequency. Further, we did not detect any genetic variants at low
allele frequency, except for TET2. We found that both BM cells andMSCs harbored TET2-S1107P in all three lymphoma patients (aged
65–71 years old).

MSC-speciﬁc mutations, and overlapping genetic variants, possibly due
to the high coverage of reads. We considered that several overlapping
mutations found in BM cells, MSC and buccal mucosa cells may be
germ-line genetic variants. Besides, shared genetic variants in BM cells
and MSC might be due to the involvement of hematopoietic cells during
the separation procedure or lineage ﬁdelity. While the VAF of BMspeciﬁc genetic variants was variable in MDS patients, possibly due toheterogeneity of specimens, it was approximately 50% in acute leukemia patients. In contrast, the MSC-speciﬁc VAF was less than 10% in
both MDS and acute leukemia, indicating that these are possible nonrandom genetic variations but not likely to be clonal. A recent reportdemonstrated non-speciﬁc genetic variation in MSCs using wholeexome analysis with coverage of ≥20 reads [13]. In contrast, we performed deep sequencing with coverage of ≥500, and therefore we
could not ignore the MSC-speciﬁc mutations with low VAF.
Several reports have demonstrated genetic abnormalities of MSCs
[11,14–17]. Flores-Figueroa et al., reported that MSCs derived from 5 of
9 MDS patients harbored abnormal karyotypes [14]. Molecular cytogenetic analysis by Blau et al., revealed that BMSCs from MDS and AML
patients exhibit chromosomal abnormalities, with the majority of cytogenetic aberrations being non-clonal and distinct from chromosomal
markers in hemopoietic cells from the same individual [15]. They also
reported MSC-speciﬁc chromosome abnormalities in 15 of 94 patientswith myeloid neoplasia, including four structural abnormalities andeleven numerical changes (e.g., −Y), although they did not detect MSCspeciﬁc mutations by conventional DNA sequencing [17]. Using ﬂuorescent in situ hybridization (FISH) and identifying genetic changes byarray-based comparative genomic hybridization (array-CGH), LopezVillar et al., demonstrated that MSCs obtained from MDS patients havegenomic abnormalities, with some linked to MDS with isolated del(5q)[16]. More recently, Huang et al. reported that MSCs derived from AML
had cytogenetic abnormalities both distinct and overlapping with theircorresponding blasts. In the current study, we did not ﬁnd clonal

4. Discussion
The interaction between cancer cells and their surrounding tissue
that constructs the tumor microenvironment has been extensively studied in solid tumors as well as hematological malignancies [26–28].
One of the major components of the tumor microenvironment is ﬁbroblasts, namely cancer-associated ﬁbroblasts (CAFs), and somatic
mutations in CAFs from whole tumor biopsies were reported in breast
cancer cells [29,30]. However, Campbell and his colleagues demonstrated that strictly isolated CAFs did not show any evidence of copy
number gain or loss, or loss of heterozygosity of any chromosomes byimprovement of technical limitation [31]. Thus, two important issues
should be addressed when dealing with genetic abnormalities of CAFs:
one is the heterogeneity of CAFs, and the other is establishing a method
to detect genetic variants using expanded CAFs in vitro.
Similar to the obstacles in solid tumors, the heterogeneity of MSCs
and methodological diﬀerences, such as depth of sequencing andlineage ﬁdelity, complicate the interpretation of genetic analyses inhematologic malignancies. For example, early passage MSCs cannot be
separated (as with microdissection), while later passage cells might be
modiﬁed by in vitro culture. Consequently, discussing genetic variationof MSCs in hematologic malignancies is challenging. Lack of germ line
controls also complicates interpretation in some cases. Considering
those complexities, we categorized several types of genetic alternations,such as BM cell-speciﬁc genetic variants reﬂecting neoplastic nature,

Table 4
Synonymous genetic variants detected in patients with acute leukemia and myelodysplastic syndrome.
Acute leukemia
Gene

UPN36
BM

SMC1A
Myelodysplastic syndrome
Gene

UPN10
BM

JAK1
JAK2
KMT2A
NF1

UPN37
MSC

I214 (0.05)

MSC

BM

MSC

UPN13
BM
MSC
E186 (0.05)
I214 (0.16)

BM

UPN38
MSC

UPN18
BM
MSC
E186 (0.05)
I214 (0.09)
E3639 (0.42) E3639 (0.48)

BM

UPN39
MSC

Bu

BM
Q391 (0.52)

BM

MSC

UPN25
BM

UPN40
BM
Q391 (0.50)

Bu
Q391 (0.49)

UPN35
MSC

Bu

I214 (0.06)
G140 (0.08)

UPN, unique patient number; BM, bone marrow mononuclear cells; MSC, bone marrow stromal cells; Bu, buccal mucosa cells; variant allele frequency is indicated in parentheses. Variant
allele frequency (VAF) in this table is shown as raw data in the parenthesis. The VAF is the proportion of reads at a site which contain the variant allele. If you have 500 reads total, and100 of them have the variant, then its frequency is 100/500 = 0.2.

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Leukemia Research 62 (2017) 23–28

K. Azuma et al.

cytogenetic abnormalities in MSC corresponding malignant cells;
however, we found both distinct and overlapping mutations by deep
sequencing. Cai et al. reported that the non-synonymous changes inhuman MSCs detected by whole-genome sequencing increased after latepassages of culture; the genetic abnormalities did exist in uncultured
cells with a low allelic frequency but reached up to 36% in passage 13[32]. Similarly, Kouvidi et al. reported that chromosomal abnormalities
in MDS-derived MSCs are more frequently detected in passage 6–8
compared to passage 2, suggesting that MDS-derived MSCs are genetically unstable [19]. Kim et al. also reported that global DNA hypomethylation, which is closely linked to genomic instability, was prominent in AML-derived BM-MSCs compared with MDS-derived BMMSCs [18]. While the earlier report by Lopez-Villar et al., demonstrated
chromosomal abnormalities in uncultured MSCs [16], genetic analysis
results (either cytogenetic or mutation analyses) may be partially
modiﬁed by in vitro culture periods.
The question still remains as to whether clonal sweeping of rare
mutations with low allelic frequency plays some roles in the leukemia
cell-stroma cell interaction. In human disease, there are conceptual
diﬃculties with the genetic coevolution of tumor cells and stroma cells,
since it means a kind of double cancer. However, a murine model by
Raaijmakers et al. demonstrated that deletion of Dicer1 speciﬁcally inmouse osteoprogenitors disrupted the integrity of hematopoiesis [20].
Since osteoprogenitors are a mesenchymal subset of stromal cells, primary stromal dysfunction due to Dicer 1 deletion leads to leukemia as
secondary malignancy. Although the biological relevance of the BMSCspeciﬁc genetic variants detected in the current study is still uncertain,
we completely rule out the possibility that clonal selection of genetically altered BMSCs, even with low allelic frequency, may modify theleukemia cell-BMSC interaction.
This study has several limitations. First, we could not obtain MSCs
from normal individuals due to ethical issues. As an alternative, weused NHL specimens with no bone marrow invasion, and except for
TET2-S1107P, did not identify any genetic variants in AL and MDS
patients. Since all lymphoma patients were aged over 60 years, TET2S1107P at low allelic frequency may reﬂect age-related genetic
changes. However, it is still uncertain whether MSCs derived fromnormal individuals exhibit rare genetic variants, and analysis using alarge number of non-myeloid malignancy controls, as well as normal
controls, must be performed. Second, we used MSCs obtained by theclassical adhesion method instead of by sorting MSCs, and therefore thecell components are heterogeneous. Hence, interpretation of shared
mutations should be performed carefully, especially when germ-line
controls are absent. Third, the number of patients is too small, andtherefore we could not discuss acute leukemia derived-MSCs and MDSderived MSCs separately. Further study should clarify these points.
In summary, our study provides ﬁndings by targeted deep sequencing that could not be identiﬁed by whole exome analysis with limited
read depth. First, we identiﬁed neoplastic cell-speciﬁc mutations, aswell as shared genetic variants, in both BM cells and MSCs. In comparison with germ-line controls, some shared mutations reﬂect pathogenic ones, while others reﬂect germ-line genetic changes. Second, we
found MSC-speciﬁc genetic variants at a very low allele frequency,
which may possibly reﬂect clonal sweeping. Since allelic frequency isvery low, these genetic variants might be undetectable by conventional
DNA sequencing. Finally, we found the TET2-S1107P mutation in BMs
and MSCs, but not germ line control cells. Non-myeloid controls also
harbor this genetic change, indicating the possibility of age-related
clonal hematopoiesis [33]. Theoretically, it would be ideal to perform
deep sequencing using singe cells without any in vitro expansion, but
this strategy is diﬃcult in practical terms. While analysis of genetic
changes of MSCs is still challenging, collecting germ line cells simpliﬁes
interpretation of the results. Another important point is the depth ofsequencing in order to evaluate genetic variants with low allelic frequency. Further studies in large cohorts are needed to clarify the molecular properties of MSCs, especially age-related genetic alterations, bydeep sequencing in addition to transcriptional and epigenetic analyses.
Conﬂicts of interest
All authors have no conﬂicts of interest to disclose.
Acknowledgments
This work was supported by the Private University Strategic
Research Based Support Project from MEXT (Ministry of Education,
Culture, Sports, Science and Technology), Tokyo, Japan [grant no.
S1311016].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.leukres.2017.09.008.
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Received: 16 March 2018
DOI: 10.1002/ccr3.1699|

Revised: 6 June 2018|

Accepted: 17 June 2018

CASE REPORT

Cardiac biopsy with intracardiac echocardiographic guidance forsuccessful diagnosis of cardiac lymphoma
Seiichiro Katagiri1
Nahoko Furuya1

Naoya Nakamura3

|

|

|

Daigo Akahane1

Hiroaki Fujimoto1

Kazuma Ohyashiki1

1

Department of Hematology, Tokyo
Medical University, Tokyo, Japan2

Department of Cardiology, Tokyo Medical
University, Tokyo, Japan3

Department of Pathology, Tokai University
School of Medicine, Kanagawa, Japan
Correspondence
Seiichiro Katagiri, Department of
Hematology, Tokyo Medical University,
6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo
160-0023, Japan (patchsei@yahoo.co.jp).

1

|

|

|

Tamiko Suguro1

Tetsushi Saito2||

Jun Yamashita2|

Key Clinical Message
The diagnosis and appropriate treatment of cardiac lymphoma are often delayed by
the difficulty in obtaining heart tissue biopsies. Intracardiac echocardiographyguided biopsy can improve the prognosis of cardiac lymphoma by decreasing postbiopsy complications and increasing biopsy quality, allowing collection of sufficientmaterial for multilateral analysis.
KEYWORDS
cardiac lymphoma, flow cytometric analysis, intracardiac echocardiography-guided biopsy

IN T RO D U C T ION

We present two cases in whom endomyocardial biopsy (EMB)
was performed safely using intracardiac echocardiography(ICE), allowing collection of sufficient material for multilateral analysis. These findings indicate that ICE-guided biopsy
can improve the prognosis of patients with cardiac lymphoma
by decreasing postbiopsy complications compared with surgery and by increasing biopsy quality compared with nonICE-guided EMB procedures.
Primary cardiac lymphoma (PCL) is rare, with an incidence of 0.1%-0.28% according to autopsy reports.1 As approximately 30%-50% of patients with cardiac lymphoma
demonstrate dyspnea, arrhythmia, and heart failure at
onset,2-4 early diagnosis and appropriate chemotherapy areimportant to improve prognosis. However, the diagnosis ofcardiac lymphoma and appropriate treatment is often delayed by the difficulty in obtaining heart tissue biopsies. ICE
using a guide for noncoronary interventional procedures canclearly show intracardiac structures and help with EMB.5
Herein, we report the details of two patients with cardiac
lymphoma in whom we successfully obtained early diagnosis

by ICE-guided biopsy with histological and flow cytometricanalyses.

2|

CASE REPORTS

Case 1 was a 68-year-old woman admitted to hospital witha chief complaint of dyspnea and pretibial pitting edema.
Chest X-ray showed cardiomegaly (cardiothoracic ratio:60%). Transesophageal echocardiography (TEE) showed a
6 × 7 cm right atrium (RA) tumor, while contrast-enhanced
computed tomography (CT) showed a cardiac tumor protruding into the RA and right ventricle (RV) wall. As ourfacility did not have positron emission tomography-CT,
gallium scintigraphy was performed and showed increased
gallium uptake in the cardiac tumor and mediastinal lymph
node. Blood tests revealed elevated levels of serum lactate
dehydrogenase (1170 U/L; normal range: 120-240 U/L),
soluble interleukin-2 receptor (2760 U/mL; normal range:
145-519 U/mL), and brain natriuretic peptide (91.6 pg/mL).
Her hemoglobin level was normal (12.2 g/dL). Case 2 wasa 71-year-old woman treated for intraocular lymphoma who

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium,provided the original work is properly cited and is not used for commercial purposes.
© 2018 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd.
Clin Case Rep. 2018;6:1681–1683.

wileyonlinelibrary.com/journal/ccr3-370-| 1681

1682|

KATAGIRI eT Al.

FIGURE 1

Intracardiac echocardiography-guided biopsy for cardiac lymphoma. A, Intracardiac echocardiography (ICE)-guided biopsy. B,
C, Posteroanterior fluoroscopic images of both patients. D, E, ICE images of both patients. RA, right atrium; RV, right ventricle; PA, pulmonary
artery; SVC, superior vena cava

had sustained complete remission for 5 years. She was admitted to hospital for atrial fibrillation. Transthoracic echocardiography showed a 5 × 6 cm RA tumor and pericardial
effusion. Contrast-enhanced CT also showed a cardiac tumor
in the RA. Gallium scintigraphy showed increased gallium
uptake in the cardiac tumor and perigastric lymph node. Her
serum lactate dehydrogenase level was 390 U/L, solubleinterleukin-2 receptor was 1240 U/mL, and brain natriuretic
peptide was 32.7 pg/mL. Her hemoglobin level was normal(13.1 g/dL).
For diagnosis, we performed EMB using ICE under shortterm heparinization in both patients. A bioptome and an
ICE catheter were inserted into the RA via the right jugularvein and right femoral vein, respectively, using fluoroscopy(Figure 1A-C). The bioptome was directed toward the cardiac tumor under ICE image guidance (Figure 1D,E). In bothcases, we obtained sufficient samples for flow cytometric,
pathological, cytogenetic, and genetic analyses. Flow cytometry showed B-cell markers, including cluster of differentiation(CD) 19 and 20 with light-chain restriction, indicating cardiac
B-cell lymphoma. Thus, we prescribed steroid therapy.
Case 1 was diagnosed with diffuse large B-cell lymphoma, which was positive for CD 20, CD10, Bcl-6, and-371-

MUM-1, but negative for CD5, indicating germinal center
B type. No metaphases were obtained in G-banding analysis. However, fluorescence in situ hybridization analysis
revealed that the lymphoma was negative for c-MYC andBCL-2 gene translocations. Case 2 was diagnosed with diffuse large B-cell lymphoma, which was positive for CD20,
Bcl-6, and MUM-1, and negative for CD10 and CD5, indicating nongerminal center B type. Cytogenetic analysis
of four metaphases revealed normal results for two andmultiple abnormalities without c-MYC and BCL-2 genetranslocations for two. Bone marrow examination using
pathological and flow cytometric analysis showed no bonemarrow involvement in either case. Cerebrospinal fluid examination and magnetic resonance imaging also revealed nocentral nervous involvement in either case. We encounteredno complications postbiopsy including thromboembolism.
Case 1 received rituximab, cyclophosphamide, vincristine,and prednisolone, while Case 2 received rituximab, cyclophosphamide, pirarubicin, vincristine, and prednisolone.
Following six cycles of rituximab-combined chemotherapy,
the tumors were undetectable by imaging tests, including
gallium scintigraphy and transthoracic echocardiography, inboth patients.

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KATAGIRI eT Al.

3|

D IS C U SSION

As approximately 95% of cardiac tumors are diagnosed
as sarcoma, cardiac lymphoma is not usually suspected.6
Previous reports suggest that only chemotherapy is able toimprove prognosis of patients with cardiac lymphoma.4 Thus,
a less invasive biopsy approach is required for cardiac lymphoma diagnosis. Generally, biopsy of cardiac lymphoma
is performed by surgical approaches, such as thoracotomyor thoracoscopic surgery.2,7 However, these invasive approaches may delay treatment because of surgical invasion.
As approximately 90% of PCL has either RA or right ventricular involvement,2-4 a transvenous EMB approach is useful. Transvenous EMB is usually safely performed underfluoroscopic guidance, although obtaining sufficient samples for diverse analysis of tumor diagnosis can be difficult.
Transvenous EMB under TEE is also useful,8 but can cause
discomfort and requires pharyngeal anesthesia. In our cases,
we performed EMB safely and obtained sufficient material
for histological and flow cytometric analyses using ICE. Flowcytometric analysis showed that the cardiac tumors had B-cellmalignancy, allowing us to start chemotherapy immediately.

ORCID
Seiichiro Katagiri

http://orcid.org/0000-0002-3031-9990

R E F E R E NC E S
1. McAllister HA, Fenoglio JJ. Tumors of the cardiovascular system. Atlas of tumor pathology, series 2. Armed Forces Institute of
Pathology; 1978. 99-100 p.
2. Carras S, Berger F, Chalabreysse L, et al. Primary cardiac lymphoma: diagnosis, treatment and outcome in a modern series.
Hematol Oncol. 2017;35:510‐519.
3. Gordon MJ, Danilova O, Spurgeon S, et al. Cardiac non-Hodgkin’s
lymphoma: clinical characteristics and trends in survival. Eur J
Haematol. 2016;97:445‐452.
4. Petrich A, Cho SI, Billett H. Primary cardiac lymphoma: an analysis of presentation, treatment, and outcome patterns. Cancer.
2011;117:581‐589.
5. Asrress KN, Mitchell AR. Intracardiac echocardiography. Heart.
2009;95:327‐331.
6. Bruce CJ. Cardiac tumours: diagnosis and management. Heart.
2011;97:151‐160.
7. Ikeda H, Nakamura S, Nishimaki H, et al. Primary lymphomaof the heart: case report and literature review. Pathol Int.
2004;54:187‐195.
8. Jurkovich D, de Marchena E, Bilsker M, et al. Primary cardiac
lymphoma diagnosed by percutaneous intracardiac biopsy withcombined fluoroscopic and transesophageal echocardiographic imaging. Catheter Cardiovasc Interv. 2000;50:226‐233.

CONFLICT OF INTEREST
None declared.

How to cite this article: Katagiri S, Akahane D,
Suguro T, et al. Cardiac biopsy with intracardiac
echocardiographic guidance for successful diagnosisof cardiac lymphoma. Clin Case Rep. 2018;6:1681–
1683. https://doi.org/10.1002/ccr3.1699

AUTHORSHIP
SK: treated the patient and wrote the manuscript; DA, TS,
NF, HF, TS, and JY: treated the patient and reviewed themanuscript; NN: performed pathological analysis and reviewed the manuscript; KO: treated the patient, reviewed themanuscript, and supervised the project.

-372-

International Journal of

Molecular Sciences
ArticleExosomal miRNA Signatures for Late-Onset AcuteGraft-Versus-Host Disease in Allogenic
Hematopoietic Stem Cell Transplantation

Seiichiro Yoshizawa 1, *, Tomohiro Umezu 1,2 ID , Yuu Saitoh 1 , Moritaka Gotoh 1 ,Daigo Akahane 1 , Chiaki Kobayashi 1 , Junko H. Ohyashiki 2 and Kazuma Ohyashiki 1,2 ID
1

2*

Department of Hematology, Tokyo Medical University, Tokyo 160-0023, Japan;t_umezu@tokyo-med.ac.jp (T.U.); yusaitoh@tokyo-med.ac.jp (Y.S.); gotohm@wj8.so-net.ne.jp (M.G.);
daigoakahane@msn.com (D.A.); chiaki-k@tokyo-med.ac.jp (C.K.); ohyashik@rr.iij4u.or.jp (K.O.)
Department of Advanced Cellular Therapy, Tokyo Medical University, Tokyo 160-0023, Japan;
junko@hh.iij4u.or.jp
Correspondence: b_perfect_simple@yahoo.co.jp; Tel.: +81-3-3342-6111 (ext. 5999)

Received: 27 July 2018; Accepted: 21 August 2018; Published: 23 August 2018


Abstract: Recent studies have demonstrated that exosomal microRNAs (miRNAs) have the potentialof facilitating molecular diagnosis. Currently, little is known about the underlying mechanism
behind late-onset acute graft-versus-host disease (LA GVHD). Identifying differentially expressed
miRNAs in exosomes should be useful for understanding the role of miRNAs in this disease.
This study was established to investigate the relevance of miRNAs in exosomes derived frompatients developing LA GVHD after allogeneic hematopoietic stem cell transplantation (HSCT).
Plasma samples were collected from patients with LA GVHD (n = 5), non-GVHD (n = 5), and controls(n = 8) for exosomal miRNA expression profiling using a TaqMan low-density array; the results werevalidated by quantitative reverse transcription polymerase chain reaction (RT-PCR). We analyzed
exosomal miRNAs differentially expressed among these three groups. MirTarBase was employed to
predict potential target genes of the miRNAs specific for LA GVHD. We detected 55 miRNAs thatwere differentially expressed with a significant change >2.0-fold between LA GVHD and non-GVHD.
Of these, we selected the 10 miRNAs (miR-423-5p, miR-19a, miR-142-3p, miR-128, miR-193b, miR-30c,
miR-193a, miR-191, miR-125b, and miR-574-3p) with the most significant differential expression.
Using quantitative RT-PCR, we further identified that miR-128 was significantly upregulated at theonset of LA GVHD compared with that in normal controls and is a promising diagnostic marker of
LA GVHD, with an area under the curve (AUC) value of 0.975. MirTarBase analysis revealed that thepredicted target genes of miR-128 are involved in the immune system and inflammation. Increased
expression of miR-128 may serve as a novel, noninvasive biomarker for early LA GVHD diagnosis.
Keywords: late-onset acute graft-versus-host disease; exosomal miRNA; hematopoietic stem celltransplantation

1. Introduction
The occurrence of severe graft-versus-host disease (GVHD) after allogeneic hematopoietic
stem cell transplantation (HSCT) leads to a poor prognosis. Recently, with this expansion in theutilization of allogeneic HSCT, such as the introduction of reduced-intensity conditioning and donor
lymphocyte infusions, late-onset acute and chronic GVHD has been reclassified based on the exact
clinical manifestation instead of the time of onset [1]. According to the new classification using
National Institutes of Health (NIH) consensus criteria established in 2005, patients with de novo

Int. J. Mol. Sci. 2018, 19, 2493; doi:10.3390/ijms19092493

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Int. J. Mol. Sci. 2018, 19, 2493

2 of 14acute GVHD occurring beyond day 100 post-transplantation are considered to have late-onset acute
GVHD (LA GVHD) [1]. In accordance with the NIH definition, 20–40% of chronic GVHD cases arereconsidered to be LA GVHD. A few reports have shown that LA GVHD is also associated withgreater nonrelapse mortality after allogeneic HSCT than non-GVHD [2,3]. However, there is still an
insufficient understanding of the association between LA GVHD and a lower incidence of relapse
caused by the induction of a beneficial graft-versus-tumor (GVT) effect. To improve long-term survival,
intensive efforts have been made to prevent GVHD without losing the GVT effect. Thus, several
molecular biomarkers for diagnosis and prediction of the severity and prognosis of GVHD have been
explored [4–7].
MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate post-transcriptional
gene expression by degrading or suppressing their target mRNA [8]. Recently, it has been widely
recognized that some circulating miRNAs are included in the content of endosome-derived
extracellular vesicles including exosomes. Exosomes are secreted by many types of cells into various
biological fluids, such as serum and plasma [9,10]. The demonstration that exosomes contain not only
proteins, RNAs, and lipids, but also functional miRNAs indicated that miRNAs could participate
in intercellular communication and elicit immune responses by their transport between cells via
exosomes [11,12]. Therefore, analysis of the alterations of exosomal miRNAs in the context of various
diseases has the potential to facilitate molecular diagnosis [13,14].
In recent studies, miRNA profiling identified a GVHD-specific miRNA signature that can control
the regulation of classic acute GVHD in allogeneic immunity. In particular, miR-155, known as an
immune-related microRNA, and miR-15a/16 and miR-17-92a were shown to be closely involved in
the pathophysiology behind the development of classic acute GVHD in a xenogeneic GVHD murine
model [15–17]. Moreover, recent studies have demonstrated that specific plasma miRNA signatures,such as miR-423, miR-199a-3p, miR-93, miR-377, and miR-586, could serve as independent biomarkers
for the prediction, diagnosis, and prognosis of hematologic diseases and classic acute GVHD [18,19].
Despite the fact that the roles of miRNAs in classic acute GVHD after allogeneic HSCT have been
extensively studied over the last few years, the relevance and expression profile of miRNAs in LA
GVHD have not yet been elucidated. Because a number of previous studies investigated the role ofmiRNAs as potential predictive biomarkers in acute GVHD, we aimed to determine the clinically
relevant exosomal miRNA profile in patients developing LA GVHD after allogeneic HSCT.
2. Results
2.1. Patient Characteristics
A summary of patients’ clinical characteristics is presented in Table 1. The median age of all
patients was 58.5 years (range, 26–70 years) and the median onset of LA GVHD was 165 days (range,
117–238 days) after allogeneic HSCT. GVHD prophylaxis included mainly calcineurin inhibitor with orwithout short-term methotrexate. Overall, four of the five of patients with LA GVHD developed severe
GVHD (grades III–IV) and no patients achieved remission despite second-line therapy. Four patientsdied from GVHD deterioration, while the remaining one patient died from post-transplantation
lymphoproliferative disorder.
2.2. miRNA Expression Profiling at LA GVHD Diagnosis
The procedure for selecting candidate miRNAs is illustrated in Figure 1. To identify candidate
miRNAs with altered expression in LA GVHD patients, miRNA expression profiling was performedon exosome fractions taken from plasma samples of 10 allogeneic HSCT patients (5 LA GVHD and 5
non-GVHD) and 8 normal subjects. We then compared exosomal miRNA expression in LA GVHD
patients with that in non-GVHD patients using a TaqMan miRNA Array (NCBI, Gene Expression
Omnibus (GEO); GSE110435). The heat map in Figure 2 demonstrates that the miRNA signature in LA
GVHD group tended to show high expression. Among these three groups, a total of 163 differentially-374-

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3 of 14
3 of 13of the LA GVHD group compared with that in the non-GVHD group. Interestingly, for all of the 55
expressed miRNAs were identified using GeneSpring software. We confined our analysis to miRNAs
selected miRNAs, the expression was upregulated in the LA GVHD group. We then evaluated the 10
for whichexpression
level exhibiteda fold change (FC) of >2.0 in exosomes of the3 ofLA
Int. J. theMol. Sci.
2018, 19, x FOR
13 GVHDmiRNAswiththehighest PEER
FC REVIEW(miR-423-5p, miR-19a, miR-142-3p, miR-128, miR-193b,
miR-30c,group compared with that in the non-GVHD group. Interestingly, for all of the 55 selected miRNAs,
miR-193a,miR-191,
miR-125b,and miR-574-3p),
as non-GVHD
shown in Table
2. Of these miRNAs,
miR-423-5p,of theLA GVHDgroup compared
withLA
thatGVHD
in thegroup.
the55
the expression
was upregulated
in thegroup. We
thenInterestingly,
evaluated fortheall10ofmiRNAs
with
miR-19a,and miRNAs,
miR-142,
were
most strikingly
differentiallyexpressed,
werethepreviously
selected
the whichexpression
was upregulated
in the LA
GVHD group. We
then evaluatedthe highest FC (miR-423-5p, miR-19a, miR-142-3p, miR-128, miR-193b, miR-30c, miR-193a,10miR-191,
miRNAsthe highest
miR-19a,miR-142-3p, miR-128, miR-193b, miR-30c,
reported
to playwith
importantrolesFCin (miR-423-5p,
acute GVHD
[18,20,21].
miR-125b,and miR-574-3p), as shown in Table 2. Of these miRNAs, miR-423-5p, miR-19a, and miR-142,
miR-193a, miR-191, miR-125b, and miR-574-3p), as shown in Table 2. Of these miRNAs, miR-423-5p,
which weremoststrikingly
expressed,
were
previouslyexpressed,
reported wereto play
important roles
miR-19a,andmiR-142, differentiallywhich were moststrikingly
differentiallypreviouslyin acutereported
GVHDto[18,20,21].
play important roles in acute GVHD [18,20,21].

Figure 1. Flow diagram for the identification of late-onset acute GVHD-specific miRNAs. The

Figure 1.
1. Flow
Flow diagram
diagram forfor thethe identification ofof late-onset
acute
GVHD-specificmiRNAs.
The
Figurelate-onset
acute
GVHD-specificmiRNAs. The
exosomes
fractions wereisolatedidentificationfrom 18 plasma samplesof HSCT
patients
and healthy volunteers,
exosomes
fractionswereisolatedfrom18
plasmasamplesof
HSCT
patients
andhealthy
volunteers,
exosomes
wereprofile
isolatedfrom18 plasma
samplesof HSCTand healthy
volunteers,
andfractions
the expressionof 381
exosomalmiRNAswere analyzed.
To patientsvalidate candidate
miRNAs,andthetheweexpression
profileof 381381
exosomalmiRNAswere
analyzed.
Tovalidatecandidate
candidatemiRNAs,performed profile
TLDA andfurthernarrowed
down themiRNAsto thoseTo
significantly
differentiallyandexpressionofexosomalmiRNAswere
analyzed.
validate
miRNAs,we performed
performed
TLDA
andfurther narrowed
narrowed down
down thethe miRNAs
miRNAs toto thosethose significantly
significantly differentiallydifferentiallyexpressedTLDA
by at least
two-fold.
weandfurther
expressed
by
at
least
two-fold.
expressed by at least two-fold.

Figure 2. miRNA expression profiling using TaqMan miRNA Array (Thermo Fisher Science, Waltham,
MA, USA). Exosome fraction taken from plasma samples of 10 patients who underwent allogeneic HSCT
(5 late-onset acute GVHD and 5 non-GVHD), with 8 normal controls being included. A differential
expression pattern was found between late-onset acute GVHD patients and non-GVHD patients. Using
SDS (versionexpression2.4) and DataAssist
software3.01,
ThermoArray
Fisher, version, Waltham,
MA, USA),
Figure2.
profilingusing(version
TaqManmiRNA
Science,
Waltham,Figure 2.miRNA
miRNA expression
profilingusing
TaqManmiRNA(Thermo
Array Fisher(ThermoFisherScience,wecalculated
theexpressionofmiRNAs
based
on
their
Ct
value
normalized
by
the
Ct
value of HSCT
MA,
USA).
Exosomefractiontaken
fromplasmasamplesof
10
patients
who
underwent
allogeneic
Waltham, MA, USA). Exosome fraction taken from plasma samples of 10 patients who underwent
ath-miR-159. Data were analyzed with GeneSpring software.

(5
late-onsetHSCT
acute(5GVHD
andacute
5 non-GVHD),
8 normal controls
being included.
A differential
allogeneic
late-onset
GVHD andwith
5 non-GVHD),
with 8 normal
controls being
included.
expression
pattern
was foundbetweenlate-onset
acutelate-onset
GVHD patientsand non-GVHD
patients.
Using
A differential
expression
pattern
was foundbetweenacute GVHD
patients andnon-GVHD
SDS(version
2.4) andsoftware(version 3.01,
Thermo(version
Fisher, version,
Waltham,MA,version,USA),patients.
Using
SDSDataAssist(version 2.4)
and DataAssist
software
3.01, Thermo
Fisher,we
calculated
expressionof miRNAs
based onCt valueby thenormalized
Ct value byofWaltham,MA,theUSA),
we calculated
the expressionof theirmiRNAsbased normalizedon their Ct value
ath-miR-159.
Data
were
analyzed
withGeneSpring
software.
the Ct value of ath-miR-159. Data were analyzed with GeneSpring software.

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-376-

ALL
ALL
AML
AML
AML

PN 6
PN 7
PN 8
PN 9
PN 10

62
26
60
62
70

33
38
57
56
60

Age (y)

M
F
M
M
F

M
M
M
F
F

GenderMatched unrelated
Matched unrelated
Mismatched
Mismatched
Mismatched

Matched unrelated
Matched related
Mismatched
Mismatched
Mismatched

Donor

6/6
6/6
4/6
5/6
4/6

6/6
6/6
5/6
4/6
4/6

HLA SeroCompatibility
Conditioning

BMBM
Umbilical cord blood
PBSC
Umbilical cord blood

Non-GVHD Group

BM
PBSC
BM
Umbilical cord blood
Umbilical cord blood
Reduced intensity
Myeloablative
Myeloablative
Reduced intensity
Reduced intensity

Myeloablative
Reduced intensity
Myeloablative
Myeloablative
Myeloablative

Late-Onset Acute GVHD Group

Stem Cell Source

CsA + st-MTX
CsA + st-MTX
Tac
Tac + st-MTX
Tac

CsA + st-MTX
CsA + st-MTX
Tac + st-MTX
Tac
Tac

GVHD
Prophylaxis

Table 1. Characteristics of hematopoietic stem cell transplantation (HSCT) patients.

+238
+180
+117
+119
+165

Days of LA
GVHD
Onset

+42
+41
+49
+41
+45

+248
+221
+136
+132
+174

Days ofSampling

Liver
Liver
Gut
Skin + gut
gut

Target
Organ

IV
IV
III
IV
II

Grade

4 of 14

AML, acute myeloid leukemia; NHL, non-Hodgkin lymphoma; ALL, acute lymphoblastic leukemia; BM, bone marrow; PBSC, peripheral blood stem cell; CsA, cyclosporine A; Tac,
tacrolimus; MTX, methotrexate.

AML
NHL
AML
ALL
ALL

PN 1
PN 2
PN 3
PN 4
PN 5

Diagnosis

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Table 2. Top 10 exosomal microRNAs differentially expressed between late-onset acute GVHD andTable
2. Top 10 exosomal microRNAs differentially expressed between late-onset acute GVHD
non-GVHD.
and non-GVHD.
Exosomal miRNAs
Fold Change
p Valuehsa-miR-423-5p
49.07
0.000198
Exosomal miRNAs
Fold Change
p Valuehsa-miR-19a
37.33
0.000013
hsa-miR-423-5p
49.07
0.000198
hsa-miR-142-3p
28.35
0.000198
hsa-miR-19a
37.33
0.000013
hsa-miR-128
24.79
0.000111
hsa-miR-142-3p
28.35
0.000198
hsa-miR-193b
16.12
0.000209hsa-miR-128
24.79
0.000111
hsa-miR-30c
14.33
0.003157
hsa-miR-193b
16.12
0.000209hsa-miR-193a
12.56
0.000111
hsa-miR-30c
14.33
0.003157
hsa-miR-191
12.312.56
0.000081
hsa-miR-193a
0.000111
hsa-miR-125b
10.712.3
0.001064
hsa-miR-191
0.000081
hsa-miR-125b
0.001064
hsa-miR-574-3p
6.1310.7
0.000198
hsa-miR-574-3p
6.13
0.000198

2.3. Comparison of Exosomal miRNAs Differentially Expressed among LA GVHD, Non-GVHD, and Healthy
2.3.
Comparison of Exosomal miRNAs Differentially Expressed among LA GVHD, Non-GVHD, and Healthy
Volunteers
Volunteers
To validate the findings on the LA GVHD-specific exosomal miRNAs, we assessed the 10
To validate
the findingson theLA highly
GVHD-specificexosomalmiRNAs,we assessed
the 10exosomal
exosomalmiRNAsthat weremost
differentiallyexpressed
among
the threegroups.
We
miRNAs
that
were
most
highlydifferentiallyexpressed
among
the
three
groups.
We
adoptedtheadopted the screening method for selecting LA GVHD-specific exosomal miRNAs as follows: (1)screeningfor inselecting
LA GVHD-specificmiRNAsas LA
follows:(1)andsignificant
significantmethod
difference
the expressionof miRNAs exosomal
is observed
between
GVHD
healthydifference
intheexpressionofmiRNAsisobserved
betweenLA
GVHD
andhealthy
volunteers
volunteers (normal control); or (2), no significant difference in the expression of miRNAs is observed(normalor (2), noandsignificant
in (normal
the expressionof miRNAs
observed between
betweencontrol);the non-GVHD
healthydifference
volunteers
control).
We firstlyis comparedsamplesthenon-GVHD
and healthy
volunteers(normalWe firstly
samples
obtainedfromobtainedfrom patientsat thediagnosisof LA control).
GVHD withthose comparedfrom healthy
volunteers(normalpatients
atthediagnosis
of
LA
GVHD
with
those
from
healthy
volunteers(normalcontrol)andfoundcontrol) and found that three miRNAs (miR-423-5p, miR-128, and miR-125b) were significantly
that
three miRNAs
andoverexpressedin theoverexpressedin the(miR-423-5p,
exosomes of miR-128,
those with
LAmiR-125b)
GVHD (p <were0.05)significantly
when comparedwith the levels
inexosomesof thosewith 3A).
LA GVHD(p < 0.05)
levels inhealthysubjects
healthy subjects(FigureMeanwhile,
nonewhenof thecomparedremainingwith
seventhemiRNAsshowedsignificant(Figure3A).inMeanwhile,of themiRNAsshowedsignificant
differences wein
differences
expression none
betweenLA remaining
GVHD andsevenhealthy
volunteers(Figure3B). Subsequently,
expression
betweenobtained
LA GVHD
healthy volunteers(Figurewe compared
compared samples
fromandnon-GVHD
patients withthose 3B).
fromSubsequently,
healthy volunteers
in Figuresamples
obtainedfrom
non-GVHD
patients
with
those
from
healthy
volunteersinFigure3A. No3A. No significant difference in the expression of miR-128 and miR-125b was observed between
thesignificant
difference
intheexpressionofmiR-128
andmiR-125b
wasobserved
betweenthe
non-GVHD
non-GVHD and healthy volunteers, while miR-423-5p showed downregulation in the non-GVHD
andhealthymiR-423-5p
showedTherefore,
downregulationin the non-GVHD
group
when
group
whenvolunteers,compared whilewith healthy
volunteers.
two miRNAs,
miR-128 andmiR-125b,comparedwithhealthy
volunteers.
Therefore,
twomiRNAs,miR-128
andmiR-125b,
werechosenaswere chosen as candidate miRNAs specific for LA GVHD; For further analysis, we focused on thecandidatemiRNAs specificfortwoLA dysregulated
GVHD; For further
analysis,
focused on the clinical implications
clinical implicationsof thesemiRNAsin LAwe
GVHD.
of these two dysregulated miRNAs in LA GVHD.

Figure 3. Cont.

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Figure 3.3.This
This
microRNA
expression
accordingto late-onset
acute(LA
GVHD
(LA
vs.
FiguremicroRNA
expression
accordingto late-onset
acute GVHD
GVHD)
vs. GVHD)
non-GVHD
non-GVHDvs.
normalcontrols
at
the
onset
of
late-onset
acute
GVHD.
Box
plots
depictthe75th
vs. normal controls at the onset of late-onset acute GVHD. Box plots depict the 75th percentile, median,percentile,
median, values;and 25th
percentile
andminimum
whiskers torepresent
minimum
to maximum
and25th percentile
andwhiskersvalues;
represent
maximum
expression.
p values
expression.
p values
calculated using
the independent
two-sample
t-test.
(A)
werecalculatedusing were
the independent
two-sample
t-test. (A) The
expression
levelsofThethreeexpression
miRNAslevels of miR-125b,
three miRNAs(miR-128, miR-125b,
and miR-423-5p)
significantlythe(miR-128,and miR-423-5p)
were significantly
higher in werethe exosomesof LAhigher
GVHDinthan
exosomes
of
LA
GVHD
than
in
normalcontrols.
The
expression
levelof
miR-423-5p
showedin normal controls. The expression level of miR-423-5p showed downregulation in the non-GVHD
downregulationin the non-GVHD
when
compared two
withmiRNAs
normal controls.
twogroupswhen comparedwith normalgroups
controls.
Accordingly,(miR-128,Accordingly,
miR-125b) were
miRNAswere chosen
candidatemiRNAsspecificfor LAofGVHD.
(B) Nochosenas (miR-128,
candidate miR-125b)
miRNAs specificfor LA as
GVHD.
(B) Nosignificant
differences
expressionofsignificant
differencesof miR-142-3p,
expression ofseven miRNAs
miR-30c,
seven
miRNAs(miR-19a,
miR-193b,
miR-30c,(miR-19a,
miR-193a,miR-142-3p,
miR-191, andmiR-193b,
miR-574-3p)
were
miR-193a,between
miR-191,
andmiR-574-3p)
werecontrols.
observed between LA GVHD and normal controls.
observed
LA
GVHD
and normal

2.4. Quantification
Quantification ofof Individual
Individual miRNA
miRNA by
by Real-Time
Real-TimeQuantitive
QuantitiveRT-PCR
RT-PCR
2.4.
Toconfirm
confirmthe
thespecificspecific
deregulated
miRNAschangeobtained
the TaqMan
low-densityToderegulated
miRNAschangeobtained
fromfrom
the TaqMan
low-density
array
array
(TLDA),
wequantified
miR-128
andmiR-125b
expressionbyquantitative
real-time
PCR(TLDA), we quantified miR-128 and miR-125b expression by quantitative real-time PCR (qRT-PCR).
(qRT-PCR).
There
were
no
significant
differencesinmiR128(Figure4A)
andmiR-125b(Figure4B)
There were no significant differences in miR128 (Figure 4A) and miR-125b (Figure 4B) expression
expression
betweenthenormalcontrol
andnon-GVHD
group(p=0.3842
and0.8336,
respectively).
between the normal control and non-GVHD group (p = 0.3842 and 0.8336, respectively). In contrast,
In contrast,
andmiR-125b
were significantly
increased
LA GVHD(LAGVHD vs.
miR-128
andmiR-128
miR-125b
weresignificantly
increased in LA
GVHDinpatients(LA patients
GVHD vs.
non-GVHD,
non-GVHD,p=
0.0027
and
0.0289;
LA
GVHD
vs.
NC,
p
=
0.0001and
0.0104,
respectively)(Figurep = 0.0027 and 0.0289; LA GVHD vs. NC, p = 0.0001 and 0.0104, respectively) (Figure 4A,B). To estimate
4A,B).
To
estimate
cut-off
valuesforthe
twomiRNAs(miR-128,
miR-125b)in
exosomesforcut-off values for the two miRNAs (miR-128, miR-125b) in exosomes for distinguishing between LA
distinguishing
betweengroups,
LA GVHDand non-GVHD
groups, characteristic
we performed
receiver-operating
GVHD
and non-GVHD
we performed
receiver-operating(ROC)
curve analysis.
characteristic
(ROC)
curve
analysis.
The
ROC
curve
for
miR-128
showedextremely
diagnostic
The ROC curve for miR-128 showed extremely high diagnostic accuracy, with an higharea undertheaccuracy,
with
an
area
under
the
ROC
curve
of
0.975
and
diagnostic
sensitivity
of
100%
[95%
ROC curve of 0.975 and diagnostic sensitivity of 100% [95% confidence interval (CI), 47.82–100%] andconfidenceofinterval(CI),CI,47.82–100%]and(Figure
specificityof 87.5% (95%
CI, curve
47.35–99.68%)(Figure5A).
specificity
87.5% (95%47.35–99.68%)
5A). Similarly,
the ROCfor miR-125b
showedSimilarly,
the
ROC
curve
for
miR-125b
showed
an
AUC
of
0.975
and
diagnostic
sensitivity
andan AUC of 0.975 and diagnostic sensitivity and specificity of 80% (95% CI, 28.36–99.49%) and 87.5%
specificityof 80% (95% CI,
28.36–99.49%)
and
87.5%
CI,values
47.35–99.68%),
5B).
(95%
CI, 47.35–99.68%),
respectively(Figure5B).
The(95%cut-off
were thusrespectively
determined(Figure
to be 3.183
The
cut-off
valueswere
thus
determined
to
be
3.183
for
miR-128
and
2.29
for
miR-125b,
which
were
for miR-128 and 2.29 for miR-125b, which were used in the following analysis. Our data suggest thatused inexpression
the following
analysis.may
Ourserve
data assuggestthat alteredexpressionof inmiRNAsmay serve as aalteredof miRNAsa diagnostic
tool forLA GVHD
HSCT practice.
diagnostic tool for LA GVHD in HSCT practice.

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Figure 4.4. (A)(A) The
The expression
expression level
level ofof miR-128
miR-128 was
was significantly
significantly upregulated
upregulated inin thethe exosomes
exosomes ofof
Figurelate-onset
acute
GVHD
when
compared
with
that
in
non-GVHD(p=
0.0027)andinhealthysubjects(plate-onset
GVHD
when comparedwith thatwas
in non-GVHD= 0.0027) andininthe
healthysubjects
Figure 4. acute(A) Theexpression
level of miR-128
significantly(p upregulated
exosomesof<
0.005).
(B)acuteThe
level
oflevelmir-125b
wasupregulated
inand
the inexosomesof late-onset(p< 0.005).
(B)expression
The expression
ofwithmir-125b
significantly
upregulated
in the exosomeslate-onset
GVHD
when
comparedthatsignificantly
inwas
non-GVHD(p = 0.0027)
healthysubjects
(pof<
acute
GVHD
when
compared
with
that
in
non-GVHD(p=
0.0289)andinhealthysubjects(p=0.0104).
late-onset
acute
GVHD
when
compared
with
that
in
non-GVHD(p=
0.0289)andinhealthysubjects
0.005). (B) The expression level of mir-125b was significantly upregulated in the exosomes of late-onset(p= 0.0104).
acute
GVHD when compared with that in non-GVHD (p = 0.0289) and in healthy subjects (p = 0.0104).

Figure 5. Association between exosomal miRNA and late-onset acute GVHD. (A) ROC curve for theidentificationof patientswith late-onset acute
GVHD versus
non-GVHDGVHD.
using miR-128.
(B)
ROCforcurve
Figure5. Association
Association
betweencurve
theFigure5.
between exosomal
exosomal miRNA
miRNA andand late-onset
late-onset acute
acute GVHD. (A)(A) ROCROC curve
for thefortheidentificationofpatients
withlate-onset
acute
GVHD
versusnon-GVHD
using
miR-125b.
identification ofofpatientslate-onset
acute
GVHD
versusnon-GVHD
usingusing
miR-128.
(B) ROC
identification
patientswith
withlate-onset
acute
GVHD
versusnon-GVHD
miR-128.
(B) curve
ROC
for
the
identificationofpatients
withlate-onset
acute
GVHD
versusnon-GVHD
using
miR-125b.
curve for the identification of patients with late-onset acute GVHD versus non-GVHD using miR-125b.

2.5. Sequential Analysis of Exosomal miRNAs Post-Allogenic HSCT

2.5. Sequential
Analysisof Exosomal
miRNAsPost-Allogenic
HSCT
To further
explorewhether miRNAs
exosomalPost-Allogenic
miRNAs canHSCT
be used as predictive biomarkers of LA
2.5. Sequential
Analysisof Exosomal
GVHD,
we
next
measuredmiR-125b
andmiR128expressions
sequentiallyin exosomesof available
To further explore whether exosomal miRNAs can be usedas predictive
biomarkers
of LA
To further explore whether exosomal miRNAs can be used as predictive biomarkers of LA GVHD,
serum samples
from miR-125b
two transplanted
patients(patient number
PN 3inand
PN 4 in of
Table1). AsGVHD,
we nexttaken
measuredand miR128
expressions
sequentially
exosomesavailable
we next measured miR-125b and miR128 expressions sequentially in exosomes of available serum
illustratedin
Figure6,weobserved
that
the
level
of
miR-128
wassignificantly
upregulated
serum samples taken from two transplanted patients (patient number PN 3 and PN 4 in Table 1). As
samplestaken fromtwo ortransplanted(patient
numberPN 3 and
PN
43in
Table
As illustrated
approximately
a weektwoobserved
weekspatients
beforeLAmiR-128
GVHD
in
PN significantly
and
PN1).
4 (Figure
6A,B).
illustrated
in Figure
6, wethat thetheonsetlevelofof
wasupregulatedin
Figure6,weobserved
that
the
level
of
miR-128
wassignificantly
upregulated
approximatelyaweek
When we useda week
the cut-off(3.183)
ROC inanalysis,thePNlevelsof miR-128
approximately
or twovalue
weeks
beforeobtained
the onsetfromof LAtheGVHD
PN 3 and4 (Figure
6A,B).
ortwo
weeks
before
the
onset
of
LA
GVHD
in
PN
3
and
PN
4
(Figure6A,B).
When
we
used
the
cut-off
expression
on days+46,value
and +70of PNobtained
3 were under
exceeded
cut-off
value atWhen
we usedthe +25,
cut-off(3.183)from thisthe value
ROC but
analysis,the thelevelsof miR-128
value(3.183)
obtainedfrom theROC
analysis,the levelsofmiR-128
expression
onmiR-128
days +25,+46, and
day
+105
in
the
prediagnosis
of
LA
GVHD
(Figure6A).
In
addition,thelevelsofexpression
expression on days +25, +46, and +70 of PN 3 were under this value but exceeded the cut-off value at+70
of PN+11
3 wereunder
this4value
but exceeded
the but
cut-off
value atday
+105 in theatprediagnosisofon daysandprediagnosis
+81
of PN
were
value
exceededthethecut-off
day expression+111 in theday+105 in theofLAunder
GVHDthat(Figure6A). In
addition,levelsvalueof miR-128
LA
GVHD(Figure6A).
In
addition,thelevelsofmiR-128
expression
on
days
+11
and
+81
of
PN
4
prediagnosisof +81LA of
GVHD(Figure6B).that
Onvaluethe otherhand, theexpression
levelin wasondays +11 andPN 4 wereunder
but exceeded
themiR-125b
cut-off value
at day +111
the
were
under
that
value
but
exceededthecut-off
value
at
day
+111
in
the
prediagnosis
of
LA
GVHD
upregulated immediately
after
HSCT,
andOn
elevatedmiR-125b
wasexpression
unable to predictLA
prediagnosis
of LA GVHD
(Figure6B).
the otherhand, expression
the miR-125b
level was(Figure6B).
On 6C,D).
the otherhand,
the miR-125b
expressionlevel
was upregulated
immediately
after
GVHD
(FigureThissuggeststhat
increased
miR-128
expressioninpatients
after
HSCT
may
upregulated immediately after HSCT, and elevated miR-125b expression was unable to predict LA
HSCT,and elevated
miR-125b
expression
was unable
toGVHD.
predict Finally,
LA GVHD(Figure 6C,D).
This suggests
be a predictive
markerprior
to thethat
onset
of LAmiR-128
themiR-128
as a
GVHD(Figure 6C,D).
This
suggests
increased
expression
in validity
patients ofafter
HSCT maythat
increased
miR-128
expressioninpatients
after
HSCT
may
be
a
predictive
marker
prior
to
the
onset
biomarker
was confirmed
by using
to discriminate
LA GVHDpatients
from non-GVHD
bea predictive
marker prior
to theit onset
of LA GVHD.
Finally,
the validityof miR-128 cases,
as aalthough
weshouldconsider
the
bias
of
stem
cell
source.
biomarker was confirmed by using it to discriminate LA GVHD patients from non-GVHD cases,
although we should consider the bias of stem cell source.
-379-

Int. J. Mol. Sci. 2018, 19, 2493

8 of 14of LA GVHD. Finally, the validity of miR-128 as a biomarker was confirmed by using it to discriminate
Int. J.GVHD
Mol. Sci.patients
2018, 19, xfrom
FOR PEER
REVIEW cases, although we should consider the bias of stem cell source.
8 of 13LA
non-GVHD

Figure 6. Bar graphs show relative expression
expression levels
levels ofof eacheach miRNA
miRNA at
at eacheach time
time point
point after
after HSCT.
HSCT.
(A,B) The
The dynamic
dynamicchanges
changesininexosomal
exosomalmiR-128
prior
to
the
late-onset
acute
GVHD
diagnosis.
miR-128 prior to the late-onset acute GVHD diagnosis. The
The
information
of3PN
was+7,
day+7,+25,
dayday
+25,
day
+46,
day
+70,
day
+105,
andTheonset.
level ofinformationof PN
was3 day
day
+46,
day
+70,
day
+105,
and
onset.
levelTheof miR-128
4 was
day
miR-128
was upregulated
over thecut-off
dayThe+105(A). The information
was upregulated
over the cut-off
level
at daylevel+105at(A).
informationof PN 4 was of
dayPN
+11,
day +81,+11,
day +81,+111,
onset.
The levelof upregulated
miR-128 wasover
upregulated
overtheatcut-off
level(B).
atday +111,
anddayonset.
Theandlevelof miR-128
wasthe cut-off
level
day +111(C,D)
The
changesof
exosomalmiR-125b
variedwidely
after
HSCT.
*
p
<
0.005,
ns:
day
+111
(B).
(C,D) The changes of exosomal miR-125b varied widely after HSCT. * p < 0.005, ns: Not significant.
Not significant.

2.6. Prediction of the Target Genes of Candidate miRNAs
2.6. Prediction of the Target Genes of Candidate miRNAs
This study indicated that upregulated miR-128 is crucial as an LA GVHD-specific miRNA. The
This
study indicatedthatinvolved
upregulated
miR-128
is crucialas an
LA GVHD-specificmiRNA.
clinicalrelevanceof miRNAsin thedevelopment
of LA
GVHD
was not evident.
Thus,
The
clinicalrelevanceofmiRNAsinvolvedinthedevelopment
of
LA
GVHD
was
not
evident.
Thus,
MirTarBase was used to find predicted target genes of miR-128 in order to determine the biological
MirTarBase
used
to findMore
predicted
targetgenes
of miR-128
in orderdetermine andthe biologicalsignificance wasof thismiRNA.
than 100
targetgenes
were extracted
byto
MirTarBase,
the target
significanceof thisstrong
miRNA.
More than
100 intargetextracted
andinthetargetgenes for whichevidence
existedthe genes
resultswereof thesearchby
areMirTarBase,summarized
Table
3.
genes
for
which
strongevidence
existedintheresultsofthesearcharesummarizedinTable
Accordingly, 12 genes, including B-cell specific Moloney murine leukemia virus integration region 3.
1
Accordingly,
12 andgenes,
including B-cell specific
Moloneymurineleukemiavirus integration(BMI1), F-box,
tryptophan-aspartic
acid (WD)repeatdomaincontaining
7 (FBXW7),region
were
1experimentally(BMI1), F-box,validated
and tryptophan-aspartic
acid (WD)domain containing
by a luciferase reporter
assay repeat
in the literature[22–27]. 7 (FBXW7), wereexperimentally validated by a luciferase reporter assay in the literature [22–27].
Table 3. Predicted target genes for miR-128 identified by miRTarBase.
Table 3. Predicted target genes for miR-128 identified by miRTarBase.
miRNA
Experimentally Validated Targets by Reporter Assay (miRTarBase)BMI1,FBXW7, DCX, Validated
RELN, WEE1,TGFBR1,
NEK2, Assay
SREBF1,SREBF2,
miRNA
Experimentally
Targets
by Reporter(miRTarBase)hsa-miR-128
ABCA1, ABCG1, RXRA
BMI1, FBXW7, DCX, RELN, WEE1, TGFBR1, NEK2, SREBF1, SREBF2,
hsa-miR-128
ABCA1, ABCG1, RXRA

3. Discussion

Recently, the definition of GVHD was revised in the NIH consensus criteria, with the new
3. Discussion
category of “late-onset” acute (LA) GVHD being established. Given the short history of thisRecently, the definition of GVHD was revised in the NIH consensus criteria, with the newcategory, the pathophysiology of LA GVHD is still poorly understood. Although the diagnosis ofcategory of “late-onset” acute (LA) GVHD being established. Given the short history of this category,
GVHD is judged based on clinical manifestations and pathological findings of the tissue sections
the pathophysiology of LA GVHD is still poorly understood. Although the diagnosis of GVHD isfrom the intestines or liver, it is difficult to provide an accurate pathological diagnosis of GVHD. For
judged based on clinical manifestations and pathological findings of the tissue sections from thethis reason, pathological findings have greatly affected various factors of radiation damage, drug
intestines or liver, it is difficult to provide an accurate pathological diagnosis of GVHD. For thisinjury, and viral infection, presenting a diagnostic dilemma. Therefore, there is an urgent need of
exploring reliable, noninvasive biomarkers of GVHD. In the context of GVHD after allogeneic
HSCT, biomarker research has progressed through a variety of techniques, such as
immunocompetent cells (CD4 helper T cells, regulatory T cells), plasma (soluble IL-2R), andinflammatory cytokines (IFN-γ, TNF-α). For example,
Paczesny et al. identified that a panel of four-380-

Int. J. Mol. Sci. 2018, 19, 2493

9 of 14reason, pathological findings have greatly affected various factors of radiation damage, drug injury,
and viral infection, presenting a diagnostic dilemma. Therefore, there is an urgent need of exploring
reliable, noninvasive biomarkers of GVHD. In the context of GVHD after allogeneic HSCT, biomarker
research has progressed through a variety of techniques, such as immunocompetent cells (CD4 helper
T cells, regulatory T cells), plasma (soluble IL-2R), and inflammatory cytokines (IFN-γ, TNF-α). For
example, Paczesny et al. identified that a panel of four biomarkers (IL-2Rα, TNFR1, IL-8, HGF) inserum was useful for the diagnosis of acute GVHD [4]. This group also found that the suppressionof tumorigenicity 2 (ST2) could distinguish treatment-resistant GVHD patients, and that ST2 levels
were associated with nonrelapse mortality [6]. Moreover, a specific plasma miRNA signature forclassic acute GVHD was recently determined by Xiao et al. [18]. To date, the identification of altered
miRNAs has been thought to contribute to the HSCT-related pathogenesis of acute GVHD; however,
the mechanism underlying LA GVHD remains to be elucidated. The above-mentioned results provided
us with a potential tool as a new liquid biopsy technique.
Because a number of previous studies investigated the role of exosomes and exosomal miRNAs
as potential predictive biomarkers in acute GVHD [20,28], we aimed to elucidate the possible role ofmiRNAs specifically in LA GVHD patients. Exosomes containing miRNAs reflect the behaviorof certain types of cells, so a survey of changes of miRNAs in exosomes should promote ourunderstanding of clinical conditions. In this context, technical advances in exosome separation andcharacterization have also recently been achieved, enabling exosomal miRNAs to be easily detectedby high-throughput techniques [29]. Additionally, Kordelas et al. reported an attractive treatmentusing mesenchymal stem cell (MSC)-derived exosomes for therapy-refractory GVHD [30]. We alsopreviously reported that many miRNAs have been identified as noninvasive, diagnostic, prognostic,
and predictive markers for hematologic malignancy [31–33]. In the current study, we explored the
LA GVHD-specific exosomal miRNA signature for LA GVHD compared with that in non-GVHD.
We also performed bioinformatic analysis to investigate the potential molecular mechanisms behind
LA GVHD and identify molecular target genes. Our results of validation by qRT-PCR showed thatmiRNA-128 significantly increased in LA GVHD patients. The miRNA miR-128 is well known asa brain-enriched miRNA, which plays a crucial role in the development of the nervous system [34].
The miRNA miR-128 has also been previously demonstrated to be dysregulated in tissues and blood
samples in several malignant tumor patients and to be correlated with tumor progression [35–37].
In the current study, it was impossible to conduct a functional analysis of miR-128. Hence, using
MirTarBase, we instead searched for potential target genes to investigate the mechanism of miR-128(Table 3).
We perfomed ROC analysis to assess the diagnostic capacity of exosomal miRNAs. Exosomal
miR-128 exhibited its potential in discriminating LA GVHD from non-GVHD groups. When theexpression level of candidate miRNAs was analyzed sequentially in a subset of LA GVHD patientsduring allogeneic HSCT, miR-128 was shown to increase over the cut-off value earlier than thedevelopment of the disease. However, our results must be interpreted with caution, because thisstudy was limited by the relatively small number of samples from LA GVHD patients and absenceof a validation cohort of any sort. After all, molecular mechanisms on the dysregulation of exosomal
miRNAs in patients with LA GVHD could not be fully provided. Also, candidate miRNAs were thenexamined longitudinally in only two patients over time. Thus, these results need to be further studied
with large cohort.
The results of the top 10 miRNAs obtained by TLDA included several characterized miRNAs(miR-423-5p, miR-19a, and miR-142) known to be dysregulated in acute GVHD [18,20,21]. In particular,
miR-423-5p, which is involved in the immune response of acute GVHD incidence, was elevated up
to 49-fold in exosomes of LA GVHD patients in our study. Crossland et al. reported differential
expression of miR-423 in exosomes at day 14, after HSCT had shown potential as a predictive
biomarker for the occurrence of acute GVHD [20]. Therefore, the pathology of LA GVHD may
resemble that of acute GVHD caused by donor T-cell alloreactivity in light of miRNAs. However,-381-

Int. J. Mol. Sci. 2018, 19, 2493

10 of 14the biology of GVHD is complex. In other words, miRNAs also regulate various molecular targets,
including normal development, differentiation, and maturation of hematopoietic cells in the immune
system [38]. The difference of miRNA expression may influence the process of immune reconstitution
after allogeneic HSCT. Moreover, the changes of immune-related miRNAs may be due to the dosageof immunosuppression. The dysregulated miRNAs when LA GVHD occurs may be repressed byimmunosuppressive regimen. However, it is difficult to evaluate whether the level of this miRNA
returns to baseline upon therapeutic intervention because of the uncontrollable nature of LA GVHD.
It is a challenge for future study to clarify the association between miRNA expression and responseto therapy. Although it is difficult to define miRNAs specific to LA GVHD in very complex medical
cases, and in the small number of transplanted patients in the current study, exosomal miR-128 may berelated to the immunoregulation of LA GVHD.
In summary, this study demonstrated that the levels of several exosomal miRNAs change at
the time of LA GVHD onset after HSCT. Although a longitudinal series of blood samples was takenfrom 2 patients only, upregulation of miR-128 expression level preceded of LA GVHD incidence. In
particular, our results suggest that biological alterations of miR-128 in exosomes may serve as potent
predictive biomarkers for close monitoring of the onset of LA GVHD. We consider that this work could
provide a foundation for revealing the importance of miRNA in the pathophysiology of LA GVHD.
GVHD remains a major cause of transplant-associated complications, which can lead not only to lethalorgan damage, but also to a decreased quality of life. Inhibition of certain exosomal miRNAs, such as
miR-128, might be considered as a novel potential treatment of LA GVHD to target the immune system
and inflammatory signaling, to control GVHD. However, further studies are needed to elucidate themolecular mechanisms behind the development of LA GVHD.
4. Materials and Methods
4.1. Ethics
The use of patient samples was approved by the Institutional Review Board of Tokyo Medical
University (IRB no. 1979, approved on 28 March 2011). Written informed consent was obtained from allof the participants before the collection of specimens, in accordance with the Declaration of Helsinki.
4.2. Diagnosis of Late-Onset Acute GVHD
The diagnosis of LA GVHD here was based on clinical symptoms or was histologically provenby biopsy in the target organs, as described previously [1]. Patients with typical manifestations ofclassic acute GVHD occurring beyond 100 days after transplantation and persistent, recurrent acute
GVHD were considered as having LA GVHD, in accordance with the new classification based on NIH
consensus criteria [1].
4.3. Isolation of Exosome Fractions from Plasma Sample of Patients
Ten patients with hematological malignancies who underwent allogeneic HSCT between February
2012 and November 2013 were included in this study, which consisted of five patients with LA
GVHD (gut, n = 2; liver, n = 2; skin and gut, n = 1) and five stable patients without GVHD
symptoms (non-GVHD) under immunosuppression. All samples were collected sequentially after
HSCT. For patients with LA GVHD, we evaluated blood samples obtained at the onset of this disease.
No treatment for LA GVHD had been given at the time of sampling. For comparison, blood samples
from HSCT patients without GVHD symptoms and other complications were used. In addition, eight
healthy controls were also randomly selected in our hospital. We extracted exosome fractions from200 µL of plasma using Total Exosome Isolation Reagent (Invitrogen, Carlsbad, CA, USA) and thenassessed exosomal miRNA expression in a subset of samples, as described previously [11].

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11 of 144.4. Exosomal miRNA Profiling
Total RNA was isolated from exosomes fractions of five patients with LA GVHD and five patients
without GVHD (non-GVHD) using the miRNeasy Mini kit (Qiagen, Hilden, Germany). Exosomes werediluted with 700 µL of QiaZol. After 5 min of incubation, 1 µL of 1 nM ath-miR-159 (Hokkaido System
Science, Hokkaido, Japan) was added to the aliquot followed by vortexing for 30 s and incubation for
5 min. Subsequent phenol extraction and centrifuge filtration were performed, in accordance with the
manufacturer’s instructions (Qiagen, Hilden, Germany). The RT reaction and pre-amplification step
were set up in accordance with the manufacturer’s instructions (Thermo Fisher Science, Waltham, MA,
USA). miRNAs were reverse-transcribed with Megaplex Prime Pools (Human Pools A v2:1; Thermo
Fisher Sciences, Waltham, MA, USA).
4.5. TaqMan Loq-Density Array Screening
The miRNA expression profiles were determined with a TaqMan miRNA Array Human Card A(Thermo Fisher Sciences, Waltham, MA, USA). Quantitative RT-PCR was performed on an Applied
Biosystem 7900 HT thermocycler, in accordance with the manufacturer’s recommended program [31].
Using SDS2.2 software and Data Assist (version 3.01, Thermo Fisher Sciences, Waltham, MA, USA),the expression of exosomal miRNAs was calculated based on cycle threshold (Ct) values normalized
by those of ath-miR-159, which was spiked in each exosomal sample. Data analysis was performed
using GeneSpring® software (Version 12.1, Agilent Technologies, Palo Alto, CA, USA) and R software(https://www.r-project.org, accessed on 27 Arpil 2017). The Benjamini–Hochberg algorithm was used
for the estimation of false discovery rates, as we reported previously [31].
4.6. Real-Time Quantitative RT-PCR for Candidate miRNAs
We determined the levels of the individual miRNAs by real-time quantitative RT-PCR witha TaqMan MicroRNA Assay (Thermo Fisher Sciences, Waltham, MA, USA) and the following
miRNA-specific stem-loop primers: hsa-miR-128 (assay ID: 002216, Ambion, Austin, TX, USA) andhsa-miR-125b (assay ID: 000449, Ambion, Austin, TX, USA). Subsequently, quantitative real-time PCR
was performed with an ABI Prism 7900 sequence detection system (Thermo Fisher Sciences, Waltham,
MA, USA). The reaction was initiated by incubation at 95 ◦ C for 2 min, followed by 50 cycles of 95 ◦ C
for 15 s and then 60 ◦ C for 1 min. All reactions were run in duplicate. Mean (Ct) values for all miRNAs
were quantified with sequence detection system software (SDS version 1.02; Thermo Fisher Sciences,
Waltham, MA, USA) [31]. The expression of all miRNAs was normalized to miR-159, which was stably
detected in all samples, yielding a −∆∆Ct value that was calculated by subtracting the −∆∆Ct value ofthe normal samples from the respective −∆Ct values of patient samples. The expression of all miRNAs
was normalized using the 2−∆∆Ct method.
Following the identification of differentially expressed miRNAs, the predicted target genes ofthese altered miRNAs were investigated using the experimentally validated miRNA–target interactions
database MirTarBase (http://mrtarbase.mbc.nctu.edu.tw/, accessed on 11 September 2017).
4.7. Statistical Analysis
Data are expressed as mean ± standard deviation (SD). Multiple groups were compared by
one-way analysis of variance (ANOVA) with post hoc Tukey’s test. Statistical analysis was performed
using GeneSpring software, and miRNAs with both a ∆Ct of either >1.0 or <−1.0 and a P value ofless than 0.05 were deemed to be differentially expressed. The area under the curve (AUC) of thereceiver-operating characteristic (ROC) curve was calculated. GraphPad Prism software (version 5cfor Macintosh; GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis.

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12 of 14

Author Contributions: S.Y., K.O. and J.H.O. designed the research and wrote the paper. T.U. and C.K. performed
the research and analyzed the data. Y.S., M.G. and D.A. provided patient samples.
Funding: This study was supported in part by the Private University Strategic Research-Based Support Project(S1311016, S1511011) from the Ministry of Education, Culture, Sports, Science and Technology (Tokyo, Japan). We
thank Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.
Acknowledgments: Kazuma Ohyashiki received research support from Celgene KK, Chugai Pharmaceutical KK,
and Janssen Pharma KK.
Conflicts of Interest: The authors declare no conflict of interest.

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Extracellular vesicle-mediated cell – cellcommunication in haematologicalneoplasms
Junko H. Ohyashiki1, Tomohiro Umezu1,2 and Kazuma Ohyashiki2

Review
Cite this article: Ohyashiki JH, Umezu T,
Ohyashiki K. 2017 Extracellular vesiclemediated cell– cell communication in
haematological neoplasms. Phil. Trans. R.
Soc. B 373: 20160484.
http://dx.doi.org/10.1098/rstb.2016.0484
Accepted: 15 April 2017
One contribution of 13 to a discussion meeting
issue ‘Extracellular vesicles and the tumour
microenvironment’.
Subject Areas:
molecular biology
Keywords:
extracellular vesicles, leukaemia, multiple
myeloma, hypoxia, bone marrow mesenchymal
stromal cells, miRNA
Author for correspondence:
Junko H. Ohyashiki
e-mail: junko@hh.iij4u.or.jp

1

Department of Molecular Oncology, Institute of Medical Science, and 2Department of Hematology, Tokyo
Medical University, Tokyo, Japan
JHO, 0000-0002-5997-2790
Crosstalk between bone marrow tumour cells and surrounding cells, including bone marrow mesenchymal stromal cells (BM-MSCs), endothelial cells
and immune cells, is important for tumour growth in haematological neoplasms. In addition to conventional signalling pathways, extracellular
vesicles (EVs), which are endosome-derived vesicles containing proteins,
mRNAs, lipids and miRNAs, can facilitate modulation of the bonemarrow microenvironment without directly contacting non-tumourous
cells. In this review, we discuss the current understanding of EV-mediatedcell –cell communication in haematological neoplasms, particularly leukaemia and multiple myeloma. We highlight the actions of tumour and
BM-MSC EVs in multiple myeloma. The origin of EVs, their tropism andmechanism of EV transfer are emerging issues that need to be addressedin EV-mediated cell –cell communication in haematological neoplasms.
This article is part of the discussion meeting issue ‘Extracellular vesicles
and the tumour microenvironment’.

1. Introduction
The tumour microenvironment (TME) plays an essential role in cancer development, metastasis and drug resistance [1]. Cancer-associated fibroblasts (CAFs)
are considered as a major component of the TME [2–5]. However, the origin of
CAFs is still controversial [2,6]. Relative to solid tumours, the bone marrow microenvironment (BME), where haematopoietic stem cells (HSCs) are located, isconsidered much more complicated (figure 1). First, HSCs reside in a specialized
microenvironment called the haematopoietic niche [7]. The endosteal niche, hallmarked by osteoblasts lining the bone marrow cavity, has been proposed as animportant source of HSC quiescence (figure 1, top right), while the vascularniche has been implicated in HSC maintenance and regeneration (figure 1,
bottom right). Second, the definition of CAFs is not generally used to indicate
the tumour-surrounding cell population in haematological neoplasms. Such cells
are referred to as bone marrow mesenchymal stromal cells (BM-MSCs) [8].
While BM-MSCs and CAFs may share biological properties, especially in multiple
myeloma (MM) [9] and chronic lymphocytic leukaemia (CLL) [10], the differences
and similarities between BM-MSCs and CAFs are not fully elucidated in leukaemias [8]. In addition to BM-MSCs, immune cells, such as T cells, B cells andnatural killer (NK) cells, also interact with tumour cells. Myeloid-derived suppressor cells (MDSCs) represent another population of regulatory cells, which impair
anti-tumour innate and adaptive immune responses [11].
It is believed that cell–cell communication between tumour cells and their surrounding cells is modulated by direct cell contact and soluble factors such as
cytokines. Emerging evidence suggests that extracellular vesicles (EVs) shedfrom either tumour cells or their surrounding cells act as a mediator of crosstalkin the TME [12] because EVs contain complex cargo including proteins, mRNAs,
lipids and microRNAs (miRNAs). The current generally recognized categories of
EVs are exosomes (the most well-known category) with a size of 30–100 nm,
microvesicles with a size of 100–1000 nm, and large oncosomes ranging in size

& 2017 The Author(s) Published by the Royal Society. All rights reserved.
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endosteal niche

bone

MDSC

tumour cell
T cellhaematopoietic
stem cells

NK cellmacrophage

mesenchymal
stem cell

endothelial cell

RBC

RBC

sinusoidbone marrow mesenchymal
stromal cell (BM-MSC)

extracellular vesicle (EV)vascular niche

Figure 1. Bone marrow microenvironment in haematological neoplasms. The bone marrow microenvironment is located in bone. Two types of niches, the endosteal
niche and vascular niche, are located adjacent to the bone cortex. Tumour cells secrete extracellular vesicles (small green circles) towards their surrounding cells such asbone marrow mesenchymal stromal cells (BM-MSCs), endothelial cells and immune cells. Various types of cell–cell communication are assumed, such as tumour cells to
BM-MSCs, tumour cells to endothelial cells, tumour cells to immune cells, and others. MDSC, myeloid-derived suppressor cell; NK, natural killer; RBC, red blood cell.
proteins, mRNAs and miRNAs, have been identified [17,21], it isdifficult to focus on a single pathway for AML. Another important issue is that AML-EVs suppress immune cells such as
NK cells [18]. In serum obtained from AML patients, AMLmicrovesicles mediate suppression of NK cell activity via thetransforming growth factor-b1 signalling pathway, and interleukin (IL)-15 protects NK cells from the adverse effects of
AML-microvesicles [18].
To date, there is no appropriate in vitro model to elucidate
the complex cell–cell interactions in the bone marrow niche
where leukaemic stem cells exist. Crosstalk via EVs between
osteoblasts and HSCs or between BM-MSCs and HSCs also
remains to be resolved. For this reason, much effort has been
made to determine the diagnostic value of circulating EVs
rather than the mechanism of cell–cell communication in
AML [23]. A recent report by Viola et al. demonstrated thatEVs derived from BM-MSCs induce tyrosine kinase inhibitor
resistance in AML [24], suggesting a new therapeutic approachtargeting BM-MSCs in AML. Considering the critical roles of
EVs in AML, understanding the mechanisms regulating signalling pathways in recipient cells may provide additional insights
into the use of EVs as therapeutic agents for treating AML.

from 1 to 10 mm [13,14]. To date, most EV studies in haematologic neoplasms refer to exosomes or microvesicles. However, a
recent study by Johnson et al. focused on the heterogeneity of
EVs and characterized large EVs in acute lymphoblastic leukaemia [15]. In this review, we highlight and discuss recent studies
of EV-mediated cell–cell communication in haematologicalneoplasms, particularly leukaemia and MM.

2. Acute myeloid leukaemia-derived extracellular
vesicles in the bone marrow
microenvironment
Leukaemia is a type of haematological neoplasm which arises
from HSCs. Leukaemia types are subdivided by cell lineage(myeloid or lymphoid) and clinical course (acute or chronic).
High throughput genomic analyses have revealed that genetic
alteration of leukaemic cells is likely a more important determinant of disease severity [16]. However, increasing evidence
suggests that leukaemic cell-derived EVs affect their surrounding
cells in autocrine and/or paracrine manners [17–19].
In acute myeloid leukaemia (AML), Kumar et al. found evidence that AML-EVs alter the BME to facilitate leukaemic cell
growth and suppress normal haematopoiesis in a mousemodel [20]. The unique miRNA profile of AML-EVs, including
miR-155, has the potential to increase leukaemic fitness by dysregulation of other cell types in the BME [21]. Myelodysplastic
syndrome (MDS) is a clonal myeloid neoplasm characterized
by ineffective haematopoiesis, and approximately 30% of
patients develop AML. In addition to AML-EVs, MDS-EVs arelinked to stromal cell dysfunction. Therefore, EV-mediatedcell–cell interaction is also involved in bone marrow failure syndrome [22]. Although various components of AML-EVs, such as

3. Bone marrow angiogenesis and extracellular
vesicles derived from chronic myeloid
leukaemia
Chronic myelogenous leukaemia (CML) characterized by the
BCR-ABL chimeric protein is another type of myeloid leukaemia,
which tends to progress more slowly than AML. Increased
microvessel density and clinicopathological correlations withbone marrow angiogenesis have been reported in CML patients-388-

Phil. Trans. R. Soc. B 373: 20160484

osteoclast

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osteoblast2

control
HUVECs24 h

20% O2

24 h

1% O2

normoxia

K562

K562

hypoxia[25]. We and others have shown that EVs secreted by CML cells
can potentially influence in vitro and/or in vivo angiogenesis bydirectly affecting the properties of endothelial cells [26,27].
Taverna and colleagues first provided direct evidence that fluorescent-labelled EVs released by K562 cells are internalized byhuman umbilical vein endothelial cells (HUVECs) during tubular differentiation on Matrigel, thereby enhancing angiogenesis[26,28]. They also demonstrated that functional transfer of
CML-EV-miR126 targets C-X-C motif chemokine ligand 12 andvascular cell adhesion molecule in HUVECs [29]. These findings
indicate that exogenous miRNAs transferred via EVs function
similarly to endogenous miRNAs in HUVECs. CML-EVs
also induce increased secretion of IL-8 in BM-MSCs, thereby
promoting leukaemic cell growth in vitro and in vivo [30].
Because hypoxia is known to be a regulator of angiogenesis,
we investigated how hypoxia triggers EV-mediated angiogenesis using the human leukaemic cell line K562. We found that
K562 cell-EVs under hypoxic conditions (1% O2 for 24 h) significantly enhance tube formation of HUVECs compared withEVs produced under normoxic conditions (20% O2 for 24 h)[31] (figure 2). These experiments employed artificial in vitro conditions for short-term exposure to hypoxia. However, we foundthat miR-210 in EVs (EV-miR-210) downregulates ephrin A3 in
HUVECs and alters EV components under hypoxic conditions,
thereby affecting the behaviour of HUVECs [31]. EVs shedfrom leukaemic cells act in the following manners. First,
leukaemia-EVs educate BM-MSCs for their own cell survival.
As a result, normal haematopoiesis is suppressed. Second, leukaemia-EVs induce immune suppression by targeting T cells, B
cells and NK cells. Third, leukaemia-EVs induce angiogenesis,
especially in CML.

4. Extracellular vesicle-mediated cell –cellcommunication in multiple myeloma
MM is a blood cancer formed by malignant plasma cells thatcreate tumour masses in bone. In contrast to leukaemia, thetumour masses are predominantly located in the bone

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Phil. Trans. R. Soc. B 373: 20160484

Figure 2. Hypoxia and angiogenesis in leukaemia. Human umbilical vein
cells (HUVECs) were cultured with or without extracellular vesicles (EVs)secreted from a human leukaemia cell line (K562). The cells were cultured
for 24 h and then EVs were collected. In normoxia, tube formation of
HUVECs was increased in the presence of K562-derived EVs compared with
control HUVECs (middle). When EVs obtained from K562 cells cultured
under hypoxic conditions (K562-EV-hypoxia) were added to HUVECs, tube formation was much more prominent (bottom). These findings indicate possibleroles of EVs, hypoxia and angiogenesis in leukaemia.

3

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marrow. Therefore, cell –cell communication in the BME has
a key role in the pathogenesis of MM. Because targeting
MM cells alone is insufficient to establish curable treatment
strategies for MM, the interaction between MM cells andtheir surrounding cells has been studied extensively [32]. In
terms of clinical practice, circulating EV-miRNAs fromnewly diagnosed MM patients are a potential biomarker for
prognosis [33]. Because MM appears to be pathophysiologically similar to solid tumours, a cell-free specimen has great
benefit for evaluating minimal residual disease. We discuss
the characteristic BME in MM, especially hypoxia, in thefollowing section. We then summarize the role of MM-EVs,
BM-MSC-EVs and the mechanism of EV transfer in MM.
Bone marrow is hypoxic in nature, and oxygen tension in
MM-infiltrated BM is lower than that in normal BM [34]. The
massive proliferation of MM cells produces hypoxic conditions in the tumour, which may lead to more rapid cell
growth, drug resistance and angiogenesis [35]. Therefore,
we focused on the effect of hypoxia in the BME andestablished three MM cell lines that could be adapted tolong-lasting hypoxia, namely hypoxia-resistant MM cells(HR-MM cells). Mutation analysis of HR-MM cells revealed
no acquired genetic variation compared with their parental
cells, while the gene expression profile of HR-MM cells was
modified during the adaptation process [36]. We consideredthat HR-MM cells may be suitable for analyses of the BME,
which mimics the in vivo state because most in vitro hypoxia
studies use short-term hypoxic exposure (3–24 h) which may
reflect acute hypoxic shock rather than the MM-BME.
Although no significant difference existed in the size of EVs
between HR-MM and parental cells, the amount of EVs
released from HR-MM cells was approximately twofold
greater. We also found that HR-MM cells enhanced in vitro
tube formation and increased angiogenesis in vivo in a Matrigel assay. It was notable that EV-miR-135b from HR-MM
cells enhanced angiogenesis by direct suppression of factorinhibiting hypoxia-inducible factor 1 (FIH-1) in endothelial
cells. It remains uncertain how other components within
HR-MM cell-EVs, such as proteins, mRNAs and miRNAs,
may be involved in EV-mediated angiogenesis.
Regarding the cargo of MM-EVs, three independent
proteomic studies revealed the significance of proteins in
EV-mediated intercellular communication [37–39]. Harshman
et al. identified 583 MM-EV proteins in total by liquid chromatography coupled with tandem mass spectrometry [37]. They
found that MM-EVs contained molecules related to a distinct
pathway, such as antigen-presenting molecules (major histocompatibility complex class I and II) and adhesion molecules(tetraspanins and integrins), in MM cell lines MM.1S andU266 [37]. MM-EVs contain the biologically active form of
CD147, which drives MM cell proliferation [38]. In addition,
MM-EVs harbour CD138, which is known to be an angiogenic
regulator [39], and the angiogenic activity has been confirmed
using mouse MM-EVs in vivo [40].
MM-EVs also interact with BM-MSCs to establish afavourable BME for MM: they promote an increase of
miR-146a in BM-MSCs, which induces more cytokines andchemokines, such as IL-6, and support MM cell growth [41](figure 3). Another example of tumour cell and BM-MSC
cooperation has been reported in another type of lymphoidmalignancy, CLL. CLL-derived EVs are internalized by
BM-MSCs and deliver functionally active components to
BM-MSCs [10]. Taken together, tumour-derived EVs have

osteolysis

MM cells

immunosuppression

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osteoclast

NK cellmacrophage

T cellsoluble factors

MDSC

IL-6, VEGF, TNF-a,
TGF-b, RANKL,CXCL12, etc.

hypoxia

FIH-1

BM-MSC

endothelial cell
HIF-1

MM cell growth, survival
and drug resistanceangiogenesis

Figure 3. Cell– cell communication by extracellular vesicles in multiple myeloma. Massive proliferation of abnormal plasma cells is shown in the centre. The centreof the mass has pale blue plasma cells, indicating the hypoxic state. In addition to conventional signalling pathways, such as direct cell adhesion and soluble factors,
either extracellular vesicles (EVs) derived from multiple myeloma (MM) cells (MM-EVs; small green circles) or EVs derived from bone marrow mesenchymal stromalcells (BM-MSC-EVs; small orange circles) modify the behaviour of recipient cells as follows. MM-EVs modulate BM-MSCs and induce a favourable microenvironmentfor MM cells. Conversely, BM-MSC-EVs also induce MM cell growth, survival and drug resistance (bottom right). MM-EVs induce angiogenesis. In a long-lasting
hypoxic state, EV-miR-135b acts as a mediator of angiogenesis through the FIH-1/hypoxia-inducible factor-1 signalling pathway (bottom left). BM-MSC-EVs interact
with myeloid-derived suppressor cells (MDSCs), and MM-EVs modulate immune cells, such as natural killer (NK) cells, resulting in immunosuppression (top right).
MM-EVs activate osteoclasts which may be linked to osteolysis in MM (top left).
cells [42]. In solid tumours, tumour-derived EVs use tropism
to transfer into organ-specific recipient cells, and this tropism
depends on distinct integrin expression patterns [43]. The
mechanism by which EVs are captured and why EVs canselect recipient cells remain to be elucidated in MM. Although
the BME in MM is controlled by either a cytokine/chemokine
network or cell adhesion molecules [32], interactions between
conventional and EV-mediated pathways are much more
complex than ever imagined.

the ability to transform BM-MSCs to CAF-like cells. Of note isthat MM-EVs induce prolonged survival of MDSCs whichhave a strong immune-suppressive activity in cancer patients[40]. Raimondi et al. demonstrated the effect of MM-EVs on
both human primary osteoclasts and murine pre-osteoclastic
RAW264.7 cells [42]. These results indicate that MM-EVs are
key regulators that educate BME-targeting endothelial cells,
BM-MSCs, MDSCs and osteoclasts.
In addition to MM-EVs, EVs derived from BM-MSCsfacilitate MM cell growth by maintaining a favourable BME
for MM. Roccaro et al. reported the difference in EV-miRNA
contents between MM-BM-MSCs and normal BM-MSCs [43].
EVs derived from MM-BM-MSCs have less tumour suppressor miR-15a and higher levels of oncogenic proteins,
cytokines and adhesion molecules, whereas normal-BMMSCs have the potential to inhibit MM cell growth. Theirfindings indicate a CAF-like ability of MM-BM-MSCs andthe possible use of normal BM-MSCs for MM cell therapy.
We also found that BM-MSCs from healthy young donors
inhibit MM angiogenesis through EV-miRNA [44]. From
the aspect of CAFs, BM-MSC-derived EVs induce proliferation, survival and drug resistance of MM cells [45], andthey are able to activate MDSCs [46]. As a result, BM-MSCderived EVs contribute to not only the BME supporting
MM cell growth but also immunosuppression related todisease progression.
Recent studies have focused on the mechanisms of EV
release and uptake in MM. EV-fibronectin has recently been
identified as a key binding ligand for heparan sulfate that
can act as a receptor for EV uptake by MM cells [41]. Conversely, Bcl-xL is an exosomal caspase-3 substrate, and its
processing is required for the uptake of exosomes by recipient

5. Perspectives
Despite the fact that most studies have focused on tumourcell- and BM-MSC-derived EVs, how EVs derived fromother types of cells act in the BME is largely unknown.
Because a category of EVs includes components other than
exosomes, including oncosomes, the type of EV and thecells from which EVs originate should be clarified to elucidate the precise mechanism of EV-mediated cell –cellcommunication. We identified EV-miRNA as an angiogenic
factor in our experimental model. However, analysis ofplasma EV-miR-135b did not simply reflect MM disease
severity. It is likely that EV-miRNA in the proximal area (i.e.
bone marrow) is not linked to EV-miRNA in remote areas
such as circulating blood. The manner of cell–cell communication in the proximal area might be different from that in
the distal area. Therefore, careful attention is needed for diagnostic use of EV-miRNA. To better understand EV-mediatedcell–cell communication in haematological neoplasms, theorigin of EVs, their tropism, and mechanism of EV transfer
are emerging issues that need to be addressed.

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Phil. Trans. R. Soc. B 373: 20160484

miR-135b

MM-EV
BM-MSC-EV

direct celladhesion4

Data accessibility. This article has no additional data.
Authors’ contributions. T.U. performed part of the study discussed in thisCompeting interests. We have no competing interests.
Funding. This review was supported by the Private University Strategic

5

review. J.H.O. drafted the manuscript. K.O. helped to draft themanuscript. All authors read and approved the final manuscript.

Research-Based Support Project (grant no. S1311016) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

rstb.royalsocietypublishing.org

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2013.141)REGULAR ARTICLE

Induction of multiple myeloma bone marrow stromal cell apoptosis by
inhibiting extracellular vesicle miR-10a secretion
Tomohiro Umezu,1,2 Satoshi Imanishi,3 Seiichiro Yoshizawa,1 Chiaki Kawana,1 Junko H. Ohyashiki,2 and Kazuma Ohyashiki1,2
1

Department of Hematology, 2Department of Advanced Cellular Therapy, and 3Institute of Medical Science, Tokyo Medical University, Tokyo, Japan.

Key Points• Inhibition of EV releaseresulted in accumulation of cellular miR-10a
and enhanced apoptosis of BMSCs derived
from patients with MM.
• Inhibition of miR-10a–enriched EVs from
BMSCs also inhibited
proliferation of MM cells.

Bone marrow stromal cells (BMSCs) interact with multiple myeloma (MM) cells in the bonemarrow and create a permissive microenvironment for MM cell proliferation and survival.
In this study, we investigated the role of extracellular vesicles (EVs) from BMSCs derived
from patients with MM (MM-BMSCs). EV-encapsulated miR-10a expression was high while
intracellular miR-10a was low in MM-BMSCs. We therefore hypothesized that miR-10a was
packaged into EVs that were actively released into the extracellular space. Inhibition of EV
release resulted in accumulation of intracellular miR-10a, inhibition of cell proliferation,and induction of apoptosis in MM-BMSCs. In contrast, proliferation and apoptosis of BMSCs
derived from healthy individuals were unaffected by inhibition of EV release. Furthermore,
miR-10a derived from MM-BMSCs was transferred into MM cells via EVs and enhanced their
proliferation. These results suggest that inhibition of EV release induced apoptosis in MMBMSCs and inhibited MM cell proliferation, indicating a possible role for MM-BMSC–targeted
therapy.

Introduction
Multiple myeloma (MM) is a B-cell neoplasm that affects immunoglobulin-producing plasma cells
and is characterized by the accumulation of monoclonal plasma cells in the bone marrow (BM).
BM stromal cells (BMSCs) interact with MM cells in vivo and create a permissive microenvironment for MM cell proliferation,1 angiogenesis, metastasis,2 and development of drug resistance.3
Targeting the tumor microenvironment is thus an attractive approach for anticancer therapy.4 Despite
improvements associated with new treatment modalities for MM, including proteasome inhibitors and/or
immunomodulators,5,6 novel approaches are needed to improve outcomes, particularly in elderly
patients. It is therefore necessary to understand the mechanisms that block the complex crosstalk
between MM cells and the BM microenvironment (BMME). Strategies targeting the tumor vasculatureand inhibiting the protumorigenic inflammatory response of the BMME have been successful, as shownby numerous agents.7-11 Inhibition of tumor cell–BMME interactions might represent another emerging
strategy.12-14
Recent advances in cancer biology revealed that extracellular vesicles (EVs) are involved in theregulation of intercellular communication, stimulating interest in their role as a potential target for cancer
therapy.15,16 EVs are membrane-wrapped structures that are secreted by most cells and exist in body
fluids.17 They can be broadly separated into 2 classes: exosomes (30-120 nm in diameter) andmicrovesicles (100-500 nm in diameter). Accumulating evidence has shown that EVs contain proteins,
lipids, DNA, messenger RNA, microRNA (miRNA), and long noncoding RNA, which can be transferredfrom producer cells to recipient cells, thus facilitating cell-to-cell communication.18-21 EVs derived
Submitted 7 May 2019; accepted 22 September 2019. DOI 10.1182/
bloodadvances.2019000403.
All data are available within the article and supplemental information files. MiRNA andmessenger RNA expression profiling data that support the findings of this study havebeen deposited in the Gene Expression Omnibus under the following accession
numbers: microarray, GSE78235 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?

acc5GSE78235); and polymerase chain reaction array, GSE78865 (https://www.
ncbi.nlm.nih.gov/geo/query/acc.cgi?acc5GSE78865).
The full-text version of this article contains a data supplement.
© 2019 by The American Society of Hematology

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from MM cells are considered mediators for BMME targeting
endothelial cells, BMSCs, myeloid-derived suppressor cells, andosteoclasts,22-24 whereas BMSC-derived EVs (BMSC-EVs) affectthe viability, survival, and drug resistance of MM cells.25,26 EV-based
cancer therapies aim to use these vesicles containing miRNA and/or anticancer drugs for targeted delivery to tumor cells.27 We
previously reported an antiangiogenic effect of reconstructed EVs
that had been rejuvenated by transfection with specific miRNAs(miR-340) from BMSCs derived from young healthy donors,28
providing new insights into EV-based cancer therapy by modification of BMSC-EVs.
The current study investigated the biological properties of EVs
and EV miRNAs derived from BMSCs with the goal of establishingnovel strategies to target the MM microenvironment. EV miRNA
expression differed between BMSCs derived from patients with
MM (MM-BMSCs) and from healthy subjects (normal-BMSCs). A
subset of miRNAs including miR-10a was selectively increased in
EVs derived from MM-BMSCs, whereas these cells showed little orno expression of intracellular miR-10a. This study focused on therole of EV–miR-10a derived from MM-BMSCs in the BMME ofpatients with MM as a potentially promising target for controlling
tumor proliferation in these patients.

Methods
EthicsThe use of patient samples was approved by the Institutional
Review Board of Tokyo Medical University (no. 2648, approved 22
April 2014). Written informed consent was obtained from allparticipants before collection of the specimens, in accordance with
the Declaration of Helsinki.
All animal experiments were conducted in compliance with theinstitutional guidelines of the Animal Experimental Center of Tokyo
Medical University/Animal Biosafety Level 2 Laboratory for Use of
Animals. The experimental protocols were approved by Tokyo
Medical University’s Institutional Animal Care and Use Committee.

MM-BMSCs
Twenty-one patients (age range, 43-82 years; median age, 68.9
years) fulfilling the International Myeloma Working Group diagnostic
criteria for MM were included (Table 1). The patients were classified
according to the International Staging System as I (n 5 8), II (n 5 6),or III (n 5 7). MM-BMSCs were isolated by using the conventional
plastic-adhesion method. Details are provided in the supplemental
Methods.

Cell culture
Human BMSCs from 2 healthy donors (aged 68 and 72 years;
normal-BMSCs) were purchased from Lonza Inc. Normal-BMSCs
and MM-BMSCs were seeded in mesenchymal stem cell basal
medium (MSCBM) supplemented with SingleQuots (Lonza) at
37°C in a humidified atmosphere containing 5% carbon dioxide.
The adherent cells were then harvested by trypsinization and either
passaged (passage 1-5) for expansion or analyzed for isolationof EVs.
Human MM cell lines (RPMI 8226, KMS-11, and U266) werepurchased from the Health Science Research Resource Bank andcultured in RPMI 1640 (Thermo Fisher Scientific) supplemented

with 10% heat-inactivated fetal bovine serum (HyClone) at 37°C in
a humidified atmosphere containing 5% carbon dioxide.

Detection of cell viability and apoptosis
BMSCs (1 3 104 cells/well) and MM cell lines (5 3 103 cells/well)
were seeded in 96-well plates and treated with FTY720 (0.1-10mM; fingolimod, sphingosine 1-phosphate [S1P] receptor modulator; Cayman Chemical) or transfected with miR-10a mimic (0.5-50nM) for 24 to 48 hours. Cell proliferation was assessed by using
WST-8 according to the manufacturer’s recommendations (CellCounting Kit-8; Dojindo Molecular Technologies, Inc.). Eachexperiment was repeated 3 times, and the data represent themean 6 standard error of 6 duplicate wells.
Two methods were used to analyze apoptosis induction. CaspaseGlo 3/7 reagent (Promega) was added to the cells after 48 hours,
and the luminescence of each sample was determined by usinga GloMax Multi microplate reader (Promega) according to the
manufacturer’s instructions. Apoptosis was also detected by using
the fluorescein isothiocyanate–Annexin V Apoptosis Detection Kit I(BD Bioscience). Details are provided in the supplemental Methods.

Isolation and characterization of EVs
BMSCs (4 3 104 cells/cm2) were cultured in 5 mL of MSCBM(Lonza) in a T-25 flask. The culture supernatants were harvested
after 48 hours of incubation, and the EV fraction was purified byusing ExoQuick-TC reagent (System Biosciences) according to the
manufacturer’s instructions. EVs were quantitated according tonanoparticle tracking analysis (NanoSight LM10; Malvern Panalytical) and observed with a transmission electron microscope (JEM1200EX; JEOL) (supplemental Methods).
For inhibition of EV secretion, BMSCs (4 3 104 cells/cm2) were
cultured in 5 mL of MSCBM with 5 mM of GW4869 (nSMase2
inhibitor; MilliporeSigma), 1 mM of manumycin A (inhibitor of Ras
farnesyltransferase; MilliporeSigma), 1 mM of JTE-013 (S1P2
antagonist; Cayman Chemical), or 1 mM of FTY720 (fingolimod,
S1P receptor modulator; Cayman Chemical). The culture medium
was collected for EV isolation.

miRNA expression profiles
Cellular and EV miRNAs were collected by using an miRNeasy kit(Qiagen), and miRNA profiling of cells and EVs was performed byusing a TaqMan low-density miRNA array (Thermo Fisher Scientific)
according to the manufacturer’s recommendations. The relative
expression level of each miRNA was calculated by using thecomparative threshold cycle (Ct) method, as described in the
supplemental Methods.

Transfection of miRNA mimics
BMSCs (3 3 104 cells/cm2) or MM cell lines (2 3 105 cells/well)
were transfected with 10 nM or 100 nM of individual miRNA mimic(has-miR-10a, has-miR-346, and has-miR-135b; Thermo Fisher
Scientific) using HiPerFect (Qiagen). To visualize the transport of
EV miRNA derived from MM-BMSCs into MM cells, miR-10a mimic
was labeled by using a Label IT siRNA Tracker Cy3 kit (Mirus Bio)according to the manufacturer’s instructions. Details are provided in
the supplemental Methods.

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Table 1. Characteristics of patients with MM
SampleSexAge, y

MM_1

M

67

MM_2

M

82

MM_4

M

MM_5

MM typeM-protein

PCBM, %

IgA k

1754 mg/dL

30

IgA k

2234 mg/dL

23

66

IgG l

5818 mg/dL

M

81

IgG k

MM_6

M

63

MM_7

M

43

MM_8

F

MM_9

M

MM_10
MM_11

MM-PC cytogenetic alterations (FISH)

CRAB

ISS

FLC, mg/L

Normal IGH Tx, and P53

2

1

NormalNormal IGH Tx; del P53: 40%

22Normal48.8

t(4;14): 61%; normal P53

2

3

62.6

3547 mg/dL

22

Normal IGH Tx, and P53

2

2

25

IgG k

2270 mg/dL

50

Normal IGH Tx, and P53

Renal

1

165

BJ k

1.6 g/d

15.2

Normal IGH Tx; del P53: 6%

Plasmacytoma

1

1 222

69

IgA l

3991 mg/dL

42

Normal IGH Tx, and P53

HyperCa, anemia, bone

2

82

IgG l

7670 mg/dL

22

Normal IGH Tx, and P53

Renal, anemia, bone

3

2 720

F

77

BJ l

6 g/d

50

Normal IGH Tx, and P53

Bone, plasmacytoma

1

2 500

F

60

IgG k

6549 mg/dL

60

Normal IGH Tx, and P53

HyperCa, anemia, bone

2

36.3

MM_12

F

74

IgA l

2790 mg/dL

19.2

t(4;14): 22%; normal P53

Renal, anemia

2

567

MM_13

M

49

BJ k

1.4 g/d36t(4;14): 38%; normal P53

Renal, bone3

16 500

MM_14

M

56

IgA l

3484 mg/dL

14.4

Normal IGH Tx; del P53: 3%

Anemia, bone

1

128

MM_15

F

70

BJ l

9.0 g/d70

Normal IGH Tx, and P53

Renal, anemia, bone

3

3 100

MM_16

M

69

BJ l

7 g/d

19

Normal IGH Tx, and P53

Plasmacytoma

1

402

MM_17

M

79

IgG k

3603 mg/dL

50

Normal IGH Tx, and P53

Anemia, bone

2

430

MM_18

M

71

BJ l

15 g/d18.8

Normal IGH Tx, and P53

Bone

3

4 110

MM_19

F

62

IgG l 1 BJ l

562 mg/dL

36.8

Normal IGH Tx, and P53

Anemia

1

550

MM_20

F

74

IgG k

3471 mg/dL

25

Normal IGH Tx, and P53

Bone

1

16.8

MM_21

F

68

IgG l

7339 mg/dL

41.6t(4;14): 79%; normal P53

HyperCa, anemia, bone

3

298

MM_22

M

77

IgG k

5669 mg/dL

54.8

Normal IGH Tx, and P53

HyperCa, renal anemia, bone

3

4 050

49.6

BJ, Bence-Jones; CRAB, hypercalcemia, renal insufficiency, anemia, and bone lesions; F, female; FISH, fluorescence in situ hybridization; FLC, free light chain; IgG, immunoglobulin G;
HyperCa, hypercalcemia; ISS, International Staging System; M, male; PCBM, myelomatous plasma cell infiltration in bone marrow; Tx, chromosomal translocation.

Western blotting
Cells were lysed in lysis buffer (Roche), and equal amounts ofprotein were separated on sodium dodecyl sulfate-polyacrylamidegels. The exosomal pellets isolated from equal volumes of culture
medium (5 mL) were lysed in 200 mL of lysis buffer. Equal volumesof lysate (30 mL) were loaded in each lane of the gels. Details
regarding antibodies are given in the supplemental Methods.

Fluorescence microscopy
Immunofluorescence analysis was performed by using antibodies
against a-smooth muscle antibody (SMA; rabbit polyclonal antiaSMA; 1:500; C-28; Santa Cruz Biotechnology), CD63 (mousemonoclonal anti-CD63; 1:100; sc-5275; Santa Cruz Biotechnology), and KDEL (mouse monoclonal anti-KDEL; 1:100; ab12223;
Abcam). Alexa Fluor 488–conjugated anti-mouse immunoglobulin
G goat serum (Molecular Probes, Thermo Fisher Scientific) wasused as secondary antibody. Nuclear staining was conducted byusing 49,6-diamidino-2-phenylindole (Abbott Diagnostics). Samples
were examined under a fluorescence microscope (Biozero BZ8000; Keyence).

In vivo evaluation of a novel, porous
3-dimensional scaffold
Hydroxylapatite-based 3-dimensional porous scaffolds (GC Corporation) were immersed in 35 mL of culture medium with BMSCs
labeled with NEO-STEM (rhodamine B isothiocyanate [RITC],
0.1 mg/mL; Biterials Co., Ltd.) (5 3 105 cells/scaffold) and RPMI
3230

UMEZU et al8226–green fluorescent protein (GFP) (5 3 105 cells per scaffold).
Details are provided in the supplemental Methods.
The mice received weekly intraperitoneal injections of 1 mg/kg
of FTY720. The specimens were harvested 8 weeks later,
frozen in Tissue-Tek OCT (Sakura Finetechnical) compound andsectioned at 8 mm by using a cryostat (Leica). The total GFP1
area (RPMI 8226–GFP) and RITC1 area (NEO-STEM–labeled
BMSCs) were quantified as the mean pixel density obtained fromimage analysis of 6 random microscopic fields by using ImageJsoftware.

Statistical analyses
Data are expressed as mean 6 standard deviation. Differences
between 2 treatment groups were analyzed by using Student t tests(2-tailed, unpaired), and differences among multiple groups wereanalyzed by analysis of variance. All statistical analyses wereconducted by using GraphPad Prism version 5c for Macintosh(GraphPad Inc.). Results were considered statistically significant
when P , .05.

Results
miR-10a expression levels were increased in EVs
derived from MM-BMSCs compared with EVs
from normal-BMSCs
MM-BMSCs were isolated from BM samples from 21 patientswith MM (aged, 43-82 years) (Table 1); the phenotypic characterization
12 NOVEMBER 2019 x VOLUME 3, NUMBER 21

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A

NormalBMSC-EVs

NormalBMSC-EVs

CD81
2
TSG101

miR-135b expression(-'Ct)

miR-377
miR-211
miR-145

*

-5
-10

M MM
SC s
M MM
SC s

cellular-miR-135b
-10

-15

-20

-15
N
B orm
M
SC als

-'Ct

N
B orm
M
SC alsEV-miR-135b0

miR-135b expression(-'Ct)

F

miR-362-3p

-15

-20

N
B orm
M
SC alsmiR-135b

B

N
B orm
M
SC als

M MM
SC s

-10
-15

miR-296-3p

8

-5

B

miR-629

*

0

M MM
SC s

miR-346

cellular-miR-346-10

B

miR-346 expression(-'Ct)

miR-489

EV-miR-346
5

miR-346 expression(-'Ct)EB

MMBMSC-EVs(n=6)
miR-10a

-17

-20

-15
N
B orm
M
SC alsNormalBMSC-EVs
(n=2)

-15

N
B orm
M
SC als

400

M MM
SC s

300

-10

cellular-miR-10a
-10

B

200

Particle size (nm)

-5

M MM
SC s

100

*

B

0

EV-miR-10a0

miR-10a expression(-'Ct)D

1

0

C

MMBMSC-EVs

CD63

miR-10a expression(-'Ct)

Nanoparticle concentration (x 108 particle/ml)

3

B

MMBMSC-EVs

Figure 1. Identification of MM-BMSC–specific EV miRNAs. (A) Representative transmission electron micrographs of EVs derived from normal-BMSCs and MM-BMSCs.
Scale bar, 50 nm. Nanoparticle concentrations and size distributions of EVs derived from normal-BMSCs (blue line) and MM-BMSCs (red line). An average of 2.1 3 105 and2.0 3 105 EVs were recovered from 1 mL of culture supernatant of normal-BMSCs and MM-BMSCs, respectively. (B) Western blotting for CD63, CD81, and TSG101 in EVs
derived from BMSCs. (C) Differential miRNA expression levels in normal-BMSC–EVs (n 5 2) and MM-BMSC–EVs (n 5 6) as a screening cohort. (D-F) EV and cellular miRNA(miR-10a, miR-346, and miR-135b) expression levels in normal-BMSCs (n 5 2) and MM-BMSCs (n 5 6) by quantitative polymerase chain reaction array. Box plot whiskers
represent minimum and maximum values. P values were calculated by using independent-sample Student t tests. *P , .01.

of BMSCs from age-matched healthy donors (normal-BMSCs) and
MM-BMSCs are shown in supplemental Figures 1 and 2. The EV
fraction was isolated from culture supernatants of normal-BMSCs and
MM-BMSCs. The size and number of EVs were similar between
normal-BMSCs and MM-BMSCs (Figure 1A). Common markers of
EVs (CD63, CD81, and TSG101) were detected in EVs from bothnormal-BMSCs and MM-BMSCs by using western blotting(Figure 1B).
EV miRNA profiles were compared by using a screening cohort of
2 normal-BMSCs and 6 MM-BMSCs selected at random from21 MM-BMSCs (National Center for Biotechnology Information

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[NCBI], Gene Expression Omnibus; GSE110271). The 2(-DCt)values of EV miRNAs with a fold change .2 between MM-BMSCs
and normal-BMSCs were ranked; 10 miRNAs were more highly
expressed in MM-BMSC EVs (n 5 6) (Figure 1C). We also
compared the expression patterns of extracellular (including EVs)
and intracellular miRNAs between normal-BMSCs (n 5 2) and MMBMSCs (n 5 6) based on the results of TaqMan miRNA arrays(NCBI, Gene Expression Omnibus; GSE110238). The 10 miRNAs
upregulated in MM-BMSC EVs were subdivided into type 1 andtype 2. Intracellular expression levels of type 1 miRNAs were wellcorrelated for most EV miRNAs; for example, expression levels ofboth cellular and EV miR-296-3p were higher in MM-BMSCs thanin normal-BMSCs (P , .01) (supplemental Figure 3). In contrast,
the type 2 subset of miRNAs showed higher expression in EVs
derived from MM-BMSCs compared with EVs from normalBMSCs, but expression levels of intracellular miRNAs, such as
miR-10a, miR-346, and miR-135b, were low or absent in both
MM-BMSCs and normal-BMSCs (P , .01) (Figure 1D-F).
We determined the expression levels of EV miR-10a, miR-346,
and miR-135b in MM-BMSCs as a validation cohort (n 5 21) byusing quantitative reverse transcription-polymerase chain reaction
and found showed that these EV miRNAs were expressed more
highly in MM-BMSCs compared with normal-BMSCs (P , .001)(supplemental Figure 4A). In contrast, there was no significant
difference in the expression levels of cellular miR-10a, miR-346, ormiR-135b between normal-BMSCs and MM-BMSCs (supplemental Figure 4B).

EV release inhibitors induced the accumulation of
intracellular miR-10a in MM-BMSCs
We determined if blocking the secretion of EVs affected theexpression of cellular miR-10a in MM-BMSCs. EV release wasdetermined by measuring levels of the EV-associated proteins
CD63, CD81, and TSG101 (Figure 2A) and quantification of total
protein (supplemental Table 1). EV markers decreased in bothnormal-BMSCs and MM-BMSCs 48 hours after treatment with 4
types of EV biogenesis/release inhibitors: GW4869 (neutral
sphingomyelinase inhibitor), manumycin A (Ras farnesyltransferase inhibitor), FTY720 (S1P receptor agonist), and JTE-013(S1P2 receptor antagonist).
EV release inhibitors also upregulated intracellular expression
levels of miR-10a, miR-346, and miR-135b in MM-BMSCs, which
exhibited little or no expression of these miRNAs without EV
release inhibition (n 5 6) (P , .01) (Figure 2B). Conversely, nosuch upregulation of intracellular miR-10a, miR-346, and miR135b expression was detected in normal-BMSCs.

EV release inhibitor inhibited cell proliferation andinduced apoptosis in MM-BMSCs
FTY720 reduced the viability of MM-BMSCs (n 5 21) but notnormal-BMSCs (P 5 .0008) (Figure 3A) in a dose- and timedependent manner (supplemental Figure 5C-D). We examined theeffect of FTY720 on apoptosis in MM-BMSCs by measuring
effector caspase activity. Caspase-3/7 expression levels increased
significantly in MM-BMSCs treated with 1 mM of FTY720 comparedwith untreated cells (P , .01) (Figure 3B), whereas no similareffect was observed in normal-BMSCs. No significant increase
in apoptosis was detected at 24 hours after treatment with
1 mM of FTY720 (supplemental Figure 6), as shown by Annexin
V–fluorescein isothiocyanate/propidium iodide flow cytometry; there was an increase, however, in early apoptosis(from 12.5% to 59.6%) after 48 hours in MM-BMSCs but not
in normal-BMSCs (Figure 3C). Furthermore, we performed
similar experiments using drugs that control exosome releaseby a different mechanism of action than FTY720. As a result,
MM-BMSCs treated with GW4869 were able to confirm theinduction of apoptosis, similar to FTY720 treatment (supplemental Figure 7).

Overexpression of miR-10a inhibited cell proliferationand induced apoptosis in MM-BMSCs
To verify if EV miRNAs played a major role in inducing apoptosis in
MM-BMSCs, we transfected normal-BMSCs (n 5 2) and MMBMSCs (n 5 21) with 10 nM miR-10a, miR-346, and miR-135b
mimics (Figure 4A-C). Similar to the results after inhibition of EVs
using FTY720 (Figure 3A), overexpression of miR-10a mimic
inhibited the viability of MM-BMSCs compared with normal-BMSCs(P 5 .0214) (Figure 4D); their viability was unaffected by miR-346
and miR-135b overexpression, however (Figure 4E-F).
Furthermore, caspase-3/7 expression was increased in MMBMSCs overexpressing miR-10a (P , .01) but not in normalBMSCs compared with the negative control (Figure 4G). Bothearly apoptosis (13.5% and 21.4%, respectively) and late
apoptosis (21.2% and 34.6%) were increased at 48 and72 hours in MM-BMSCs overexpressing miR-10a mimic compared with cells overexpressing a negative control miRNA (NegamiR) (Figure 4H). Similar to the results for caspase-3/7,
apoptosis of normal-BMSCs was unaffected by miR-10a overexpression at both 24 and 72 hours (supplemental Figure 8).
We performed additional experiments to obtain direct evidenceof apoptosis induction by FTY720 inhibition of EV release andaccumulation of miR-10a in the BMSCs. Induction of apoptosis in
MM-BMSCs treated with FTY720 was blocked by transfection ofanti–miR-10a inhibitor (supplemental Figure 9).

FTY720 resulted in greater intracellular accumulation of thesemiRNAs than other EV-release inhibitors, including manumycin
A and JTE-013. To confirm the inhibition of EV release by
FTY720, we treated normal-BMSCs and MM-BMSCs with 1 mM
of FTY720 and observed the accumulation of CD63 1 microvesicles in the cytoplasm of these BMSCs after 48 hours(Figure 2C). Cellular miR-10a, miR-346, and miR-135b in MMBMSCs accumulated in a dose- and time-dependent manner
after FYT720 exposure (supplemental Figure 5A-B). Subsequent
EV inhibition experiments were therefore performed by using
FTY720.

To clarify the role of EV-mediated miR-10a secreted from MMBMSCs in the BMME, the EV fraction extracted from conditioned
medium from MM-BMSCs (n 5 21) was added to RPMI 8226
MM cells. The EV fraction enhanced the proliferation of RPMI 8226
cells compared with adding the EV fraction from normal-BMSCs(P , .05) (Figure 5A). The enhanced MM cell proliferation inducedby the MM-BMSC EV fraction was canceled by FTY720 treatment of the MM-BMSCs (P , .01). We also visualized the uptake of3232

12 NOVEMBER 2019 x VOLUME 3, NUMBER 21

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EV-mediated transfer of miR-10a promoted
proliferation of MM cells

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A

CD63

CD63

CD81

CD81

TSG101

TSG101

GW4869Man. AFTY720
JTE-013

–
–
–
–

+
–
–
–

–
+
–
–

–
–
+
–

–
–
–
+

GW4869Man. AFTY720
JTE-013

–
–
–
–

+
–
–
–

cellular-miR-10a

Relative expression level(normalized to RNU6B)

30

cellular-miR-135b
30
*

*

20

*
*

10

0
GW4869Man. AFTY720
JTE-013–––+cellular-miR-346
30

*

20

–
–
+
–

MM-BMSCs

Normal-BMSCs

B

–
+
–
–

*
*

10

20
*

*

10

*
*

0
–
–
–
–

+
–
–
–

–
+
–
–

–
–
+
–

NormalBMSCs
(n=2)

C

–
–
–
+

–
–
–
–

+
–
–
–

–
+
–
–

–
–
+
–

–
–
–
+

0
–
–
–
–

MMBMSCs(n=6)+ – – –
– + – –
– – + –
– – – +
NormalBMSCs
(n=2)

–
–
–
–

–
–
–
–

+ – – –
– + – –
– – + –
– – – +MMBMSCs(n=6)+ – – –
– + – –
– – + –
– – – +
NormalBMSCs
(n=2)

–
–
–
–

+ – – –
– + – –
– – + –
– – – +MMBMSCs(n=6)

MM-BMSCs

control

controlNormal-BMSCs

CD63/KDEL/DAPI

CD63

CD63/KDEL/DAPI

CD63

CD63/KDEL/DAPI

CD63

CD63/KDEL/DAPIFTY720(1 PM)FTY720(1 PM)

CD63

Figure 2. EV release inhibitors GW4869, manumycin A, FTY720, and JTE-013 induced accumulation of intracellular miRNAs in MM-BMSCs. (A) Western blot
analysis of EV markers CD63, CD81, and TSG101 verified that the inhibitors reduced EV release. (B) TaqMan low-density miRNA array analysis of cellular miRNA expression(miR-10a, miR-346, and miR-135b) in normal-BMSCs (n 5 2) and MM-BMSCs (n 5 6) treated with EV release inhibitors. *P , .01 vs control without EV release inhibitors.
(C) Immunofluorescence staining of CD63 (green) in normal-BMSCs and MM-BMSCs to visualize the accumulation of EVs after treatment with FTY720. KDEL (red) and
49,6-diamidino-2-phenylindole (DAPI; blue) were used as cytoplasmic and nuclear counterstains, respectively. Scale bars, 50 nm.

Cy3-labeled miR-10a via EVs into MM cells, and noted dots
indicating Cy3–miR-10a in the cytoplasm of RPMI 8226, KMS-11,
and U266 cells (Figure 5B; supplemental Figure 10A).
To identify the function of EV-transferred miR-10a in MM
cells, 3 MM cell lines were transfected with miR-10a mimic(Figure 5C). We found that the proliferation rates of RPMI
8226, KMS-11, and U266 cells were significantly enhanced at

3 to 7 days after miR-10a overexpression compared with cells
overexpressing Nega-miR (P , .01 and P , .001) (Figure 5D).
Additional experiments were conducted to clarify whether miR-10a
contained in EVs has an important role in MM cell proliferation. First,
EVs were collected from the conditioned medium of MM-BMSCs
transfected with anti–miR-10a inhibitor. Addition of EVs to RPMI
8226 did not induce their proliferation (Figure 5E), suggesting that

12 NOVEMBER 2019 x VOLUME 3, NUMBER 21

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Figure 3. FTY720 inhibited proliferation and induced

B

A

BMSCs (n 5 21) were cultured with or without FTY720 (1 mM).

8(A) Cell viability after 48 hours. Values represent proliferation
rate normalized by control (FTY7201/FTY720–) 6 standard de-Relative caspase 3/7 activity(normalized to cell number)

Cell viability normalized to control(FTY720+/FTY720-)

1.5

apoptosis in MM-BMSCs. Normal-BMSCs (n 5 2) and MM-*

P = 0.0008

1.0

0.5

viation. (B) Caspase-3/7 activity measured after 48 hours. Data

6

represent fold increase compared with control (without FTY720)
6 standard deviation. (C) Rates of apoptosis in normal-BMSCs
and MM-BMSCs treated with 1 mM of FTY720 for 48 hours,

4

determined by Annexin V and propidium iodide (PI) staining
and flow cytometry. *P , .01 vs control (without FTY720).

2

0

0.0
MMBMSCs(n=21)

NormalBMSCs
(n=2)

C

–

FTY720(1 PM)+

Normal-BMSCs

FTY720 –

107

Q2–UL
0.2%

105

105

104

104

103

103

10

1

101

PI

10

FTY720 +

107
106

Q2–LL
85.1%

7

102

Q2–LR
11.8%

103

104

105

Q2–UL
0.2%

106

102
10

1

107

Q2–UR
4.0%

10
10

105

105

104

104

103

103

101

Q2–UL
0.2%

Q2–UR
3.1%

Q2–LL
84.2%

Q2–LR
12.5%

101
7

106

102

+

MMBMSCs(n=21)

MM-BMSCs
Q2–UR
2.9%

106

102

–

Normal
BMSCs
(n=2)

102

103

104

105

106

107

Q2–UL
0.1%

Q2–UR
5.8%

Q2–LL
34.4%

Q2–LR
59.6%

6

Q2–LL
80.5%

101

102

Q2–LR
15.4%

103

104

105

106

107

102
101

101

102

103

104

105

106

107

Annexin V
miR-10a derived from MM-BMSCs was involved in the regulation of MM cell proliferation via EVs. Second, we examined
the involvement of FTY720 pro-apoptotic activity and miR-10a
induction in MM cells. The increase in cellular miR-10a expression, as observed in MM-BMSCs, was not confirmed in MM cells
treated with FTY720 because MM cells and their EVs originally
had low miR-10a expression (supplemental Figure 10B). We
further performed knockdown experiments for EV–miR-10a. EVsderived from MM-BMSCs were collected, and anti–miR-10a
inhibitor was then directly transfected into MM-BMSC–EVs byusing the Exo-fect Exosome Transfection Kit (System Biosciences). Knockdown of miR-10a in EVs derived from MM-BMSCs
abolished the induction of MM cell proliferation (Figure 5F).

3234

12 NOVEMBER 2019 x VOLUME 3, NUMBER 21

UMEZU et al

Target pathways of miR-10a differ between
MM-BMSCs and MM cells
We explored the mechanism by which the accumulation of intracellular miR-10a induced apoptosis in MM-BMSCs (Figure 5D,G-H)
and affected MM cell proliferation (Figure 6D) by examining

-399-

B

Nega-miR
miR-10a mimic

20

20

20

10

15
10
5

0
NormalBMSCs(n=2)

NormalBMSCs
(n=2)

E

P = 0.0214

1.5

0MMBMSCs(n=21)

Cell viability normalized to control(+miR-346/+Nega-miR)

Cell viability normalized to control(+miR-10a/+Nega-miR)

25

Relative expression level(miR-135b/RNU6)

Relative expression level(miR-346/RNU6)

Relative expression level(miR-10a/RNU6B)

40

0

1.0

0.5

0.0

MMBMSCs(n=21)

F

1.5

1.0

0.5

3

24 h
107

Q2–UL
0.2%

Nega-miR

106

2

1

48 h
Q2–UR
3.1%

107
106

PI
MMBMSCs(n=21)

miR-10a

NormalBMSCs
(n=2)

106
105
104

103

103
Q2–LL
84.9%

Q2–LR
11.8%

Q2–UL
0.4%

106

– Nega+ miR-10a

107

104

102
101

101 102 103 104 105 106 107

+
–

72 h
Q2–UR
3.6%

105

107

0

Q2–UL
0.1%

104

101

–
+

MMBMSCs(n=21)

0.5

105

102

+
–

NormalBMSCs
(n=2)

1.0

MMBMSCs(n=21)H*

MMBMSCs(n=21)

0.0
NormalBMSCs(n=2)

MMBMSCs(n=21)

G

NormalBMSCs
(n=2)

1.5

0.0
NormalBMSCs(n=2)

Relative caspase 3/7 activity(normalized to cell number)

Nega-miR
miR-135b mimic

30

60

D

C

Nega-miR
miR-346 mimic

Cell viability normalized to control(+miR-135b/+Nega-miR)

A

Q2–UR
7.0%

106

Q2–LR
13.5%

102
101

Q2–UL
0.9%

Q2–UR
21.2%

106

105

105

104

104

104

10

10

Q2–LL
80.3%

10

2

102.6

Q2–LR
12.3%

104

105

106

107

3

10

2

Q2–LL
83.2%

Q2–LR
13.4%

101 102 103 104 105 106 107
107

105

3

Q2–UR
3.3%

103
Q2–LL
82.9%

101 102 103 104 105 106 107
107

Q2–UL
0.1%

Q2–LL
64.4%

102.6

104

Q2–LR
13.5%

105

106

103

Q2–UL
0.8%

Q2–UR
34.6%

Q2–LL
43.2%

Q2–LR
21.4%

102
107
102.6

104

105

106

107

Annexin V
Figure 4. Overexpression of miR-10a inhibited cell proliferation and induced apoptosis in MM-BMSCs. Expression levels of miR-10a (A), miR-346 (B), and miR135b (C) in BMSCs after transfection with the respective mimics, measured by using quantitative reverse transcription-polymerase chain reaction. Data represent fold increase
compared with control (Nega-miR) 6 standard deviation. Cell viability 72 hours after transfection with miR-10a (D), miR-346 (E), and miR-135b (F) mimics. Values represent
proliferation rates normalized by control (with Nega-miR) 6 standard deviation. (G) Caspase-3/7 activity in normal-BMSCs and MM-BMSCs transfected with 10 nM of miR-10a
mimic for 72 hours. Data represent fold increases compared with control (with Nega-miR) 6 standard deviation. *P , .01 vs control (with Nega-miR). (H) Time course of
apoptosis induced by miR-10a transfection in MM-BMSCs determined by Annexin V and PI staining and flow cytometry.

the global gene expression profiles (NCBI, Gene Expression
Omnibus; GSE108915 and GSE118282) of miR-10a–overexpressing
MM-BMSCs and MM cell lines (RPMI 8226, KMS-11, and U266).

A total of 228 genes were commonly downregulated by miR-10a
overexpression in MM-BMSCs. These downregulated genes
included predicted target genes and experimentally validated

12 NOVEMBER 2019 x VOLUME 3, NUMBER 21

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A

B

*P  0.05

RPMI8226

P  0.01

#

2.5
2.0
1.5
1.0
0.5
0.0

C

D

150

Nega-miR100

50+ – + – + –miR-10a– + – + – +

3

**
**

2
*
1

66

KMS-11

3

**

2

3

5

**
*

1

**

2
*

1

0
1

7

U266

3

0

0
1

U2

11
SKM

RPMI8226

miR-10a mimic

3

5

1

7

Days

Days

3

5

7

Days

R

PM

I8

22

6

0
Nega-miR

Cell number (×1000 cells/Pl)

4

Relative expression level(miR-10a/RNU6B)

Calcein AM/miR-10a/DAPI

MMMMBMSC-EVs BMSCs/FTY720(n=21)-EVs(n=21)

Cell number (×1000 cells/Pl)

NormalBMSC-EVs(n=2)

Cell number (×1000 cells/Pl)

Relative cell proliferationof RPMI8226

P = 0.627

EP  0.005

Relative cell proliferationof RPMI8226

P  0.05
2.5
2.0
1.5
1.0
0.5

N
-B eg
M a-m
SC i
-E RV
N
or A
m nti
al -m
-B iR
M -1
SC 0
-E aV
M
M Ne
-B g
M a-m
SC i
-E RV
A
M nt
M i-m
-B iR
M -1
SC 0
-E aV

0.0

m
or
N

P  0.005

3
2
1
0
+

–

Nega-miR–+

Anti-miR-10a

MMBMSC-EVs(n=21)al

Relative cell proliferationof RPMI8226

F

P = 0.3375

Figure 5. EV miR-10a derived from MM-BMSCs transferred to MM cells and enhanced MM cell proliferation. (A) Viabilities of RPMI 8226 cells cultured with EV
fractions isolated from conditioned medium of normal-BMSCs (normal-BMSC–EVs, n 5 2), MM-BMSCs (MM-BMSC–EVs, n 5 21), or MM-BMSCs treated with 1 mM of
FTY720 (MM-BMSC/FTY720–EVs, n 5 21) for 48 hours. Data represent fold increases compared with normal-BMSC–EVs 6 standard deviation. *Normal-BMSC–EVs vs
MM-BMSC–EVs, P , .05. #MM-BMSC–EVs vs MM-BMSC/FTY720–EVs, P , .01. (B) RPMI 8226 cells treated with EVs directly transfected with Cy3–miR-10a mimic for 48
hours. Nuclear and cytoplasmic staining with DAPI (blue) and calcein AM (green), respectively. Scale bar, 20 mm. (C) Expression levels of miR-10a in RPMI 8226, KMS-11,
and U266 MM cells transfected with negative control-miR or miR-10a mimic for 48 hours, measured by using quantitative reverse transcription-polymerase chain reaction. Data
represent fold increases compared with control (Nega-miR) 6 standard deviation. (D) Numbers of MM cells transfected with miR-10a mimic on days 1, 3, 5, and 7 according
to flow cytometry. Data are mean concentrations of viable cells 6 standard deviation determined in 3 separate experiments. *P , .01, **P , .001 vs control (with Nega-miR).
(E) Viabilities of RPMI 8226 cells cultured with EV fractions isolated from conditioned medium of normal-BMSCs (normal-BMSC–EVs, n 5 2) or MM-BMSCs(MM-BMSC–EVs, n 5 21) transfected with 10 nM of negative control-miR (Nega-miR) or 10 nM anti–miR-10a inhibitor for 48 hours. Data represent fold increases comparedwith Nega-miR–transfected normal-BMSC–EVs 6 standard deviation. (F) Viabilities of RPMI 8226 cells treated with EVs directly transfected with anti–miR-10a inhibitor for 48
hours. Data represent fold increases compared with anti–miR-10a inhibitor-transfected EVs of MM-BMSCs 6 standard deviation.

targets of miR-10a presented in miRTarBase (release 7.0: http://
mirtarbase.mbc.nctu.edu.tw/php/index.php) and TargetScan (Release 7.2: http://www.targetscan.org), including phosphatase andtensin homolog (PTEN), annexin A7 (ANXA7), cyclin-dependent
kinase 6 (CDK6), and transferrin receptor protein 1 (TFRC). The

gene expression profiles for 3 MM cell lines revealed that 50
genes, including CCR3, XCL1, SMAD4, and PRKX, were downregulated by miR-10a overexpression. Functional annotation
analysis using the Database for Annotation, Visualization andIntegrated Discovery (DAVID) tool identified the most-affected3236

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A
• NP-labeled BMSCs• RPMI8226-GFP• 1mg/kg FTY720• weekly i.p. injections• 8 weeks after implantation,
the specimens were
harvested.
Nude mice model

Porous scaffolds
Normal-BMSCs

MM-BMSCs

H&E staining

RPMI8226-GFP

RPMI8226-GFP+
MM-BMSCs

RPMI8226-GFP+
Normal-BMSCs

C

D

E

F

G

H

I

J

K

FTY+

FTY−

B

GFP/RITC/DAPI

L
FTY–

FTY+

RITC positive cells(fold of normal-BMSCs/FTY−)

*

3

*

2
1
0
NormalBMSCs

MMBMSCs

RPMI8226-GFP

RPMI8226-GFP+
MM-BMSCs

RPMI8226-GFP+
Normal-BMSCs

M
GFP positive cells(fold of RPMI8226/FTY−)

FTY–

**

FTY+

**

4
3
2
1
0
NormalBMSCs

MMBMSCs

RPMI8226-GFP

RPMI8226-GFP+
MM-BMSCs

RPMI8226-GFP+
Normal-BMSCs

Figure 6. FTY720 indirectly and directly inhibited the proliferation of MM cells in vivo. (A) Schematic of in vivo 3-dimensional coculture model. Scale bars, 1 mm.
(B-K) Fluorescence microscopic examination of RITC1 cells (nanoparticle-labeled BMSCs; red) and GFP1 cells (RPMI 8226-GFP; green) 8 weeks after implantation of scaffolds impregnated with normal-BMSCs (B,G), MM-BMSCs (C,H), RPMI 8226–GFP (D,I), RPMI 8226–GFP1 MM-BMSCs (E,J), and RPMI 8226–GFP1 normal-BMSCs (F,K).
Nuclear counterstaining was performed by using DAPI (blue). Scale bars, 200 mm. (L) Quantitative data for RITC1 cells in the scaffold determined according to pixel density.
*P , .01 (Student t test). Values are mean 6 standard deviation. (M) Quantitative data for GFP1 cells in the scaffold determined according to pixel density. **P , .001
(Student t test). Values are mean 6 standard deviation. FTY1, mice treated with 1 mg/kg of FTY720; FTY2, mice without FTY720; H&E, hematoxylin and eosin.

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biological pathways of the genes downregulated by overexpression of miR-10a, including cell proliferation and regulation of theapoptotic process (supplemental Tables 2 and 3). Overall, theseresults suggest that miR-10a has 2 functions involving growth
inhibition of MM-BMSCs and tumor progression in MM cells,
reflecting the different targeting mechanisms in MM-BMSCs andMM cells.

FTY720 indirectly and directly inhibited theproliferation of MM cells in vivo
To investigate the potential therapeutic targeting value of EV
secretion by MM-BMSCs, we used an in vivo 3-dimensional
coculture model to reproduce the BMME in patients with MM.
Although this model could not exclude the effect of directinteractions between MM cells and BMSCs, it was neverthelessuseful for evaluating the effects of FTY720 administration. We
subcutaneously implanted a porous scaffold loaded with RPMI
8226–GFP MM cells with or without fluorescent silica nanoparticlelabeled BMSCs into nude mice (Figure 6A). The number of normalBMSCs was unaffected by FTY720 administration at 3 weeks after
implantation (Figure 6B,G-H,L), whereas FTY720 reduced thenumber of MM-BMSCs in the scaffolds (Figure 6C,H,L). Furthermore, the proliferation of RPMI 8226–GFP cells in the scaffolds
was enhanced by coexistence with MM-BMSCs, and this enhanced
proliferation was cancelled by FTY720 (Figure 6D-E,M). In contrast,
there was no significant increase in RPMI 8226–GFP cells in
scaffolds with normal-BMSCs (Figure 6F,K,M). Notably, FTY720
inhibited the MM-BMSC–induced proliferation of RPMI 8226–GFP
tumor cells (Figure 6J,M) but had little effect on the proliferation of
RPMI 8226 cells themselves (Figure 6I,M). We performed a further
experiment using MM-derived cell lines other than RPMI 8226
(KMS-11 and U266) and primary CD1381 myeloma cells. Similar to
the experiment using RPMI 8226 cells, we determined that FTY720
influences the antitumor effect by causing a decrease in the numberof MM-BMSCs (supplemental Figure 11).

Discussion
Specific miRNAs are enriched in EVs in a cell type–dependent
fashion.18,29 However, the mechanisms by which miRNAs are
sorted into EVs and the significance of miRNA transfer to recipient
cells remain largely unclear. miRNAs can be sorted into EVs byvarious process mechanisms. Previous reports showed that a subset of miRNAs is loaded into EVs by a selective mechanism involving
RNA-binding proteins, including AGO2 and Y-box protein 1.29,30
Santangelo et al31 showed that RNA-binding proteins cooperatewith each other during miRNA sorting into EVs via specific binding
to a common extra-seed sequence, called the “EXO motif.” Thecurrent study identified a subset of miRNAs including miR-10a thatwas selectively increased in EVs derived from MM-BMSCs, andoverexpression of miR-10a mimic inhibited the viability of MMBMSCs. We have not yet fully elucidated the mechanism by which
miR-10a is sorted into MM-BMSC–EVs, but our results suggestthat low expression of cellular miR-10a might be important forsurvival of MM-BMSCs.

EV–miR-10a released by MM-BMSCs regulated MM cell proliferation. A single miRNA can coordinate multiple pathways
involving the regulation of multiple genes, and the status of miR10a in tumor progression remains controversial. Upregulation of
miR-10a has been found in several different tumor types, and it has
been shown to enhance migration, invasion, and growth in solid
tumors, including pancreatic cancer,32 lung cancer,33 and thyroid
carcinoma,34 via suppression of HOXA132 and PTEN.33 Similarly,
Ovcharenko et al35 and Bryant et al36 reported that aberrant miR10a overexpression was associated with NPM1 mutation and
MDM4 downregulation in intermediate-risk acute myeloid leukemia.
In contrast to the pro-oncogenic function of miR-10a in acute
myeloid leukemia, negative regulation of the proto-oncogene BCL6
by miR-10a suppressed proliferation and promoted apoptosis ofdiffuse large B-cell lymphoma cells.37 Together, these results showthat miR-10a expression can fine-tune the control of cell proliferation and apoptosis depending on the type of cancer.
The role of miR-10a in MM growth has not yet been reported,
however. The current study therefore examined the global
messenger RNA expression profiles of miR-10a–transfected MM
cell lines, raising the possibility that miR-10a can target various
suppressors of MM progression. Our findings indicate that miR-10a
might act as an “onco-miR” in MM. We also searched miRTarBase
and TargetScan for direct target genes of miR-10a in MM cells.
From the search results, we extracted 4 candidate genes, includingTAK1. The expression of candidate miR-10a target factors in MM
cells overexpressing miR-10a was analyzed by using western
blotting. Of the 4 factors analyzed, SF2 and TRA2B expression was
not significantly different, but interestingly, TAK1 and bTRC
expression was elevated by miR-10a overexpression. Although
not affected by the TAK1 inhibitor, treatment with the bTRC
inhibitor canceled the promotion of cell proliferation by miR-10a
overexpression in MM cells (supplemental Figure 12A).
In contrast, overexpression of miR-10a promoted apoptosis of MMBMSCs. We therefore also analyzed the gene expression profiles of
miR-10a–transfected BMSCs to explore miR-10a–related pathways in MM-BMSCs. The genes that were downregulated inmiR-10a–overexpressing MM-BMSCs, including PTEN, ANXA7,
and CDK6, are all known regulators of the cell cycle andapoptosis. In contrast, cell death was not induced by miR-10a
overexpression in normal-BMSCs. We then searched for directtarget genes of miR-10a in MM-BMSCs by using TargetScan.
Among the genes whose expression was decreased in MM-BMSCs
by miR-10a overexpression, we extracted candidate genes withconserved miR-10a–binding sites. Furthermore, we found that
ST6GALNAC1 was specifically expressed in MM-BMSCs compared with normal-BMSCs among the candidate genes. ST6GALNAC1 is a critical regulator of cell–cell interactions with theextracellular matrix and the function of intracellular molecules. In
addition, apoptosis induction by overexpression of miR-10a in MMBMSCs was attenuated by knockdown of ST6GALNAC1 by smallinterfering RNA (supplemental Figure 12B).

We showed that miR-10a, which negatively affects the survival of
MM-BMSCs, is secreted by EVs and transported into MM cells,
promoting their proliferation. miR-10a–enriched EVs might thus
contribute to the function of MM-BMSCs to create a permissive
microenvironment for MM cell proliferation and survival. Notably,

MM-BMSCs were treated with 4 different EV secretion inhibitors in
the current study. GW4869 is the most widely used pharmacological agent for blocking exosome generation,19,38 and it inhibits theceramide-mediated inward budding of multivesicular bodies andrelease of mature exosomes from multivesicular bodies. FTY720(fingolimod) is an S1P modulator, and FTY720 treatment inhibits

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UMEZU et al-403-

S1P signaling on multivesicular endosomes, which in turn blocks
exosomal multivesicular endosome maturation.39 The EV releaseinhibitory effect of FTY720 was higher than that of the otherinhibitors in the current study, and the resulting EV release inhibition
had the dual effect of inducing apoptosis in MM-BMSCs andinhibiting the support of tumor growth by MM-BMSCs.
Furthermore, FTY720 directly inhibited the proliferation of RPMI
8226 MM cells. FTY720 is currently approved by the US Food andDrug Administration and the European Medicines Agency for thetreatment of multiple sclerosis and has also been shown to induce
apoptosis in various neoplastic cells, including solid tumors,40 acutelymphoblastic leukemia,41 and chronic myelogenous leukemia.42
FTY720 was also shown to inhibit MM cell proliferation by inducing
mitochondria-associated apoptosis.43 Overall, these results suggest that FTY720 might be a candidate anti-MM agent able to exert
synergistic effects by targeting MM cells directly and via the BM
milieu.
This study had some limitations per the small cohort of samples
used. We were unable to determine why BMSCs derived frompatients with MM differed from those from healthy donors. It also
remains unknown if the transformation of MM-BMSCs observed inthis study is unique to MM or is similar among other hematopoietic
malignancies. However, our results indicated that inhibition of EV
release from MM-BMSCs had potentially novel anti-MM activity. To
the best of our knowledge, this study is the first to investigate direct
BMSC-targeting strategies in “soil”-targeted therapy. Although theprecise mechanisms underlying the loading of specific miRNAs,
such as miR-10a, into EVs require further clarification, the current
results provide a preclinical rationale for the therapeutic applicationof FTY720 in MM and shed light on a possible role for EV releaseinhibitors in targeting MM tumor load in the BMME.

Acknowledgments
The authors thank Susan Furness and H. Nikki March, from Edanz
Group (www.edanzediting.com/ac), for editing a draft of themanuscript.
This study was supported in part by the Private University
Strategic Research–Based Support Project (S1511011) and inpart by a grant-in-aid (16K07183 and 18K07244) from the Ministryof Education, Culture, Sports, Science, and Technology and the
Takeda Science Foundation.

Authorship
Contribution: T.U., K.O., and J.H.O. designed the research and wrote
the article; T.U., S.I., and C.K. performed the research and analyzed
data; and S.Y. provided patient samples.
Conflict-of-interest disclosure: K.O. received research support
from Celgene KK, Chugai Pharmaceutical KK, and Janssen Pharma
KK. The remaining authors declare no competing financial interests.
The current affiliation for T.U. is the Department of MolecularPathology, Tokyo Medical University, Tokyo, Japan.
ORCID profiles: T.U., 0000-0002-0123-8921; K.O., 00000003-0845-5758.
Correspondence: Tomohiro Umezu, Tokyo Medical University, 6-7-1 Nishi-shinjuku, Shinjuku, Tokyo 160-0023, Japan;
e-mail: t_umezu@tokyo-med.ac.jp.

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Corrected: Author Correction

ARTICLEhttps://doi.org/10.1038/s41467-019-09710-z

OPEN

1234567890():,;

Lineage-speciﬁc RUNX2 super-enhancer activates
MYC and promotes the development of blastic
plasmacytoid dendritic cell neoplasm
Sho Kubota 1, Kenji Tokunaga2, Tomohiro Umezu 3, Takako Yokomizo-Nakano1, Yuqi Sun1,
Motohiko Oshima4,5, Kar Tong Tan6, Henry Yang6, Akinori Kanai7, Eisaku Iwanaga2, Norio Asou8,
Takahiro Maeda9, Naomi Nakagata10, Atsushi Iwama4,5, Kazuma Ohyashiki3, Motomi Osato6,11,12 &Goro Sashida 1

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive subtype of acuteleukemia, the cell of origin of which is considered to be precursors of plasmacytoid dendritic
cells (pDCs). Since translocation (6;8)(p21;q24) is a recurrent anomaly for BPDCN, wedemonstrate that a pDC-speciﬁc super-enhancer of RUNX2 is associated with the MYC
promoter due to t(6;8). RUNX2 ensures the expression of pDC-signature genes in leukemic
cells, but also confers survival and proliferative properties in BPDCN cells. Furthermore, thepDC-speciﬁc RUNX2 super-enhancer is hijacked to activate MYC in addition to RUNX2
expression, thereby promoting the proliferation of BPDCN. We also demonstrate that thetransduction of MYC and RUNX2 is sufﬁcient to initiate the transformation of BPDCN in micelacking Tet2 and Tp53, providing a model that accurately recapitulates the aggressive humandisease and gives an insight into the molecular mechanisms underlying the pathogenesis of
BPDCN.

1 Laboratory of Transcriptional Regulation in Leukemogenesis, International Research Center for Medical Sciences (IRCMS), Kumamoto University, 2-2-1

Honjo, Chuo Ward, Kumamoto 860-0811, Japan. 2 Department of Hematology, Kumamoto University, 1-1-1 Honjo, Chuo Ward, Kumamoto 860-8556, Japan.

3 Department of Hematology, Tokyo Medical University, 6-7-1 Nishi-Shinjuku, Shinjuku, Tokyo 160-0023, Japan. 4 Department of Cellular and Molecular

Medicine, Chiba University, 1-8-1 Inohana, Chuo Ward, Chiba 260-8670, Japan. 5 Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology
and Regenerative Medicine, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato, Tokyo 108-8639, Japan. 6 Cancer Science
Institute of Singapore, National University of Singapore, Singapore 119077, Singapore. 7 Department of Molecular Oncology, Research Institute for Radiation
Biology and Medicine, Hiroshima University, Hiroshima 739-0046, Japan. 8 Department of Hematology, International Medical Center, Saitama Medical
University, Saitama 350-1298, Japan. 9 Department of General Medicine, Nagasaki University, Graduate School of Biomedical Science, Nagasaki 852-8523,
Japan. 10 Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, 2-2-1 Honjo, Chuo Ward,
Kumamoto 860-0811, Japan. 11 Laboratory of Runx Biology, International Research Center for Medical Sciences (IRCMS), Kumamoto University, 2-2-1 Honjo,
Chuo Ward, Kumamoto 860-0811, Japan. 12 Center for Metabolic Regulation of Healthy Aging (CMHA), Kumamoto University, Chuo Ward, Kumamoto
860-0811, Japan. These authors contributed equally: Sho Kubota, Kenji Tokunaga. Correspondence and requests for materials should be addressed to
M.O. (email: csimo@nus.edu.sg) or to G.S. (email: sashidag@kumamoto-u.ac.jp)
NATURE COMMUNICATIONS | (2019)10:1653 | https://doi.org/10.1038/s41467-019-09710-z | www.nature.com/naturecommunications

-406-

1

ARTICLE

B

NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09710-z

lastic plasmacytoid dendritic cell neoplasm (BPDCN) is arare and aggressive hematological malignancy, characteristic of skin lesions followed by hematopoietic organ
dissemination, and is highly resistant to conventional chemotherapies, resulting in a poor outcome1,2. BPDCN was previously classiﬁed as blastic NK-cell lymphoma or CD4+CD56+hematodermic neoplasm because of the expression of the NK
cell marker, CD56; however, subsequent phenotypic (e.g. BDCA2, IL3-Ra/CD123) and molecular studies identiﬁed plasmacytoid
dendritic cells (pDCs) as a normal cellular counterpart of
BPDCN3. Recent genetic studies identiﬁed the activation of the
RAS signaling pathway, inactivation of tumor suppressor genes(e.g. TP53 and CDKN2A), and loss-of-function mutations in
epigenetic modiﬁers (e.g. TET2)4, which are commonly mutatedin myeloid malignancies5, whereas chromosomal translocation(6;8)(p21;q24) was exclusively observed in patients with
BPDCN6–8.
RUNX2, located on chromosome 6p21, is strongly expressed in
pDCs and is also required for the differentiation and migration of
pDCs through its regulation of the expression of pDC-signature
genes9,10. CD34+stem/progenitor cells in BPDCN patients havebeen shown to have signiﬁcantly higher expression levels of
RUNX2 than acute myeloid leukemia (AML)11. BPDCN cells
were recently reported to harbor super-enhancers of RUNX2 aswell as TCF4, which are known to be critical for the developmentof pDCs12. The TCF4 transcription factor maintains pDC identity
by positively regulating the expression of transcription factors,
including RUNX2, which, in turn, regulate TCF4 during thedifferentiation of pDCs9,13.
Super-enhancers are large clusters of enhancers that are densely occupied by transcription factors and mediators, such as
BRD4, which drive the transcriptional activation of critical genesthat deﬁne cell identity through their regulation of differentiation14. BRD4 is a member of the bromodomain and extraterminal(BET) subfamily, which binds to acetylated histones and facilitates transcriptional activation in normal as well as malignant
cells15,16. BRD4 inhibitors, such as JQ1 and iBET, preferentiallyinhibit the function of super-enhancers, leading to the selective
repression of potent oncogenes in various tumors17–19. The
inhibition of BRD4 was shown to signiﬁcantly abrogate theproliferative capacity of BPDCN, at least in part, due to the
suppression of TCF4 transcription12,20. Thus, these lineagesurvival transcription factors appear to utilize the activation ofsuper-enhancers from precursors of and/or mature pDCs andconfer transformation properties in BPDCN.
The function of MYC, located on chromosome 8q24, is critical
for the development of various tumors21,22, and the expression of
MYC is activated by a gene ampliﬁcation and the disrupted
regulation of tissue-speciﬁc enhancers of MYC (e.g. colon cancer,
T-ALL)23–25. Translocation-induced enhancer hijacking has beenshown to activate the expression of oncogenes including MYC(e.g. IgH-MYC in lymphoma, TCR-LMO2 in T-ALL)15,26. Previous studies reported that t(6;8)(p21;q24) involved adjacentregions to MYC and RUNX2 in the cells of BPDCN patients;7,8,27
however, the biological function of t(6;8) currently remains
unclear. Based on these ﬁndings, we successfully demonstrated
that t(6;8) juxtaposed the MYC and pDC-speciﬁc RUNX2 superenhancer in BPDCN cells. The deletion of the mutant-allele
super-enhancer of RUNX2 signiﬁcantly reduced the expression of
MYC and impaired the proliferative capacity of BPDCN cells,
indicating that the pDC-speciﬁc RUNX2 super-enhancer activates
the transcription of MYC. In addition, we investigated how MYC
and RUNX2 promote the transformation of BPDCN in micelacking Tet2 and Tp53.
We herein demonstrate that the pDC-speciﬁc RUNX2 superenhancer is hijacked to activate expression of MYC via t(6;8) in
2

BPDCN cells, and unveil the molecular mechanisms underlying
the pathogenesis of BPDCN, which originates from a precursor of
pDCs by utilizing a mouse model.
Results
Enhanced expression of MYC in BPDCN cells harboring t(6;8).
Since the super-enhancer of RUNX2 has been detected in BPDCN
cells harboring t(6;8), which involves a region adjacent to
MYC12,27, we initially examined the expression levels of RUNX2
and MYC in leukemic cells harboring t(6;8) in patients and thecell line, CAL-1, which has a loss-of-function mutation in TET2(Supplementary Fig. 1)6. The expression levels of RUNX2 weresigniﬁcantly higher in BPDCN cells than in AML cells and U2OS
and Saos2 osteosarcoma cells as Saos2 has higher expression levelof RUNX2 than normal counterpart cells and promotes the cell
growth28, while the expression levels of RUNX2 were lower in
BPDCN cells than in mature pDCs isolated from healthy donors(Fig. 1a). We found that BPDCN cells had markedly higher MYC
expression levels among these malignant cells, whereas normal
pDCs only negligibly expressed MYC (Fig. 1b). Furthermore,
CAL-1 cells strongly expressed the MYC and RUNX2 proteins(Fig. 1c). Notably, a recent study reported that 22 out of 118
BPDCN patients harbored t(6;8) and enhanced expression of
MYC, compared to BPDCN cells without t(6;8)8. Therefore, theexpression of MYC are increased in BPDCN cells harboring t(6;8).
In order to identify the breakpoint of t(6;8)(p21;q24), we
performed ﬂuorescent in situ hybridization (FISH) utilizing three
distinct probes against either the 5′ region of MYC (8q24), 1.7Mb 3′ downstream region of MYC (8q24), which contains bloodcell-speciﬁc MYC enhancers in normal cells and AML cells24,29,30,
or a coding region of the RUNX2 gene (6p21) in a patient andCAL-1 cells (Fig. 1d). We found an associated signal between
RUNX2 and the 3′ enhancer region of MYC on derivative
chromosome 6 (der(6)), and a single signal of the 5′ region of
MYC on der(8) in BPDCN cells (Fig. 1e, and Supplementary
Fig. 2). Consistent with the ﬁnding of a fusion point between thedownstream region of MYC and upstream region of RUNX2 inother patients (Fig. 1d)7,8, we performed whole-genome sequencing of CAL-1 cells, and identiﬁed a fusion point of chromosometranslocation between chromosome 8:128,817,726 (hg19) in 69
kilobases (kb) downstream of MYC and chromosome 6:
44,657,458 (hg19) (Fig. 1d), which was 58 kb centromeric to along and clustered enhancer of RUNX2 (791 kb upstream of
RUNX2) deﬁned by chromatin immunoprecipitation (ChIP)
sequencing using anti-H3K27ac or anti-BRD4 antibodies(Fig. 1f)12. We analyzed the H3K27ac peak rank order for
super-enhancers using the ROSE algorithm15,16, and conﬁrmed
super-enhancers of RUNX2 and TCF4 in CAL-1 cells (Supplementary Fig. 3), as previously reported12, while we found smalland scattered peaks of H3K27ac and BRD4 in blood cell-speciﬁc
MYC enhancers also known as BENC (blood enhancer cluster;
Supplementary Fig. 4). Murine pDCs contained a long andclustered enhancer of Runx2 (543 kb upstream of Runx2)
equivalent to that in humans, whereas multipotent progenitors(MPPs), common dendritic cell progenitors (CDPs), and conventional dendritic cells (DCs) did not (Supplementary Fig. 5)31,
suggesting that BPDCN cells utilize the super enhancer of RUNX2(seRUNX2 -791kb), which is activated in normal pDCs. In orderto clarify whether the super-enhancer of RUNX2 was physicallyassociated with the MYC promoter on der(8), we performed achromatin conﬁguration capture (3 C) analysis and a target
sequencing to detect the expected chromosomes 6 and 8
regions using CAL-1 cells and Jurkat T-ALL cells lacking t(6;8)(Fig. 1g and Supplementary Fig. 6). The results revealed that the

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a

b

RUNX2

Relative expression(fold increase to HEL)

800
600
400
200
0

0.9

antiRUNX2

0.6

antiMYC

0.3

antiβ-Actin

50

37

M MO HE
O L L
N M
O -1
-M 3
AC
-1
t(6 CAL;8 t(6 ) N 1;8 o.1
)N
o.
2
U
2O
N S S
or ao
m s
al 2
pD
C

H
E
LM L
O -1
-M 3
AC
-1
C
t(6 A
;8 L-1t(6 ) No
;8 .1
)N
o.
U 2
2O
N Sa S
or
m os2
al
pD
C
O

FISH

69 kb

T-ALL MYC super-enhancer

MYC

FAM49B
Der(6)

Ch 6p2144M-45.5M

58 kb

FISH

FISH

PVT1(del)Der(8) Centromere

Telomere
CDC5L

MYC
MYC enhancer (BENC)

69 kb 58 kb
T-ALL MYC super-enhancer

RUNX2 super-enhancer

Centromere

Telomere
FAM49B

SUPT3H RUNX2

PVT1(del)
FISH
RUNX2

Merge

Ch8

Ch6

Der(8)

Der(6)

RUNX2 super-enhancer
and RUNX2 promoter

–ΔCt

3

0

0

No ligation

super-enhancer of RUNX2 was signiﬁcantly associated with thepromoter of MYC and the promoter of RUNX2 in CAL-1 cells,
but not in Jurkat cells showing undetectable RUNX2 protein level(see below). These results indicate that BPDCN cells enhanced
the expression of MYC following a juxtaposition of MYC and thepDC-speciﬁc seRUNX2 (-791kb) due to the translocation.

6

3
at

RUNX2

9

6

Jurk

SUPT3H12

9

Ligation

No ligation

-1

20 kb

***

15

12

at

BRD4
Ceribelli et al. 2016

–ΔCt

H3K27ac[0–10]*

15-1[0–20]

RUNX2 super-enhancer
and MYC promotert

791 kbCALg

hg19: Ch6: 44500 kb–45600 kb

Jurka

Der(6)Ch6

CAL

Der(6)

Der(8)

Ch8

-1

Ch8

Jurk

MYC

CAL

MYC_Enhancer

FISH

-1

Translocated alleles

FISH

SUPT3H RUNX2

CDC5L
RUNX2 super-enhancer

Der(6)

MYC enhancer (BENC)

PVT1

CAL

M

N

O

M

Normal alleles

Der(8)

f

50
50

0

Ch 8q24
128M-131M

e

(kDa)

Jurk
at

Relative expression(fold increase to HEL)

1200

H

1.2

1500

d

c

MYC

EL
M
O
LM
-1
M
3
O
N
O
C
AL MA
C
-1
-1
U
2O
S
Sa
os
2

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Ligation

MYC and RUNX2 collaborate to promote the tumor cell
growth. Based on the enhanced expression of MYC in BPDCN
cells harboring t(6;8), we examined whether MYC and/or RUNX2
promoted the cell growth of BPDCN in vitro. We knocked down
the expression of MYC using two distinct MYC-directed shRNA
vectors in CAL-1 cells and found that both MYC-directed

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Fig. 1 Enhanced expression of MYC in BPDCN cells harboring t(6;8). a Expression levels of RUNX2 mRNA in acute leukemia cell lines (HEL, MOLM13, andMONO-MAC1), CAL-1, bone marrow mononuclear cells harboring t(6;8) isolated from two patients (#1 and #2), and osteosarcoma cell lines (Saos2,
U2OS) examined by quantitative RT-PCR (q-PCR) compared to those in normal pDCs isolated from healthy donors (n = 5). b Expression levels of MYC
mRNA examined by q-PCR in the same cells described in Fig. 1a. c Expression levels of RUNX2 and MYC proteins in acute leukemia cell lines (HEL,
MOLM13, and MONO-MAC1), CAL-1, and two osteosarcoma cell lines (Saos2, U2OS). Levels of β-Actin were used as loading controls. d Maps showing
chromosomal regions of human 8q24 (129M-131M) (black line) and 6p21 (44M-47M) (blue line) (upper panel) and derivative chromosome regions of Der(6) and Der(8) (lower panel). Red line indicates a fusion point of t(6;8)(p21;q24) in CAL-1 cells identiﬁed by whole genome sequencing in this study. Red,
green, and aqua bars indicate the target regions of FISH probes in Fig. 1e. e Association between the 3′ enhancer region of MYC (green signals) and the
RUNX2 gene (aqua signals) observed in Der(6) in a patient (#2) assessed by FISH. Arrows and arrow heads show Der(6) and Der(8), respectively. f CAL-1
cells showing a long and clustered enhancer of RUNX2 (hg19: 44500kb−45600kb) assessed by ChIP sequencing utilizing either an anti-H3K27ac in thisstudy or anti-BRD4 antibody12. g CAL-1 cells showing a signiﬁcant association between the super enhancer of RUNX2 and the promoter of either MYC or
RUNX2 assessed by a 3C-qPCR assay. DNA ligase non-treated samples were used as negative controls. Data are representative of three independent
experiments. a, b, g Bars show the mean±SD, *p < 0.05, and ***p < 0.001 by the Student’s t-test

shRNA-transduced cells had signiﬁcantly lower expression levelsof MYC mRNA and protein than control vector-transduced cells(Fig. 2a, b). Given the potent oncogenic functions of MYC invarious tumors, colony formation capacities were signiﬁcantly
weaker in MYC knocked down (KD) cells than in control vectortransduced cells (Fig. 2c). Thus, MYC functioned as an oncogene
and enhanced the clonogenic capacity of BPDCN cells.
We also transduced two distinct RUNX2-directed shRNA
vectors into CAL-1 cells, and conﬁrmed that the mRNA andprotein expression levels of RUNX2 were signiﬁcantly lower in
RUNX2-directed shRNA-transduced cells than in control vectortransduced cells (Fig. 2d, e). The number of RUNX2-directed
shRNA-transduced cells was signiﬁcantly lower than that ofcontrol vector-transduced cells in an in vitro liquid culture, andcolony formation capacities were weaker in the former than in thelatter (Fig. 2f, g). Consistent with the impaired cell growth
capacities of RUNX2 KD cells, RUNX2 KD cells showedsigniﬁcantly lower BrdU incorporation than did the control cells(Fig. 2h). Furthermore, RUNX2 KD cells had signiﬁcantly more
Annexin V-positive cells than control vector-transduced cells(Fig. 2i), indicating that RUNX2 enhanced proliferative capacities, but also suppressed inappropriate apoptosis in BPDCN
cells. In addition, when we knocked down the expression of both
MYC and RUNX2 in CAL-1 cells, MYC and RUNX2-double KD
cells showing signiﬁcantly weaker clonogenic capacity than either
single KD cells (Fig. 2j). Therefore, MYC and RUNX2 collaborate
to promote the cell growth capacity of BPDCN in vitro.
RUNX2 functions as a lineage-survival factor in BPDCN. Since
RUNX2 is required for the differentiation of pDCs9, in order toclarify the mechanisms by which RUNX2 promotes the cell
growth of BPDCN, we analyzed the gene expression proﬁles of
two distinct RUNX2 KD CAL-1 cells and control CAL-1 cells
using a microarray analysis. These two RUNX2 KD cells showedsigniﬁcant overlapping of genes that were either up- or downregulated in comparison with the control cells (Fig. 3a). Upregulated and down-regulated genes were listed in Supplementary
Data 1. A gene set enrichment analysis (GSEA) revealed that
RUNX2 KD cells showed the negatively enriched expression ofpDC signature genes, which was deﬁned by genes expressed in
pDCs (HLA-DR+CD123+CD11c−) compared with myeloid
dendritic cells (mDCs) (HLA-DR+CD123−CD11c+) in humans(Fig. 3b)32. Quantitative RT-PCR conﬁrmed that RUNX2 KD
cells had signiﬁcantly lower expression levels of pDC-signature
genes, such as TCF4, TLR7, TLR9, and IL-3RA, than control cells(Fig. 3c), indicating that RUNX2 maintained pDC-signature gene
expression in BPDCN cells. Furthermore, RUNX2 KD cells had
signiﬁcantly decreased expression levels of the BPDCNcharacteristic surface markers, the CD56 and CD123 proteins(Fig. 3d), which was accompanied by impaired proliferation and4enhanced apoptosis upon the knockdown of RUNX2 (Fig. 2h, i).
Our results also demonstrated that RUNX2 KD cells had signiﬁcantly decreased expression levels of canonical MYC target
genes (Fig. 3e) accompanied with the decreased levels of MYC
mRNA expression (Fig. 3f), indicating that RUNX2 facilitated theactivation of MYC targets in this context. We found that ectopic
expression of MYC partially rescued the reduced colony formation capacity in RUNX2 KD CAL-1 cells (Supplementary Fig. 7);
which is consistent with the ﬁndings that RUNX2 required MYC
to promote the survival of cancer cells28,33. These results indicatethat RUNX2 ensures the expression of pDC-signature genes, butalso confers survival and proliferative properties in leukemic cells,in part, due to the activated function of MYC.
RUNX2 activates the function of enhancers in BPDCN. Since
the super-enhancer of RUNX2 appeared to promote the development of BPDCN by activating the expression of MYC andRUNX2, we examined whether the BRD4 inhibitor JQ1 abrogatedthe proliferation of CAL-1 cells in vitro. As expected, JQ1-treated
CAL-1 cells had signiﬁcantly lower expression levels of MYC andRUNX2 mRNA than DMSO-treated CAL-1 cells (Fig. 4a). We
also conﬁrmed that JQ1 markedly reduced the expression levels of
the RUNX2 and MYC proteins in CAL-1 cells, while Jurkat TALL cells showed a mild change in MYC protein levels after the
JQ1 treatment (Fig. 4b). Consistent with the reduced expression
levels of these oncogenes, our results revealed that CAL-1 cells
showed higher sensitivity to JQ1 in a dose-dependent manner,
whereas Jurkat cells were resistant to this treatment (Fig. 4c). In
addition, the colony formation capacities of CAL-1 cells wereabolished by the treatment with JQ1 (Fig. 4d). The JQ1 treatment
modestly decreased level of H3K27ac expression in CAL-1 cells(Fig. 4e), and H3K27ac ChIP sequencing revealed that H3K27ac
modiﬁcation levels were reduced at the region of RUNX2 superenhancer after the JQ1 treatment (Fig. 4f). Thus, the inhibition of
BRD4 signiﬁcantly impaired the growth of BPDCN cells following the reduced expression of MYC and RUNX2.
In order to clarify if RUNX2 and MYC were responsible for theformation of BPDCN due to activating the function of enhancers,
we examined whether the ectopic expression of RUNX2 and/or
MYC rescued impaired cell growth after the JQ1 treatment. We
conﬁrmed the successful transduction of RUNX2 and/or MYC in
CAL-1 cells (Fig. 4g). We found that neither RUNX2 alone, MYC
alone, nor RUNX2 plus MYC overexpression promoted signiﬁcant changes in growth capacities without the JQ1 treatment(Fig. 4h). While the ectopic expression of MYC alone causedslight increases in the growth of CAL-1 cells after the JQ1treatment, RUNX2 alone and RUNX2 plus MYC overexpression
signiﬁcantly sustained the numbers of CAL-1 cells after the
JQ1 treatment (Fig. 4h), indicating that RUNX2 plays a pivotal
role in transcriptional networks for survival upon the insult of

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shLuc
MYC/β-Actin 1.00

0.50

50

antiβ-Actin

0.41

antiβ-Actin

R
sh

sh

#1
U

N

X2

X2

Lu

c

0
N

R
sh

#2

#1
N

sh

R

U

U
R
sh

**

120

X2

Lu

X2

N

sh

3
ay
D

2
D

ay

1
ay

c

0

0

10

sh

10

U

2

20

R

4

20

***

30

sh

BrdU positive (%)

6**
Annexin V positive (%)

***

30

shRUNX2 #2

#2

U

U
R

R

i

shRUNX2 #1

D

N

N

X2

sh
L

X2

U

U
R
sh

sh

h

shLuc

8

#1

30
0

37

#2

60

N

N

X2

#1

uc

0

90

uc

0.2

0.28

120

#1

50

RUNX2/β-Actin 1.00

Number of colonies

0.4

150

(kDa)

antiRUNX2

***
***

#2

shRUNX2 #2

shRUNX2 #1

shLuc

0.6

10

YC

f

sh
M

sh
M

sh
M

YC

sh
L

#2

#1

sh
L

sh
M

YC

*

0.8

sh
L

30
0

e

RUNX2

g

**
80
40

sh YC
#
+ RU
sh N 1
M X2
YC #
#1 2

M

sh

N
X2

Lu
sh

R
U
sh

#2

0
c

Number of colonies

60

uc

0

1.0

j

0.42

90

#2

0.2

*
Relative expression

50

120

YC

0.4

d

Fold changes(kDa)

antiMYC

0.6

***
***

150

X2

0.8

uc

Relative expression

1.0

Number of colonies***cshMYC #2b

MYC

shMYC #1

aFig. 2 MYC and RUNX2 collaborate to promote the formation of BPDCN cells. a Expression levels of MYC mRNA in CAL-1 cells transduced with two
distinct MYC-directed shRNA vectors and a control luciferase-directed shRNA vector. b Expression levels of the MYC protein in MYC KD CAL-1 cells.
Levels of β-Actin were used as loading controls. c Impaired colony formation capacities in MYC KD CAL-1 cells from those in control cells (n = 3).
d Expression levels of RUNX2 mRNA in CAL-1 cells transduced with two distinct RUNX2-directed shRNA vectors and a control luciferase-directed shRNA
vector. e Expression levels of the RUNX2 protein in RUNX2 KD CAL-1 cells. Levels of β-Actin were used as loading controls. f Impaired colony formation
capacities in RUNX2 KD CAL-1 cells from those in control cells (n = 3). g Decreased cell growth in RUNX2 KD CAL-1 cells (squares and triangles) underin vitro liquid culture conditions from that in control CAL-1 cells (circles) (n = 3). h Reduced proliferative capacities in RUNX2 KD CAL-1 cells assessed by
BrdU incorporation (n = 3). i Enhanced apoptosis in RUNX2 KD CAL-1 cells over that in control CAL-1 cells (n = 3). j Reduced colony formation capacity in
MYC and RUNX2 double KD CAL-1 cells from those in single KD cells (n = 3). a, c, d, f, g–j Bars show the mean±SD, *p < 0.05, **p < 0.01, and ***p < 0.001
by the Student’s t-test. Data are representative of 2–3 independent experiments

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NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-09710-zabUp-regulated genes
shRUNX2 #1
shRUNX2 #2

159

59

0.4
0.3
0.2
0.1
0

113

Representation factor : 43.3
p <1×10–82

Down-regulated genes
shRUNX2 #1
shRUNX2 #2

9330

GSE29618_PDC_VS_MDC_UP
shLuc versus shRUNX2 #1

NES = 1.989
FDR = 0.000

shLuc versus shRUNX2 #2

NES = 2.195
FDR = 0.000

0.4
0.3
0.2
0.1
0

22

Representation factor : 121.6
p <1×10–57

1.0

*

***
*

*
***

***
*

TCF4

TLR7

TLR9

IL3-RA

*

0.8
0.6
0.4
0.2
0

d

CD123

CD56

***
***

***
***
Relative MFI

0.8
0.6
0.4
0.2
0

e

Day3

Day6

0.8

shLuc
shRUNX2 #1
shRUNX2 #2

0.6

f

0.4

MYC

0.2
0

HALLMARK_MYC_TARGETS_V1
shLuc versus shRUNX2 #1
NES = 2.349
FDR = 0.000

***
***

**
Day3

Day6

shLuc versus shRUNX2 #2
NES = 2.199
FDR = 0.000

0.4
0.3
0.2
0.1
0

*

1.0
0.8
0.6
0.4
0.2
0

sh

0.5
0.4
0.3
0.2
0.1
0

1.0

***
***

s
R hL
U
uc
N
sh
X2
R
#
U
N 1
X2
#2

1.0
Relative MFI

shLuc
shRUNX2 #1
shRUNX2 #2

Relative expression

Relative expressioncFig. 3 RUNX2 functions as a lineage-speciﬁc transcription factor in BPDCN. a Venn diagrams of signiﬁcant overlaps in up-regulated and down-regulated
genes between two distinct RUNX2 KD CAL-1 cells relative to control CAL-1 cells. b GSEA for pDC signature genes, which was deﬁned by up-regulated
genes in normal pDCs versus mDCs (GSE29618), in two RUNX2 KD CAL-1 cells relative to control CAL-1 cells. c q-PCR showing the reduced expression ofpDC signature genes, such as TCF4, TLR7, TLR9, and IL3-RA, in RUNX2 KD CAL-1 cells (gray and black bars). d Decreased mean ﬂuorescence intensities(MFI) of CD56 and CD123/IL3-RA expression in RUNX2 KD CAL-1 cells (gray and black bars) 3 and 6 days after transduction examined by FACS (n = 3).
e GESA for canonical MYC target genes (Hallmark MYC targets V1) in two RUNX2 KD CAL-1 cells relative to control CAL-1 cells. f Expression levels of
MYC mRNA examined by q-PCR in RUNX2 KD CAL-1 cells (gray and black bars) and control CAL-1 cells (opened bar). c, d, f Bars show the mean±SD, *p <0.05,**p < 0.01, and ***p < 0.001 by the Student’s t-test. Data are representative of three independent experiments

BRD4 inhibition. Indeed, RUNX2 ChIP sequencing revealed
that RUNX2 were signiﬁcantly enriched in the RUNX2 superenhancer region showing at least 5 peaks (Fig. 4f), and RUNX2binding sites were markedly overlapped with super-enhancers
rather than conventional enhancers (Supplementary Fig. 8). In
addition, a motif enrichment analysis on DNA sequences of
H3K27ac ChIP sequencing revealed that super-enhancers
enriched the binding sequence of RUNX2 signiﬁcantly morethan conventional enhancers in CAL-1 cells (Supplementary
6

Fig. 9). Thus, RUNX2 and MYC promote the proliferation of
BPDCN, at least in part, due to the RUNX2-dependent direct
activation of super-enhancers, which is reversed by the inhibitionof BRD4.
RUNX2 super-enhancer is hijacked to activate MYC expression. In order to clarify whether the RUNX2 super-enhancer
directly activated the expression of MYC in a cis manner, we

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0.2
0

SO

M

D

0

25

1

0.2
0

SO

M

D

0

25

nM

JQ

DMSO

40
Anti-Histone
H3K27Ac

SO

M

D

50

1

nM

6
3
0

1.00

0.34

Day1

50
50

Day2

Day3

Jurkat

12
9
6
3
0

Day1

Day2

DMSO

0.52

Day3

100 nM JQ1

250 nM JQ1

AntiHistone H3

JQ

0.39

9

(kDa)
15

H3K27Ac/H3 1.00

N.D.

0

1.00

CAL-1

***

20

50

AntiMYC
MYC/β-Actin 0.77 0.48
Antiβ-Actin

1

e
60

(kDa)

RUNX2/β-Actin N.D. N.D.

JQ

nM

DMSO

0.4

d
Number of colonies

AntiRUNX2

0.6

Relative cell numbers

0.4

0.8

CAL-1

12

Relative cell numbers

0.6

1.0

JQ1 250 nM

0.8

Relative expression

Relative expression

*

*

c

CAL-1

DMSO

MYC

RUNX2
1.0

Jurkat

250 nM JQ1b

250 nM JQ1

a

15

2

[0–20]

H3K27ac

AntiRUNX2[0–20]

H3K27ac_JQ1

[0–1]

(kDa)
50

RUNX2/β-Actin 1.00 1.35

50

MYC/β-Actin 1.00 2.02
RUNX2

RUNX2

MYC

Relative cell numbers

Relative cell numbers

80
60
40
20
1

Day1

Day3

Day5

2.0

20
15
10
5
1

37

MYC + RUNX2

JQ1 100 nM

DMSO

Relative cell numbers

Control

0.04 0.52

Antiβ-Actin

RUNX2

h

0.25 0.81

AntiMYCRUNX2
SUPT3H250 nM JQ1Control

Control

DMSO

MYC +
RUNX2

g

791 kbhg19: Ch6: 44500 kb-45600 kb

MYC +
RUNX2

f

Day1

Day3

Day5

JQ1 200 nM

1.5
1.0
0.5
0

Day1

Day3

Day5

Fig. 4 MYC and RUNX2 promote the formation of BPDCN by activating the function of enhancers. a Expression levels of RUNX2 and MYC mRNA examinedby q-PCR in CAL-1 cells 1 day after the 250 nM JQ1 treatment (n = 3). b CAL-1 cells showing reduced levels of the RUNX2 and MYC proteins 1 day after the
250 nM JQ1 treatment. Levels of β-Actin were used as loading controls. c Decreased cell growth capacities in CAL-1 cells after the JQ1 treatment at 100 nM(light blue line) and 250 nM (blue line) from that in cells treated with DMSO (green line), but not in Jurkat cells (n = 3). d Impaired colony formation
capacities of CAL-1 cells treated with 250 nM JQ1 from that in cells treated with DMSO (n = 3). e Decreased level of H3K27ac expression in CAL-1 cells
1 day after the 250 nM JQ1 treatment. f RUNX2 binding to the super-enhancer of RUNX2 that reduced level of H3K27ac post JQ1 treatment in CAL-1 cells
assessed by H3K27ac- and RUNX2-ChIP sequencing. g Successful transduction of RUNX2 and MYC in CAL-1 cells with or without the JQ1 treatment at
250 nM examined by western blotting. Levels of β-Actin were used as loading controls. h RUNX2 alone and RUNX2 plus MYC transductions restoring theimpaired cell growth post JQ1 treatment. Empty controls (red lines), RUNX2 alone (green lines), MYC alone (blue lines), and RUNX2 plus MYC vectors(black lines) were transduced in CAL-1 cells, which were then treated with JQ1 at 100 nM and 200 nM under liquid culture conditions (n = 3). a, c, d, h Bars
show the mean±SD, *p < 0.05 and ***p < 0.001 by the Student’s t-test. Data are representative of 2–3 independent experiments

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Super-enhancer of RUNX2

58 kb

69 kb

Control
Clone 3

(bp)
1000

RUNX2

Clone 2Ch6

Control

791 kbsgRNA #1

sgRNA
#1 + #3

sgRNA#1 + #2

Clone 4bClone 1

a

(bp)
1000

500

500

100

100

Der(8)
sgRNA #1

MYC

sgRNA #2 sgRNA #3

c

Ch6
WT
Ch6 origin
Clone 1
Clone 2104.6 kbp deletion
Ch6 origin

Ch8 origin

Clone 3sgRNA #1 + #3 Clone 4

C

lo

ne

C

lo

***

2
C

lo

ne

3
C

lo

ne

4

**
**

0

**

1
3
2
4
ne one one one
l
l
l
C
C
C

l

ne

1

0.5

tro

ne one one one
l
l
l
lo
C
C
C
C

4

***

**

on

3

*** ***

0

l

2

40

RUNX2
1.0

C

C

1

80

tro

tro

l

0

**

**

on

0.5

**

f

120

C

**

Number of colonies

e

MYC
1.0

on

Relative expressiond111.6 kbp deletion

Relative expression

sgRNA #1 + #2

Ch8 origin

loCFig. 5 RUNX2 super-enhancer-driven MYC expression promotes the formation of BPDCN. a Schematic illustration of the RUNX2 super-enhancers on
chromosome 6 (seRUNX2 (-791kb); a back line) and derivative chromosome 8 (seRUNX2der8; a black and red fused line). sgRNA vectors were directed a
distal region of the super-enhancer (sgRNA #1; chr6: 44,557,984-44,558,006) and two distinct downstream regions of MYC (sgRNA #2;
chr8:128,812,545-128,812,567 and sgRNA #3; chr8:128,805,565-128,805,587). Arrows indicate primers for genomic PCR utilized in Fig. 5b. b Detecting
successful deletions of the seRUNX2der8 by genomic PCR using primers on distinct chromosomes of 6 and 8. c Sequences of seRUNX2der8-deleted clones
elucidated by Sanger sequencing and sizes of deletion in those clones. d Expression levels of MYC mRNA in seRUNX2der8-deleted CAL-1 clones examinedby q-PCR. e Impaired colony formation capacities in seRUNX2der8-deleted CAL-1 clones from those in control cells (n = 3). f Expression levels of RUNX2mRNA in seRUNX2der8-deleted CAL-1 clones examined by q-PCR. d–f Bars show the mean±SD, **p < 0.01 and ***p < 0.001 by the Student’s t-test

attempted to genetically delete the mutant-allele super-enhancer of RUNX2 on der(8) (seRUNX2der8), but not that on
chromosome 6 (seRUNX2 (−791kb)), in BPDCN cells using a
CRISPR-Cas9 system (Figs. 1d and 5a). We generated sgRNACRISPR-Cas9 vectors targeted for the distal region of the
RUNX2 super-enhancer on chromosome 6 (sgRNA #1) and two
distinct downstream regions of the MYC gene on chromosome
8 (sgRNA #2 and sgRNA #3; Fig. 5a), infected vectors into
CAL-1 cells, and then selected single cell clones. We successfully established four distinct clones of CAL-1 cells from two
sgRNA vectors (#1 plus #2, and #1 plus #3), and conﬁrmed adeletion of seRUNX2der8 by ampliﬁcation with genomic PCRutilizing primers beyond the fusion point between chromosomes 6 and 8 (Fig. 5b). The Sanger sequences of PCR products
revealed the expected 104 kb or 111 kb deletion including theseRUNX2der8 in these clones (Fig. 5c). After establishing single
cell clones, we examined the expression levels of MYC using
quantitative PCR, and found that the expression levels of MYC
were signiﬁcantly lower in seRUNX2der8-deleted clones than incontrol-vector transduced cells (Fig. 5d). All seRUNX2-mutdeleted clones showed markedly impaired colony formation
capacities from those in control CAL-1 cells (Fig. 5e). We notedthat these clones showed between 20 to 60% reductions in
RUNX2 expression levels (Fig. 5f), suggesting that MYC positively regulates the expression of RUNX2 in a direct and/or an
indirect manner. While the reduced expression of MYC andRUNX2 may further inhibit colony formation capacities of8these clones, the mutant-allele super-enhancer of RUNX2
directly activates the expression of MYC to promote the formation of BPDCN.
Transduction of MYC promotes the in vitro colony formation.
Since MYC and RUNX2 promote the formation of BPDCN due
to the RUNX2 super-enhancer in vitro, we examined whether
the over-expression of MYC and RUNX2 was sufﬁcient for the
initiation of BPDCN in the absence of Tet2 and Tp53, because
loss-of-function mutations in TET2 and TP53 are the most frequently detected in patients with BPDCN regardless the presenceof t(6;8)4. In order to achieve this, we generated Tet2wt/wt;
Tp53wt/wt;Cre-ERT2 and Tet2ﬂox/ﬂox;Tp53ﬂox/ﬂox;Cre-ERT2 compound mice and deleted Tet2 and Tp53 via an injection oftamoxifen at 8 weeks old, hereafter referred to as wild-type (WT)and Tet2Δ/Δp53Δ/Δ double knockout (DKO) mice, respectively.
We puriﬁed Lineage-Sca-1+c-Kit+ (LSK) hematopoietic stem/
progenitor cells from WT and DKO mice 1 month after thetamoxifen injection and infected them with the MYC-IRES-NGFR
and/or RUNX2-IRES-GFP retrovirus vectors (Fig. 6a). When we
induced the differentiation of LSK cells into CD11b−CD11c+
B220+ pDCs under liquid culture conditions with the Flt3-ligand
and TPO (Fig. 6b)10. MYC plus RUNX2-expressing DKO cells
produced more pDCs than the other cells following an additional
treatment with lipopolysaccharide (LPS), which induced theterminal differentiation of pDCs (Fig. 6c). Furthermore, we found

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a

wt/wt

Tet2

Tet2

fl/fl

;p53

;p53

RUNX2-GFP
MYC-NGFR

wt/wt

fl/fl

;Cre-ERT2;Cre-ERT2Infection

LSK sorting

Tamoxifen

In vitro culture
FLT3-ligand, TPOb

MYC + RUNX2-DKO

Competitive transplantationin lethally-irradiated mice

c

CD11b

WT

In vitro differentiation assay
In vitro colony formation assay

***

32.5%

10.5%

B220

CD11c
69.7%

35.9%

Numbers of pDCs

10,000
8000
6000
4000
2000

O
2DK
O
+
-D RU
KO N
X2
NX

20
O
+
-D RU
KO N
X2

DK

KO

2M

YC

NX

O
DK

-D
RU

YC

N.D.

YC

2NX

RU

M

N.D.

M

N.D.

W
T
+
R
-W U
T NX
2

N.D.

T

N.D.

T

0

W
MYC + RUNX2 - DKO

M
YC

40

YC
MYC + RUNX2 - WT

DKO

RU

60

M
WT

O

80

GFP
Number of colonies

f

DK

e

-W

MYC + RUNX2-DKO

Brightfield

WT

Number of colonies

d

-D
K

CD11c

M
YC

M
YC

-W

W

T

RU
T
NX
M
2YC
W
T
+
-W RU
N
T
X2

0

80
60
40
20
0

N.D.

N.D.

1st

N.D. N.D. N.D.

2nd

N.D. N.D. N.D.

3rd

Fig. 6 Transduction of MYC and RUNX2 promote the production of pDCs with the serial replating property. a Experimental scheme utilizing MYC andRUNX2 vectors and Tet2/p53 DKO mice for in vitro and in vivo assays. b Representative ﬂow cytometric proﬁles of the CD45+CD11b−-gated CD11c+B220+
cells. c Absolute numbers of CD11b−CD11c+B220+ pDCs among 1000 LSK cells-derived CD45+ hematopoietic cells under liquid culture conditions (n =6). d Representative colony images (top columns) and GFP (bottom columns) of WT and DKO cells expressing MYC plus RUNX2 in the ﬁrst plating.
e Increased colony-forming properties of DKO cells expressing MYC and DKO cells expressing MYC plus RUNX2 (n = 3). f Enhanced serial re-plating
properties of DKO cells expressing MYC plus RUNX2 (black bars) from those of WT cells (opened bars), WT cells expressing MYC plus RUNX2 (graybars) and DKO (dark gray bars) (n = 3). c, e, f Bars show the mean±SD, *p < 0.05 and **p < 0.01 by the Student’s t-test. N.D. not detected. Data arerepresentative of 2–4 independent experimentsthat MYC-expressing DKO cells and MYC plus RUNX2-expressing DKO cells showed higher numbers and larger colonies cultured with the Flt3-ligand and TPO (Fig. 6d, e). MYC plus
RUNX2-expressing DKO cells subsequently gained serial replating capacities, whereas the other cells had severely diminishedcolonies (Fig. 6f). Thus, the overexpression of MYC and RUNX2
promoted the production of pDCs and the MYC overexpression
was critical for the colony forming properties of DKO cells under
this in vitro pDC differentiation setting.

MYC and RUNX2 initiates the transformation of BPDCN
in vivo. We next examined the in vivo BPDCN-initiating capacityof MYC plus RUNX2 expressing DKO cells. After a 9-day culture
promoting the differentiation of pDCs, we transplanted empty
vector-transduced WT (WT), MYC- and RUNX2-transduced WT(MYC+RUNX2-WT), empty vector-transduced DKO (DKO),
and MYC- and RUNX2-transduced DKO (MYC+RUNX2-DKO)
CD45.2+ cells into lethally irradiated recipient mice together withwild-type competitor CD45.1+ bone marrow cells (Fig. 6a).

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***

0

0
W
T
+
-W RU
N
T
X2

DK
O
+
-D RU
KO N
X2
M
or
ib
un
d

20

c

***

100

100
75

Percent survival

80
60
40
20
0

MYC + RUNX2-DKO (N = 15)

25

2nd transplantation
MYC + RUNX2-DKO (N = 10)

O
+
-D RU
KO N
X2

2

60

YC

DK

+
-W RU
T NX

T
M

M

YC

W

15
30
45
Days after transplantation

e

***

***

800
Spleen weights (mg)

2

1

f

BM

PB

h

×100

×400

600
400
200

B220+ gated

CD11b

Liver

Bst2

B220

GFP+NGFR+ gatedSpleen

DK
YC
O
+
-D RU
KO N
X2
M

YC

W
T
+
R
-W U
T NX
2

YC
O
+
-D RU
KO N
X2

DK
M

W

YC
M
GFP (RUNX2)

CD45.2+ gated

M

0

0
T
+
-W RU
N
T
X2

Bone marrow cell numbers (× 107)

DKO (N = 8)

50

0

3

NGFR (MYC)

CD11c

While WT, MYC+RUNX2-WT, and DKO mice did not show
signiﬁcant changes in blood counts in peripheral blood (PB)
(Fig. 7a) due to the negligible populating capacities of CD45.2+
cells 3 weeks post-transplantation (Fig. 7b), MYC+RUNX2-DKO
moribund mice showed robust leukocytosis, anemia, andthrombocytopenia (Fig. 7a) following the emergence andexpansion of CD45.2+ cells (Fig. 7b). All MYC+RUNX2-DKO
10

MYC + RUNX2-WT (N = 9)

0

d

g

WT (N = 10)***

CD45.2 frequencies in PB (%)

b

40

4

M
YC

M
YC

W
T
+
-W RU
T NX
2

0

8

W
T
+
-W RU
T NX
2

2

60

***
***

M
YC

4

12
× 104/μL

g/dL

6

80

M
YC

× 104/μL

8

16

DK
O
+
-D RU
KO N
X2
M
or
ib
un
d

**

10

Platelets

DK
O
+
-D RU
KO N
X2
M
or
ib
un
d

Hemoglobin

**

M
YC

White blood cells

M
YC

a

mice died 1 month post-transplantation (Fig. 7c), whereas there
were no lethal hematological malignancies in MYC+RUNX2-WTor DKO mice 6 months post-transplantation. At the time ofkilling, MYC + RUNX2-DKO moribund mice had signiﬁcantly
lower bone marrow cell counts and larger spleens than the othergenotype mice (Fig. 7d, e). A detailed analysis of moribund mice
revealed the expansion of immature leukemic blasts with a high

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Fig. 7 Transduction of MYC and RUNX2 promote the development of BPDCN in vivo. a Complete blood cell counts of WT (n = 10), MYC+RUNX2-WT(n = 9), DKO (n = 8), and MYC+RUNX2-DKO (n = 15) mice 3 weeks after transplantation and moribund MYC+RUNX2-DKO leukemic mice (n = 15) at
the time of killing. b Proportions of CD45.2+ cells in the PB of WT, MYC+RUNX2-WT, DKO, and MYC+RUNX2-DKO mice (n = 8–10) 3 weeks after
transplantation and moribund MYC+RUNX2-DKO leukemic mice (n = 15) at the time of killing. c Signiﬁcantly shorter median survival of primarytransplanted MYC-RUNX2-DKO mice (n = 15) (red line) than other genotype mice (n = 8–10) (25 days versus undetermined, ***p < 0.0001 by the Logrank test) and a shorter median survival of the secondary-transplanted MYC-RUNX2-DKO mice (n = 10; broken red line). d Bone marrow cells counts
isolated from 1 femur and 1 tibia of WT, MYC-RUNX2-WT, and DKO mice 6 months after transplantation (n = 7–8) and MYC-RUNX2-DKO leukemic miceat the time of killing (n = 11). e Spleen weights of WT, MYC-RUNX2-WT, and DKO mice 6 months after transplantation (n = 7–8) and MYC-RUNX2-DKO
leukemic mice at the time of killing (n = 13). f Immature leukemic cells in the BM and the PB of MYC+RUNX2-DKO mouse observed by May-Grüenwald
Giemsa staining. Scar bar, 20 μm. g Representative ﬂow cytometric proﬁles of GFP+NGFR-APC+-gated Bst2+B220+CD11b-CD11c+ leukemic cells in a
MYC+RUNX2-DKO mouse. h Representative histology of the spleen and liver of a MYC+RUNX2-DKO leukemic mouse observed by hematoxylin-eosin
staining. Scar bars, 100 μm or 20 μm. a, b, d, e Bars show the mean±SD, *p < 0.05, **p < 0.01, and ***p < 0.001 by the Student’s t-test. Data are combinedfrom two independent experiments

nucleo-cytoplasmic ratio, dispersed chromatin, and microvacuoles in the PB and BM (Fig. 7f). A FACS analysis showed thatthese leukemic blasts were CD11b−CD11cmid/+B220+Bst2+(Fig. 7g), which was consistent with the murine pDCs
immunophenotype, and also massively inﬁltrated the spleen
and liver tissues (Fig. 7h), whereas the leukemic cells expressed
neither myeloperoxidase nor lysozyme; markers of myeloid cells(Supplementary Fig. 10). MYC+RUNX2-DKO leukemic cells
were transplantable in secondary recipient mice with the same
CD11b−CD11cmid/+B220+Bst2+ immunophenotype, and recipient mice died earlier at 2 weeks post-transplantation (the mediansurvival, 23 days; Fig. 7c) due to severe anemia and thrombocytopenia (Supplementary Fig. 11). These results indicate that theoverexpression of MYC and RUNX2 initiates the transformationof BPDCN in mice lacking Tet2 and p53.
BPDCN activates oncogenes but impedes pDC-signature genes.
In order to elucidate the molecular mechanisms underlying thepathogenesis of MYC+RUNX2-DKO BPDCN, we performed an
RNA sequencing analysis of CD11cmid/+B220+Bst2+ leukemic
cells isolated from distinct primary- and secondary-recipient micein addition to those in LSK Flt3+ lymphoid primed MPPs
(LMPPs), macrophage-dendritic cell progenitors (MDPs),
granulocyte–monocyte progenitors (GMPs), and pDCs isolated
from WT mice. RNA sequencing from MYC+RUNX2-DKO
leukemic cells revealed 1445 up-regulated genes and 1526 downregulated genes from WT pDCs (Fig. 8a and Supplementary
Data 2). In order to determine whether the gene expression
proﬁle of MYC+RUNX2-DKO leukemic cells reﬂected that inhuman BPDCN cells, we utilized published data sets comparing
CAL-1 cells and normal pDCs in humans (Supplementary
Data 2)34, and found that up-regulated and down-regulated genes
both signiﬁcantly overlapped between these comparisons inpatients and mice (Fig. 8a), conﬁrming the relevance of thismouse model for human BPDCN.
A principal component analysis showed that primary andsecondary BPDCN cells clustered together, and these BPDCN
cells were located closer to WT LMPPs and MDPs but apart from
GMPs (Fig. 8b). In addition, a hierarchical clustering analysis
using differentially expressed genes between BPDCN cells andpDCs located BPDCN cells closer to WT LMPPs and MDPs than
GMPs (Fig. 8c), supporting a distinct origin of BPDCN from theleukemic GMPs of AML35. Given the transduction of MYC in
DKO leukemia cells, GSEA showed the positive enrichment ofcanonical MYC target genes in BPDCN cells from that in WT
MDPs or WT pDCs (Fig. 8d and Supplementary Table 1), and agene ontology analysis revealed signiﬁcant enrichments inribosome and translation-related pathways, which may be
activated by MYC. Since human BPDCN cells showed lower
expression levels of pDCs-signature genes than mature pDCs (e.g.

TCF4, TLR7, and TLR9; Supplementary Fig. 12), BPDCN cells
showed the signiﬁcant down-regulation of pDCs-signature genes
expressed in WT pDCs, but the positive enrichment of thesegenes from those in LMPPs, MDPs, and GMPs (Fig. 8e andSupplementary Table 1). Unsupervised GSEA showed thatinterferon α and inﬂammatory responses were signiﬁcantly andnegatively enriched in BPDCN cells, which compromised theimmunological function activated in mature pDCs (Fig. 8f and
Supplementary Table 1). Quantitative RT-PCR conﬁrmed thatthat the expression levels of pDC-signature genes, such as Tcf4,
Tlr7, and Tlr9, were slightly increased in MYC+RUNX2-DKO
leukemic cells and markedly increased in pDCs (Fig. 8g),
indicating that BPDCN blocked terminal differentiation into
pDCs by inhibiting the expression of pDC-signature genes tolower levels in mature pDCs. To examine the in vivo BPDCNinitiating capacity of precursor cells, we transplanted MDPs andpDCs puriﬁed from cultured MYC+RUNX2-DKO cells into
lethally-irradiated recipient mice (Supplementary Fig. 13). While
the pDCs did not repopulate in mice, the MDPs expanded inmice and developed lethal BPDCN with the same phenotype ofCD11b−CD11cmid/+B220+Bst2+ following leukocytosis andthrombocytopenia (Supplementary Fig. 14). All MYC+RUNX2DKO MDPs-transplanted mice died 70 days post-transplantation(median survival: 42 days versus undetermined in MYC+RUNX2-DKO pDCs mice) (Supplementary Fig. 15). Thus,
MDPs were at least one of the cells of origin of BPDCN in thiscontext.
The pDC-speciﬁc RUNX2 super-enhancer appears to play animportant role in the differentiation and function of normal
pDCs via increasing the transcription of RUNX2, but in theleukemic cells harboring t(6;8), the RUNX2 super-enhancer on
the derivative 8 is hijacked to robustly activate the transcription of
MYC, while the BENC enhancer does not appear to activate thatof MYC (Fig. 8h). In addition, RUNX2 transcription factor binds
to and helps the seRUNX2der8 activate the expression of MYC in
BPDCN cells (Fig. 8h). Therefore, the expression of MYC andRUNX2 functions to confer survival and proliferative effects on
leukemic precursors such as MDPs that initiated BPDCN in vivo,
but also impedes terminal differentiation into pDCs to promote
the development of BPDCN.
Discussion
BPDCN is a rare and aggressive hematological malignancy, andshows a poor clinical outcome due to inappropriate chemotherapies because its pathogenesis has not yet been elucidated
in detail. Since t(6;8)(p21;q24) is speciﬁc to BPDCN, using
functional genomic assays combined with a mouse model, we
elucidated the molecular mechanisms responsible for the initiation and progression of BPDCN, which are driven by the MYC
oncogene and pDC-lineage transcription factor RUNX2.

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Fig. 8 BPDCN cells activate MYC-mediated oncogenic pathways but impede the activation of pDC-signature genes. a Venn diagrams of signiﬁcant
overlaps in up-regulated genes and down-regulated genes in comparisons of MYC+RUNX2-DKO leukemic cells with murine pDCs and CAL-1 cells withhuman pDCs examined in GSE6201434. b A principal component analysis based on total gene expression in LMPPs (red dots), MDPs (blue dots), GMPs
(green dots), and pDCs (yellow dots) isolated from wild-type mice and MYC+RUNX2-DKO leukemic cells isolated from two distinct primary mice (pinkdots) and secondary transplanted mice (purple dots). c Hierarchical clustering based on differentially expressed genes between leukemic cells and wildtype pDCs (e.g. Myc, Tcf4, Tlr7, and Tlr9) in the same cells utilized in Fig. 8b. d GSEA plots for canonical MYC target genes comparing MYC+RUNX2-DKO
leukemic cells to MDPs and pDCs isolated from WT mice. Normalized enrichment score (NES) and false discovery rate (FDR) q-values are indicatedin Fig. 8d–f. e GSEA plots for pDC-signature genes, which were deﬁned by the expression levels of genes in wild-type pDCs (≥3-fold change, p <0.001 signiﬁcantly different from the levels of other fractions), comparing MYC+RUNX2-DKO leukemic cells to MDPs and pDCs isolated from WT mice.
f GSEA plots for inﬂammatory response and interferon α response comparing MYC+RUNX2-DKO leukemic cells to WT pDCs. g Quantitative RT-PCR
analysis of the expression of Tcf4, Tlr7, and Tlr9 in LMPPs, MDPs, GMPs, and pDCs isolated from wild-type mice and MYC+RUNX2-DKO leukemic cells(n = 3). Bars show the mean±SD. h Model showing the functions of seRUNX2 (−791 kb) in normal pDCs and seRUNX2 der8 in BPDCN cells

cells29, but also known as the Brd4-mediated enhancers of Myc inmurine AML cells30. However, CAL-1 cells showed scattered
BRD4 bindings in the BENC enhancers and did not change levelsof BRD4 post the JQ1 treatment. Although the BENC enhancer
may be able to increase the transcription of MYC on the
WT allele and that of RUNX2 on the mutant allele in BPDCN
cells, we demonstrated that the speciﬁc deletion of theseRUNX2der8 signiﬁcantly attenuated the transcription of MYC
and resulted in the abolished cell growth capacities in vitro.
Therefore, the RUNX2 super-enhances appear to dominantly
activate the transcription of both MYC and RUNX2 in BPDCN
cells (Fig. 8h).
Super-enhancers are critical for development and tissue
homeostasis because they tightly regulate the expression of
lineage-speciﬁc transcription factors in somatic stem cells, andthe disruption of super enhancer regulation appears to contributeto malignant transformation14. The emergence and activation of
the super-enhancer of RUNX2 (−791 kb) appears to occur during
the differentiation of pDCs with the subsequent induction of
RUNX2 expression, which is critical for the maturation andmigratory function of pDCs9,10. Although BPDCN showed lower
expression levels of RUNX2 than pDCs, BPDCN cells had signiﬁcantly higher expression levels of RUNX2 than other subtypesof AML cells, and the knockdown of RUNX2 signiﬁcantly abrogated proliferative capacity following the attenuation of pDCsignature gene expression in BPDCN cells. In addition, theknockdown of RUNX2 reduced the expression of the BPDCNspeciﬁc markers, CD56 and CD123, and inhibited the expressionof canonical MYC target genes, indicating that RUNX2 executes
the malignant transcriptional program in collaboration with
MYC in BPDCN. TCF4, a master regulator of pDC differentiation, which is known to mutually regulate RUNX2, was recently
identiﬁed as a critical transcription factor for the survival of
BPDCN cells by RNAi screening12. We found that the knockdown of RUNX2 reduced the expression of TCF4 in BPDCN cells
and this was accompanied by enhanced apoptosis. Thus, RUNX2dependent transcriptional programs for the differentiation of
pDCs were maintained in order to provide the survival propertiesof malignant cells, at least in part, due to the regulation of TCF4,
but were also reprogramed to favor the propagation of BPDCN.
Since BPDCN cells have a higher sensitivity to the inhibition of
BRD4, which has been shown to depend on TCF412, we also
demonstrated that RUNX2 is one of the crucial targets in thesetting of BRD4 inhibition in BPDCN cells. The inhibition of
BRD4 signiﬁcantly reduced the expression of RUNX2 and MYC
in BPDCN cells, and the ectopic expression of RUNX2 alone, but
not MYC alone, signiﬁcantly restored impaired cell growth by the
inhibition of BRD4, suggesting that MYC requires BRD4 andother factors to activate the enhancers and promote the cell
growth, consistent with previous ﬁndings that ectopic expressionof MYC did not restore JQ1-mediated impairments in cell growth

in BPDCN and prostate cancer cells12,38. We also found that the
RUNX2-binding DNA motif was dominantly enriched in superenhancers rather than in conventional enhancers in CAL-1 cells.
Furthermore, RUNX2 itself bound to the region of the seRUNX2der8, and the knockdown of RUNX2 signiﬁcantly reduced
H3K27ac modiﬁcation levels at the super-enhancer with subsequent reductions in MYC, suggesting a pioneering role for
RUNX2 in the activation of super-enhancers during the transformation of BPDCN. Thus, RUNX2 plays a dominant role incontrolling transcription networks in BPDCN, which provide thelineage-dependent survival signal and enhance the transformation capability driven by the expression of MYC.
MYC and RUNX transcription factor families both have theability to activate the expression of p19ARF, which stabilizes thep53 protein, and MYC is known to induce apoptosis in p53dependent and independent manners. One decade ago, RUNX2
and MYC were shown to collaborate in order to generate T-cell
lymphoma in a CD2 promoter-dependent manner in mice
because RUNX2 suppressed MYC-dependent apoptosis in lymphoma cells in vivo33. Consistent with this ﬁnding, we demonstrated that the knockdown of RUNX2 enhanced the levels of
apoptosis in CAL-1 BPDCN cells harboring wild-type TP53, andthe double knockdown of RUNX2 and MYC markedly reduced
the clonogenic capacity of CAL-1 cells. In addition, the transduction of RUNX2 and MYC transformed p53 null/Tet2 nullcells, but not wild-type cells into BPDCN in vivo. These results
imply that RUNX2 exerts a survival effect by suppressing MYC/
p53-dependent apoptosis and, in turn, collaborates with MYC in
this speciﬁc type of leukemia.
Since loss-of-function mutations in TET2 and TP53 are frequently detected in BPDCN patients, we revealed that the overexpression of MYC and RUNX2 initiated the transformation of
BPDCN-like disease lacking Tet2 and p53 in mice. Although we
did not detect the expression of CD4, CD56 or CD123 in murine
tumors, which are commonly detected in BPDCN patients, theseleukemic cells showed the murine pDC phenotype of CD11b−CD11cmid/+B220+Bst2+, accompanied by disseminative andtransplantable capacities in secondary recipient mice. Interestingly, we observed that two out of 10 BPDCN mice showed focal
invasions of leukemic cells on the skin and subcutaneous tissues.
Since BPDCN-like tumor cells placed transcription proﬁles closer
to LMPPs and MDPs than to pDCs and GMPs, we found that
MYC- and RUNX2-expressing p53 null/Tet2 null MDPs repopulated in mice and developed lethal BPDCN disease, indicating
leukemic MPDs being one of the cells of origin of BPDCN, whichis also supported by a ﬁnding that MYC- and RUNX2-expressing
p53 null/Tet2 null LSK cells and GMP cells occasionally developed AML in a non-pDC-differentiation setting. Therefore, thismouse model accurately recapitulates the phenotype of aggressive
BPDCN and gives an insight into the molecular mechanisms
underlying the pathogenesis of BPDCN.

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A result of the present study is that translocation juxtaposes thepDCs-speciﬁc RUNX2 super-enhancer with MYC, resulting in thedevelopment of BPDCN, and our mouse model recapitulates
aggressive BPDCN lacking p53 and Tet2 in mice. We also
demonstrated how MYC and RUNX2 collaborate for the initiation and progression of BPDCN by activating the function ofsuper-enhancers that may be reversed through the inhibition of
BRD4, as previously suggested12,20. These results support a
rationale for combined applications of a BRD4 inhibitor withagents targeting MYC and/or RUNX2-dependent pDC-signature
genes in patients (e.g. BCL2)39.
Methods
Cells. CAL-1, HEL, MOLM-13, MONO-MAC-1, and Jurkat cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (FBS) with penicillin/
streptomycin in a humidiﬁed incubator. U2OS and Saos2 cell lines were cultured in
DMEM containing 10% FBS. The CAL-1 cell line was established from a patientwith BPDCN as previously described6, and it is available from the author upon
reasonable request. The CAL-1 was neither authenticated nor tested for mycoplasma contamination. The Jurkat was obtained from RIKEN (Japan), the othercell lines were obtained from ATCC.
BPDCN patient samples. All samples were obtained at Kumamoto University andTokyo Medical University after written consent was obtained from patients. Patientanonymity was ensured, and the study was approved by the Institutional Review
Committees at Kumamoto University (Kumamoto, Japan) and Tokyo Medical
University (Tokyo, Japan).
Virus vectors and infections. MYC was constructed from cDNA encoding human
MYC, and subcloned into the retrovirus vector pGCDNsam IRES-NGFR. RUNX2
was constructed from cDNA encoding human RUNX2 and subcloned into theretrovirus vector pGCDNsam IRES-EGFP. shRNA directed against either MYC or
RUNX2 was expressed using the lentivirus vectors, pCS-H1-shRNA-EF-1-EGFP
and pCS-H1-shRNA-EF-1-mRFP40. Target sequences were designated as follows:
shRUNX2 #1; 5′-AAGGTTCAACGATCTGATTTG-3′, shRUNX2 #2; 5′-ACAA
GGACAGAGTCAGATT-3′41,42, shMYC #1; 5′-CGATTCCTTCTAACAGA
AATG-3′, shMYC #2; 5′-GATGAGGAAGAAATCGATG-3′12. To produce therecombinant virus, virus vectors was transfected together with packaging vectors
into 293T cells. Hematopoietic cells were then spinoculated with virus supernatantin 10 μg/ml protamine sulfate for 30 min, and infected cells were further
incubated43.
Mice. All mice were in the C57BL/6 background. Tp53 conditional KO (p53ﬂox/ﬂox) mice were previously generated44. Tet2 conditional knockout (Tet2ﬂox/ﬂox)
mice were previously generated45. These mice were crossed with Rosa26:Cre-ERT2

mice (Taconic) for conditional deletion. C57BL/6 mice congenic for the Ly5 locus(CD45.1) were purchased from Sankyo-Lab Service. All experiments using thesemice were performed in accordance with our institutional guidelines for the use oflaboratory animals and approved by the Review Board for Animal Experiments ofKumamoto University (Kumamoto, Japan). All mice experiments were performed
without randomization and blinding.
pDCs differentiation and transplantation assays. Puriﬁed murine hematopoietic
stem/progenitor cells (Lineage−Sca-1+c−Kit+) were cultured in RPMI 1640 supplemented with 10% FBS, 50 μM 2-mercaptoethanol, 20 ng/ml human TPO, and
100 ng/ml Flt3-ligand for 9 days10. On day 3 of the in vitro culture, 3000 puriﬁed
GFP+NGFR+ virus vector-infected MDP cells (Lineage-CD11c-c-Kit+CD115+CD135+) were injected into lethally irradiated CD45.1+ mice (8–12 weeks old)
together with 2×105 CD45.1+ bone marrow cells. On day 9 of the in vitro culture,
6000 puriﬁed GFP+NGFR+ virus vector-infected cells were injected into lethally
irradiated CD45.1+ mice together with 2×105 radioprotective CD45.1+ bonemarrow cells. Colony formation assays were performed using Methocult M3234(Stem Cell Technologies) supplemented with 20 ng/ml human TPO and 100 ng/ml
Flt3-ligand with 1000 puriﬁed GFP+NGFR+c-Kit+ cells. In replating assays,colonies were counted on day 14 and 3×104 pooled cells were plated into the same
medium. Terminal pDC differentiation was induced by the administration of 1 ng/
ml LPS for one day on day 9 of the culture with TPO and Flt3-ligand. In transplantation assays, 3000 puriﬁed GFP+NGFR+ virus vector-infected pDC cells(CD11b−CD11c+B220+) were injected into lethally irradiated CD45.1+ mice
together with 2×105 CD45.1+ bone marrow cells, and blood was analyzed using a
Celtac-α (NIHON KODEN). Mice were excluded from the analysis if blood cells
were not recovered 2 weeks post transplantation due to injection failure.
Flow cytometry and immunohistochemistry antibodies. Flow cytometry and cellsorting were performed by utilizing the following anti-human antibodies (clone
14

and catalog numbers): CD271 (ME20.4, 345108), CD45 (HI30, 25-0459), CD4(OKT4, 50-0048), CD56 (MY31, 60-0564), CD123 (6H6, 306011), and TCL1 (1-21,
330506), as well as the following anti-murine antibodies (clone and catalog
numbers): CD45.2 (104, 109820), CD45.1 (A20, 110730), Gr1 (RB6-8C5, 108404),
CD11b/Mac1 (M1/70, 101208), CD11c (N418, 117304), NK1.1 (PK136, 108704),
Ter119 (116204), CD127/IL-7Rα (A7R34, 121104), Bst2 (927, 127010), B220 (RA36B2, 103212), CD3e (145-2C11, 100320), CD4 (L3T4, 100526), CD8α (53-6.7,
100714), CD56 (809220, FAB7820A), CD117/c-Kit (2B8, 105812), Sca1 (D7,108114), CD34 (MEC14.7, 11-0341-85), CD123 (5B11, 106005), CD135 (A2F10,135306), CD115 (AFS98, 135510), and FcγRII-III (93, 101308). These antibodies
were purchased from BioLegend, eBioscience, R&D systems or TONBO biosciences. The lineage mixture solution contained biotin-conjugated anti-Gr1, B220,
CD4, CD8α, Ter119, and IL-7Rα antibodies. Apoptotic cells were stained with ananti-Annexin V–APC antibody (BD 550474) and propidium iodide. In order toevaluate cell cycle progression, cells were cultured with BrdU, ﬁxed and permeabilized according to the manufacturer’s instructions, and then detected using theanti-BrdU-APC antibody (BD). All ﬂow cytometric analyses and cell sorting were
performed on FACSAriaII or FACSCantoII (BD 557892). Formalin ﬁxed parafﬁnembedded bone marrow cells were stained with anti-MPO antibody (DAKO
A0398) or anti-Lysozyme antibody (DAKO L1828).
Chromatin IP sequencing. CAL-1 cells were ﬁxed by 0.5% paraformaldehyde at
37 °C for 2 min and then lysed. Cells were sonicated at 50% amplitude for 10 s 15
times. Samples were incubated with anti-RUNX2 antibody- (MBL; 8G5) or antiH3K27Ac antibody-conjugated (Active motif; MABI 0309) Sheep anti-Rabbit IgG
Dynabeads at 4 °C overnight. Input and immunoprecipitates were incubated at 65 °C for 4 h for reverse cross-linking, and DNA was puriﬁed using the MinElute PCR
Puriﬁcation Kit (Qiagen). ChIP-seq libraries were generated using the ThruPLEXDNA-seq kit (Rubicon Genomics). Bowtie2 (version 2.2.6;default parameters) wasused to map the reads to the reference genome (UCSC/hg19). HOMER (version
4.9) was used for de novo motif discovery in the peaks of super enhancers andenhancers. ChIP-sequencing data were deposited in the DDBJ database under theaccession number DRA006440 and DRA007469.
Next-generation sequencing. Next-generation sequencing was performed on
CAL-1 cells using the Illumina HiSeq X Ten platform with a 150-bp paired-end
read protocol according to the manufacturer’s instructions. DNA-seq paired reads
were aligned towards the UCSC hg19 reference genome using BWA-MEM. Genefusions was detected by utilization of both soft clipping mapping and fragment
information derived based on pair-end reads. Estimation of breakpoints for a genefusion was performed after pileup of soft clipped reads for both sides. Genome
sequencing data were deposited in the DDBJ database under the accession number
DRA006440.
RNA sequencing. Total RNA was extracted using an RNeasy Plus Micro kit(Qiagen), and cDNA was synthesized using the SMARTer Pico PCR cDNA
Synthesis kit (Clontech). ds-cDNA was fragmented and cDNA libraries weregenerated using the KAPA HyperPlus Library Preparation kit (KAPA Biosystems)
and FastGene Adaptor kit (Fastgene). Sequencing was performed using NextSeq500 (Illumina) with a single-read sequencing length of 60 bp, and the CLC
genomic Workbench was used to analyze and visualize sequencing data. RNA
sequencing data have been deposited in the DDBJ database under the accession
number DRA006565.
Microarray. Twenty nanograms of total RNA was mixed with spike-in controls
using the Agilent One Color Spike Mix Kit (Agilent), ampliﬁed, and labeled with
Cyanine 3 using the Low Input Quick Amp Labeling Kit (Agilent) according to the
manufacturer’s instructions. A microarray analysis using the SurePrint G3 Human
GE 8 × 60 K Microarray (Agilent) was performed according to the manufacturer’s
instructions. Microarray raw data were deposited in Gene Expression Omnibus
under the accession number GSE110140.
Chromosome conformation capture (3C)-quantitative PCR. Cells were ﬁxed by
1% formaldehyde at 25 °C for 10 min. Nuclei samples were digested with EcoRI at
37 °C 16 h and then ligated using T4 DNA ligase (NEB)46. Quantitative PCRprimers were designed to detect the closest EcoRI target sites of the promoters andenhancers of RUNX2 and MYC (Supplementary Table 2).
CRISPR. In order to delete the RUNX2 super enhancer from the translocated allele
using the CRISPR-Cas9 system, the target sequence of gRNA was designed using
sgRNA Designer47 and CRISPR direct (Supplementary Table 2)48. Designed
gRNAs were subcloned into the lentiCRISPRv2 vector49, and transfected into
293T cells together with packaging plasmids. CAL-1 cells were infected using a
sgRNA lentivirus and cultured in RPMI 1640 medium supplemented with 1 μg/ml
puromycin in order to select sgRNA-vector-transduced clones. Uncropped gel
images are available in Supplementary Fig. 16.

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FISH. CAL-1 cells were treated with 0.05 µg/ml colcemid solution for 3 h and 75
mM KCl for 60 minutes, and were then ﬁxed in 3:1 methanol:acetic acid solution.
MYC and the enhancer region of MYC were detected using Vysis LSI-MYC dual
color probes (Abbott Laboratories). The RUNX2 probe was generated by thebacterial artiﬁcial chromosome clone RP11-1019C24 (Empire Genomics) andlabeled with DY-415-dUTP: aqua dye (Enzo Life Sciences). Probe-hybridized slides
were washed, and cells were counterstained with 4′,6-diamidino-2-phenylindole(DAPI) and then analyzed using the BZ8000 System (Keyence).
Quantitative RT-PCR and western blotting. Total RNA was isolated using the
RNeasy Mini kit and then reverse-transcribed by the ThermoScript RT-PCR system (Invitrogen) with an oligo-dT primer. Quantitative-RT-PCR was performed
with LightCycler 480 (Roche) and SYBR Premix ExTaq (Takara). Expression levels
were normalized to ACTB/β-actin expression. Primers for PCR are listed in Supplementary Table 2. The following antibodies were used for Western blotting:
RUNX2 (MBL 8G5; 1:200 dilution), Actin (Santa Cruz C4; 1:1000 dilution), MYC(Santa Cruz 9E10; 1:100 dilution), H3 (Abcam ab1791; 1:500 dilution), andH3K27ac (Active motif MABI 0309; 1:1000 dilution). Uncropped immunoblot
images are available in Supplementary Fig. 16.
Statistical analysis. All statistical tests were performed using Graph Pad Prism
version 7 (GraphPad Software). The signiﬁcance of differences was measured by an
unpaired two-tailed Student’s t-test or Mann–Whitney non-parametric test. A P
value of <0.05 was considered signiﬁcant. No statistical methods were used topredetermine sample size for animal studies.
Reporting Summary. Further information on experimental design is available in
the Nature Research Reporting Summary linked to this article.

Data availability
All data are available within the article and Supplementary Information ﬁles. Sequencing
data that support the ﬁndings of this study have been deposited in DDBJ or GEO under
the accession numbers below. Whole-genome sequencing; DRA006440. Microarray;
GSE110140. ChIP-sequencing; DRA006440 and DRA007469. RNA-sequencing;
DRA006565

Received: 17 April 2018 Accepted: 26 March 2019

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Acknowledgements
We thank the members of the Sashida and Osato Laboratories for their discussions
during the preparation of this manuscript and Ms. Mihoko Iimori, Ms. Noriko Yamamoto, Dr. Hirotaka Matsui, Mr. Shinji Kudoh, and Dr. Takaaki Ito for their technicalhelp. This work was supported in part by a grant from the Takeda Science Foundation (S.
K.), the Uehara Memorial Foundation (G.S.), Japanese Society of Hematology (M.O., G.
S.), Grants-in-Aid for Scientiﬁc Research (16KT0113, 18H02842, 15H04312, 16H06276,
18KT0026, and 18K16090) and Strategic Research Foundation for Private Universities(S1511011) from the Ministry of Education, Culture, Sports, Science and Technology(MEXT) of Japan, and NCIS Centre Grant Seed Funding Program, Singapore Ministry of
Education’s AcRF Tier 3 grant (MOE2014-T3-1-006).

Author contributions
S.K. performed experiments, analyzed results, and wrote the manuscript; K.T. performedexperiments and provided reagents; T.U., T.YN. and Y.S. performed experiments; M.O.,
K.T.T., H.Y., A.K. and E.I. analyzed results; N.A., T.M. and N.N. provided reagents; A.I.
analyzed results; K.O. provided reagents; M.O. designed the research and analyzed results; G.S.
designed the research, performed experiments, analyzed results, and wrote the manuscript.

16

Additional information
Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467019-09710-z.
Competing interests: The authors declare no competing interests.
Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/
Journal peer review information: Nature Communications thanks the anonymous
reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports
are available.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional afﬁliations.

Open Access This article is licensed under a Creative Commons
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© The Author(s) 2019

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ONCOLOGY LETTERS 19: 2053-2061, 2020

Downregulation of extracellular vesicle microRNA‑101
derived from bone marrow mesenchymal stromal cells in
myelodysplastic syndrome with disease progression
YUU SAITOH1,2*, TOMOHIRO UMEZU1,3*, SATOSHI IMANISHI4, MICHIYO ASANO1, SEIICHIRO YOSHIZAWA1,
SEIICHIRO KATAGIRI1, TAMIKO SUGURO1, HIROAKI FUJIMOTO1, DAIGO AKAHANE1,CHIAKI KOBAYASHI-KAWANA1, JUNKO H. OHYASHIKI4,5 and KAZUMA OHYASHIKI1,5
1

Department of Hematology, Tokyo Medical University, Tokyo 160-8402; 2Department of Hematology, Shizuoka General
Hospital, Shizuoka 420-8527; 3Department of Molecular Pathology, Tokyo Medical University, Tokyo 160-8402;
4
Institute of Medical Sciences; 5Department of Advanced Cellular Therapy, Tokyo Medical University, Tokyo 160‑0023, Japan
Received July 4, 2019; Accepted November 11, 2019
DOI: 10.3892/ol.2020.11282
Abstract. To evaluate the mechanism underlying the communication between myeloid malignant and bone marrow (BM)microenvironment cells in disease progression, the current
study established BM mesenchymal stromal cells (MSCs)
and assessed extracellular vesicle (EV) microRNA (miR)
expression in 22 patients with myelodysplastic syndrome(MDS) and 7 patients with acute myeloid leukemia andmyelodysplasia-related changes (AML/MRC). Patientswith MDS were separated into two categories based on therevised International Prognostic Scoring System (IPSS-R),
and EV-miR expression in BM-MSCs was evaluated using a
TaqMan low-density array. The selected miRs were evaluatedusing reverse transcription-quantitative PCR. The current
study demonstrated that the expression of BM-MSC-derived
EV-miR was heterogenous and based on MDS severity, theexpression of EV-miR-101 was lower in high-risk group andpatients with AML/MRC compared with the control andlow-risk groups. This reversibly correlated with BM blast
percentage, with which the cellular miR-101 from BM-MSCs

Correspondence to: Professor Kazuma Ohyashiki, Departmentof Advanced Cellular Therapy, Tokyo Medical University,
6‑7‑1 Nishishinjuku, Shinjuku‑ku, Tokyo 160‑0023, Japan
E-mail: ohyashik@rr.iij4u.or.jp*

Contributed equally

Abbreviations: BM, bone marrow; BM-MSC, bone marrowmesenchymal stromal cell; miR, microRNA; MDS, myelodysplastic
syndrome; AML, acute myeloid leukemia; AML/MRC, acute
myeloid leukemia with myelodysplasia-related changes; TLDA,
TaqMan‑low density array; CAF, cancer‑associated fibroblast;
IPSS-R, revised International Prognostic Scoring System

Key words: myelodysplastic syndrome, bone marrow mesenchymal
stroma cells, extracellular vesicle, microRNA-101, disease progression

or serum EV-miR-101 expression exhibited no association.
Database analyses indicated that miR-101 negatively regulated cell proliferation and epigenetic gene expression. The
downregulation of BM-MSC-derived EV-miR-101 may be
associated with cell-to-cell communication and may accelerate the malignant process in MDS cells.
Introduction
The hematopoietic microenvironment comprises a mixtureof bone marrow mesenchymal stromal cells (BM-MSCs),
fibroblasts, vascular endothelial cells, etc. BM‑MSCs support
the proliferation and differentiation of hematopoietic stem
cells, and themselves undergo differentiation into various
functional cells, including adipocytes, osteocytes, and cells
from the chondrogenic lineage (1,2). Studies on hematologic
malignancies have demonstrated genetic abnormalities inneoplastic cells; however, the role of the hematopoietic microenvironment in the pathogenesis of human myeloid neoplasia
is still unclear, therefore we attempted to assess BM-MSCs.
Considering the anatomical architecture, BM-MSCs arecomponents of the hematopoietic niche, and hematopoietic
cells lie in close proximity to the bone endosteal surface,which is in direct contact with endothelial cells (1) and hence
linked to drug sensitivity (3). BM-MSCs also act as immunosuppressive agents by inhibiting the proliferation of immune
cells (4,5). Therefore, BM-MSCs may have an important effecton adjacent hematopoietic cells and immune control system inhematologic neoplasia.
Recent studies have demonstrated that neoplastic cells shed
extracellular vesicles (EVs), including exosomes, and endow
BM-MSCs to facilitate progression of neoplastic cells (6,7).
Exosomes are EVs, about 50-150 nm in size, and containgenetic elements such as messenger RNA, microRNA (miR),
and DNA (8-10). EVs act as cargos involved in cell-to-cellcommunication and are associated with the transformation of
adjacent fibroblasts into cancer‑associated fibroblasts (CAFs),
which are involved in cancer progression (11,12). Tumor
cell-derived EVs, including exosomes, also act as angiogenesis

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SAITOH et al: EV-miR‑101 IN BM‑MSC DERIVED FROM MYELODYSPLASTIC SYNDROMEA

agents in the cancer microenvironment (10). Thus, certain
types of tumors have the ability to reconstruct the surrounding
environment to facilitate the progression of neoplastic cells.
On the contrary, BM-MSCs affect functions of neoplastic cells
associated with therapy resistance and disease progression (4).
Therefore, cell-to-cell communication may exist between
tumor cells and the surrounding environment, including fibroblasts, microvessels, and other adjacent tissues.
miRs are very short non-coding RNAs around 20 bp inlength and are detected in various cell types and body fluids,
including serum, urine, and saliva (9,10). miRs are consideredas the candidates of liquid biopsy. In the body fluids, miRs areusually encapsulated in EVs in the stable forms and are transported to other cells, wherein they control cellular functions.
As BM-MSCs are major players in the hematopoietic tissue andtheir genetic alterations in myelodysplastic syndrome (MDS)
is plausible, we attempted to search EV-miRs in the BM-MSCs
obtained from patients with MDS and acute myeloid leukemiawith myelodysplasia-related changes (AML/MRC). We foundthat the expression of EV-miR-101 in BM-MSCs was downregulated and related to MDS progression.
Materials and methods
Patients. Twenty-nine consecutive patients with myeloid
malignancies (22 with MDS and 7 with AML/MRC)
were enrolled in this study (Table I). The diagnoses were
established as per the World Health Organization criteria,
and included three patients with MDS with single lineage
dysplasia (SLD), seven with multilineage dysplasia (MLD),
two with unclassifiable MDS, one with 5q-syndrome, fivewith excess blast (EB)-1, and four with EB-2. Risk analysis
was carried out based on the revised International Prognostic
Scoring System (IPSS-R) (13) and risk categorization was
performed as per the NCCN guideline (14). Four patientswith MDS with intermediate risk were tentatively includedin the low-risk MDS group. Written informed consents wereobtained from all patients before the collection of specimens
according to the Declaration of Helsinki. The present study
was validated by the internal review boards of Tokyo Medical
University (no. 2648, approved 22 April, 2014). As controls,
human BM-MSCs from healthy donors (normal BM-MSCs)
were purchased from Lonza Inc. Control serum was obtained
from age-matched healthy individuals.
Culture of BM‑MSCs. BM-MSCs from patients with MDS and
AML/MRC were isolated using the conventional plastic adhesion method with a minor modification (15). Briefly, 0.5 to 1 mlof freshy obtained heparinized BM aspirates were cultured
in equivalent volumes of Roswell Park Memorial Institute(RPMI)‑1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1%
penicillin‑streptomycin (P/S; Thermo Fisher Scientific, Inc.)and Dulbecco's modified Eagle's medium (DMEM) (Thermo
Fisher Scientific) containing 10% FBS (GE Healthcare), 1%
of P/S, and 1% non-essential amino acids (NEAAs; Thermo
Fisher Scientific, Inc.). Cells were cultured for 3 to 5 days, andthe medium was changed to DMEM (with 10% of FBS, 1% of
P/S and 1% of NEAAs) for non-hematopoietic expansion after
the removal of non-adherent cells. Cultured BM-MSC popula-tion was identified as CD34 −/CD45 −/CD73+/CD90+/CD105+
with flow cytometry with <5% CD34+ and CD45+ (15).
Isolation and miR profiling of BM‑MSCs. BM-MSCs(4x104 cells/cm 2) were cultured in 5 ml of DMEM and theculture supernatants were harvested after 48 h of incubation. EV fraction was purified with Exoquick-TC regent(System Biosciences) according to the manufacturer's instructions. EVs were quantitated with a nanoparticle tracking
analysis (NanoSight LM10; Malven) (16-18). Isolation of
BM-MSC-derived EV-miRs or cellular-miR and serum
EV-miR was performed using the miRNeasy kit (Qiagen).
Two-hundred microliters of EV were diluted with 700 µl of
QIAzol (Qiagen). After 5 min incubation, 1 nM ath-miR-159(Hokkaido System Science) was added [18]; the mixture
was vortexed for 30 sec and incubated on ice for 10 min.
Phenol extraction and cartridge filtration were subsequently
performed according to the manufacture's instruction.
Screening of candidate miRNA. miRNA profiling was
carried out using a TaqMan low-density miRNA array(TLDA; Thermo Fisher Scientific, Inc.), as previously
reported (16,17). PCR was done on a Applied Biosystem 7900
HT thermocycler (Carlsberg) according to the manufacturer's
recommended program (the reaction was first incubated at
95˚C for 2 min, followed by 50 cycles of 95˚C for 15 sec
and 60˚C for 1 min) using SDS2.2 software and Data Assist(Thermo Fisher Sciences, Inc.). The expression of miRNAs
was calculated based on cycle threshold (Ct) values using
the 2-ΔΔCq method (19) normalized to those of ath-miR-159,
which was spiked in each sample. Data analysis was carriedout using GeneSpring software (Agilent Technologies). The
Benjamin-Hochberg algorithm was used for the estimation offalse discovery rates, as previously reported (16,17).
Validation of candidate miRNA expression by qRT‑PCR. To
compare selected miRNA expression in various fractions(EV-miRNA, cellular miRNA, and serum EV-miRNA), quantitative real-time PCR was performed by TaqMan® MicorRNA
Assays (ThermoFisher Scientific, Inc.) using an ABI Prism
7900 sequence detection system (Applied Biosystem)
according to the manufacturer's instruction.
The microRNA specific stem‑loop primers (has‑miR‑101,
cat. no. 000438; ath-miR-159, cat. no. 000338) were purchasedfrom ThermoFisher Scientific, Inc. The reaction was first
incubated at 95˚C for 2 min, followed by 50 cycles of 95˚C for
15 sec and 60˚C for 1 min, as we have reported previously (18).
Statistical analysis and evaluation of miR‑101 targets.
Data were expressed as means ± standard deviation (SD).
Mann-Whitney U and chi-square tests were used to determine
statistical significance for comparisons between the control
and test groups. Multiple groups were compared with one-way
analysis of variance (ANOVA). Statistical analysis was carriedout using R and GraphPad Prism software (v. 5c for Macintosh;
GraphPad Software Inc.).
miRs with a ΔCt value >1.0 or <‑1.0, and P‑values <0.05
were considered to exhibit differential expression. Following
identification of differentially expressed miRs, the predicted
target genes for these altered miRs were subjected to

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51
58
55
78
68
82

77

47
22
82
46
84

50

72
38
71
66
72

56
68
87

68
77
72
61

33
54
59
82
95
93

101

102
103
107
64_2
141

115

8
12
58
22
44

38
120
108

175
4
30
13

UPN

Age
(Year)

-424-

M
M
M
M

M
F
M

M
M
M
M
M

M

F
M
F
F
M

M

M
F
M
M
M
M

M/F

2014/8/8
2014/8/26
2015/1/22
2014/11/27
2015/1/22

2016/10/11

2015/9/30
2015/10/9
2015/10/22
2016/3/8
2016/5/19

2015/9/17

2014/10/10
2014/12/16
2015/1/27
2015/4/30
2015/8/20
2015/8/11

Date ofmaterial
obtained

MDS-EB1
2016/2/3
AML with MRC 2014/4/2
AML with MRC 2014/10/8
AML (M6A)
2014/9/10
with MRCMDS‑EB2
2014/10/14
MDS‑EB2
2016/3/10
MDS‑U with > 2015/10/26
1% PB blasts

MDS‑EB1MDS‑EB2
MDS-EB1MDS‑EB1MDS‑EB2

MDS‑U basedon defining
cytogenetic
abnormality
MDS‑MLD
MDS‑MLD
MDS‑MLD
MDS‑MLDMDS withisolateddel(5q)
MDS‑MLDMDS-SLDMDS‑EB1
MDS‑MLD
MDS‑MLD
MDS‑SLD
MDS‑SLD

Dx
(WHO)

Yes

No
Yes
No

No
No
No
Yes
Yes

No

No
No
No
No
No

No

No
No
No
No
No
No

Transition
to acute
leukemia

Dead
Dead
Unknown
Dead

Dead
Dead
Dead

Unknown
Unknown
Unknown
Unknown
Dead

Unknown

Alive
Unknown
Alive
Alive
Alive

Alive

Alive
Alive
Alive
Unknown
Unknown
Dead

Outcome

Int-2

Int‑2
High
in‑2

Int‑1
Int‑2
Int-2
Int‑1
High

Int‑1

Int‑1
Low
Low
Low
Int‑1

Low

Int-1
Int‑1
Low
Int‑1
Low
Low

IPSS

6600

2800
2600
6100
4500
3500

4600

5200
2400
4700
1900
10200
2200

WBC

High

2000
4500
800
1300

High
5500
Very high 1100
High
11400

High
1700
High
3000
Very high 2900
High
2000
Very high 16300

Low

Low
Int
Low
Very lowInt

Very lowInt
Int
Very low
Very low
Low
Very low

IPSS‑R

1110
393
248
338

4895
374
10864

408
1410
2233
910
2853

5214

1274
845
4148
2835
1295

2852

4030
1080
2961
1267
7905
1606

Neutrophils

Plt

30

8.8
8.9
7.7
7.6

8.3
7.8
8.1

9
10.3
8.3
7.6
9.1

4.3

30
23
50
17

164
19
65

78
95
31
16
97

42

8.3 15
6.3 21
11.2 187
13.1 299
9.5 34410

7.6 135
9.3 7413.7 56
10.6 57
12.1 18
10.4 105

Hb

Table I. Hematologic features of myelodysplastic syndrome patients of whom bone marrow mesenchymal stroma cells were obtained.

2
17
2
12

0
0
1.5

0
0.5
0
0
5

0.5

0
0
0.5
0
0

0

0
0
0
0
0
0

PBblasts (%)

4.4
49.5
21
12.8

12.4
17.2
4.8

5.2
10
9.2
7.6
16.8

2

0
0
0.4
2
0.5

0

0.8
5.6
0
2
1.2
0

BM-blasts(%)

46,XY,del(11)(q14)46,XY
45,XY,‑7complex
46,XY
46,XY,‑7,+m/46,
XY,idem,del(12)
(p13)
46,XY
complex
46,XY,del(20)(q11)/46,XY,
idem,-7,+8
complexcomplex
45,X,‑Ycomplex

46,XX
46,XY
47,XX,+846,XX
46,XY,del(5q)complex
46,XX
46,XY
45,X,‑Y
46,XY
46,XY,del(20)(q11)
45,X,‑Y

Karyotype

ONCOLOGY LETTERS 19: 2053-2061, 2020

2055

Dead
Alive
Dead
Dead
AML with MRC 2014/9/16
AML with MRC 2016/4/11
AML with MRC 2016/4/26
AML with MRC 2015/9/9
F
M
F
F
67
64
67
42
18
127
133
98

M/F
UPN

UPN, unique patient number; M/F, male/female; Dx, diagnosis; IPSS, International Prognostic Scoring System; IPSS‑R, revised IPSS; WBC, while blood cell count (x109/l); Hb, hemoglobin g/dl; Plt,
platelets (x1012/l); PB, peripheral blood; BM, bone marrow; MDS, myelodysplastic syndrome; AML with MRC, acute myeloid leukemia with myelodysplasia-related changes; Unknown, due to transfer
to another hospital.

66
23.2
34
21
2.5
0
8
1.5
7.9
7
9.3 78
7.3 2511.3 175
520
240
400
270
2000
1300
1400
2000

KaryotypeBM-blasts(%)
PBblasts (%)
Plt
Hb
Neutrophils
WBC
IPSS‑R
IPSS
OutcomeTransition
to acute
leukemia
Date ofmaterial
obtained
Dx
(WHO)
Age
(Year)

Table I. Continued. Hematologic features of myelodysplastic syndrome patients of whom bone marrow mesenchymal stroma cells were obtained.

45,XX,‑746,XY
45,XX,‑18
46,XX,inv(16)(p13;q22)/47,
XX,idem+22

SAITOH et al: EV-miR‑101 IN BM‑MSC DERIVED FROM MYELODYSPLASTIC SYNDROMEA

2056

experimental validation using miR-target interaction database
MiRTarBase (http://mirtarbase.mbc.nctu.edu.tw/). In addition, functional annotation of target genes was carried out
with Database for Annotation, Visualization, and Integrated
Discovery (DAVID Bioinformatics tools v6.7) (http://david.
abcc.ncifcrf.gov).
Results
Downregulation of BM‑MSC‑derived EV miR‑101 in patientswith high‑risk MDS. To identify the association between
EV-miRs from BM-MSCs and risk in patients with MDS,
we used TLDA to screen the miR expression profile between
low-risk group (n=13: Very low, low, and intermediate risk;
9 males/4 females, mean age 61.5 years (range 22 to 84 yearsold)) and high-risk group (n=9: High and very high risk;
8 males/1 females, mean age 66.4 years (range 38 to 87 yearsold)) separated by the IPSS-R. Seven patients with AML-MRC
included 4 males and 3 females, mean age 64.3 years
with ranging 42 to 77 years old). We extracted nine miRs
(has-miR-375, has-miR-101, has-miR-424, has-miR-548c-3p,
has-miR-15b, has-miR-485-3p, has-miR-579, has-miR-195,
and has-miR-369-3p) that were differentially expressed
between low-risk and high-risk groups using R software-T test
(Figs. 1 and S1) (GSE133276). We then compared the expression patterns of BM-MSC-derived EV-miRs detected withTLDA between normal controls (Lonza) and low-risk groupor high-risk group. Of the nine EV-miRs, EV-miR-101 showedno significant difference between normal control and low‑riskgroups (P=0.5824), while its expression was downregulated in
the high-risk group (P=0.0012). We separated 13 MDS patientsas low-risk group, including 5 with very low MDS by IPSS-R
with marrow blasts <2%, therefore, we considered the characterof BM-MSC may overlap of those from normal control. On
the other hand, EV-miR-15b expression was downregulated inboth the low-risk (P=0.0004) and high-risk (P=0.0001) groups
as compared with that in the control BM-MSCs, suggestingthat EV-miR-15b could serve as a biomarker for MDS rather
than that for disease progression (Fig. 1).
To confirm the observation of low expression level of
BM-MSC-derived EV-miR-101 in high-risk group and patientswith AML/MRC, we quantified the expression of EV‑miR‑101
with qRT‑PCR. As a result, we found no significant difference in EV-miR-101 expression level in BM-MSCs fromnormal control and low-risk groups (P=0.2579). In contrast,
EV‑miR‑101 expression significantly decreased in the high‑riskgroup (P<0.0001) and patients with AML/MRC (P<0.0001)(Fig. 2A), indicating that BM‑MSC‑derived EV‑miR‑101
expression level was associated with disease severity inpatients with MDS.
Expression pattern of miR‑101 in BM‑MSCs and serum
EV‑miR‑101. We assessed miR-101 level in BM-MSCs using
qRT-PCR, as the pattern of BM-MSC-derived EV-miR-101
was related to MDS disease progression. The relative expression level of miR-101 in control BM-MSCs (Lonza) was lower
than that reported in the BM-MSCs from the high-risk group(P=0.0058) and patients with AML/MRC (P=0.0095). No
significant difference was reported between normal control
and low-risk groups (P=0.8713). Therefore, the expression-425-

ONCOLOGY LETTERS 19: 2053-2061, 2020

2057

Figure 1. Expression of extracellular vesicle miR derived from BM‑MSCs
using TaqMan low‑density Array (Thermo Fisher Scientific, Inc.). miR
expression according to normal control (NC) vs. low-risk MDS group (Low),
high-risk MDS group (High) and AML/MRC group (AML). Box plots depict
the 75th percentile, median and 25th percentile values; and whisker plots
represent minimum to maximum expression. P values were calculated using
the independent two-sample t-test. (A) The expression of EV-miR-101 wassignificantly higher in the low‑risk group compared with the high‑risk group.
The expression of miR‑101 in the low‑risk group indicated no significant difference compared with the normal controls. (B) Significant differences in theexpression of miR-15b were observed between low-risk and normal control
groups. miR, microRNA; BM-MSCs, bone marrow mesenchymal stromalcells; NC, negative control; MDS, myelodysplastic syndrome; AML/MRC,
acute myeloid leukemia with myelodysplasia-related changes.

profile of cellular miR‑101 seemed to be a mirror image of the
EV‑miR‑101 obtained from BM‑MSCs (Fig. 2B).
We next assessed EV-miR-101 level obtained from theserum using qRT-PCR. Serum was obtained from identicalpatients with MDS assessed by TLDA, and normal control
serum was obtained from age-matched healthy individuals(n=4). The expression level of serum EV-miR-101 wassignificantly downregulated in low-risk group (n=10) as
compared with that in healthy control (P=0.0070), while nosignificant difference was observed in high‑risk group (n=7)(P=0.3232) or patients with AML/MRC (n=6) (P=0.2731) as

Figure 2. EV miR101 obtained from BM‑MSCs. (A) Expression of miR‑101
derived from BM-MSCs (B) or serum EV-miR-101 expression (C) as assessedusing reverse transcription-quantitative PCR. The expression of EV-miR-101
in the BM‑MSCs was significantly higher in the low‑risk MDS group compared with the high-risk MDS group, while cellular miR-101 expression was
significantly increased in the high‑risk group compared with the low‑riskgroup or normal controls. EV, extracellular vesicle; miR, microRNA;
BM-MSCs, bone marrow mesenchymal stromal cells; MDS, myelodysplastic
syndrome.

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SAITOH et al: EV-miR‑101 IN BM‑MSC DERIVED FROM MYELODYSPLASTIC SYNDROMEA

EV-miR-101 nor BM-MSC-derived cellular-miR-101 level(data not shown).
MiR‑101 expression level and BM blast percentage. We
assessed the association between miR-101 expression levels
and percentage of blasts in the BM. The BM-MSC-derived
EV-miR-101 level negatively correlated with the occupancyof blasts in the BM (P= 0.0185) (Fig. 3A). No significant
correlations were observed between BM blast percentage and
BM‑MSC‑derived cellular miR‑101 level (P=0.1457) (Fig. 3B)or serum EV‑miR‑101 level (P=0.3909) (Fig. 3C).
Target for miR‑101. MiRTarBase was used to identify thepredicted target genes of miR-101 to determine its biologicalsignificance. More than 50 target genes were extracted by
MiRTarBase, and the target genes that showed a strong
evidence are summarized in Supplementary Table I. The
epigenetic regulator genes, including enhancer of zester
homolog 2 (EZH2), part of polycomb repressive complex
subunit (SUZ12), ten-eleven translocation oncogene family
member 2 (TET2), and proto-oncogenes (c‑FOS and c‑MYC),
were experimentally validated with a luciferase reporter assay
in the literature. Functional annotation analysis with DAVID
revealed the most affected biological pathways of these genes
downregulated in the high-risk group, including cell-cycle
related pathway and epigenetic regulation pathway (Table II).
Discussion

Figure 3. Correlation between ΒΜ blast percentage and (A) BM‑MSC‑derived
EV-miR-101, (B) cellular miR-101 from BM-MSCs or serum (C) EV-miR-101.
ΒΜ, bone marrow; BM‑MSCs, bone marrow mesenchymal stromal cells;
miR, microRNA.

compared with healthy subjects (Fig. 2C). Serum EV‑miR‑101
expression pattern correlated with neither BM-MSC-derived

The present study demonstrates that EV-miR-101 expression
level in BM-MSCs was different depending on MDS severity.
High‑risk group and patients with AML/MRC showed significantly lower expression of BM-MSC-derived EV-miR-101
than the low-risk group or normal controls. In contrast, theexpression of miR-101 derived from BM-MSCs was upregulated in patients from the high-risk group, indicative of the
dissociation between miR-101 expression level and EV from
BM-MSCs in patients with MDS. Although no relationship wasobserved between BM blast percentage and BM-MSC-derived
cellular-miR-101 or serum EV-miR-101 expression level,
EV-miR-101 level from BM-MSCs exhibited reverse correlation with BM blast percentage. These findings suggest thepossibility that some specific miRs from BM-MSCs may
be interrupted from translocation into EVs (or intra-cellularaccumulation) and this phenomenon may be linked with theincrease in the number of blasts in patients with MDS. The
dissociation between cellular miR and EV-miR (or exosomal
miR) has been observed in neoplastic cells (9), including
leukemia cells (18). However, the current findings deny thepossible utility of serum miR measurement as liquid biopsy
for various diseases. The importance of BM-MSC-derived
EV-miR may indicate the nature of EV-miR, which may
participate in the communication between adjacent cells, especially between BM-MSCs and hematologic malignant cells.
Hematologic neoplasia-derived exosomes affect the microenvironment in the BM. For instance, chronic lymphocytic
leukemia (CLL) cell-derived exosomes induce the transition
of BM stroma cells into CAFs (20). These present enrichedmiR-150 and miR-146a in CLL-derived exosomes and anelevated miR-150 and miR-146a in BM-MSCs after co-culture-427-

ONCOLOGY LETTERS 19: 2053-2061, 2020

2059

Table II. The gene ontology terms enriched in targets of miR‑101. The terms with P‑value <0.05 and false discovery rate <0.05
were listed.
Term

P-value

Benjamini

Positive regulation of transcription from RNA polymerase II promoter
Negative regulation of transcription from RNA polymerase II promoter
Positive regulation of gene expression
Negative regulation of cell proliferation
Positive regulation of transcription, DNA-templated
Response to estradiol
Positive regulation of neuroblast proliferation
Cochlea morphogenesis
Branching morphogenesis of an epithelial tube
Positive regulation of mesenchymal cell proliferation
Protein phosphorylation
Positive regulation of epithelial to mesenchymal transition
Epithelial to mesenchymal transition
Response to lipopolysaccharide
Coronary artery morphogenesis
Cellular response to hypoxia
Heart development
Thymus development
Positive regulation of apoptotic process
Response to drug
Positive regulation of cellular component movement
Negative regulation of epithelial cell differentiation
Negative regulation of gene expression, epigenetic
Regulation of apoptotic process
Positive regulation of protein phosphorylation
Positive regulation of epithelial cell proliferation

9.90x1010
4.10x107
2.00x106
5.00x106
5.70x106
1.20x105
3.60x105
4.90 x105
5.60 x105
8.20x105
1.10x104
1.70x104
1.90x104
2.00x104
2.20x1042.70x104
3.30x104
3.70x104
4.30x104
4.60x104
4.70x104
5.70x104
5.80x104
6.50x104
7.90x104
1.00x103

1.30x106
2.60x1048.70x104
1.60x103
1.50x103
2.50x103
6.60x103
7.80 x103
8.00 x103
1.00x1021.30x102
1.80x102
1.80x102
1.80x102
1.80x1022.20x1022.40x1022.60x1022.90x1022.90x1022.80x1023.30x1023.20x1023.40x102
4.00x102
4.80x102

miR, microRNA.

with primary CLL cells. CLL exosomes induced the proliferation and migration of BM-MSCs with angiogenesis (20). We also
reported that EV-miR-135b from multiple myeloma cells transfers
and induces angiogenesis (18). In solid tumors, the phenomenonof transfiguration of the surrounding fibroblasts into CAF has
been reported, and this change is known to affect cancer, leading
to tumor progression and drug resistance (21). In hematologic
neoplasia, the similar situation (CAF induction of BM‑MSCs)
could be plausible; however, such phenomenon is only limited
in lymphoid neoplasia, i.e., CLL and multiple myeloma (20,22).
Ozdogan et al demonstrated the low expression levelof DICER1 gene and miR dysregulation in the BM-MSCs
obtained from patients with MDS and AML along with thedownregulated expression of miRs (miR-30d-5p, miR-222-3p
and miR-30a-3p) and overexpression of miR-4426 in
MDS-derived BM-MSCs (23). Downregulation of DICER1
expression in the BM-MSCs from patients with MDS promoted
cellular senescence and decreased the supportive capabilityof hematopoietic stem cells along with the downregulationof the expression of miR-17 family (miR-17-5p, miR-20a/b,
miR-106a/b, and miR-93) (24). Studies on BM-MSC-derived
EV (including exosomes) have focused on limited fields ofhematologic neoplasia. Wang et al demonstrated that mouse
BM-MSC (5T33)-derived exosomes increased the viability
and proliferation and decreased the apoptosis of mouse
myeloma cells (5T33 MM model) in response to bortezomib
resistance (3). Moreover, these authors showed that human
myeloma BM-MSC-derived exosomes increased the viabilityof human myeloma cells (RPMI-8226) in a dose-dependent
manner (3). Umezu et al also reported that the BM-MSC-derived
EV-miR-340 from normal young donor (Lonza), rather
than old donor, inhibited myeloma-induced angiogenesis
via hepatocyte growth factor/c-MET signaling (25). In thisstudy, no particular correlation was observed in the expression level between BM-MSC-derived EV-miR-101 andserum EV-miR-101, suggesting that the BM-MSC-derived
EV-miR-101 may affect adjacent cells, including MDS cells.
miR-101 is known to exert anti-neoplastic effects and facilitate apoptosis (26). For instance, miR‑101 exhibited suppressive
effects on bladder cancer via c‑FOS (27), colorectal cancer via
EZH2 (28), and breast cancer via EYA1 (29). miR-101 is also
known to exert suppressive effects on vascular endothelial
growth factor-C (30,31). Recent reports found that miR-101
regulates the development and progression of myeloid- and

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SAITOH et al: EV-miR‑101 IN BM‑MSC DERIVED FROM MYELODYSPLASTIC SYNDROMEA

T-lymphoid lineage leukemia (32,33) and suppresses theproliferation of neoplastic cells by targeting genes related toepigenetic regulation (Tables II and SI). Thus, we speculate thepossibility that EV-miR-101 from BM-MSCs transfer to MDS
cells and control their proliferation via epigenetic regulation.
In patients with high-risk MDS, the decrease in the expression
level of EV-miR-101 may result in the release of the anti-proliferative effect on MDS cells. Another plausible possibility is thatthe more malignant clone during MDS progression may induce
BM-MSCs to suppress EV-miR-101 expression. miR-101
antisense transfection assay showed enhanced proliferationof human myeloid leukemia (HL-60) cells with an increase in
c‑FOS expression (unpublished observation), thus suggestingthat the downregulation of EV-miR-101 expression in patientswith high-risk MDS may affect the proliferation of MDS cells.
Development of MDS is known to be associated with theaccumulation of genetic abnormalities by aging, so-called‘Clonal Hematopoiesis of Indeterminate Potential (CHIP)’,and additional mutations in critical genes, including epigenetic
regulator genes, accelerate AML (34). In addition to genetic
abnormalities in MDS cells, BM-MSCs are also shown to havesynonymous or non-synonymous mutations (15,35,36) such as‘genetic injury.’ The unresolved issue is whether demethylating
agents available for patients with MDS could exert any effect on
BM-MSCs, especially BM-MSC-derived EV, as miR-101 targets
many epigenetic regulator genes. Poon et al demonstrated that ahypomethylating agent (azacytidine) could also target BM-MSCs
in dysplastic MDS and contribute to the restoration of active
hematopoiesis, whereas patients with MDS that failed to respondto hypomethylating treatment had disease progression (37). We
could not reproduce the peculiarity of BM-MSC-derived miRconcerning the anatomical architecture of MDS.
In conclusion, the current study demonstrates that the
BM-MSCs obtained from patients with MDS were heterogenous with respect to the expression of EV-miR. Patientswith high-risk MDS and AML/MRC had lower expression of
BM-MSC-derived EV-miR-101 than those with low-risk MDS.
This tendency was reversible as compared with the miR-101
level from BM-MSCs; the serum EV-miR-101 expression
showed no association. Although we failed to clarify the exact
mechanism underlying the downregulation of EV-miR-101
expression from BM-MSCs in patients with high-risk MDS,
targeting miR-101 may regulate neoplastic proliferation. Thus,
the downregulation of EV-miR-101 expression may affectadjacent cell-to-cell communication and accelerate the malignant process in MDS cells.
Acknowledgements
Not applicable.
Funding
The present study was supported by the Platform Project for
Practical Research for Innovative Cancer Control from theJapan Agency for Medical Research and Development (grantno. 15Ack0106073h0002), the Private University Strategic
Research-Based Support Project (grant no. S1311016) from
the Ministry of Education, Culture, Sports, Science andTechnology and JSPS KAKENHI (grant no. 19K16811).

Availability of data and materials
The datasets generated and/or analyzed during the present
study are available from the Gene Expression Omnibus
repository, https://www.ncbi.nlm.nih.gov/geo/query/acc.
cgi?acc=GSE133276.
Authors' contributions
YS, MA, SY, SK, TS, HF, DA and KO treated the patients; TU
and CKK performed TLDA, PCR and data analysis; SI established BM-MSCs; TU contributed essential reagents or tools;
YS and KO wrote the manuscript; KO and JHO designed theresearch and supervised the project. All authors contributed todata analysis and drafting of the manuscript.
Ethics approval and consent to participate
Patient data was used in the present study according to the
ethical principles for medical research involving humansubjects of The Declaration of Helsinki (38). The study protocols were approved by The Regional Ethics Review Boardof Tokyo Medical University (Tokyo, Japan), and all patientsprovided written informed consent prior to participation.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

Repression of Smad3 by Stat3 and c-Ski/SnoN induces geﬁtinib
resistance in lung adenocarcinoma
Yojiro Makino a, 1, Jeong-Hwan Yoon b, c, 1, Eunjin Bae b, 1, Mitsuyasu Kato d,
Keiji Miyazawa e, Tatsuo Ohira a, Norihiko Ikeda a, Masahiko Kuroda b,
Mizuko Mamura b, c, f, *a

Department of Surgery, Tokyo Medical University, Tokyo, Japan
Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan
Biomedical Research Institute, Kyungpook National University Hospital, Daegu, Republic of KoreadDepartment of Experimental Pathology, Graduate School of Comprehensive Human Sciences and Faculty of Medicine, University of Tsukuba, Ibaraki, Japane
Departments of Biochemistry, University of Yamanashi, Yamanashi, JapanfPhysician, Student and Researcher Support Center, Tokyo Medical University, Tokyo, Japanbca r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 January 2017
Accepted 19 January 2017
Available online 20 January 2017

Cancer-associated inﬂammation develops resistance to the epidermal growth-factor receptor (EGFR)
tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR
mutations. Stat3-mediated interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-b(TGF-b) signaling pathways play crucial regulatory roles in cancer-associated inﬂammation. However,mechanisms how these pathways regulate sensitivity and resistance to EGFR-TKI in NSCLCs remain
largely undetermined. Here we show that signal transducer and activator of transcription (Stat)3 represses Smad3 in synergy with the potent negative regulators of TGF-b signaling, c-Ski and SnoN,
whereby renders geﬁtinib-sensitive HCC827 cells resistant. We found that IL-6 signaling via phosphorylated Stat3 induced geﬁtinib resistance as repressing transcription of Smad3, whereas TGF-b enhanced
geﬁtinib sensitivity as activating transcription of Smad3 in HCC827 cells with geﬁtinib-sensitizing EGFR
mutation. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad2/3/4induced transcription of the Smad3 gene. Smad3 was found to be an apoptosis inducer, which upregulated pro-apoptotic genes such as caspase-3 and downregulated anti-apoptotic genes such as Bcl-2. Our
results suggest that derepression of Smad3 can be a therapeutic strategy to prevent geﬁtinib-resistancein NSCLCs with geﬁtinib-sensitizing EGFR mutation.
© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords:
Cell cycle
Geﬁtinib resistanceLung cancer
Smad3
Stat3
c-Ski/SnoN

1. Introduction
Although EGFR-TKIs have remarkably improved the therapeutic
efﬁcacy for NSCLCs harboring the sensitive EGFR mutation [1], drug
resistance inevitably develops [2]. Genetic changes such as secondary mutations in EGFR (T790M) and ampliﬁcation of the MET
receptor tyrosine kinase are the main mechanisms of acquired drug

* Corresponding author. Department of Molecular Pathology, Tokyo Medical
University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo, 160-8402, Japan; Biomedical
Research Institute, Kyungpook National University Hospital, 50 SAMDUK-2GA,
Jungu, Daegu, 41944, Republic of Korea. Tel.: þ81 3 3351 6141; fax: þ81 3 3351 6173.
E-mailaddresses:
mamumik@tokyo-med.ac.jp,
mikoeyo@gmail.com(M. Mamura).
1
These authors contributed equally to this work.

resistance [3,4]. Unknown mechanisms of EGFR-TKI resistance inthe remaining cases [4] could be attributed to chronic inﬂammation, which plays critical roles in tumorigenesis [5]. It is also
possible that chronic inﬂammatory responses induce DNA damages
and mutations [6].
IL-6 is the pivotal pro-inﬂammatory cytokine in inﬂammation
including cancer-associated inﬂammation [7,8], which affects
treatment effectiveness [9]. IL-6 and EGF share Stat3 for theirsignaling pathways, which promote carcinogenesis throughinﬂammation [10,11]. In NSCLCs, correlation between elevation of
IL-6 and phosphorylation of Stat3 are signiﬁcantly correlated[12,13].
In contrast with IL-6, TGF-b is the principal immunosuppressive
cytokine secreted in the tumor microenvironment [14]. Elevation of
TGF-b is frequently observed in NSCLCs, which is correlated with

http://dx.doi.org/10.1016/j.bbrc.2017.01.093
0006-291X/© 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

2.3. Immunocytochemistry
Abbreviations
EGFR
TKIs
NSCLCs
TGF-b
IL-6
STAT

epidermal growth-factor receptor
tyrosine kinase inhibitors
non-small cell lung cancers
transforming growth factor-binterleukin-6
signal transducer and activator of transcription

poor prognosis and metastasis [15]. Cytoplasmic proteins called
Smads mediate the canonical pathway for signal transduction fromthe TGF-b receptor (TbR) serine threonine kinases. Activated TbRI
phosphorylates serine residues in MH2 domains of receptorregulated Smads (R-Smads), Smad2 and Smad3, which oligomerize with the common-mediator Smad, Smad4 to regulatetranscription of the target genes [16,17]. Smad2 and Smad3 arehighly homologous molecules with distinct effects in contextdependent manners, however, detailed mechanisms of involvement of Smad2 versus Smad3 in carcinogenesis remains largely
undetermined [18].
Transmodulation between the STAT and SMAD signaling pathways has been implicated as the basis for the antagonism between
TGF-b and pro-inﬂammatory cytokines such as interferon-g, IL-1b
and TNF-a [19,20]. It has been reported that TGF-b-dependent IL-6
secretion and subsequent Stat3 activation induce EGFR-TKI resistance in NSCLCs [21]. We previously reported that Smad2 andSmad3 oppositely regulate Th17 differentiation as Stat3 cofactors[22]. However, the mechanisms whether and how Stat3-mediatedIL-6 signaling and Smad-mediated TGF-b signaling interact toregulate EGFR-TKI resistance in NSCLCs remain largely unknown.
In this study, we examined the mechanisms how R-Smads andStat3 regulate the sensitivity and resistance to geﬁtinib in HCC827
lung adenocarcinoma cells with geﬁtinib-sensitizing EGFR
mutation.
2. Materials and methods
2.1. Cell culture and transfection
Human lung adenocarcinoma HCC827 cells (ATCC) were
cultured in RPMI 1640 (Hyclone) supplemented with 10% heatinactivated FBS (Gibco) and 1% penicillin/streptomycin (ThermoScientiﬁc) with or without the combinations of human IL-6 (R&D,
10 ng/ml), TGF-b1 (R&D, 5 ng/ml) and geﬁtinib (AstraZeneca, 2 mM)
for 4 h after serum starvation and grown at 37 � C with 5% CO2. No
mutations or copy number variations of TGF-b signaling molecules
are observed in HCC827 cells (http://cancer.sanger.ac.uk/cell_lines/
sample/overview?id¼1240146). HCC827 cells were seeded a day
before transfection and transfected with Smad3 cDNA, Smad3
siRNA, Stat3 siRNA or control siRNA (GE Dharmacon) using Lipofectamine LTX & Plus Reagent (Invitrogen) following manufacture's protocol as previously described [22].
2.2. Cell cycle analysis
DNA content was determined by propidium iodide (PI; BD
Pharmingen). Brieﬂy, HCC827 cells were ﬁxed by cold 80% ethanol
overnight at �20 � C, washed twice with phosphate-buffered saline(PBS) containing 1% bovine serum albumin (BSA), and then resuspended in PI solution in PBS containing RNase A (0.1 mg/ml; SigmaAldrich). Stained samples were acquired by FACSCalibur (BDBioscience) and analyzed by FlowJo (Tree Star).

Proximity ligation assay (PLA) was performed as previously
described [22]. HCC827 cells were ﬁxed with 4% formaldehyde.
Fixed slides were permeabilized by 0.1% Triton X-100 in PBS for
staining with Duolink in situ PLA probes anti-rabbit PLUS and PLA
probe anti-rabbit MINUS or PLA probe anti-mouse PLUS and PLA
probe anti-mouse MINUS and detection reagents (Sigma-Aldrich),rabbit or mouse antibodies against Smad2, Smad3, phospho-Stat3
Y705, phospho-Stat3 S727 (Cell Signaling Technology), Stat3, cSki and SnoN (Santa Cruz). Nucleus was stained with DAPI. Slides
were observed using a confocal microscope, LSM700 (Carl Zeiss).
PLA signals were quantiﬁed using BlobFinder software (http://
www.cb.uu.se/~amin/BlobFinder/).
2.4. RNA isolation and quantitative RT-PCR
Total RNA was extracted from HCC827 cells using Trizol according to the manufacturer's instructions (Invitrogen). RNA wasreverse transcribed with a cDNA RT kit (Invitrogen). Human cDNA
was quantitated by SYBR green (Applied Biosystems) using ABI
7900 (Applied Biosystems). Each experiment was performed intriplicate. The following primers were used: GAPDH 50 - CTCAAGACCTTGGGCTGGG-30 , 50 -TCGAACAGGAGGAGCAGAGA-30 , Smad3
50 -GAGTTGAGGCGAAGTTTGGG-30 , 50 -AGTGAAAGGCAGGATGGACG30 , Bcl2 50 -AACATCGCCCTGTGGATGAC-30 , 50 -CAGGGCCAAACTGAGCAGAG-30 , Bbc3 50 -ACTCACCAAACCAGAGCAGG-30 , 50 -ACAAATCTGGCAGGGGACC-30 , Bnipl 50 -AACCAGCTCCCCAACAACTC-30 , 50 ATGTCTCAGGGCTGCTCCTA-30 , Caspase2 50 -CTTGCTGCCTAAGAGG
GGTC-30 , 50 - CTCACACACCGGAAAAGGGA-30 , Caspase3 50 -CTCTGGT
TTTCGGTGGGTGT-30 , 50 -TCCAGAGTCCATTGATTCGCT-30 , Cidec3 50 50 -CACAGAGGTACGCACTCAGAGCCAGGGGATGAGAAA-30 ,GACA-30
2.5. Luciferase reporter assay
The 2000 bp promoter region of Smad3 was generated by PCRfrom genomic DNA using the primers: 50 -GATCACGCGTTTGGGTTCAAATTCCAGCTC-30 and 50 -GATCCTCGAGGCAGCAGAAGTTTGGGTT
TC-30 . The product was veriﬁed by sequencing and was subcloned
into pGL3 Basic ﬁreﬂy luciferase construct (Promega) using Mlu1
and XhoI sites. The promoter construct was transfected in various
combination with Stat3, Flag-tagged Stat3Y705F, Stat3 S727A(Addgene, submitted by J. Darnell), Flag-tagged Smad2/3/4, Flagtagged c-Ski, SnoN, TGIF or empty pcDNA3 plasmid in
HCC827 cells using Lipofectamine (Invitrogen). Twenty-four hours
after transfection, HCC827 cells were lysed using lysis buffer for themeasurement using luciferase assay kit (Promega) by luminometer.
Each experiment was performed in triplicate.
2.6. Chromatin immunoprecipitation (ChIP)
Chromatin was prepared from HCC827 cells. Immunoprecipitation was performed with antibodies against Smad2, Smad3, trimethyl histone H3 Lys4 and tri-methyl histone H3 Lys27 (Cell
Signaling Technology), Smad4, c-Ski, SnoN and STAT3 (Santa Cruz)using ChIP kit (Cell Signaling Technology) according to the manufacturer's protocol. Immunoprecipitated DNA released from thecross-linked proteins was quantitated by real time PCR using theprimers: �1700 to �1534 50 -AATGCCAAGTAAGGCACAGG-30 50 CTCCCTTCCACTTGCTGCTA-30 , �1559 to �1410 50 -TCTCTAGCAG50 -GCAGCTTGTCAGGGGTTGT-30 ,�1196
CAAGTGGAAGG-30
0
to �1003 5 -TAGCCTGATAGGGAGGCTGA-30 50 -CCGGAGAGGACTCGAGAAGT-30 , �524 to �372 50 -GAGCTTTTCTGAACCCCTCA-30 50 -AC
�220
to
�28
50 CGGACTCCTGGGGACT-30 ,-432-

Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

CTGGGAAGGAGGCTGCAC-30 50 -AAACTTTGCTGGCCTGGAT-30 �107
50 -CGAto
þ63
50 -CAGAGGAGGAGGAGGAGGAG-30
GACTCCAAGTGGCAGTAG-30 and was normalized to input DNA.
Each experiment was performed in triplicate.
3. Results
3.1. Stat3-mediated IL-6 signaling induces geﬁtinib resistance in
HCC827 lung adenocarcinoma cells

271(Fig. 3B, lower), which was epigenetically inactive with trimethylated histone H3 lysine 27 (Fig. 3C, right). PLA showed that TGF-b
inhibited, whereas IL-6 enhanced the interactions between Stat3
and c-Ski/SnoN (Fig. 3D) in accordance with Stat3 phosphorylation
status (Fig. 1B). These results indicate that phosphorylated Stat3
cooperates with c-Ski and SnoN to repress Smad-induced transcription of the Smad3 gene.
3.4. Smad3 induces apoptosis and restores IL-6-induced geﬁtinib
resistance

We used HCC827 lung adenocarcinoma cells with EGFR746E750A deletion to examine the mechanisms how IL-6 and its
downstream Stat3 signaling induce geﬁtinib resistance in thisstudy. TGF-b and geﬁtinib induced apoptosis with synergistic proapoptotic effect, whereas IL-6 suppressed TGF-b/geﬁtinib-induced
apoptosis (Fig. 1A). IL-6 enhanced phosphorylation of Stat3 attyrosine 705 (pY705) and serine 727 (pS727), whereas TGF-b andgeﬁtinib suppressed Stat3 phosphorylation (Fig. 1B). Consistent
with the previous report [23], we conﬁrmed that Stat3 deletion by
RNAi accelerated, whereas Stat3 overexpression inhibited TGF-b/
geﬁtinib-induced apoptosis (Fig. 1C, Supplementary Fig. 1). Thus,
Stat3-mediated IL-6 signaling induced resistance to TGF-b/geﬁtinib
in HCC827 cells.
3.2. Stat3-mediated IL-6 signaling represses Smad3 in HCC827 cells
We next examined the status of TGF-b receptor-regulated Smads(R-Smads), Smad2 and Smad3 in HCC827 cells treated with TGF-b
and geﬁtinib with or without IL-6. We found that IL-6 abolished theexpression of Smad3 protein, whereas Smad2 remained expressed(Fig. 2A). IL-6 completely inhibited the expression of Smad3 mRNA
(Fig. 2B), indicating that IL-6 repressed the transcription of the
Smad3 gene. By contrast, TGF-b and geﬁtinib upregulated Smad3
mRNA (Fig. 2B). Luciferase reporter spanning 2 kilobase upstreamof the ﬁrst exons of the Smad3 genes showed that IL-6 repressed,
whereas TGF-b and geﬁnitib induced the Smad3 gene promoter
activity (Fig. 2C). Canonical TGF-b signaling molecules, R-Smads:
Smad2/3 and common Smad: Smad4 synergistically induced the
Smad3 gene promoter activity (Fig. 2D). By contrast, Stat3 repressed
the Smad3/4-induced Smad3 gene promoter activity (Fig. 2E).
Consistently with the phosphorylation status of Stat3 (Fig. 1B) andthe expression levels of Smad3 (Fig. 2A), Stat3 mutations at Y705 or
S727 completely abolished this repression (Fig. 2E), indicating that
Stat3 phosphorylation at Y705 and S727 is essential for Stat3induced repression of the Smad3 gene. Overexpression of Stat3
signiﬁcantly suppressed, whereas Stat3 knockdown upregulated
the expression of Smad3 protein in HCC827 cells (Fig. 2F). These
results show that Smad-mediated TGF-b signaling induces,whereas Stat3-mediated IL-6 signaling represses the transcriptionof the Smad3 gene.
3.3. c-Ski and SnoN cooperate with Stat3 to repress Smad3
We sought to determine how Stat3 represses the Smad3 gene.
We screened the representative Smad co-repressors: Ski/SnoN and
TGIF [17,24e26] for Stat3 co-repressors, and found that c-Ski and
SnoN, but not TGIF showed synergy with Stat3 to repress the
Smad3/4-induced Smad3 gene promoter activity (Fig. 3A). c-Ski and
SnoN are the potent negative regulators of TGF-b signaling [24e26].
ChIP showed that Smad2/3/4 were bound to the same site in the
Smad3 promoter (�1700 to �1534) upon TGF-b stimulation (Fig. 3B,
middle), which was epigenetically active with methylated histone
H3 lysine 4 (Fig. 3C, left). By contrast, Stat3, c-Ski and SnoN werebound to the Smad3 promoter (�1559 to �1410), overlapped or
proximal to the Smad2/3/4 biding site upon IL-6 stimulation-433-

We examined the physiological relevance of IL-6/Stat3-induced
repression of Smad3 to geﬁtinib resistance by overexpression or
knockdown of Smad3 in HCC827 cells. Forced expression of Smad3
signiﬁcantly induced, whereas knockdown of Smad3 by RNAi
signiﬁcantly inhibited spontaneous and geﬁtinib/TGF-b-induced
apoptosis of HCC827 cells (Fig. 4A). IL-6-induced geﬁtinib resistance was completely abolished by forced expression of Smad3(Fig. 4A, right third row). Smad3 overexpression upregulated proapoptotic genes such as Caspase3, Caspase2 and Cidec (cell deathinducing DNA fragmentation factor-like effector c), while
repressed anti-apoptotic genes such as Bcl2 (b-cell leukemia/lymphoma 2 protein), Bbc3 (BCL2 Binding Component 3) and Bnipl(BCL2 Interacting Protein Like) (Fig. 4B). By contrast, Smad3
knockdown showed the opposing patterns (Fig. 4B). These data
show that Smad3 enhances geﬁtinib/TGF-b-induced apoptosis andrestores IL-6-induced geﬁtinib resistance by upregulating proapoptotic genes and downregulating anti-apoptotic genes in
HCC827 cells.
4. Discussion
Various mechanisms lead to inevitable development of EGFR TKI
resistance including the secondary mutations to a drug-resistantstate, activation of alternative signaling pathways, impairment of
EGFR-TKI-mediated apoptosis and histologic transformation orepithelial-mesenchymal transition (EMT) [27]. Chronic inﬂammatory responses in tumor microenvironment not only induce drug
resistance but also induce DNA damages and mutations [6,21,23].
Thus, it is important to investigate the inﬂammatory mechanisms
to induce EGFR TKI resistance. The representative proinﬂammatory cytokine in cancer-associated inﬂammation, IL-6 isthe potent inducer of EGFR TKI resistance in NSCLCs [23]. TGF-b isessential for erlotinib resistance, EMT and increased activation of
IL-6 axis in resistant H1650 cells [21]. Unlike resistant NSCLCs, TGFb exerted synergistic pro-apoptotic effect with geﬁtinib in case ofhighly EGFR TKI-sensitive HCC827 cells [28], in which both TGF-b
and geﬁtinib suppressed Stat3 phosphorylation at Y705 and S727 in
the absence of IL-6. The most signiﬁcant difference between highly
EGFR TKI-sensitive HCC827 cells and resistant NSCLC cell lines is IL6-induced repression of Smad3 in HCC827 cells. By contrast, Smad3
remained expressed and highly phosphorylated in resistant
H1650 cells with augmented IL-6 secretion [23]. It has been reported that high expression of Smad3 and proﬁlin-2 is linked topoor survival rate in lung cancer patients [29]. Because TGF-b
switches from tumor suppressor at early stages of carcinogenesis to
pro-oncogene at later stages of disease leading to metastasis [16],our ﬁndings suggest that combined anti-inﬂammatory therapies
with EGFR-TKI at early stages may prevent cancer progression.
Although aberrant TGF-b signaling such as the mutations/deletion of Smad4 and the TGF-b type II receptor is frequent incancers, the mutations/deletion of Smad2 and Smad3 are rarely
found [30]. Loss of Smad2 or Smad3 by post-transcriptional modulations has been reported in various cancers [18]. However, precise molecular mechanisms how their expression is selectively

Fig. 1. IL-6 and STAT3 render HCC827 adenocarcinoma cells geﬁtinib-resistant. (A) HCC827 cells were treated with various combinations of geﬁtinib, IL-6 and TGF-b. (B) STAT3
phosphorylated at Y705 or S727 was detected by PLA. Signals were quantiﬁed using BlobFinder software (scale bars: 10 mm, nucleus: black, cytoplasm: white, n ¼ 10 ﬁelds). Data aremean þ s.d. (C) HCC827 cells were transfected with STAT3 cDNA, STAT3 siRNA or control siRNA and treated with various combinations of geﬁtinib, IL-6 and TGF-b. DNA contents
were determined by PI staining using ﬂowcytometry (A, C). Data are representative of three independent experiments.

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Fig. 2. STAT3-mediated IL-6 signaling represses Smad3 in HCC827 adenocarcinoma cells. HCC827 cells were treated with geﬁtinib and TGF-b in the presence or absence of IL-6. (A)Expression of Smad2 and Smad3 proteins in HCC827 cells was determined by PLA. (B) The expression of Smad3 mRNA relative to GAPDH in HCC827 cells was determined byquantitative RT-PCR. (C) HCC827 cells transfected with the Smad3 promoter reporter construct were treated with geﬁtinib and TGF-b in the presence or absence of IL-6. (D)HCC827 cells were transfected with the Smad3 promoter reporter construct with the indicated combinations of Smad2, Smad3 and Smad4. (E) HCC827 cells were transfected with
the Smad3 promoter reporter construct with the indicated combinations of Smad3, Smad4, STAT3, STAT3 mutants: Y705F or S727A. Fireﬂy luciferase activity was measured with aluminometer and normalized to b-galactosidase activity. Each experiment was performed in triplicate. (F) Smad3 protein in HCC827 cells transfected with STAT3 cDNA, STAT3 siRNAor control siRNA was detected by PLA. Signals were quantiﬁed using BlobFinder software (scale bars: 10 mm, nucleus: black, cytoplasm: white, n ¼ 10 ﬁelds). Data are mean þ s.d.
Data are representative of three independent experiments.

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Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

Fig. 3. Phosphorylated STAT3 represses the transcription of the Smad3 gene in synergy with c-Ski and SnoN. (A) HCC827 cells were transfected with the Smad3 promoter reporter
construct with the indicated combinations of Smad3, Smad4, STAT3, c-Ski, SnoN and TGIF. Fireﬂy luciferase activity was measured with a luminometer and normalized to bgalactosidase activity. Bindings of Smad2/3/4, STAT3, c-Ski and SnoN to (B) the Smad3 proximal promoter region and (C) trimethylated histone H3 Lys4 (H3K4me3) and trimethylated histone H3 Lys 27 (H3K27me3) in HCC827 cells were determined by ChIP. Each experiment was performed in triplicate. (D) Protein interactions between STAT3 and c-Ski,
STAT3 and SnoN in HCC827 cells treated with or without TGF-b or IL-6 were detected by PLA. Signals were quantiﬁed using BlobFinder software (scale bars: 10 mm, nucleus: black,cytoplasm: white, n ¼ 10 ﬁelds). Data are mean þ s.d. Data are representative of three independent experiments.

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Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

regulated remain largely unknown. Downregulation of either of RSmads could be one of the mechanisms to distinguish their distinct
functions [18]. Smad3 is downregulated through decreased transcription in lung epithelial cells [31] or through decreased geneactivation as well as increased protein degradation in glomerularmesangial cells [32]. Here, we show that IL-6-Stat3 signaling represses transcription of Smad3, thereby inducing geﬁtinib/TGF-b
resistance in HCC827 cells. Similar and synergistic effect of geﬁtinibwith TGF-b on HCC827 cells might be attributed to induction of
TGF-b by geﬁtinib [33]. Pro-inﬂammatory cytokine signaling
pathways antagonize TGF-b through upregulation of inhibitory
Smad, Smad7 [19,20]. Recent report shows that direct crosstalk275

between Stat3 and Smad3 antagonizes TGF-b [34]. Ski and theclosely related SnoN are oncogenes, which act as transcriptional corepressors in TGF-b signaling through interaction with Smads[24e26]. We show that c-Ski/SnoN cooperate with Stat3 to antagonize TGF-b by repressing Smad2/3/4-induced transcription of the
Smad3 gene. Future studies are required to clarify the relevance ofrepression of Smad3 to the interactions of c-Ski/SnoN, Stat3 andSmads in inﬂammation and carcinogenesis.
Stat3 activated through IL-6/JAK1 promotes cell survival anderlotinib resistance in EGFR-addicted NSCLC cell lines [23]. TGF-b
also plays pivotal roles in cell survival [16e18]. Smad3 sensitizes
hepatocytes to apoptosis through down-regulation of Bcl-2 [35] orFig. 4. Smad3 enhances geﬁtinib-induced apoptosis of HCC827 cells. (A) HCC827 cells were transfected with Smad3 cDNA, Smad3 siRNA or control siRNA and treated with geﬁtinibwith or without TGF-b or IL-6. DNA contents were determined by PI staining using ﬂowcytometry. (B) The expression of Caspase2, Caspase3, Cidec, Bcl2, Bbc3 and Bnipl mRNA
relative to GAPDH in HCC827 cells transfected with Smad3 cDNA, Smad3 siRNA or control siRNA was determined by quantitative RT-PCR. Each experiment was performed intriplicate. Data are mean þ s.d. Data are representative of three independent experiments.

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Y. Makino et al. / Biochemical and Biophysical Research Communications 484 (2017) 269e277

through cleavage of Bcl-2-associated death promoter (BAD) protein[36]. TGF-b/Smad3 induces apoptosis through inducing and activating caspase-3 [37]. Our ﬁndings that Smad3 signiﬁcantly prevented IL-6-induced geﬁtinib resistance in HCC827 cells as
upregulating caspase3 and downregulating Bcl2 are consistent
with these previous reports. Stat3 also targets similar set of
apoptosis-related genes [38]. Involvement of crosstalk between
Smads and Stat3 in regulation of apoptosis in IL-6-induced geﬁtinib
resistance is yet to be determined.
In summary, present study elucidated the novel mechanism
how IL-6/Stat3 induce geﬁtinib resistance in highly sensitive
HCC827 cells. IL-6 enhanced phosphorylation of Stat3 at Y705 andS727, which interacted with the Ski family of proto-oncoproteins, cSki and SnoN to repress Smad2/3/4-induced transcription of the
Smad3 gene. Repression of Smad3 resulted in resistance to
geﬁtinib-induced apoptosis, whereas forced expression of Smad3
restored IL-6-induced geﬁtinib resistance. Smad3 induced
apoptosis by upregulating pro-apoptotic genes as downregulating
anti-apoptotic genes. Our ﬁndings suggest that derepression of
Smad3 might prevent inﬂammation-induced geﬁtinib-resistance atearly stages of NSCLCs with geﬁtinib-sensitizing EGFR mutation
while they retain sensitivity to tumor suppressor effects of TGF-b.
Declaration of interest
NI receives support from AstraZeneca. Other authors declare nocompeting interests.
Acknowledgements
The work was supported by Basic Science Research Program
through the National Research Foundation of Korea(2015R1A1A3A04001051), the Pioneer Research Center Program
through the National Research Foundation of Korea funded by the
Ministry of Science, ICT and Future Planning (2015-001937 and2015-001923) to MM, Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1D1A1B03931785) to JHY and NRF(2014R1A1A2057903) to EB, supported in part by Grants-in-Aid forscientiﬁc research (B: 26293088), a Grant-in-Aid for Exploratory
Research (15K15100) from Japan Society for the Promotion of Science (JSPS) Strategic Research Foundation Grant-aided Project forPrivate Universities from the Ministry of Education, Culture, Sports,
Science and Technology of Japan to MK.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2017.01.093.
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Tumor volume determines the feasibility of cell-free
DNA sequencing for mutation detection in non-small
cell lung cancer
Tatsuo Ohira,1,4 Kazuko Sakai,2,4 Jun Matsubayashi,3 Naohiro Kajiwara,1 Masatoshi Kakihana,1 Masaru Hagiwara,1Masaaki Hibi,2 Koichi Yoshida,1 Junichi Maeda,1 Keishi Ohtani,1 Toshitaka Nagao,3 Kazuto Nishio2 and Norihiko
Ikeda1
1
3

Department of Surgery, Tokyo Medical University, Tokyo; 2Department of Genome Biology, Kindai University Faculty of Medicine, Osaka-Sayama;
Department of Anatomic Pathology, Tokyo Medical University, Tokyo, Japan

Key words
Cell-free DNA, mutation, non-small cell lung cancer,
sequencing, tumor volume
Correspondence
Kazuto Nishio, Department of Genome Biology, Kindai
University Faculty of Medicine, 377-2 Ohno-higashi,
Osaka-Sayama, Osaka 589-8511, Japan.
Tel: +81-72-366-0221; Fax: +81-72-367-6369;
E-mail: knishio@med.kindai.ac.jp
Funding Information
Applied Research for Innovative Treatment of Cancer(14525177) Ministry of Health, Labor, and Welfare ofJapan.
4

These authors contributed equally to this study.

Received April 7, 2016; Revised August 24, 2016; Accepted
August 27, 2016
Cancer Sci 107 (2016) 1660–1666
doi: 10.1111/cas.13068

Next-generation sequencing (NGS) and digital PCR technologies allow analysis of
the mutational profile of circulating cell-free DNA (cfDNA) in individuals withadvanced lung cancer. We have now evaluated the feasibility of cfDNA sequencing for mutation detection in patients with non-small cell lung cancer at earlier
stages. A total of 150 matched tumor and serum samples were collected from
non-small cell lung cancer patients at stages IA–IIIA. Amplicon sequencing withDNA extracted from tumor tissue detected frequent mutations in EGFR (37% of
patients), TP53 (39%), and KRAS (10%), consistent with previous findings. In contrast, NGS of cfDNA identified only EGFR, TP53, and PIK3CA mutations in three,five, and one patient, respectively, even though adequate amounts of cfDNA
were extracted (median of 4936 copies/mL serum). Next-generation sequencing
showed a high accuracy (98.8%) compared with droplet digital PCR for cfDNA
mutation detection, suggesting that the low frequency of mutations in cfDNA
was not due to a low assay sensitivity. Whereas the yield of cfDNA did not differ
among tumor stages, the cfDNA mutations were detected in seven patients atstages IIA–IIIA and at T2b or T3. Tumor volume was significantly higher in the
cfDNA mutation-positive patients than in the negative patients at stages T2b–T4(159.1  58.0 vs. 52.5  9.9 cm3, P = 0.014). Our results thus suggest that tumor
volume is a determinant of the feasibility of mutation detection with cfDNA as
the analyte.

L

ung cancer is the leading cause of death related to cancer
worldwide, with non-small cell lung cancer (NSCLC) being
the most common form of this disease.(1) Targeted therapeutics
such as tyrosine kinase inhibitors of the epidermal growth factor
receptor (EGFR) or anaplastic lymphoma kinase (ALK) haverecently been introduced into clinical practice for individuals
with NSCLC positive for actionable mutations of EGFR or ALK
fusions, respectively. Molecular profiling that is able to predictthe response to such drugs has thus become an important therapeutic strategy, allowing selection of the most appropriate treatment for individual patients.(2) This strategy is limited, however,by the difficulty of obtaining tumor specimens, the collection ofwhich often requires invasive procedures.
Sequencing of circulating cell-free DNA (cfDNA), a noninvasive approach to the detection of aberrant tumor-derived
DNA in blood, has the potential to allow early identificationand management of solid tumors as well as prediction of drug
sensitivity or resistance. Several studies have evaluated cfDNA
as a potential biomarker in NSCLC patients, who tend tohave a higher plasma cfDNA concentration than healthy
individuals.(3–5)
Tumors at an advanced stage often shed cfDNA into the circulation, and mutations in this cfDNA can be detected with

PCR-based(6–10) or sequencing-based(5,11–13) assays. Deep
sequencing of amplicons has proved feasible for the detectionof somatic mutations in cfDNA if the total number of reads
exceeds 300 000.(13) Digital PCR is also a highly sensitivetechnology that allows the detection of mutations in cfDNA
with a high accuracy relative to those in tumor cell DNA in
individuals with advanced lung cancer.(10) The clinicopathologic factors that are associated with the feasibility of mutation
identification in cfDNA remain unknown, however. We have
now compared the mutation profiles of surgically resected
tumor specimens from patients with NSCLC of stage IA to
IIIA with those of matched serum samples in order to ascertain
the feasibility of mutation detection in cfDNA at such early
disease stages as well as its determinant factors.

Cancer Sci | November 2016 | vol. 107 | no. 11 | 1660–1666© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and distribution
in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made.

Materials and Methods
Patients and specimen collection. Matched lung cancer tissue
and serum specimens were collected from 150 patients who
underwent surgery for NSCLC at Tokyo Medical University
Hospital (Tokyo, Japan) from January 2013 to July 2014. All
tissue samples were immediately flash-frozen in liquid nitrogenand stored at 80°C until analysis. Tumor volume was-440-Original Article
Ohira et al.

www.wileyonlinelibrary.com/journal/cas

calculated as length 9 width 9 height during surgery. Blood
samples were also collected during surgery and were centrifuged at 1400 g for 10 min, with serum being storedat 80°C until analysis. All patients provided written informed
consent to participate in the study, including the collection oftumor and serum specimens for analysis. The study protocol
was approved by the institutional ethics committees of Kindai
University Faculty of Medicine (Osaka-Sayama, Japan;
approval no. 25-135) and Tokyo Medical University Hospital(approval no. 2541).
DNA was isolated from the frozen tumor tissue with the useof an AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA,
USA). The quality and quantity of the DNA were verified withthe use of a NanoDrop 2000 device (Thermo Fisher Scientific,
Waltham, MA, USA) and PicoGreen dsDNA Assay Kit (LifeTechnologies, Foster City, CA, USA). Cell-free DNA was
purified from 0.52 to 1.0 mL serum with the use of a QIAamp
Circulating Nucleic Acid Kit (Qiagen), and its copy number
was determined with an RNaseP copy number assay (LifeTechnologies).
Sample processing. Sequencing analysis. Tumor DNA andcfDNA samples were subjected to analysis with next-generation
sequencing (NGS) panels for mutation detection. For library
preparation, tumor DNA (10 ng) and cfDNA (maximum of 3000
copies) were subjected to multiplex PCR amplification with theuse of an Ion AmpliSeq Library Kit 2.0 (Life Technologies) and
Ion AmpliSeq Colon and Lung Cancer Panel version 2 (LifeTechnologies), the latter of which targets mutational hotspot
regions of 22 cancer-associated genes: AKT1, ALK, BRAF,CTNNB1, DDR2, EGFR, ERBB2, ERBB4, FBXW7, FGFR1,
FGFR2, FGFR3, KRAS, MAP2K1, MET, NOTCH1, NRAS,PIK3CA, PTEN, SMAD4, STK11, and TP53. The PCR products
were then ligated to Ion Xpress Barcode Adapters (Life Technologies) and purified with the use of Agencourt AMPure XP
beads (Beckman Coulter, Brea, CA, USA). The purified libraries
were pooled and then sequenced with an Ion Torrent Proton
instrument, Ion Proton Hi-Q Sequencing Kit, and Ion PI version
3 Chip (all from Life Technologies). DNA sequencing data wereaccessed through the Torrent Suite version 4.4 program (LifeTechnologies). Reads were aligned with the hg19 humanreference genome, and variants were called with the use of Variant Call Format version 4.4. Samples with a median coverage of<1000 per amplicon were not accepted for evaluation. For
detection of mutations in tumor tissue DNA, raw variant calls
were filtered with the following annotations: quality score of<100 and depth of coverage of <19. Germline mutations were
excluded with the use of the Human Genetic Variation Database(http://www.genome.med.kyoto-u.ac.jp/SnpDB).(14) For cfDNA
mutation detection, the nucleotide count for each target baseposition from each allele count file in the variant calling report
was used. The presence of mutant nucleotides was determinedwith the use of Poisson distribution statistics. The nucleotide
count for each base position was applied to the Poisson function
with the use of Microsoft Excel 2013 (Redmond, WA, USA).
The value of the baseline error rate (non-reference nucleotide
count divided by the total nucleotide count) plus three standard deviations calculated from 10 normal blood DNA samples was used for the Poisson parameter lambda (k) ranging
from 0.00045 to 0.11859. If the nucleotide count of themutant was significantly higher (a = 2 9 105) than that of
the Poisson probability, the nucleotide was judged to have amutation-positive status. We established the a value with reference to a previous study.(11) The minimum detection limit
was calculated for a read number per site of 30 000 and a of

0.00002, with all values being shown in Table S1. The Ion
AmpliSeq Colon and Lung Cancer Panel version 2 isdesigned to amplify 92 amplicons covering 1205 hot spot
mutations. The theoretical minimum detection limit ranged
from 12.7% to 0.103% and was <0.5% for 84.4% (1017/
1205) of hot spot sites.
Digital PCR analysis. Digital PCR was carried out with a
QX100 Droplet Digital PCR System (Bio-Rad, Hercules,
CA, USA). The primers and probes for detection of the
EGFR L858R, KRAS G12C, and PIK3CA E545K and
H1047R mutations were obtained from Bio-Rad. The
cycling conditions included an initial incubation at 95°C for
10 min, 40 cycles of 94°C for 30 s and 55°C for 60 s, andenzyme inactivation at 98°C for 10 min. After thermal
cycling, the plates were transferred to a Droplet reader(Bio-Rad), and the digital PCR data were analyzed with the
Quanta Soft analytic software package (Bio-Rad). The cutoff values for mutations were determined with the use ofplasma cfDNA and normal blood DNA derived from 10
healthy volunteers. Each cut-off was set at 3 copies per one
reaction because no background noise (0 copies per one
reaction) was detected for any of the mutants in plasma
cfDNA and normal blood DNA derived from the 10 healthy
volunteers.(10)
Statistical analysis. Statistical analysis was carried out using
the two-tailed Student’s t-test (Prism software; GraphPad Software, San Diego, CA, USA). A P-value of < 0.05 was considered statistically significant.

Cancer Sci | November 2016 | vol. 107 | no. 11 | 1661

© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

Results
Somatic mutations detected in lung cancer tissue by NGS. Frozen surgically resected tumor tissue and matched serum
samples were obtained from 150 NSCLC patients (Table 1).

Table 1. Characteristics of patients with non-small
carcinoma (n = 150) who participated in this studycellCharacteristic

Classification

No. (%)Median (range)
<65
≥65
Male
FemaleYes
No
Unknown
IA
IB
IIA
IIB
IIIA
T1a
T1b
T2a
T2b
T3
T4
N0
N1
N2
NX

69 (23–85)
51 (34.0)
99 (66.0)
80 (53.3)
70 (46.7)
97 (64.7)
48 (32.0)
5 (3.3)63 (42.0)
32 (21.3)
20 (13.3)
10 (6.7)
25 (16.7)
39 (26.0)
36 (24.0)
44 (29.3)
10 (6.7)
20 (13.3)
1 (0.7)104 (69.3)
19 (12.7)
21 (14.0)
6 (4.0)Age, years

Sex
Smoking status

Pathological stage

Tumor size (T stage)

Lymph node metastasis(N stage)

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All tumor DNA samples were successfully sequenced with
the Ion Proton platform. The median read number per
amplicon was 15 632, with a range of 1272 to 323 280(Fig. S1). We identified TP53 mutations in 58 cases(38.7%), EGFR mutations in 56 (37.3%), KRAS mutations in
15 (10.0%), CTNNB1 mutations in 7 (4.7%), ERBB2,PIK3CA, BRAF, and PTEN mutations in 3 each (2.0%), and
ERBB4, MET, ALK, FGFR2, NRAS, AKT1, and FBXW7
mutations in 1 each (0.7%) (Fig. 1a). No mutation was
detected in 22.0% (33/150) of the samples. Mutation profiles
for patients harboring at least one mutation are shown in
Figure 1(b).
Somatic mutations detected in cfDNA by NGS. Serum cfDNA
was extracted for all 150 patients, with a median yield (copynumber) of 4936 (range, 572–373 658) (Fig. S2). A total of
149 of the 150 (99.3%) cfDNA samples were successfully
sequenced with the Ion Proton platform, with sequencing failure being due to an insufficient read number per amplicon(case no. 200, 197 reads) in the one unsuccessful case. The
median read number per amplicon for the 149 successfully
sequenced cfDNA samples was 33 982, with a range of 1563–
63 601 (Fig. S1).
We identified only a small number of non-synonymous
somatic mutations in cfDNA, including TP53 mutations in 5
cases (3.3%), EGFR mutations in 3 cases (2.0%), and a
PIK3CA mutation in 1 case (0.7%) (Fig. 1a). The mutation
profiles for patients harboring cfDNA mutations are shown in
Figure 1(b). Compared with tumor tissue samples, the sensitivity and specificity for mutation detection in serum were 5.8%
(9/155) and 100% (33/33), respectively, with an accuracy of
22.3% (42/188).

Accuracy of somatic mutation detection by NGS and digital
PCR. To exclude the possibility of false negative results gener-

ated by NGS, we applied digital PCR to confirm mutations
detected with NGS. The L858R mutation of EGFR, G12C of
KRAS, and E545K and H1047R of PIK3CA accounted for a
substantial proportion of the mutations detected in the tumor
samples with NGS. The 42 tumor samples determined to bepositive for these mutations by NGS and the paired serum
samples were therefore analyzed for the same mutations withdigital PCR (Table 2). For the 42 tumor samples, all of themutations detected by NGS were also detected by digital PCR.
For the corresponding serum samples, the two mutations(EGFR L858R in case no. 79 and PIK3CA E545K in case no.
65) detected by NGS were confirmed with digital PCR; only
one mutation (KRAS G12C in case no. 215) was detected in
the remaining 40 serum samples by digital PCR. Comparedwith digital PCR, the sensitivity and specificity of NGS for
mutation detection were 97.8% (44/45) and 100% (39/39),
respectively, with an accuracy of 98.8% (83/84).
A comparison of variant allele frequency revealed a highcorrelation (R2 = 0.992) between NGS and digital PCR for 30
tumor specimens positive for EGFR L858R (Fig. 2). We further examined the ability of NGS to detect mutant alleles bycomparing the fraction of variant reads for these 30 tumor
specimens positive for EGFR L858R with the corresponding
allele copy number determined by digital PCR. The twoparameters showed a good linear correlation (R2 = 0.893)(Fig. S3), suggesting that the power of NGS for mutation
detection was equivalent to that of digital PCR.
To clear the baseline error rate in NGS assay, we measured
the rate of two sample cohorts: 36 cfDNA samples in lungFig. 1. Mutation profiles for matched tumor and serum specimens of patients with non-small cell lung carcinoma who participated in thisstudy. (a) Incidence of mutations by gene in the study cohort as determined by next-generation sequencing with tumor DNA and serum cfDNA
samples. (b) Mutation profiles for the patients harboring at least one mutation. Black and red boxes indicate mutations detected in tumor samples alone or in both tumor and serum samples, respectively.
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Table 2. Concordance for detection of major somatic mutations intumor tissue and serum samples from patients with non-small celllung carcinoma using next-generation sequencing (NGS) and digital
PCR

Mutation

EGFR L858R

KRAS G12CPIK3CA E545K
PIK3CA H1047R

Case no.

55
66
71
74
79
82
86
103
105
114
117
118
126
135
142
143
155
156
161
164
168
171
176
177
192
204
208
209
212
220
73
78
120
123
149
162
194
206
215
65
184
138

NGS (% ofvariant reads)

Digital PCR(copy number)

Tumor

Serum

Tumor

Serum

16.3
45.8
24.2
33.4
100.0
18.5
35.7
43.6
15.1
26.8
23.6
15.6
41.8
26.2
10.4
7.8
25.6
28.7
19.9
18.0
33.5
27.9
32.2
57.1
4.3
49.0
35.7
25.7
11.8
4.5
4.9
24.2
6.6
15.8
4.2
12.8
30.9
12.0
32.4
58.6
31.2
24.9

n.d.
n.d.
n.d.
n.d.
0.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3.5
n.d.
n.d.

2520
4880
2280
2240
16660
1208
4160
3720
1150
1740
3040
1064
4000
1900
680
566
2340
2340
2000
1286
6240
2280
3320
8720
186
5460
3240
1580
1100
238
278
2200
858
1528
352
1228
2540
882
2560
11260
2660
1499

n.d.
n.d.
n.d.
n.d.
74
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4
43
n.d.
n.d.

Fig. 2. Correlation between next-generation sequencing (NGS) anddigital PCR data in a comparison between mutant allele frequency in
EGFR L858R (c.2573T>G) mutation positive tumors. The correlation
coefficient (R2) is indicated.

Allele frequency of EGFR L858R detected by digital PCR
assay was 0.00 in all but one cfDNA sample of patient withno mutations in tumor (Fig. S4). These results indicate that theallele frequency was lower than the baseline error rate of NGS
analysis.
Together, these results indicated that NGS performed wellfor mutation detection with both tumor tissue and serum
samples.
Clinicopathologic features of cfDNA mutation-positive patients.

n.d., not detected.

cancer patients with KRAS or EGFR exon 19 deletion mutation(KRAS/EGFRex19del) in tumor and 32 cfDNA samples inpatients without any mutation in the tumor. Median allele frequency of cfDNA samples at position EGFR L858R(chr7:55259515) of NGS assay was 0.00631 (range, 0–0.11177) and 0.01046 (range, 0–0.04392) in tumor KRAS/
EGFRex19del-positive and no mutation groups, respectively.
In addition, we examined the EGFR L858R mutation in
cfDNA samples by digital PCR assay in these two groups.
Cancer Sci | November 2016 | vol. 107 | no. 11 | 1663

The study subjects included patients with surgically resectable
NSCLC of stage IA–IIIA (Table 1), with more than half of thepatients having a disease stage of IA or IB (95/150, 63.3%).
We next examined the possible association of clinicopathologic features with serum cfDNA status. Determination of
DNA copy number for cfDNA by quantitative PCR analysis
revealed no statistically significant association between cfDNA
yield and either disease stage (Fig. S2b) or T factor (Fig. S2c).
The seven cases for which mutations were detected in cfDNA
by NGS included two patients at stage IIA (T2b/N0/M0), three
at stage IIB (two T3/N0/M0 and one T2b/N1/M0), and two atstage IIIA (T2b/N2/M0 and T3/N2/M0) (Fig. 3). Among
patients with mutation-positive tumors, mutations weredetected in cfDNA for 2 of 17 patients at stage IIA, 3 of 5
patients at stage IIB, and 2 of 20 patients at stage IIIA, withno mutations being detected in cfDNA of any patient at stage
IA or IB (Fig. 4a). With regard to T factor of the TNM classification, no mutation was detected in cfDNA of patients at
T1a–T2a, with all such mutations being found in those at T2b
or T3 (Fig. 4b). Mutations in cfDNA were detected forpatients at N0, N1, or N2 of the TNM classification (Fig. 4c).
These results thus suggested that detection of mutations in
cfDNA of patients with disease at stage IA or IB or at T2a orlower is difficult, and that the feasibility of mutation detection
with cfDNA may depend on the T factor rather than the N factor. We therefore compared tumor volume determined pathologically between patients positive or negative for cfDNA
mutation among the subpopulation of patients with mutationpositive tumors and a T factor of T2b–T4. Tumor volume inthe cfDNA mutation-positive group was significantly greaterthan that in the cfDNA mutation-negative group (159.1  58.0
vs. 52.5  9.9 cm3, P = 0.014) (Fig. 4d). The maximum© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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Fig. 3. Mutation profiles and clinicopathologic characteristics for 42 mutation-positive patients with non-small cell lung carcinoma with apathological stage of IIA–IIIA. Black and red boxes indicate mutations detected in tumor samples alone or in both tumor and serum samples,
respectively. cfDNA, cell-free DNA; MTD, maximum tumor diameter evaluated by diagnostic imaging; TV; tumor volume calculated during pathological examination.

tumor diameter calculated at diagnosis was also larger in the
cfDNA mutation-positive group than in the cfDNA mutationnegative group (5.3  0.7 vs. 4.1  0.3 cm, P = 0.050)(Fig. 4e). These results suggested that tumor volume is a determining factor for the feasibility of mutation detection with
cfDNA.

Discussion

Mutation profiling by NGS with tumor biopsy specimens has
recently become established, with EGFR, TP53, and KRAS
genes having been found to be frequently mutated in lung cancer specimens from Japanese patients.(2) In the present study,© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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Fig. 4. Relation between cfDNA mutation incidence and clinicopathologic features in patients with non-small cell lung carcinoma. (a) Incidenceof cfDNA mutations at each pathological stage for patients with mutation-positive tumors. (b) Incidence of cfDNA mutations for each T factor
among patients with mutation-positive tumors and a pathological stage of IIA–IIIA. (c) Incidence of cfDNA mutations for each N factor among
patients with mutation-positive tumors and a pathological stage of IIA–IIIA. (d) Dot plot for tumor volume in cfDNA mutation-positive (n = 7) or
cfDNA mutation-negative (n = 16) patients with mutation-positive tumors and a T factor of T2b–T4. (E) Dot plot for maximum tumor diameterat diagnosis in cfDNA mutation-positive (n = 6; unknown for one patient) and cfDNA mutation-negative (n = 16) patients with mutation-positive
tumors and a T factor of T2b–T4. Mean  SD values as well as P-values determined by Student’s t-test are also shown in (d,e).

mutations of EGFR, TP53, and KRAS were also frequently
detected in NSCLC specimens, with those in KRAS and EGFR
being mutually exclusive. Mutations in the genes analyzed
were detected in 78.0% of tumor samples, with the mutation
profiles being consistent with those observed previously.(15,16)
The purpose of the present study was to investigate themutation status for cfDNA isolated from serum of patientswith NSCLC at an early stage. In comparison with matchedtumor tissue, the sensitivity for mutation detection in serum by
NGS was low (5.8%), consistent with previous results.(17,18) It
thus appears to be difficult to detect mutations in cfDNA for
patients with NSCLC at an early stage. In contrast, previous
studies have reported a sensitivity of 70–80% for detection of
EGFR mutations in plasma DNA compared with matchedtumor DNA of patients with advanced NSCLC by beaming or
digital PCR.(5,8,9) We previously found the sensitivity for
detection of activating mutations of EGFR in plasma cfDNAof such patients by digital PCR to be 66.7%.(10)The feasibility of cfDNA sequencing is thought to be limitedby the copy number of the DNA extracted from serum or
plasma samples. The median copy number for the total of 150
samples in the present study was 4936, and library preparation
and NGS were successful for 149 of these samples (99.3%). It
is therefore unlikely that an insufficient amount of cfDNA was
responsible for the low mutation frequency of cfDNA in ourcohort. However, amplicons for NGS are derived from wildtype as well as variant alleles, with a low frequency of mutant
alleles compared with the wild-type allele making it difficult
to detect the former. The amplicon sequencing system wasthus a possible limiting factor in our NGS assay. We therefore
applied digital PCR to exclude this technical issue. The mutational status of cfDNA determined by NGS was confirmedwith digital PCR, with the two approaches showing a highconcordance. We therefore conclude that the NGS assay had
sufficient power to detect a low frequency of mutations inserum, and that the discordance between the results obtainedwith tumor DNA and serum cfDNA was not due to the sensitivity of the NGS technology. The proportion of serum cfDNA
that is derived from tumor cells thus appears to be much lower
for patients with NSCLC at the resectable stage than for thosewith such tumors at an advanced stage. Our findings are consistent with those reported for different types of cancer including primary brain, renal, prostate, and thyroid tumors.(19)We examined whether clinicopathologic features of thestudy patients might have influenced the mutation detection
rate with cfDNA. The seven cfDNA mutation-positive cases
detected by NGS were all stage T2b or higher, suggesting thattumor volume is related to the rate of mutation detection inserum. The relation between the T factor and mutation detection in cfDNA, including the clinical relevance of mutation
detection in cfDNA derived from patients with an advanced T
factor, even at an early pathological stage, requires further validation. Lymph node metastasis (N stage) did not appear to berelated to the rate of cfDNA mutation detection, suggestingthat the lymphatic system might not play a major role in the
spread of tumor cells into the circulation.
In conclusion, we found that 78.0% of resected NSCLC
tumors harbored at least one mutation as detected by NGS.
The incidence of cfDNA mutations in patients with early stage
NSCLC was extremely low, however. Next-generation
sequencing showed a high accuracy compared with digital
PCR for detection of mutations, indicating that NGS technology is similarly useful for the monitoring of mutant alleles.
Given the multiplex nature of NGS, it is likely to become
more widely adopted for monitoring of tumor progression andrecurrence with cfDNA as the analyte.

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Mutation detection with cell-free DNA in early-stage NSCLC

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Acknowledgments

Disclosure Statement

We thank K. Nagase, Y. Hosono, T. Miyazaki, and A. Kurumatanifor cooperation and assistance. This work was supported by
Applied Research for Innovative Treatment of Cancer (subject
no. 14525177 to K.N.) from the Ministry of Health, Labor, andWelfare of Japan.

M.H. is an employee of SRL Inc. (Tokyo, Japan). K.N. has received lecture fees from Chugai Pharmaceutical, Daiichi Sankyo, and Sumitomo
Bakelite. N.I. has received research grants from Ono Pharmaceutical,
Japan Mediphysics, Eli Lilly Japan, Pfizer, Taiho Pharmaceutical, Chugai
Pharmaceutical, and Bayer. The other authors have no conflict of interest.

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1 Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA Cancer J Clin
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5 Couraud S, Vaca-Paniagua F, Villar S et al. Noninvasive diagnosis ofactionable mutations by deep sequencing of circulating free DNA in lungcancer from never-smokers: a proof-of-concept study from BioCAST/IFCT1002. Clin Cancer Res 2014; 20: 4613–24.
6 Kimura H, Fujiwara Y, Sone T et al. EGFR mutation status in tumourderived DNA from pleural effusion fluid is a practical basis for predicting
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DNA for non-invasive detection of drug resistance mechanisms in EGFR

Supporting Information
Additional Supporting Information may be found online in the supporting information tab for this article:
Fig. S1. Median next-generation sequencing read number per amplicon for all 150 non-small cell lung carcinoma tumor and serum samples.
Fig. S2. Yield of cell-free DNA (cfDNA) for all 150 serum samples from patients with non-small cell lung carcinoma.
Fig. S3. Correlation between next-generation sequencing (NGS) and digital PCR data.
Fig. S4. Allele frequency at EGFR L858R position using next-generation sequencing and digital PCR assay.
Table S1. Poisson parameter (k) and theoretical minimum detection limit (MDL) for next-generation sequencing with the Ion AmpliSeq Colonand Lung Cancer Panel version 2 (Life Technologies, Foster City, CA, USA).

© 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltdon behalf of Japanese Cancer Association.

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Editorial

Comment on prognostic value of epidermal growth factor
receptor mutation subtypes in surgically resected non-small celllung cancer
Yujin Kudo1, Yoshihisa Shimada1, Hisashi Saji1,2, Norihiko Ikeda1
1

Department of Thoracic Surgery, Tokyo Medical University, Tokyo, Japan; 2Department of Chest Surgery, St. Marianna University School of

Medicine, Kanagawa, Japan
Correspondence to: Yujin Kudo, MD, PhD. Department of Thoracic Surgery, Tokyo Medical University. 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 1600023, Japan. Email: yjnkudo@gmail.com.
Provenance: This is an invited Editorial commissioned by the Section Editor Gang Shen, MMSC (The Second Afﬁliated Hospital Zhejiang University
School of Medicine, Hangzhou, China).
Comment on: Isaka T, Nakayama H, Yokose T, et al. Epidermal Growth Factor Receptor Mutations and Prognosis in Pathologic N1-N2 Pulmonary
Adenocarcinoma. Ann Thorac Surg 2016;102:1821-8.
Submitted Fed 27, 2017. Accepted for publication Feb 27, 2017.
doi: 10.21037/jtd.2017.03.62
View this article at: http://dx.doi.org/10.21037/jtd.2017.03.62

was lost after adjusting for other confounding prognostic
factors (4). Several other studies in the literature corroborate
this ﬁnding (5,6). Additionally, EGFR mutation status andthe use of EGFR-TKI therapy particularly affected the
postoperative recurrence survival (PRS), which is survival
after disease recurrence, among patients who underwent
surgical resection for lung cancers (7). The results could
also affect OS in general.
Exon 19 deletion (Ex19) and the L858R point mutationin exon 21 (Ex21) of EGFR comprise approximately 90%
of all EGFR mutation positive lung adenocarcinomas (8)and are strongly associated with robust responses to
EGFR-TKI. Patients with Ex19 advanced NSCLC haveconsistently shown improved outcomes with afatinib vs.
chemotherapy compared with those with Ex21 NSCLC (9).
The cause for these differences in response to EGFRTKIs among EGFR mutation subtypes is not known.
However, another trial that demonstrated survival curvesof patients with Ex19 and Ex21 advanced NSCLC showedno statistically significant differences between afatinib or
geﬁtinib and the EGFR mutation subtypes (10).
In the study by Isaka et al. published in the Annals ofThoracic Surgery, the authors demonstrated that different
EGFR mutation subtypes were associated with different
prognoses among patients with surgically resected
pathologic N1-N2 lung adenocarcinoma (11). This

Personalization of lung cancer treatment requires predictive
biomarkers that have been validated by correlation
between tumor features and outcomes after therapy.
Several mutations have been identified in the epidermal
growth factor receptor (EGFR) in non-small cell lungcancer (NSCLC). Mutations in this gene are considered
an important predictor of response to EGFR tyrosine
kinase inhibitors (TKIs) with 70–80% of NSCLC patients
receiving substantial beneﬁts from this targeted therapy (1).
The EGFR mutation is both a predictive and prognostic
factor of EGFR-TKI therapy outcome (1,2). Testing forthese mutations in all patients with recurrent or metastatic
lung adenocarcinoma is therefore recommended forstandard practice.
Several studies have reported that EGFR mutations
have an impact on prognosis after surgical resection of
NSCLCs (3,4). Izar et al. demonstrated that the mutation
status of EGFR can serve as an independent prognostic
marker associated with decreased recurrence and improved
progression-free survival and overall survival (OS) in patientswith stage I lung adenocarcinoma (3). This study was
restricted to patients who had no adjuvant or neoadjuvant
systemic therapy administered and focused solely on the
postoperative prognostic differences among different
genotypes. On the other hand, Kosaka et al. reported thatthe significant prognostic impact of EGFR mutations© Journal of Thoracic Disease. All rights reserved.

jtd.amegroups.com-447-

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E500

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Table 1 Summary of EGFR mutation subtypes (exon19 deletion vs. L858R point mutation in exon 21) as a prognostic factor in surgically resectedlung cancer patients

Authors

Journal

N (Ex19
vs. Ex21)

Multivariate analysis for
EGFR mutation subtypes(Ex19 vs. Ex21) as aprognostic factor

Stage

5 y-DFS, median DFS,
comment

5 y-OS, MST,
comment

3 y-OS; Ex19, 93.3%; No data
Ex21, 60.9% (P=0.01)

Ex19: better prognosis
W Liu, et al. (14) Med Oncol,
2014

30 vs. 23

Stage
I–IIIA

Ex19, 46.2 mo; Ex21,
21.9 mo (P=0.056)

T Isaka,
et al. (11)55 vs. 41

N1-2

Ex19, 38.8%; Ex21, 11.8% Ex19, 78.3%; Ex21,(P=0.001)
48.3% (P=0.123)

DFS: HR 2.25, P=0.011;
OS: HR 1.48, P=0.299

Ann Thorac
Surg, 2016

Ex21: better prognosis
YJ Lee,et al. (15)J Cancer Res
Clin Oncol,
2009

38 vs. 11

Stage
I–IIIA

Ex19 had higher
recurrence rate than Ex21
(HR 4.13, P=0.03)

No data

No data

T Nishi,
et al. (16)Asia Pac J Clin 55 vs. 85
Oncol, 2016

Stage INo significant difference inStage IA (P=0.681); DFS
better in Ex21 than in Ex19
in Stage IB (P=0.008)

No significant
difference in Stage IA
or IB (P=0.503 and0.134, respectively)

No data

T Okamoto,et al. (17)Anticancer
Research,
2016

27 vs. 45

Stage I–III

Ex19, 53.3%; Ex21, 84.4% Ex19, 95.0%; Ex21,(P=0.027)
84.8% (P=0.70)

H Shigematsu,
et al. (18)J Natl Cancer
Inst, 2005

31 vs. 31

Stage
I–IV

No data

No significant difference (P No data
value unknown)

No data

DFS: HR 0.41, P=0.067

Ex21 has betterNo datasurvival rate than Ex19
(P=0.05) in patients
without EGFR-TKI
therapy

No difference between Ex19 and Ex21
N Nose,et al. (19)J Clin Oncol,
2009

74 vs. 92

Stage
I–IV

Y Jin, et al. (20)

Scientific
Reports, 2016

53 vs. 51

Stage I–III

Ex19, 29.4 mo; Ex21, 25.7
mo (P=0.941, analysis
includes wild-type)

No data

No dataJL Marks,et al. (5)J Thorac
Oncol, 2008

19 vs. 19

Stage I–III

No data

No significant
difference (P=0.499)

No data

T Kosaka,
et al. (4)J Thorac
Oncol, 2009

65 vs. 80

Stage
I–IV

No data

No significant
difference (P=0.4144)

No data

K Sugio,
et al. (21)Br J Cancer,
2006

52 vs. 70

Stage
I–IV

No data

No significant
difference (P=0.5625)

No data

EGFR, epidermal growth factor receptor; Ex19, exon19 deletion of EGFR; Ex 21, L858R point mutation in exon 21 of EGFR; DFS, disease
free survival; MST, median survival time; HR, hazard ratio; mo, months; EGFR-TKI, EGFR tyrosine kinase inhibitor.

stage (stage II or III) were determined to not be statisticallyinformative prognostic factors for DFS using multivariate
analysis (HR 1.71; P=0.353, or HR 0.75; P=0.627,
respectively). Additionally, patients with Ex21 tumors

meaningful work showed that patients with Ex19 displayed
statistically significant better 5-year disease-free survival(DFS) rate (38.8%) than those with Ex21 (11.8%, P=0.001).
In this study, pathologic N status (N1 or N2) or pathologic© Journal of Thoracic Disease. All rights reserved.

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of the patients in other studies. Moreover, it might benecessary to interpret that DFS were affected by death fromother cause as well as lung cancer recurrence or death andby follow-up assessment schedule after operation.
The current study demonstrated the association between
EGFR mutation subtype and prognosis, especially DFS
among patients with pN1–N2 lung adenocarcinoma.
This study could provide additional evidence to support
EGFR mutation analysis for surgically resected lungadenocarcinoma. Large prospective and multicenter studies
with appropriate EGFR mutation detection methods
are warranted to validate the prognostic effect of EGFR
mutation subtypes.

displayed a signiﬁcantly higher prevalence of pN2 (70.7%)
than those with Ex19 tumors (45.5%, P=0.014). However,in this case, the EGFR mutation subtype was a prognostic
factor for DFS using multivariate analysis (HR 2.25; 95%
CI, 1.21–4.20, P=0.011). Therefore, they hypothesizedthat tumors with Ex19 might possess lower proliferative
potential, making them more susceptible to cell death thanthose tumors with Ex21.
Meanwhile, two conflicting in vivo assessments have beenreported. Carey et al. showed that the growth rate of theNR6-EGFR del [746-752] cell line was more aggressivein vivo than the NR6-EGFR L858R cell line (12). Thisdata might support the results and hypothesis of Isaka’s
study. However, Politi et al. demonstrated that lung tumors
from EGFR L858R expressing mice developed faster andexhibited a preponderance of lepidic growth with a moreaggressive nature compared with lung tumors derived
from EGFR del [L747-S752] expressing mice. It is to be
noted that both EGFR mutation subtypes promoted lungadenocarcinomas with lepidic growth features in transgenic
mice (13).
Table 1 summarizes ten studies that reported a
relationship between EGFR mutation subtypes andprognosis (4,5,11,14-21). In focusing on the association
between DFS or recurrence and EGFR mutation subtype,Liu et al. also reported that patients with Stage I-IIIA
lung adenocarcinoma harboring Ex19 had a better DFS
rate than those with Ex21 (P=0.056) (14). Conversely,
Okamoto et al. demonstrated that patients with Stage I-III
lung adenocarcinoma harboring Ex21 showed statisticallysigniﬁcant better DFS than those with Ex19 (P=0.027), andthat the EGFR mutation subtype was a potential prognostic
factor for DFS (HR 0.41, P=0.067) (17). In the study
reported by Nishi et al., whose institution was the same as
Isaka et al., the DFS rate of Stage IB lung adenocarcinoma
harboring Ex21 was better than that of Ex19 (P=0.008),
with no differences in DFS observed among patients withstage IA adenocarcinoma (P=0.681) (16). Furthermore,
several other groups reported there to be no significant
differences of DFS between tumors with Ex19 and thosewith Ex21 (19,20).
Possibilities for the discrepancies in survival might bedue to clinicopathological factors, studies containing small
cohorts of patients, or selection bias in the examinationof EGFR mutation analysis. For instance, Isaka et al.
determined the EGFR mutation status in their cohort andidentiﬁed mutations in 72.9% of 277 patients with pN1–2,
and EGFR mutation status was also examined for not all© Journal of Thoracic Disease. All rights reserved.

Acknowledgements
Funding: This work was supported by the Grant-in-Aid for
Scientific Research, Japan Society for the Promotion of
Science (24592104), Ministry of Education, Culture, Sports,
Science and Technology, Japan.
Footnote
Conflicts of Interest: The authors have no conflicts of interest
to declare.
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Kosaka T, Yatabe Y, Onozato R, et al. Prognostic
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Marks JL, Broderick S, Zhou Q, et al. Prognostic andtherapeutic implications of EGFR and KRAS mutations
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adenocarcinomas induced in mice by mutant EGF
receptors found in human lung cancers respond to atyrosine kinase inhibitor or to down-regulation of thereceptors. Genes Dev 2006;20:1496-510.
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15. Lee YJ, Park IK, Park MS, et al. Activating mutations
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16. Nishii T, Yokose T, Miyagi Y, et al. Prognostic value of
EGFR mutations in surgically resected pathological stage I
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18. Shigematsu H, Lin L, Takahashi T, et al. Clinical andbiological features associated with epidermal growth factor
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19. Nose N, Sugio K, Oyama T, et al. Association between
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factor receptor mutation in the postoperative prognosis ofadenocarcinoma of the lung. J Clin Oncol 2009;27:411-7.
20. Jin Y, Chen M, Yu X. Differences among lesions with exon
19, exon 21 EGFR mutations and wild types in surgicallyresected non-small cell lung cancer. Sci Rep 2016;6:31636.
21. Sugio K, Uramoto H, Ono K, et al. Mutations within thetyrosine kinase domain of EGFR gene speciﬁcally occur
in lung adenocarcinoma patients with a low exposure of
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2008;3:111-6.
Kim YT, Seong YW, Jung YJ, et al. The presence ofmutations in epidermal growth factor receptor gene is not
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7. Kudo Y, Shimada Y, Saji H, et al. Prognostic Factors for
Survival After Recurrence in Patients With Completely
Resected Lung Adenocarcinoma: Important Roles of
Epidermal Growth Factor Receptor Mutation Status
and the Current Staging System. Clin Lung Cancer
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8. Shigematsu H, Gazdar AF. Somatic mutations of epidermal
growth factor receptor signaling pathway in lung cancers.
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10. Paz-Ares L, Tan EH, O'Byrne K, et al. Afatinib versus
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11. Isaka T, Nakayama H, Yokose T, et al. Epidermal Growth
Factor Receptor Mutations and Prognosis in Pathologic
N1-N2 Pulmonary Adenocarcinoma. Ann Thorac Surg
2016;102:1821-8.
12. Carey KD, Garton AJ, Romero MS, et al. Kinetic analysis
of epidermal growth factor receptor somatic mutant
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Cite this article as: Kudo Y, Shimada Y, Saji H, Ikeda N.
Comment on prognostic value of epidermal growth factor
receptor mutation subtypes in surgically resected non-small celllung cancer. J Thorac Dis 2017;9(5):E499-E502. doi: 10.21037/
jtd.2017.03.62© Journal of Thoracic Disease. All rights reserved.

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J Thorac Dis 2017;9(5):E499-E502

Original Article

Prognostic value of tumor solid-part size and solid-part volume inpatients with clinical stage I non-small cell lung cancer
Yoshihisa Shimada1, Hideyuki Furumoto1, Kentaro Imai1, Ryuhei Masuno2, Jun Matsubayashi3,
Naohiro Kajiwara1, Tatsuo Ohira1, Norihiko Ikeda1
1

Department of Thoracic Surgery, 2Department of Radiology, 3Department of Anatomic Pathology, Tokyo Medical University, Tokyo, Japan

Contributions: (I) Conception and design: Y Shimada; (II) Administrative support: N Ikeda; (III) Provision of study materials or patients: Y Shimada,
H Furumoto, K Imai, R Masuno, J Matsubayashi; (IV) Collection and assembly of data: Y Shimada, H Furumoto, K Imai; (V) Data analysis andinterpretation: Y Shimada; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.
Correspondence to: Yoshihisa Shimada, MD, PhD. Department of Thoracic Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku,
Tokyo 160-0023, Japan. Email: zenkyu@za3.so-net.ne.jp.

Background: This study aimed to predict the malignant potential of clinical stage I non-small celllung cancer (c-I NSCLC) by semiautomatic three-dimensional (3D) volumetric measurement of a tumor(3D-data) and the axial computed tomography (CT) data derived from a 3D volumetric dataset (2D-data).
The predictive performance was evaluated in terms of overall survival (OS), disease-free survival (DFS), andpathological invasive factors (positive lymphatic invasion, blood vessel invasion, pleural invasion, or lymph
node metastasis).
Methods: We identified 252 patients (122 male; mean age, 68 years; range, 23–84 years) with c-I NSCLC
who underwent high resolution CT and reconstruction of 3D imaging, followed by complete resection
between January 2012 and December 2015. In this study, 2D-data including whole tumor size (WTS) andsolid-part size (SPS) and 3D-data including whole tumor volume (WTV) and solid-part volume (SPV)
acquired by a 3D volume rendering software were analyzed.
Results: The area under the receiver operating characteristic (ROC) curve for WTS, SPS, WTV, SPV
relevant to recurrence was 0.667, 0.727, 0.654, and 0.751 while analyses of ROC curves revealed optimal
WTS, SPS, WTV, and SPV cut-off values to predict recurrence of 2.48 cm, 2.03 cm, 3,258 mm 3 and1,889 mm3, respectively. The association between SPS and SPV was the coefficient of determination(R2) =0.59. Multivariate analysis showed that SPV >1,889 mm3 (P=0.016) and male (P=0.041) were significant
predictors of OS whereas SPV >1,889 mm3 (P=0.001), male (P=0.003), and the serum carcinoembryonicantigen value (P=0.041) were significantly correlated with DFS. SPS, SPV as well as the combination of
SPS and SPV were all significantly correlated with the prediction of OS and DFS, and the incidence of
pathological invasive factors.
Conclusions: SPV and the integrated use of SPS and SPV was highly beneficial for the prediction of
postoperative prognosis in c-I NSCLC.
Keywords: Non-small cell lung cancer (NSCLC); volume; solid-part; three-dimensional CT (3DCT); stage I
Submitted Mar 09, 2018. Accepted for publication Oct 11, 2018.
doi: 10.21037/jtd.2018.11.08
View this article at: http://dx.doi.org/10.21037/jtd.2018.11.08

Introduction

prognosis by evaluating the high-resolution computed

Recent advances in imaging technology in the field of lungcancer have enabled the stratification of peripheral nonsmall cell lung cancer (NSCLC) patients according to theirtomography (HRCT) features of their lung tumor (1-5).

© Journal of Thoracic Disease. All rights reserved.

The ratio of the maximum consolidation diameter to the
maximum tumor diameter is widely accepted for predicting

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Shimada et al. Solid-part size and volume of stage I NSCLC

emission tomography/computed tomography (FDG-PET/
CT). The protocols for data collection and analysis wereapproved and the need to obtain written informed consent
from each patient was waived by the Institutional Review
Board of Tokyo Medical University.

the outcome of patients who underwent not only pulmonary
resection but also stereotactic body radiotherapy (4,6).
Patients with a smaller proportion of solid-part size in lungcancer on HRCT had a significantly lower recurrence rate
regardless of the operative procedures, suggesting that the
separate measurements of the solid-part size and the wholetumor size are highly beneficial for tailored treatment
planning (2,4). Although decreased CT-measured solid-partsize of lung cancer, especially adenocarcinoma, is reported
to be a better prognostic factor, small lung lesions with
an irregularly shaped solid part in the greatest dimension
are often encountered and can cause intraobserver andinterobserver variability in practice.
To address the issue, we preoperatively take advantageof three-dimensional (3D) visualization using volume
rendered imaging technique (3D-VRT) not only for
surgical simulation but also for possible prediction of thepathobiological features of a lung tumor and prognostic
outcomes by conducting volumetric quantification of
the solid and non-solid parts separately using a 3DCT
workstation (Fujifilm Synapse Vincent system; Fujifilm
Corp., Tokyo, Japan) (7-11). Semiautomatic volumetricmeasurement of a tumor presents a pixel-wise segmentationof the 3DCT volume depending on the attenuation ofeach single voxel. As the calculation of the solid-part andnon-solid-part volume based on 3D-VRT is consistent,
the volumetric quantification is more likely to reflect thepatient’s prognosis.
This study aimed to investigate the clinical outcomesand pathological malignant grade of patients with resectedclinical stage I NSCLC by comparing preoperative HRCT
and 3D-VRT oriented radiologic parameters such as solidpart size and solid-part volume.

HRCT and 3DCT evaluation for primary tumors
The patients underwent contrast-enhanced CT imaging
using a 64-channel multidetector CT system (Light Speed
VCT, GE Medical systems, Milwaukee, WI, USA). Axial
sections (1.25 mm in thickness) of the whole lung werereconstructed at intervals of 1.0 mm, and images wereviewed on standard lung windows (level, −600 HU; width
1,500 HU) with the sharp kernel of the version “LUNG”
and mediastinal windows (level, 30 HU; width 400 HU).
Each CT image was acquired within 1 breath hold of about
5 s, after a delay of 70 s during which the contrast media
injection took effect.
HRCT scans were evaluated by at least 2 surgeons(Y Shimada, H Furumoto, K Imai) and 1 radiologist(R Masuno) in consensus. We defined the soli-part size as
the maximum dimension of the solid component of thelung windows excluding non-solid-part and measured the
maximum diameter of tumor on axial CT slices (Figure 1).
Non-solid-part representing ground glass opacity wasdefined as a misty increase in lung attenuation that did not
obscure the underlying vascular markings.
The presented CT scan protocol has been used forboth standard staging for lung cancer patients amenable
to contrast radiography and 3D image conversion using
the Synapse Vincent system. After digital imaging andcommunication in medicine data were transferred to aworkstation using the indicated software, 3D-VRT were
performed (Figure 1). In terms of volumetric measurementof a lung tumor, a density level of −300 HU is the thresholdlevel that separate solid-part from non-solid-part by thesoftware algorithm. This 3D tumor analysis provided thewhole tumor volume, solid-part volume, and non-solid-partvolume separately. The semiautomated volumetric analysis
performed by the software needed to be adjusted manually.
The results were confirmed in consensus (2 or 3 surgeons,Y Shimada, H Furumoto, K Imai, 1 radiologist, R Masuno).

Methods
Patients
We identified 252 patients with clinical stage I NSCLC
who underwent complete resection between January
2012 and December 2015 at our department. HRCT and
3DCT were preoperatively performed for all patients using
the Fujifilm Synapse Vincent system. The Tumor Node
Metastasis (TNM) stage was determined in accordancewith the 8th edition of the TNM classification of malignant
tumors (12). Preoperative evaluation included physicalexamination, chest radiography, chest and abdomen CT,
blood examination, and F-18 fluorodeoxyglucose-positron© Journal of Thoracic Disease. All rights reserved.

Definition of pathological invasive factors and patient
outcomes
The available pathology reports for all the 252 surgicaljtd.amegroups.com-452-

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A

6493

B

Whole tumor volume: 1273 mm3Solid-part volume: 268 mm3

Figure 1 Representative comparative images of a lesion’s whole tumor size and solid-part size on high-resolution computed tomography(HRCT), and whole tumor volume and solid-part volume measured by the Synapse Vincent system. An HRCT image presenting wholetumor size of 1.28 cm and solid-part size of 0.42 cm (A), and a three-dimensional (3D) volume rendered images of a tumor presenting wholetumor volume of 1,273 mm3 and solid-part volume of 268 mm3 with serial sections of the display (B). The 3D volume rendered images resultin the detection of a solid-part with red and a ground glass opacity part with green.

comparison was performed using the Pearson chi-squared
test for discrete data and Student t-test for continuous data.
Pearson correlation coefficient was used to determine thecorrelation between solid-part size and solid-part volume
and the value of the coefficient of determination (R2) was
also provided. HRCT and 3DCT parameters measured bythree observers were recorded, and interobserver variability
by using a random sample of 100 patients was calculated for
intraclass correlation coefficient (ICC). Univariate analysis
for OS and DFS was performed using the Cox proportional
hazards regression model whereas multivariate analysis was
conducted with a stepwise variable selection for the Coxmodel. All tests were two-sided, and P values of less than
0.05 were considered to indicate a statistically significant
difference. SPSS statistical software package (version 24.0;
DDR3 RDIMM, SPSS Inc., Chicago, IL, USA) was used
for statistical analysis.

specimens were reviewed in this study. Blood vessel invasion(V) was identified by the presence of erythrocytes in thelumen or an endothelial cell lining, or the presence ofelastic tissue around large vessels while lymphatic invasion(Ly) was determined to be present when tumor cells floating
in lymphatic vessels with no supporting smooth muscles
or elastic fibers were identified. Resected lymph nodes
were sectioned thicker than 2 mm and correctly embedded
to ensure that the pathologists can identify all macrometastases. At least single or 2 sections from blocks wereprepared for diagnosis. Pathological invasive factors were
defined as positive Ly, V, pleural invasion (PL), or lymph
node metastasis. Overall survival (OS) was measured fromthe date of surgery to the date of death from any cause or
the date on which the patients were last known to be alive.
Disease-free survival (DFS) was measured from the date ofsurgery until the first event (relapse or death from any cause
or the last follow-up visit).

Results
Statistical analysis

The characteristics of the patients were shown in Table 1.
The median follow-up time for survivors was 1,233 days
(range, 16 to 2,227 days). During the study period,
recurrences occurred in 29 patients (12%), some of whom
eventually died from these relapses (n=12, 5%). Only
1 patient died from subarachnoid hemorrhage during thestudy period. The clinical TNM stage showed 186 patients

The receiver operating characteristic (ROC) curves of eachparameter were used to predict recurrence. The area under
the ROC curve (AUC) with its 95% confidence interval(CI) was measured. OS and DFS curves were plotted using
the Kaplan-Meier method, and differences in variables
were determined using the log-rank test. Categorical© Journal of Thoracic Disease. All rights reserved.

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Shimada et al. Solid-part size and volume of stage I NSCLC

Table 1 Patient characteristics
Variable
Age, mean [range] (years)

Value (n=252) (%)
15000

68 [23–84]

R2=0.59Male

122 [48]

Female130 [52]

Solid-part volume (mm3)SexcTNM stage
Stage IA

186 [74]

Stage IB66 [26]

10000

5000

Lymphatic invasion
Positive

83 [33]

Negative

169 [67]0
0.0

Blood vessel invasion
Positive81 [32]

Negative

171 [68]

1.0

2.0

3.0

4.0

Figure 2 Correlation between solid-part size and solid-part volume
as determined using Pearson correlation coefficient (R2 =0.59).

Pleural invasion
Positive48 [19]Negative

204 [81]tumor volume, and solid-part volume were 2.18 cm,
1.56 cm, 3,063 mm 3 and 1,275 mm 3, respectively. The
pathological findings indicated that the numbers of patientswith Ly, V, PL, and lymph node metastasis were 83 (33%),
81 (32%), 48 (19%), and 34 (13%), respectively.
We analyzed the correlation between solid-part size andsolid-part volume using Pearson correlation coefficient(R2 =0.59; Figure 2), and this coefficient value showed astrong correlation. Interobserver agreements for volumetricmeasurement of a tumor by the dedicated software(ICC =0.977) was higher than the measurement of solidtumor size on HRCT (ICC =0.925, Table 2).
To investigate the impact of HRCT and 3D volumetricparameters for prognostic outcomes, we analyzed the ROC
curves for recurrences (Figure 3). AUC and the optimal
cut-off value relevant to recurrence for whole tumor size,
solid-part size, whole tumor volume, and solid-part volume
were 0.667 and 2.48 cm (Figure 3A), 0.727 and 2.03 cm(Figure 3B), 0.654 and 3,258 mm3 (Figure 3C), and 0.751
and 1,889 mm 3 (Figure 3D), respectively. Univariate
and multivariate analyses were performed to examine
the association between OS or DFS and clinical factors
including HRCT and 3D volumetric radiologic features(Tables 3,4). Univariate analysis identified the following as
significant predictors of unfavorable OS: age (P=0.032),
male (P=0.019), the presence of smoking history (P=0.037),
whole tumor volume >3,258 mm 3 (P=0.044), and solidpart volume >1,889 mm3 (P=0.008). Multivariate analysis

Lymph node metastasis
Positive

34 [13]

Negative

218 [87]

Histology
Adenocarcinoma

221 [88]

Non-adenocarcinoma

31 [12]Whole tumor size,
range (mean ± SD) (cm)0.50–4.00 (2.23±0.75)

Solid-part size,
range (mean ± SD) (cm)

0–3.91 (1.67±0.99)

Whole tumor volume,
range (mean ± SD) (mm3)51–24,393 (4,239±3,975)

Solid-part volume,
range (mean ± SD) (mm3)0.4–16,344 (2,350±2,761)

TNM, pathological tumor, node and metastasis; SD, standard
deviation.

(74%) with stage IA and 66 patients (26%) with stage
IB. The mean whole tumor size, solid-part size, wholetumor volume, and solid-part volume were 2.23±0.75 cm,
1.67±0.99 cm, 4,239±3,975 mm3, and 2,350±2,761 mm3
while the median whole tumor size, solid-part size, whole© Journal of Thoracic Disease. All rights reserved.

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Table 2 Analysis of interobserver reproducibility of solid-tumor size and solid-tumor volume
Parameter

Patients, n

ICC (95% CI)

P value

Solid-tumor size on HRCT

100

0.925 (0.881–0.956)<0.001

Solid-tumor volume by volumetric measurement with 3D-VRT

100

0.977 (0.959–0.987)<0.001

ICC, intra-class correlation coefficient; CI, confidence interval; HRCT, high-resolution computed tomography; 3D-VRT, three-dimensional
volume rendered imaging techniques.

A

B

100

Sensitivity (%)

Sensitivity (%)

100

50

50

AUC =0.727
95% CI: 0.645−0.809
Cut-off value: 2.03 cm

AUC =0.667
95% CI: 0.571−0.764
Cut-off value: 2.48 cm
0

C

0

50
100-specificity (%)

D

50

0

50
100-specificity (%)

100

50

AUC =0.654
95% CI: 0.558−0.751
Cut-off value: 3,258 mm3050
100-specificity (%)

100

Sensitivity (%)

100

Sensitivity (%)

0
0

100

AUC =0.751
95% CI: 0.671−0.831
Cut-off value: 1,889 mm3
0

100

0

50
100-specificity (%)

100

Figure 3 The area under the receiver operating characteristic (ROC) curve for recurrence as determined using whole tumor size (A),soli-part size (B), whole tumor volume (C), and solid-part volume (D) in patients with stage I non-small cell lung cancer patients. AUC, area
under the curve; CI, confidence interval.

>3,258 mm3 (P=0.005), and solid-part volume >1,889 mm3(P<0.001). Multivariate analysis revealed the following as
independently associated with DFS (Table 4): sex (P=0.003),
the serum CEA level (P=0.041), and solid-part volume>1,889 mm3 (P=0.001).
We further assessed whether the integrated use of bothsolid-part size and solid-part volume was beneficial forthe prediction of postoperative prognosis. Patients wererevealed the following as independently associated with OS(Table 3): sex (P=0.041) and solid-part volume >1,889 mm3(P=0.016). Univariate analysis identified the following as
significant predictors of unfavorable DFS: sex (P=0.001),
the presence of smoking history (P=0.001), the serum
carcinoembryonic antigen (CEA) value (P=0.001), stage
IB (P=0.027), whole tumor size >2.48 cm (P=0.001),
solid-part size >2.03 cm (P<0.001), whole tumor volume© Journal of Thoracic Disease. All rights reserved.

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Table 3 Univariate and multivariate analyses for overall survival
Univariate analysis

Variable

Multivariate analysis

Hazard ratio (95% CI)

P value

Hazard ratio (95% CI)

P valueAge

1.083 (1.007–1.166)

0.032

–

–

Sex (male vs. female)

6.060 (1.340–27.027)

0.019

4.852 (1.069–22.016)

0.041

Smoking history (yes vs. no)4.953 (1.098–22.349)

0.037

–

–

CEA

1.055 (0.959–1.160)

0.272

–

–

cTNM stage (IB vs. IA)

1.255 (0.386–4.079)

0.705

–

–

Whole tumor size (>2.48 vs. ≤2.48 cm)

2.730 (0.893–8.347)

0.078

–

–

2.907 (0.951–8.889)

0.061

–

–

Whole tumor volume (>3,258 vs. ≤3,258 mm )3.756 (1.033–13.548)

0.044

–

–

Solid-part volume (>1,889 vs. ≤1,889 mm3)

7.733 (1.714–34.889)

0.008

6.452 (1.420–71.190)

0.016

Solid-part size (>2.03 vs. ≤2.03 cm)
3Table 4 Univariate and multivariate analyses for disease-free survival
Univariate analysis

Variable

Multivariate analysis

Hazard ratio (95% CI)

P value

Hazard ratio (95% CI)

P valueAge

1.030 (0.990–1.071)

0.141

–

–

Sex (male vs. female)

4.632 (1.893–11.332)

0.001

3.965 (1.614–9.740)

0.003

Smoking history (yes vs. no)4.739 (1.815–12.346)

0.001

–

–

CEA

1.094 (1.040–1.150)

0.001

1.064 (1.002–1.129)

0.041

cTNM stage (IB vs. IA)

2.237 (1.086–4.608)

0.027

–

–

Whole tumor size (>2.48 vs. ≤2.48 cm)

3.571 (1.669–7.634)

0.001

–

–

3.861 (1.805–8.264)<0.001––3.226 (1.435–7.246)

0.005

–

–

5.952 (2.433–14.493)<0.0014.464 (1.783–11.236)

0.001

Solid-part size (>2.03 vs. ≤2.03 cm)
3Whole tumor volume (>3,258 vs. ≤3,258 mm )3

Solid-part volume (>1,889 vs. ≤1,889 mm )CI, confidence interval; CEA, carcinoembryonic antigen; TNM, pathological tumor, node and metastasis.

OS (P=0.050; Figure 4A) and DFS (P<0.001; Figure 4D)
whereas solid-part volume was also significantly correlatedwith OS (P=0.002; Figure 4B) and DFS (P<0.001; Figure 4E).
Subgroup analysis with the combination revealed that the
4-year OSs of 96.6%, 91.2%, and 87.4% and the 4-year
DFSs of 93.4%, 88.4%, and 73.4% for group A, B, andC, respectively (Figure 4C,F and Table 5). The differencesin OS were statistically significant between group A and
C (P=0.005), and between group A and B (P=0.018). The
differences in DFS between group A and C was statisticallysignificant (P<0.001) while that between group A and B was
marginally significant (P=0.093). Together, group C had themost unfavorable OS and DFS compared to group A, B as

divided into three groups; group A, patients with solid-partsize ≤2.03 cm and solid-part volume ≤1,889 mm3; groupB, patients with either solid-part size >2.03 cm or solidpart volume >1,889 mm 3; group C, patients with solidpart size >2.03 cm and solid-part volume >1,889 mm3. The
predictive performance of the pathological invasive factors,
and OS and DFS outcomes by using the cut-off values were
demonstrated in Table 5. Both solid-part size (P<0.001) andsolid-part volume (P<0.001) showed a significant correlation
with the incidence of pathological invasive factors. Group C
also exhibited high proportion of invasive factors comparedto Group A (P<0.001). The Kaplan-Meier curves for OS
and DFS (Figure 4) showed that solid-part size influenced© Journal of Thoracic Disease. All rights reserved.

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Table 5 Prognostic impact of preoperative computed tomographic imaging factors
Invasive factor (negative/positive)

Variable (n=252)

N [%]Solid-part size, cm

P value

4-year OS%<0.001

≤2.03
>2.03

P value

4-year DFS
%

0.050

<0.001111 [69]/50 [31]

95.2

93.6

30 [33]/61 [67]

88.7

76.1

3

Solid-part volume, mm<0.001

0.002<0.001≤1,889104 [72]/41 [28]

96.8

93.8

>1,88937 [35]/70 [65]

87.4

75.9

Integrated use of solid-partsize and volume

A vs. B: P<0.001;
A vs. C: P<0.001;
B vs. C: P=0.536

P value

A vs. B: P=0.018;
A vs. C: P=0.005;
B vs. C: P=0.845

A vs. B: P=0.093;
A vs. C: P<0.001;
B vs. C; P=0.129A

100 [75]/34 [25]

96.6

93.4

B

15 [39]/23 [61]

91.2

88.4

C

26 [33]/54 [67]

87.4

73.4

Invasive factor: positive lymphatic invasion, vascular invasion, pleural invasion, or lymph node metastasis. OS, overall survival; DFS,
disease-free survival; A, patients with solid-part size ≤2.03 cm and solid-part volume ≤1,889 mm3; B, patients with either solid-partsize >2.03 cm or solid-part volume >1,889 mm3; C, patients with solid-part size >2.03 cm and solid-part volume >1,889 mm3.

surgical procedures since all these factors are responsible forincreased recurrence rate and unfavorable prognosis (12-17).
A greater number of studies have shown a carefulobservation of HRCT-based features such as the calculationof consolidation to tumor ratio of lung cancer, and paying
attention to the solid-part size of small peripheral lungcancer helps in predicting prognosis and pathologicalmalignancy (2-4,18-20). At present, conducting HRCT
before any treatment to evaluate small peripheral NSCLC
is mandatory as the maximum diameter of the solid-partserves as a determinant for the latest TNM classification.
Although those parameters are very important, it isoccasionally difficult to even judge whether the lesion ofinterest is a predominantly solid or non-solid-predominant
tumor because of the irregularity of the solid-part shape.
A 3D virtual lung surgery planning workstation provides
surgeons with an effective tool for safe and rational
surgery (7-9). In terms of its extensive role, 3D-VRT
provides not only visual information for surgical simulations
but also quantitative information such as the volume of thelungs and tumor. Takenaka et al. reported that the solidpart volume as quantified by the Synapse Vincent system
was a prominent predictive factor for DFS compared withthe whole tumor volume or solid-part size on HRCT (21).
Comparative analysis of interobserver reproducibility ofwell as individual use of either solid-part size or sold-partvolume.
Discussion
In this study, we evaluated the survival outcomes andassociation between CT variables and pathological findings
by comparing the predictive performance of whole tumor
size, solid-part size, whole tumor volume, and solidpart volume in patients with clinical stage I NSCLC.
Multivariate analyses for the prediction of unfavorable OS
and DFS showed that solid-part volume had a significant
correlation with both outcomes. The combined use ofsolid-part size and solid-part volume also contributed to theexcellent stratification of patients on prognosis.
The ability to preoperatively estimate survival outcomes
and the presence of prognostic pathological factors on
imaging modalities is crucial to the determination of
the appropriate treatment strategy. Lymphatic invasion
and blood vessel invasion in patients with NSCLC havebeen reported to be a strong independent predictor forrecurrence (13-16). Preoperative prediction of pathologicalinvasive factors in the current study including lymphatic
invasion, blood vessel invasion, pleural invasion, and lymph
node metastasis is of importance to select appropriate© Journal of Thoracic Disease. All rights reserved.

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Shimada et al. Solid-part size and volume of stage I NSCLC

B

P=0.05000365 730 1095 1460 1825
Time (days)

Disease-free survival probability

52
30

E
≤2.03 cm
>2.03 cm50

P<0.00100

365 730 1095 1460 1825
Time (days)

At risk≤2.03 cm 161 155 149 99>2.03 cm 91 80 71 51

50
26≤1889 mm
>1889 mm350

P=0.002
0
0

15
10

94
66

48
34

F
≤1,889 mm3>1,889 mm350

P<0.00100

365 730 1095 1460 1825
Time (days)

At risk≤1,889 mm3 145 140 135>1,889 mm3 107 95 85

92
58

46
30

B
C

50

0

A vs. B; P=0.018
A vs. C; P=0.005
B vs. C; P=0.845
0365 730 1095 1460 1825
Time (days)

At riskA
134 133 129B
38 36 32C
80 78 71

15
12

100

A

100

365 730 1095 1460 1825
Time (days)

At risk≤1,889 mm3 145 142 137>1,889 mm3 107 104 94

15
12

100

100

Overall survival probability

50

At risk≤2.03 cm 161 159 152 101>2.03 cm 91 87 79
59

D

Overall survival probability≤ 2.03 cm> 2.03 cm

3

15
10

Disease-free survival probability

100

C

Disease-free survival probability

Overall survival probability

A

88
20
53

48
5
30

15
1
12

100
B

A
C

50
A vs. B; P=0.093
A vs. C; P<0.001
B vs. C; P=0.129
00

365 730 1095 1460 1825
Time (days)

At riskA
134 131 127
B
38 34 31C
80 71 63

86
20
45

46
5
26

15
1
10

Figure 4 Overall survival (OS) and disease-free survival (DFS) curves of patients with clinical stage I non-small cell lung cancer. All thepatients were divided into three groups due to the cut off values of solid-part size and solid-part volume; group A, patients with solid-partsize ≤2.03 cm and solid-part volume ≤1,889 mm3; group B, patients with either solid-part size >2.03 cm or solid-part volume >1,889 mm3;group C, patients with solid-part size >2.03 cm and solid-part volume >1,889 mm3. (A,B) OS curves according to solid-part size (A) andsolid-part volume (B) that were dichotomized at each cut-off value, 2.03 cm and 1,889 mm3; (C) OS curves according to the three groups.
(D,E,F) DFS curves according to solid-part size (D), solid-part volume (E), and the three groups (F).

solid-part volume in the current study (Figure 2), solidpart size and solid-part volume resulted in the confounding
factors one another. That could be the reason why solidpart size was not independent predictive factors for OS
and DFS on our multivariate analyses. The measurementof solid-part size on HRCT is supposed to be fundamental
to determine T factor, and simpler and easier method
to predict postoperative survival and recurrence. On thecontrary, although surgical simulations by 3D-VRT offer us
to do better surgical planning and more deeply understandlung cancer pathobiology, this process can be time- andcost-intensive compared with the conducting HRCT alone.
Therefore, the proposed application of this data to clinicalsolid-tumor size and volume (Table 2) revealed that theworkstation semiautomatically provided reproducible
and consistent volumetric information of the tumor ofinterest with ICC of 0.977. Even though the volumetricquantification of a tumor by 3D-VRT is time consuming
in contrast to tumor size measurements on conventional
axial CT slices, the measurement of solid-part volume
for estimating prognostic outcomes such as OS, DFS,
and pathological invasive factors supports the successfulplanning of an appropriate surgical strategy and cancontribute to individualized surgical approach including theindication of limited resection for stage I NSCLC. Since
there was a strong correlation between solid-part size and© Journal of Thoracic Disease. All rights reserved.

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scenarios of early-staged lung cancer unless 3D-VRT
can be performed routinely would be to add volumetricquantification of lung tumor for a tailored surgical approach
when the shape of the solid-part in greatest dimension on
HRCT is too irregular and heterogenous to measure inconsensus.
The limitations of this study are its retrospective natureand potential bias. The cut-off values of the ROC curves
that dichotomized the two groups could be an arbitrarily
value and not available universally. We measured tumor
volume by initially connecting the end-to-end dimensionsof a tumor with a straight line in an axial plane on theworkstation, which was similar to the methodology on
HRCT. However, once we delineate the end-to-end line,
whole tumor volume, solid-part volume, and non-solid-partvolume were separately generated semiautomatically withexcellent interobserver agreement.
In conclusion, solid-part volume was significantly
associated with the prediction of OS, DFS, and thepresence of pathological invasive factors. The measurementof not only solid-part size but also solid-part volume isuseful in patients with clinical stage I NSCLC when themeasurement of the solid-part on HRCT is too complicated
due to the chance of inconsistent or irregular-shaped
findings.

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Ann Thorac Cardiovasc Surg 2013;19:1-5.
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and segmentectomy: results of preoperative assessment of
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surgerydagger. Eur J Cardiothorac Surg 2014;46:e120-6.
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based on high-quality three-dimensional lung modellingfrom computed tomography images. Interact Cardiovasc
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10. Furumoto H, Shimada Y, Imai K, et al. Prognostic impactof the integration of volumetric quantification of the solidpart of the tumor on 3DCT and FDG-PET imaging inclinical stage IA adenocarcinoma of the lung. Lung Cancer
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2.

Acknowledgements
The authors are indebted to the medical editors of the
Department of International Medical Communicationsof Tokyo Medical University for editing the English
manuscript. We also thank Ms. Mami Murakami for helping
with the statistical analyses of this work.
Footnote
Conflicts of Interest: The authors have no conflicts of interest
to declare.
Ethics Statement: This study was approved by theInstitutional Ethics Board of Tokyo Medical University (No.
3907).
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Cite this article as: Shimada Y, Furumoto H, Imai K, Masuno
R, Matsubayashi J, Kajiwara N, Ohira T, Ikeda N. Prognostic
value of tumor solid-part size and solid-part volume in patientswith clinical stage I non-small cell lung cancer. J Thorac Dis
2018;10(12):6491-6500. doi: 10.21037/jtd.2018.11.08© Journal of Thoracic Disease. All rights reserved.

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J Thorac Dis 2018;10(12):6491-6500

Original Article

Page 1 of 10

Clinicopathological characteristics and treatment strategies foryoung lung cancer patientsR e m i Yo n e y a m a 1 , 2 , H i s a s h i S a j i 1 , 3 , Ya s u f u m i K a t o 1 , 4 , Yu j i n K u d o 1 , 5 , Yo s h i h i s a S h i m a d a 1 ,Masakazu Kimura2, Masaru Hagiwara1, Masatoshi Kakihana1, Kuniharu Miyajima1,2, Naohiro Kajiwara1,
Tatsuo Ohira1, Harubumi Kato2, Norihiko Ikeda1
1

Department of Surgery, Tokyo Medical University, Tokyo, Japan; 2Department of Thoracic Surgery, Niizashiki Central General Hospital, Saitama,

Japan; 3Department of Chest Surgery, St. Marianna University School of Medicine, Kanagawa, Japan; 4Department of Thoracic Surgery, Kanto
Central Hospital, Tokyo, Japan; 5Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston,
TX, USA
Contributions: (I) Conception and design: H Saji; (II) Administrative support: H Saji; (III) Provision of study materials or patients: R Yoneyama; (IV)Collection and assembly of data: R Yoneyama; (V) Data analysis and interpretation: H Saji; (VI) Manuscript writing: All authors; (VII) Final approvalof manuscript: All authors.
Correspondence to: Remi Yoneyama, MD, PhD. Department of Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku, Tokyo 160-0023,
Japan. Email: remiyone@tokyo-med.ac.jp.

Background: The reported age-specific survival rates of lung cancer patients have been largely
inconsistent. Management strategies for younger patients and treatment outcomes are not well characterized.
Methods: Out of the 4,697 lung cancer patients with treatment history at Tokyo Medical University
Hospital between January 2000 and December 2014, 266 patients were <49 years of age. Patientcharacteristics were investigated, and the association of overall survival (OS) with age, sex, stage, andhistological type were investigated.
Results: The 1-, 3-, and 5-year survival rates in the ≤49 years age group were 82.9%, 64.6%, and 57.0%.
Among surgical cases, the survival rate of patients in the ≤49 years age group was significantly better thanthat in the 50–69 and ≥70 years age groups (P=0.29 and P<0.0001, respectively). In comparison with the OS
rate with clinical stages, I, II, and III (but not with clinical stage IV) in the older than 50 years age group,
the rates in the ≤49 years age group were better. The 1-, 3-, and 5-year OS rates of females were higher thanthose of their males. The 1-, 3-, and 5-year OS rates for lung adenocarcinoma patients were higher than thatof lung non-adenocarcinoma patients.
Conclusions: Despite the higher proportion of advanced disease, the postoperative survival rate of theyounger was higher than that of the older. Aggressive multimodality treatments, including surgery, are morefeasible and effective for younger patients as compared with that in older patients.
Keywords: Young lung cancer patients; lung cancer; survival; adenocarcinoma
Submitted Sep 09, 2018. Accepted for publication Jan 16, 2019.
doi: 10.21037/atm.2019.01.69
View this article at: http://dx.doi.org/10.21037/atm.2019.01.69

Introduction

Labor, and Welfare, an estimated 146,000 people sufferedfrom respiratory cancer and 73,396 deaths were attributed

Lung cancer is one of the leading causes of cancer-related
deaths worldwide regardless of sex (1). In the US, it is aparticularly prominent disease in the elderly population;
people <45 years of age account for <2% of all cases of lungcancer (2). According to the Japanese Ministry of Health,© Annals of Translational Medicine. All rights reserved.

to respiratory cancer in 2014. According to a Japanese
Lung Cancer Registry Study in 2004, patients younger than
50 years (data available since 1994) (3) accounted for
between 5.0% and 8.2% of all lung cancer patients who

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Page 2 of 10

and the World Health Organization classification (11).
The hospital charts of all patients were reviewed to collectclinicopathological data, including age; sex; smoking
history; clinical T, N, and M factors; histological type;surgical procedures; epidermal growth factor receptor(EGFR) mutation status; and overall survival (OS). OS wasdetermined as the duration from the day of initial diagnosis
until the day of death from all causes. Data pertaining
to patients who were alive and showed no evidence ofrecurrence at the end of the follow-up period were censoredfrom the analysis.

underwent surgical resection. Several studies have shownthat post-therapeutic survival rates in younger patientswith lung cancer are higher than those in their older
counterparts (4-6); however, other studies have shown
comparable survival outcomes among patients in different
age groups (7,8). Some investigators have suggested that thecourse of lung cancer in young patients is more aggressive
than that in older patients (9). Owing to the small numberof young patients with lung cancer, their clinicopathologicalcharacteristics are not well characterized; moreover,there is no consensus on the most effective management
strategies for these patients. Therefore, this study aimed toelucidate the clinicopathological characteristics and the best
therapeutic strategies for lung cancer in young patients andcompared them with elderly patients.

Statistical analysis
Categorical variables are presented as frequencies andproportions, whereas continuous variables are presented
as mean ± standard deviation. Patient characteristics
were compared using the chi-squared test or Fisher exact
test for categorical outcomes of continuous variables, as
appropriate. OS was estimated using the Kaplan-Meiermethod, and between-group differences were assessed with
log-rank test. P values and hazard ratios in the multivariate
analyses were calculated using the Cox regression model.
Between-group differences associated with P values <0.05
were considered statistically significant. All statisticalanalyses were performed using SPSS statistical software(version 21.0; SPSS, Inc., Chicago, IL, USA).

Methods
Patient selection
We retrospectively reviewed 4,733 consecutive patients withpathologically confirmed lung cancer at the Tokyo Medical
University Hospital from January 2000 to December 2014.
Of these, 36 patients who did not undergo therapeutic
management were excluded. The remaining 4,697 patientswere divided into the following three groups by age:
<50 years (n=266), between 50 to 69 years (n=2,360), and≥70 years (n=2,071). The diagnosis and treatment of nonsmall cell lung cancer (NSCLC) and small cell lung cancer(SCLC) were performed in accordance with the thenguidelines of the Japan Lung Cancer Society. The protocols
for data collection and analyzes were approved by theinstitutional review board of the Tokyo Medical University.
The requirement for informed consent from the patientswas waived off due to the retrospective nature of the study.
Confidentiality of patient data was maintained throughout
the study.

Results
Patient characteristics

Pretreatment evaluation and treatment management
included physical examination, blood examination, chest
radiography, and chest and abdomen computed tomography(CT). Brain CT or magnetic resonance imaging andpositron emission tomography-CT were performed if
clinically indicated. Staging and pathological findings forlung cancer were determined according to the seventh
TNM Classification for Lung and Pleural Tumors (10)Clinicopathological characteristics of patients with lungcancer from 2000 to 2014, disaggregated by age groups(≤49, 50–69, and ≥70 years) are shown in Table 1. In the≤49 years age group, there were 169 (63.5%) men and 97(36.5%) women; no significant between-group difference
was observed with respect to sex distribution (P=0.809).
Approximately 80% of patients with therapeutic lung cancer
in the ≤49 and 50–69 years age groups had performancestatus 0–1, which was better than that of patients in the≥70 years age group. A majority of the patients in all threegroups were consulted us because of an abnormal chest
shadow detected on routine health checkup. The proportionof “never smokers” in the ≤49 years age group (36.5%) was
significantly higher than that in the other age groups. The
proportion of patients with stable disease in the ≤49 years
age group ranged from 5.4% to 5.9%. Regarding clinical© Annals of Translational Medicine. All rights reserved.

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Table 1 Characteristics of the patients (n=4,697)
Variables≤49 years old (n=266)

50–69 years old (n=2,360)≥70 years old (n=2,071)

n

n

n

%

%

%

Sex

P
0.809

Female

97

36.5

820

34.7

733

35.4

Male

169

63.5

1,540

65.3

1,338

64.6

Performance status

0.0001

0

188

70.7

1,706

72.3

1,345

64.9

1

24

9.0

210

8.9

358

17.3

2

4

1.5

31

1.3

47

2.3

3/4

1

0.4

21

0.9

14

0.7

No data

49

18.4

392

16.6

307

14.8

Symptoms

0.822

Present

66

24.8

516

21.9

429

20.7

Absent

16

6

152

6.4

141

6.8

Abnormal shadow in routine health check

164

61.7

1,502

63.6

1,330

64.2

No data

20

7.5

190

8.1

171

8.3

Current smoker

68

25.6

726

30.8

465

22.5

Ex-smoker

78

29.3

827

35

855

41.3

Never smoker

97

36.5

592

25.1

580

28.0

No data

23

8.6

215

9.1

171

8.3

Smoker0.0001

Year of diagnosis

0.444

2000–2004 (n=1,166)

63

5.4

601

51.5

502

43.1

2005–2009 (n=1,917)

108

5.6

979

51.1

830

43.3

2010–2014 (n=1,614)

95

5.9

780

48.3

739

45.8

Clinical stage

0.053

Stage I

131

49.2

1,134

48.1

1,003

48.4

Stage II

10

3.8

228

9.7

218

10.5

Stage III

69

25.9

573

24.3

494

23.9

Stage IV

56

21.1

425

18.0

356

17.2

Pathology

0.0001

Adenocarcinoma

202

75.9

1,551

65.7

1,195

57.7

Squamous cell carcinoma

18

6.8

453

19.2

529

25.5

Large cell carcinoma

0

0

15

0.6

13

0.6

Non-small cell carcinoma, NOS

24

9.0

187

7.9

164

7.9

Adenosquamous cell carcinoma

2

0.8

12

0.5

16

0.8

Small cell carcinoma

4

1.5

128

5.4

147

7.1

Carcinoid

9

3.4

11

0.5

6

0.3

Others

7

2.6

3

0.1

1

0

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Table 1 (continued)
Variables≤49 years old (n=266)

50–69 years old (n=2,360)≥70 years old (n=2,071)

n

n

n

%

%

%

EGFR mutation status

0.327

Exon 19 deletion

24

0.9

128

5.4

82

4.0

Exon 21 L858R mutation

15

5.6

141

6.0

112

5.4

Minor mutation

4

1.5

13

0.6

11

0.5

Wild type

67

25.2

388

16.4

305

14.7

Unknown

156

58.6

1,690

71.6

1,561

75.4

Initial therapy

0.0001

Surgery

110

41.4

884

37.5

870

42.0

Surgery plus adjuvant therapy

42

15.8

506

21.4

266

12.8

Induction therapy plus surgery

16

6.0

88

3.7

26

1.3

Surgical procedures

0.0001

Lobectomy

146

84.4

1,298

85.6

978

82.4

Sublobar resection

20

11.6

149

9.8

180

15.2

Pneumonectomy

2

1.2

31

2.0

9

0.8

Others

5

2.9

39

2.6

20

1.7

98

36.8

882

37.4

909

43.9

Chemotherapy or others

P

NOS, not otherwise specified; EGFR, epidermal growth factor receptor.

stage, 125 patients in the ≤49 years age group had advanced
stage tumors at presentation, which exhibited a trend ofbeing higher than that in the other groups (P=0.053). The
percentage of adenocarcinoma and carcinoid tumors was
significantly higher in the group aged ≤49 years whereasthat of squamous cell carcinoma was significantly lower
compared to the other groups. Squamous cell carcinoma
was higher in the group aged ≥70 years than in the younger
groups. Furthermore, we analyzed EGFR mutation status
in a limited number of patients; the rate of patients with
an EGFR mutation (particularly exon 19 deletion) in the≤49 years age group was significantly higher than thatin the other groups (12). Regarding initial therapeuticstrategies, patients in the ≥70 years age group underwent
more nonsurgical treatments as compared with those in theyounger age groups (P=0.0001). Subsequently, sublobar
resection and other less invasive surgical procedures weremore frequently selected in these patients (P=0.0001).

The median follow-up time of all patients was 43.1 months(range, 0.3–189.0 months). The median follow-up timein the ≤49, 50–69, and ≥70 years age groups was 46.3,
46.6, and 38.7 months, respectively. The median followup time in the ≥70 years age group was significantly lower
than that in the ≤49 and 50–69 years age groups (P=0.001
and P=0.045). The 1-, 3-, and 5-year survival rates in the≤49 years age group were 82.9%, 64.6%, and 57.0%(Figure 1A). The 1-, 3-, and 5-year survival rates of patientsin the ≤49 years age group who underwent surgery were
97.6%, 88.2%, and 81.5%, respectively (Figure 1B). The 1-,3-, and 5-year survival rates of patients in the ≤49 years age
group who did not undergo surgery were 55.9%, 21.1%,
and 12.8%, respectively (Figure 1C).
The postoperative survival rate in the ≤49 age group was
significantly higher than the other 50–69 and ≥70 years age
groups (P=0.29 and P<0.0001, respectively) (Figure 1B). In
the entire study population, although the survival rate inthe ≤49 years age group was significantly higher than that inthe ≥70 years age group (P=0.0001), it was not significantly
different from that in the 50–69 years age group (P=0.439)(Figure 1A). Moreover, among patients who did not undergo
surgery, the survival rate of those in the ≤49 years age group© Annals of Translational Medicine. All rights reserved.

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Annals of Translational Medicine, Vol 7, No 5 March 2019

A

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B

All cases

Surgical cases

1.0

0.8

0.8

Overall
Overallsurvival
survival

Overall
Overallsurvival
survival

1.0

0.6

0.6

0.4

0.4

0.2

0.2

P=0.0001

0.0
0.000

20.00040.000Time (months)

60.00080.0001-yearsurvival

3-yearsurvival

5-yearsurvival≤49-years old
n=266

82.9%

64.6%

57.0%

+

50–69-years old
n=2,360

84.6%

64.9%

55.3%

+

≥70-years old
n=2,070

78.9%

56.2%

46.3%

C

0.000

Time (months)+

P=0.0001

0.0
20.00040.00060.00080.000Time(months)
Time (months)

100.000

1-yearsurvival

3-yearsurvival

5-yearsurvival+≤49-years old
n=168

97.6%

88.2%

81.5%

+

50–69-years old
n=1,478

96.2%

86.0%

77.3%

+

≥70-years old
n=1,161

93.6%

80.5%

70.6%

Non-surgical cases

1.0

Overall
Overallsurvival
survival

0.8

0.6

0.4

0.2
P=0.066

0.0

0.000 10.000 20.000 30.000 40.000 50.000 60.000
Time (months)

Time (months)
1-yearsurvival

3-yearsurvival

5-yearsurvival+≤49-years old
n=168

55.9%

21.1%

12.8%

+

50–69-years old
n=1,478

64.2%

27.0%

15.7%

+

≥70-years old
n=1,161

59.6%

24.0%

15.0%

Figure 1 Kaplan-Meier curves for 1-, 3-, and 5-year overall survival of lung cancer patients according to (A) all patients (P=0.0001), (B)surgical cases (P=0.0001), and (C) non-surgical cases in the age group of ≤49, 50–69, and ≥70.

was not significantly different from the survival rates of
those in the other age groups (P=0.066) (Figure 1C).
The detailed survival data of patients with NSCLC ofdifferent TNM stages are summarized in Figure 2. From thepoint of clinical staging, 1-, 3-, and 5-year survival rates ofpatients with stage I lung cancer in the ≤49 years age groupwere 98.5%, 95.0%, and 88.4%, respectively (Figure 2A).
The 1-, 3-, and 5-year survival rates of patients with stage© Annals of Translational Medicine. All rights reserved.

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Yoneyama et al. Outcome of young lung cancer patients

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A

B

0.8

0.8

Overall Overall
survival
survival

B1.0

Overall Overall
survival
survival

A 1.0
0.6

0.6

0.4

0.4

0.2

0.2

P=0.0001

0.0
0.000

20.00040.00060.00080.0001-yearsurvival

3-yearsurvival

5-yearsurvivalTime(months)Time(months)

Stage I

P=0.0001

0.0+≤49-years old
n=131

98.5%

95.0%

88.4%

+

50–69-years old
n=1,134

97.9%

90.4%

82.9%

+

≥70-years old
n=1,003

95.2%

83.3%

73.0%

C

0.000

20.00040.00060.00080.0001-yearsurvival

3-yearsurvival

5-yearsurvival

Time Time(months)(months)

Stage II+≤49-years old
n=10

100.0%

90.0%

90.0%

+

50–69-years old
n=228

90.1%

74.3%

63.8%

+

≥70-years old
n=217

86.8%

62.9%

49.0%

D

C1.0

1.0

0.8

OverallOverall
survival
survival

OverallOverall
survival
survival

0.8

0.6
0.4
0.2
0.0

P=0.0001
0.000

20.000Stage III40.00060.000TimeTime(months)(months)

0.6

0.4

0.2
P=0.1200.0

80.0001-yearsurvival

3-yearsurvival

5-yearsurvival+≤49-years old
n=69

76.6%

44.4%

30.3%

+

50–69-years old
n=573

77.3%

41.3%

28.7%

+

≥70-years old
n=494

64.5%

26.5%

16.5%

0.000

20.00040.000Time
Time(months)(months)

Stage IV

60.0001-yearsurvival

3-yearsurvival

5-yearsurvival+≤49-years old
n=56

50.1%

8.4%

5.6%

+

50–69-years old
n=425

54.5%

19.3%

8.2%

+

≥70-years old
n=356

46.8%

13.3%

8.1%

Figure 2 Kaplan-Meier curves for 1-, 3-, and 5-year overall survival of lung cancer patients according to (A) stage 1 (P=0.0001), (B) stage 2(P=0.0001), (C) stage 3 (P=0.0001), and (D) stage 4 in the age group of ≤49, 50–69, and ≥70.

II lung cancer in the ≤49 years age group were 100.0%,
90.0%, and 90.0%, respectively (Figure 2B). The 1-, 3-, and5-year survival rates of patients with stage II lung cancer inthe ≤49 years age group were 76.6%, 44.4%, and 30.3%,

respectively (Figure 2C). The 1-, 3-, and 5-year survival
rates of patients with stage IV lung cancer in the ≤49 years
age group were 50.1%, 8.4%, and 5.6%, respectively(Figure 2D). The OS rates of lung cancer patients with

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Annals of Translational Medicine, Vol 7, No 5 March 2019B

Sex and age

Histology and age

1.0

1.0

0.8

0.8
Overall survival

Overall survival

A

Page 7 of 10

0.6
0.4

0.6
0.4
0.2

0.2
P=0.0001

0.0

0.000

0.000 20.000 40.000 60.000 80.000 100.000
Time (months)
1-yearsurvival

3-yearsurvival

5-yearsurvival+≤49-years old (women)
n=97

84.3%

74.9%

65.1%

+

≤ 49-years old (men)
n=169

80.9%

59.1%

52.6%

+

50-69-years old (women)
n=820

91.4%

77.7%

66.5%

+

50-69-years old (men)n=1,540

80.8%

58.0%

49.3%

+

≥70-years old (women)
n=733

87.8%

69.2%

60.1%

+

≥70-years old (men)n=1,337

73.8%

56.2%

48.9%

P=0.0001

0.0
20.00040.000 60.000
Time (months)

80.000100.000

1-yearsurvival

3-yearsurvival

5-yearsurvival+≤49-years old (women)
n=202

86.9%

64.9%

56.9%

+

≤ 49-years old (men)
n=64

70.1%

63.3%

57.2%

+

50-69-years old (women)
n=1,551

88.1%

73.0%

63.1%

+

50-69-years old (men)
n=809

77.7%

58.0%

49.2%

+

≥70-years old (women)
n=1,194

84.7%

66.2%

56.5%

+

≥70-years old (men)
n=876

70.8%

42.1%

32.2%

Figure 3 Kaplan-Meier curves for 1-, 3-, and 5-year overall survival of lung cancer patients according to (A) sex and age (P=0.0001) and (B)histology and age (P=0.0001) in the age group of ≤49, 50–69, and ≥70.

clinical stages I, II, and III in the ≤49 years age group were
better than those of their counterparts older than 50 years.
Regarding sex differences, the 1-, 3-, and 5-year OS rates
in females were higher than that in males (Figure 3A).
The 1-, 3-, and 5-year OS rates of patients withadenocarcinoma were better than that of patients with nonadenocarcinoma. In the non-adenocarcinoma group, the 3and 5-year OS rates of younger patients were higher thanthose of older patients (Figure 3B).

Lung cancer mostly occurs in patients of older age (13).
Therefore, lung cancer in young patients is recognized tobe relatively rare and to comprise a distinct oncologicalentity.
Owing to the reported increase in the incidence oflung cancer among young patients in Europe and Japan(14,15), there is an urgent need to characterize younglung cancer patients. Several studies have compared theclinicopathological features and prognoses of young
NSCLC patients of different ages (9,16-19). In general,
epidemiological observations have been explained by thetime period required for the environmental toxins to induce
genetic damage that ultimately leads to cancer. Therefore,
many studies have suggested that lung cancer in the young
may constitute a distinct clinicopathological entity with adifferent sex distribution, stage at diagnosis, pathologicalfeatures, and prognosis, although the reported data arefrequently discordant (3,8,9,16-20).
At our institution, a total of 266 patients in the ≤49 years
age group were registered between 2000 and 2014, which
accounts for 5.7% of all cases (Table 1). This is consistent
with a previous nationwide Japanese cohort (6) that was
stable over the last 15 years. The young patients included
169 men and 97 women; the male:female ratio of 1.74:1(Table 1) is similar to that in a Japanese nationwide cohort
reported in 2004 (3), and comparable with that in the older
age groups in the present study. We also found that thehistological type in almost 80% of the younger patients was© Annals of Translational Medicine. All rights reserved.

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adenocarcinoma (Table 1). It is possible that there are lesscases of smoking-related squamous carcinoma or small cell
carcinoma in young patients.
The therapeutic strategy is sometimes constrained inolder patients because of existing comorbidities, which may
not be the case with younger patients.
Regarding the clinical TNM staging of young lungcancer patients in this series, there were 131 stage I, 10 stageII, 69 stage III, and 56 stage IV patients; the proportionof patients with stages III–IV advanced lung cancer was
higher than that in the older age groups (P=0.053) (Table 1).
In comparison with elderly patients, it has suggested thatlung cancer in young patients tends to be more aggressive
in nature; however, the performance status of these young
patients tends to be better (6,19,21,22).
The present study suggests that surgical resection was
more frequently selected as the first-line treatment foryounger patients as compared with that in the other age
groups. The higher rates of neoadjuvant chemotherapy
in the younger group may reflect the higher proportionof patients with operable stage III advanced disease,which is a prime indication for induction therapy (6);moreover, young patients tend to show better tolerance
for such treatment owing to better functional status oforgans. The rate of neoadjuvant chemotherapy in theyounger group was more than two-fold higher thanthat in the older group. Radzikowska et al. also reported
similar results with more aggressive treatment in younger
patients (4). These results indicate that planned active
multimodal strategies in younger patients result in good
performance status. In addition, among patients who
underwent surgical resection, the 5year survival rates inthe ≤49 years age patients group was the highest at 81.5%;
the corresponding rates in the 50–69 and ≥70 years age
groups were significantly lower at 77.3% and 70.6%,
respectively (P=0.0001) (Figure 1B). Therefore, moreaggressive multimodality treatments, including surgery,are feasible for young patients with lung cancer and arehighly recommended in our daily practice.
The tendency for a higher survival rate in female
patients and in patients with adenocarcinoma was similar
to that seen with the analyses of all ages (6) (Figure 3).
Adenocarcinoma in situ showing ground-glass opacity on
CT, which is currently classified as adenocarcinoma in situ
or minimally invasive adenocarcinoma (23), is generally
considered as a slow-growing, low-grade adenocarcinoma
and a unique subtype associated with a never-smoking
history and female sex (24). Although CT data were notanalyzed in the present study cohort, higher proportionsof adenocarcinoma and females in the young patient group
might affect the patients' characteristics, with a higher rateof adenocarcinoma in situ with lower malignant behavior.

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Limitations
This single institutional study of a large number ofsurgically and non-surgically treated lung cancer patientshas several limitations. As it is a retrospective study, we
cannot clarify the prognostic effects of multimodal therapy
in young patients owing to limited comprehensive data,
including data pertaining to chemotherapy regimens ormolecular targeted therapy during their entire treatment
course. Other limitations are the persistence of patientselected treatment bias and time-to-detection bias that may
have worsened prognostic outcomes in elderly patients.
Moreover, we could not analyze the treatment outcomes
after recurrence in the present study; young patients withgood performance status might have more chance to receive
secondary or more chemotherapy in combination withradiation or immunotherapy. Further prospective, multiinstitutional investigations and substantial clinical analyzes
are required to clarify the cause underlying the better
prognosis of younger patients who receive treatment ascompared with patients in the other age groups (25).
Conclusions
We retrospectively reviewed 4,697 consecutive patients withpathologically confirmed lung cancer who received surgicaland nonsurgical treatment at the Tokyo Medical University
Hospital between January 2000 and December 2014.
We compared the clinicopathological characteristics andsurvival outcomes of younger patients and older patients.
We interestingly found better postoperative survival ofthese young lung cancer patients, although the proportionof patients with advanced disease was higher compared withthat in the older group.
Clinical practice points
 Especially to postoperative patients, the survival rate is
better than older group.
 Longer-term follow-up is essential for primary cancer
and associating health care problems (26-28).
 Data storage system including specimen sample is needed
expecting to treatment options in future.

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Acknowledgements

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in young patients. Respir Med. 1992;86:499-502.
19. Subramanian J, Morgensztern D, Goodgame B, et al.
Distinctive characteristics of non-small cell lung cancer(NSCLC) in the young: a surveillance, epidemiology, andend results (SEER) analysis. J Thorac Oncol 2010;5:23-8.
20. Thomas A, Chen Y, Yu T, et al. Trends and Characteristics
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whole tumor size and solid component size on highresolution computed tomography in the prediction of

Medical English writing assistance was provided by Crimson
Interactive Pvt. Ltd. The authors are fully responsible forthe content and editorial decisions for this manuscript.
This study was supported by a Grant-in-Aid for Scientific
Research, from the Japan Society for the Promotion of
Science, Ministry of Education, Culture, Sports, Scienceand Technology, Japan.
Footnote
Conflicts of Interest: The authors have no conflicts of interest
to declare.
Ethical Statement: The protocols for data collection andanalyzes were approved by the institutional review boardof the Tokyo Medical University. The requirement for
informed consent from the patients was waived off due tothe retrospective nature of the study.
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prescription rates of anxiolytics and hypnotics tosurvivors of cancer in childhood, adolescence, and young
adulthood-A population-based study. Pediatr Blood
Cancer 2018;65:e26860.

the degree of pathologic malignancy and the prognostic
outcome in primary lung adenocarcinoma. Acta Radiol
2015;56:1187-95.
24. Kudo Y, Matsubayashi J, Saji H, et al. Association
between high-resolution computed to-mography findings
and the IASLC/ATS/ERS classification of small lungadenocarcinomas in Japanese patients. Lung Cancer
2015;90:47-54.
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Practice Guidelines in Oncology. J Natl Compr Canc
Netw 2018;16:66-97.
26. Loren AW, Mangu PB, Beck LN, et al. Fertility

Cite this article as: Yoneyama R, Saji H, Kato Y, Kudo Y,
Shimada Y, Kimura M, Hagiwara M, Kakihana M, Miyajima
K, Kajiwara N, Ohira T, Kato H, Ikeda N. Clinicopathologicalcharacteristics and treatment strategies for young lungcancer patients. Ann Transl Med 2019;7(5):100. doi: 10.21037/
atm.2019.01.69© Annals of Translational Medicine. All rights reserved.

atm.amegroups.com-470-

Ann Transl Med 2019;7(5):100

Received: 16 January 2019

| Revised: 28 March 2019 | Accepted: 2 April 2019

DOI: 10.1111/cas.14016

ORIGINAL ARTICLE

Performance of Oncomine Fusion Transcript kit for formalin‐
fixed, paraffin‐embedded lung cancer specimens
Kazuko Sakai1 | Tatsuo Ohira2 | Jun Matsubayashi3 | Azusa Yoneshige4 | Akihiko Ito4 |Tetsuya Mitsudomi5 | Toshitaka Nagao3 | Emi Iwamatsu6 | Jin Katayama7 |Norihiko Ikeda2 | Kazuto Nishio1
1
Department of Genome Biology, Kindai
University Faculty of Medicine, Osaka,Japan
2

Department of Surgery, Tokyo Medical
University, Tokyo, Japan3
Department of Anatomic Pathology, Tokyo
Medical University, Tokyo, Japan
4

Abstract
Gene fusions play an important role in the carcinogenesis of lung adenocarcinoma.
The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, aslung tumor predictive biomarkers has increased the need for precision medicine. We
used formalin‐fixed, paraffin‐embedded tissue samples of non‐small cell lung cancer

Department of Pathology, Kindai University
Faculty of Medicine, Osaka, Japan

from 150 EGFR mutation‐negative cases and 10 fusion status‐known cases and com‐

5
Department of Thoracic Surgery, Kindai
University Faculty of Medicine, Osaka,Japan

FISH break‐apart for the detection of ALK, RET, and ROS1 fusion genes. RNA was ex‐

6Life Technologies Japan Ltd, Tokyo, Japan

7

Diagnostic Partnering, Clinical Sequencing
Division, Thermo Fisher Scientific, Tokyo,
Japan
Correspondence
Kazuto Nishio, Department of Genome
Biology, Kindai University Faculty of
Medicine, Osakasayama, Japan.
Email: knishio@med.kindai.ac.jp

pared the performance of the Oncomine Dx Fusion Transcript Test (ODxFT) withtracted from the paraffin‐embedded tissue samples with or without macrodissection
under hematoxylin and eosin staining, and the ALK fusion gene was independently
determined using these assays. Fusion detection analyses were successfully carriedout using ODxFT in 150 cases, with only one invalid case. ALK fusion genes weredetected at a frequency of 7.3% (11/150) in the lung cancer specimens. Concordance
rate between the ODxFT and ALK‐FISH analyses was 99.3% (148/149). Sensitivity
and specificity were 91.7% and 99.3%, respectively. All the samples with a known
fusion status were accurately matched between the two assays. Our results show ahigh concordance rate between the ODxFT and ALK‐FISH analyses. ODxFT was thus
validated as an effective method for detecting clinically significant ALK fusion genes
in paraffin‐embedded tissue samples.
KEYWORDS

ALK fusion gene, companion diagnosis, FISH, next‐generation sequencer, non‐small cell lungcancer

1 | I NTRO D U C TI O N

genes such as ALK,4 ROS1, 5 RET, 6,7 and NTRK18 have also been
described. These fusion events, which can involve a variety ofNon‐small cell lung cancer (NSCLC) is characterized by gene al‐partner genes, result in the formation of chimeric fusion kinases

terations. Activation of driver oncogenes such as EGFR, BRAF,

capable of oncogenic transformation and the induction of onco‐

and ERBB21‐3 increases the sensitivity of tumors to tyrosine ki‐

gene dependency within neoplastic cells. The individual preva‐

nase inhibitors. Gene fusions involving tyrosine kinase receptor

lence of each of these chromosomal rearrangements ranges from

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
2044|

wileyonlinelibrary.com/journal/cas

Cancer Science. 2019;110:2044–2049.

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| 2045SAKAI et Al.

1% to 7% in NSCLC,4,6,9,10 and, together, these rearrangements canbe identified in approximately 5%‐9% of NSCLC.7,11,12 Lung can‐

2.2 | RNA extraction

cers harboring these fusion genes show oncogene addiction to the

RNA was extracted from each of the clinical research samples using

respective fusion genes and are therefore sensitive to treatment

the AllPrep DNA/RNA FFPE Kit (Qiagen, Valencia, CA, USA) basedwith tyrosine kinase inhibitors (TKI), such as crizotinib, alectinibon the respective standard extraction procedures. RNA was quanti‐(CH5424802), and ceritinib (LDK378).

fied using the Nanodrop 2000 instrument (Thermo Fisher Scientific,

Fluorescence in situ hybridization, immunohistochemistry (IHC),
and real‐time PCR can be used to detect fusion genes in clinical tissue

Wilmington, DE, USA) and the RiboGreen RNA Assay Kit (Thermo
Fisher Scientific).

samples. The FISH break‐apart assay is the gold standard for com‐panion diagnosis. Development of the above‐mentioned agents8,9,13
has driven a rapid need for systematic and sensitive assays to de‐tect fusion genes. The massively parallel nature of next‐generation

2.3 | Oncomine Solid Tumor Fusion Transcript
kit design

sequencing (NGS) allows rapid characterization of point mutations,

Primers spanning 72 fusions (37 ALK, 9 RET, 15 ROS1, and 11 NTRK1)

small insertions, and deletions as well as the detection of chromo‐

were designed in order to to span all previously described fusions (assome rearrangements in a large set of genes through the targetedat time of development) of ALK, ROS1, RET, and NTRK1 by a researchsequencing of fusion junctions. In the present study, we validated

team at Thermo Fisher Scientific. Sources used for the curation of

the use of a new library kit, Oncomine Dx Fusion Transcript Test

all known fusions included the COSMIC and NCBI databases and a(ODxFT), for the characterization of the most frequent fusion genes

review of current medical literature. Targeted fusion genes and num‐in lung adenocarcinoma using NGS. This library kit is based on thebers of subtypes are shown in Tables 1 and 2. The multiplex primer

high‐multiplexing capabilities of PCR and focuses on the identifica‐

mixes also included primers for the amplification of five housekeep‐tion of 72 different fusion genes. Herein, we report the clinical per‐ing genes: HMBS, ITGB7, LMNA, MYC, and TBP. Additionally, prim‐

formance of this assay for the detection of fusion genes implicateders designed to amplify the 5′ and 3′ regions of ALK, RET, ROS1, andin the occurrence of NSCLC.

NTRK1 were included in the primer mix. Amplification of these re‐gions for each gene of interest allowed for the comparison of expres‐sion levels between the 3′ end of the gene, which was part of the

2 | M ATE R I A L S A N D M E TH O DS

resulting fusion, and the non‐involved 5′ end of the gene, but was
not used for the determination of fusion gene detection.

2.1 | Samples
A total of 160 lung cancer samples were collected from Tokyo
Medical University Hospital and Kindai University Hospital. All the

2.4 | Detection of fusions

samples were from resections that had been formalin‐fixed and par‐Total RNA was reverse transcribed using the SuperScript VILO

affin‐embedded (FFPE) and had the wild‐type EGFR genotype. Ten of

cDNA Synthesis Kit (Life Technologies Japan, Tokyo, Japan) fol‐these samples had been previously tested for ALK, RET, or ROS1 fu‐

lowed by library preparation using ODxFT (Life Technologies Japan).

sion genes using an Ion AmpliSeq RNA Lung Cancer Research Fusion

Reverse‐transcribed cDNA was amplified using the supplied rea‐

Panel for research purposes. All the patients enrolled in the study

gent. The PCR product was digested and ligated with a barcode

provided written informed consent for the use of their resected

adaptor followed by purification (all from supplied reagent). The

tissue. This study was approved by the ethics committee of Kindai

purified libraries were quantified using the Ion Library TaqMan

University Faculty of Medicine (Authorization Number: 27‐228) and

Quantitation Kit (Life Technologies Japan), followed by a pooled andTokyo Medical University (Authorization Number: 3380).

prepared sequencing template using the Ion OneTouch Dx Template

TA B L E 1

Partners for ALK, RET, ROS1, and NTRK1Target gene

ALK

RET

ROS1

NTRK1

Partner gene

EML4

KIF5B

CD74

CEL

KIF5B

CCDC6

SDC4

NFASC

KLC1

CUX1

SLC34A2IRF2BP2

HIP1

EZR

TFG

TPR

TPM3

SQSTM1

LRIG3

SSBP2

GOPC

CD74
DYNC2H1
MPRIP

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|

TA B L E 2

SAKAI et Al.

TA B L E 3 Concordance between ODxFT and FISH analysis for
ALK fusion gene

Constitution of subtypes

Fusion genes

No. of subtypes

EML4‐ALK18

KIF5B‐RET7

KIF5B‐ALK

4

SDC4‐ROS1

4

Positive

11

0

0

11

SLC34A2‐ROS1

4

Negative

1

137

0

138

CD74‐ROS1

2

0

1

0

1

GOPC‐ROS1

2

12

138

0

150

HIP1‐ALK2MPRIP‐NTRK1

3

Other

1

ALK FISH
Positive

Negative

Invalid

Total

ODxFT

Invalid
Total

ODxFT, Oncomine Dx Fusion Transcript Test.

were 91.7% and 99.3% when the FISH results were assumed to betrue. No statistically significant difference was observed between

Kit and an Ion OneTouch Dx Instrument (Life Technologies Japan).

the correlations of ODxFT and ALK‐FISH (κ = 0.95, 95% CI 0.79‐1.11).

The sequencing template was purified using an Ion OneTouch ES Dx

Detailed findings for all the clinical samples are shown in Table S1.

Instrument followed by sequencing on an Ion PGM Dx Sequencer

Representative FISH data and Integrative Genomics Viewer (IGV) di‐

using the Ion PGM Dx Sequencing Kit and the Ion 318 Dx Chip Kit

agram from ALK‐positive samples are shown in Figure 1A.

(all from Life Technologies Japan).
Data analysis was carried out using an Ion Torrent T430 Server.
This assay determined the fusion gene to be positive if the detectionof the fused sequence was greater than the cutoff value.

3.2 | Detection of ROS1 and RET fusion transcripts
Oncomine Dx Fusion Transcript Test is a hotspot panel designed
to detect ALK, ROS1, RET, and NTRK1 fusion transcripts. ODxFT
detected five RET and three ROS1 fusions. Panel results for ROS1

2.5 | Fluorescence in situ hybridization

and RET were concordant in 5/5 samples (100%) for ROS1 and in 3/3

Formalin‐fixed and paraffin‐embedded tissues sectioned at a thick‐samples (100%) for RET when compared with the respective FISH

ness of 4 μm and placed on glass slides were subjected to FISH using

findings (Figure 1B,C). No NTRK1 fusion transcripts were detecteda break‐apart probe for the ALK gene (Vysis ALK break‐apart FISH

using ODxFT.

probe; Abbott Molecular, Des Plaines, IL, USA), RET gene (RET Split
Dual Color FISH probe; GSP Research Inc.), or ROS1 gene (ROS1
Split Dual Color FISH probe; GSP Research, Inc., Kobe, Japan). FISH
positivity was defined as the presence of >15% split signals in thetumor cells.

3.3 | Detection of fusion genes in 10 fusion status‐
known cases
Because ALK, RET, or ROS1 fusion genes are not common oncogenic
events in patients with NSCLC, we examined an additional 10 posi‐tive samples that had been previously tested using an Ion AmpliSeq

2.6 | Statistical analysis

RNA Lung Cancer Research Fusion Panel for research purposes (8The kappa statistic and associated 95% confidence intervals were

EML4‐ALK, 1 KIF5B‐RET, and 1 SLC34A2‐ROS1 cases). All the geneused to measure agreement among the assays.

fusions were detected by ODxFT and FISH. The results of ODxFTand FISH matched exactly.

3 | R E S U LT S
4 | D I S CU S S I O N

3.1 | Method correlation agreement analysis for thedetection of ALK fusion transcripts

In the present study, we showed the feasibility of using the ODxFT

Of the 150 clinical research samples tested, 149 (99.3%) passed theassay to detect fusion genes in lung cancer tissues with a high ratequality control requirement. One failure case could not be analyzed

of success. Discordance between ALK FISH and other methods has

using ODxFT because of the low yield of library.

been previously reported.14,15 A recent study using the Ion AmpliSeq

ALK fusion genes were detected in 11/149 (7.4%) and 12/150

RNA Lung Cancer Research Fusion Panel for fusion detection re‐(8.0%) samples using ODxFT and ALK‐FISH, respectively (Table 3).

ported 100% concordance between this method and other meth‐

The ODxFT results were 99.3% concordant (148/149 samples) withodologies.16 This study and ours confirm the clinical performancethe FISH analysis. The invalid test rate for ODxFT was 0.7% (1/150).

of inhouse NGS tests using RNA samples obtained from small tissue

Positive predictive value and negative predictive value for ODxFT

samples.

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| 2047SAKAI et Al.

F I G U R E 1 FISH analysis (left) and Integrative Genomics Viewer (IGV) diagram (right) of ALK, RET, and ROS1 fusion cases. A‐C,
Representative FISH images for an ALK fusion‐positive specimen (A), a RET fusion‐positive specimen (B), and a ROS1 fusion‐positive
specimen (C). Arrowhead, bold rightwards arrow, and the fine arrow show the pseudocolor signals of the 5′/3′ probe, the 5′ probe, and the3′ probe, respectively
The simultaneous detection of fusions has important clinical(10 ng). This advantage is particularly attractive for assays target‐

implications for lung cancer patients in terms of turnaround time

ing lung cancers, as these samples are often obtained by biopsy andand cost. This method requires a very small amount of input RNA

contained a limited amount of tissue. In clinical practice, lung cancer

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2048

|

SAKAI et Al.

fusions have been detected using FISH, IHC, or RT‐PCR. Althoughtesting is the first gene screening test that is carried out because

FISH is considered to be the gold standard, especially for ALK testing

the prevalence of EGFR gene mutation positivity is relatively highbecause of the availability of an FDA‐approved ALK FISH assay, FISH

(40%‐50%). Therefore, it is reasonable to screen for fusion genes

analysis for multiple targets per sample can be costly and may po‐

in a single assay using ODxFT after the confirmation of a wild‐type

tentially extend the time needed to rule out all relevant fusion genes.

EGFR genotype.

IHC staining offers a cheaper alternative; however, this methodology

In conclusion, the RT‐PCR NGS assay described here offers manyis subjective, and accurate interpretations are sometimes difficult.17

advantages for laboratory testing in lung adenocarcinoma samples.

An RT‐PCR kit to detect ROS1 fusions is available as a companion di‐

The single‐assay format potentially allows for a faster turnaroundagnosis. The main limitation of traditional RT‐PCR is that this method

time and a lower cost than carrying out the assays separately.

typically focuses on only the most common fusion events and is thus

Furthermore, the small amount of input RNA that is required is very

limited when it comes to detecting rarer exon combinations and isadvantageous for this type of sample. However, ODxFT primarily

unable to detect fusions involving novel 5′ partners.18 In contrast to

targets known fusion genes. Last, efforts to periodically update

FISH or IHC, detection of ALK, RET, ROS1, and NTRK1 fusions can be

the primer pool as additional partner genes for ALK, ROS1, RET, andcombined into a single assay using ODxFT. This allows the detection

NTRK1 fusions are identified would aid in the continuing utility ofof fusions that might otherwise remain undetected.

this assay.

In the present study, we had one case with sequencing failure(K013) and one case discordant with FISH analysis (T060). Sample
K013 had a sufficient amount of RNA (10 ng RNA) for the library

AC K N OW L E D G M E N T

preparation, but the library concentration was low. Hence, we spec‐

The authors wish to thank Ayaka Kurumatani of the Department ofulate that the NGS analysis of K013 failed as a result of poor‐quality

Genome Biology, Kindai University Faculty of Medicine and KumikoRNA. One discordant case was shown to be ALK fusion negative inNagase of Department of Surgery, Tokyo Medical University for

ODxFT but was positive in FISH. There are several fusion partners

their technical support.

besides the partners contained in the ODxFT panel such as GCC2,
DCTN1, and CLIP1.19 As the number of sequencing reads and align‐
ment quality were sufficiently valid, it is possible that the partner

D I S C LO S U R EThis study was supported by a contract research fund from Life

gene was not included in the ODxFT panel.
Oncomine Dx Fusion Transcript Test includes a multiplexing ofTechnologies Japan Ltd. Emi Iwamatsu and Jin Katayama are em‐

primers for 72 different fusion combinations and is thus not lim‐

ployees of Life Technologies Japan Ltd and Thermo Fisher Scientific.

ited to only the most common fusions. For traditional RT‐PCR, one

Akihiko Ito, Norihiko Ikeda, and Kazuto Nishio received the researchmust have previous knowledge of all possible relevant fusions. The

fund from Life Technologies Japan Ltd. Kazuko Sakai, Tatsuo Ohira, Jun

ODxFT assay addresses this problem in two ways. First, during the

Matsubayashi, Azusa Yoneshige, Tetsuya Mitsudomi, and Toshitaka

analysis of the sequenced reads, all reads that are initially unaligned

Nagao have no conflicts of interest with any companies or organiza‐

to the reference sequence are split in half and allowed to realign.

tions whose products or services were discussed in this article.

This step fosters the detection of novel fusions involving existing
primers. Second, the assay includes a method for detecting fusions
involving unknown partners using a 3′/5′ imbalance calculation. This
step analyzes the expression levels of the 3′ and 5′ ends of each

ORCID
Kazuto Nishio

https://orcid.org/0000‐0002‐8275‐0846

driver gene. For genes involved in a fusion event, the 3′ end of thegene is now under different regulatory control and typically shows
overexpression relative to the 5′ end of the gene. Another recently

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13. Gainor JF, Shaw AT. Novel targets in non‐small cell lung cancer:
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S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found online in the
Supporting Information section at the end of the article.

How to cite this article: Sakai K, Ohira T, Matsubayashi J, et al.
Performance of Oncomine Fusion Transcript kit for formalin‐
fixed, paraffin‐embedded lung cancer specimens. Cancer Sci.
2019;110:2044–2049. https://doi.org/10.1111/cas.14016-476-

ONCOLOGY LETTERS 17: 5125-5131, 2019

Frequency and significance of epidermal growth
factor receptor mutations detected by PCR methods
in patients with non‑small cell lung cancer
EIJI NAKAJIMA1, MICHIO SUGITA2, KINYA FURUKAWA1, HIDENOBU TAKAHASHI3,OSAMU UCHIDA3, YOUHEI KAWAGUCHI4, TATSUO OHIRA4, JUN MATSUBAYASHI5,NORIHIKO IKEDA4, FRED R. HIRSCH6 and WILBUR A. FRANKLIN2
1

Department of Thoracic Surgery, Tokyo Medical University Ibaraki Medical Center, Ibaraki 300‑0395, Japan;
2
Department of Pathology, University of Colorado Health Sciences Center, Aurora, CO 80045, USA;3
Department of Thoracic Surgery, Tokyo Medical University Hachioji Medical Center, Tokyo 193‑0998;
Departments of 4Thoracic Surgery and 5Pathology, Tokyo Medical University Hospital, Tokyo 160‑0023, Japan;
6
Division of Medical Oncology, University of Colorado Health Sciences Center, Aurora, CO 80045, USA
Received September 2, 2018; Accepted February 7, 2019
DOI: 10.3892/ol.2019.10157
Abstract. Epidermal growth factor receptor (EGFR) is themost important driver gene of non-small cell lung cancer(NSCLC) as EGFR mutations determine the efficacy of EGFR
tyrosine kinase inhibitor (EGFR‑TKI) therapy. In the present
study, the comprehensive ability of widely used polymerase
chain reaction (PCR) methods to detect EGFR mutations wasdetermined. Among the 35 EGFR mutations detected via
the direct sequencing of 73 patients with NSCLC, 11 types
were identified in exons 18, 19 and 21. Among the 11 mutation types, all exon 18 and 21 mutations were identified by
2 widely used PCR methods, namely, Scorpion‑Amplification
Refractory Mutation System and cobas v2. However, among
the 9 different exon 19 deletions, 3 types were not identified by
the 2 methods. In addition, 25 samples with EGFR mutations
were analyzed by the 2 methods, including a sample from apatient with an unidentified exon 19 deletion, the T751_I759
deletion and insertion S; this patient had long-term disease
control as a result of EGFR‑TKI therapy. The 2 methods could

Correspondence to: Dr Eiji Nakajima, Department of Thoracic
Surgery, Tokyo Medical University Ibaraki Medical Center, 3‑20‑1
Chuo, Ami, Inashiki, Ibaraki 300‑0395, JapanE‑mail: nakajime@tokyo‑med.ac.jp
Abbreviations: EGFR, epidermal growth factor receptor;
NSCLC, non‑small cell lung cancer; TKI, tyrosine kinase inhibitor;
PCR, polymerase chain reaction; IVD, in vitro diagnostics;
Scorpion‑ARMS, Scorpion Amplification Refractory Mutation
System; FFPE, formalin‑fixed and paraffin‑embedded; COSMIC,
the catalog of somatic mutations in cancer; PR, partial response;CR, complete response; SD, stable disease; RR, response rate; DCR,
disease control rate
Key words: non-small cell lung cancer, epidermal growth factor
receptor, tyrosine kinase inhibitor, PCR, exon 19 deletion

not detect this unidentified deletion, whereas sizing capillaryelectrophoresis for the comprehensive detection of exon 19
deletions detected this deletion. It is generally thought thatpatients with exon 19 mutations have higher response rates
to EGFR‑TKI therapy than patients with exon 21 mutations.
The present study confirmed the EGFR mutation status
by comparing the mutations with the Catalog Of Somatic
Mutations In Cancer, which is the world's largest and mostcomprehensive resource for analyzing the effects of somatic
mutations in human cancers. The predicted frequency of
EGFR mutations identified by the 2 methods was 85%. The
frequency of mutations detectable by the 2 methods was lessfor exon 19 than exon 21. Therefore, the results of the present
study suggest that decreasing false‑negative detection ofexon 19 deletions is crucial for the clinical testing of EGFR
mutations.
Introduction
Epidermal growth factor receptor (EGFR) mutations areimportant factors in non-small lung cancer (NSCLC), as
EGFR tyrosine kinase inhibitor (EGFR‑TKI) treatment has
exhibited high efficacy in NSCLC patients with EGFR mutations (1-6). Polymerase chain reaction (PCR) methods areused clinically for the detection of EGFR mutations. Two
widely used PCR methods are approved as in vitro diagnostic (IVD) methods, namely, the Scorpion Amplification
Refractory Mutation System (ARMS; QIAGEN therascreen®
EGFR; Qiagen, Inc., Valencia, CA, USA) and the cobas®
EGFR Mutation Test v2 (Roche Diagnostics, Indianapolis,
IN, USA) (7,8). These 2 methods are a real‑time PCR test for
the qualitative detection of defined mutations of the EGFR
gene in DNA derived from formalin‑fixed paraffin‑embedded(FFPE) tumor tissue from NSCLC patients. The test isintended to aid in identifying patients with NSCLC whose
tumors have defined EGFR mutations and for whom safety
and efficacy of EGFR‑TKI have been established. The first

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NAKAJIMA et al: FREQUENCY OF EGFR MUTATIONS DETECTED BY PCR METHODS IN NSCLCEGFR‑TKI is gefitinib, which was approved from July 2002
in Japan. Erlotinib, afatinib, dacomitinib and osimertinib
are subsequently approved as EGFR‑TKIs. Dacomitinib isa second‑generation, irreversible EGFR‑TKI. In NSCLC
patients with EGFR mutations detected by Scorpion‑ARMS
method, dacomitinib significantly improved progression‑freesurvival over gefitinib in first‑line treatment (5). Osimertinib
is a third‑generation, irreversible EGFR‑TKI. In the first‑line
treatment of EGFR mutation-positive advanced NSCLC
identified by cobas v2, osimertinib showed efficacy superior
to that of gefitinib or erlotinib with a similar safety profile andlower rates of serious adverse events (6). In addition, cobas v2
can be used with plasma samples, as a companion diagnostic
for NSCLC therapy. The Scorpion‑ARMS and the cobas v2
are useful, rapid and cost-effective methods as a companion
diagnostic. However, they can only identify a small proportionof the different types of mutation, including common exon 19
deletions and exon 21 L858R. The present study therefore
analyzed the frequency of detectable EGFR mutations and theclinical significance of mutations that are not detected by these2 methods.
Materials and methods
Patients. The present study included a cohort of 73 Japanesepatients with NSCLC, from whom written informed consent
was obtained for the use of their samples in this research.
These patients presented with recurrent disease following
surgery between 1992 and 2004. The response of patients with
EGFR mutations to EGFR‑TKI treatment, which was a daily
dose of gefitinib (250 mg) administered between April 2002
and October 2005, was evaluated. During this period, only
gefitinib was approved as an EGFR‑TKI therapy for NSCLC
patients in Japan (Table I). The present study received ethics
approval from the Institutional Review Board of Tokyo
Medical University (Tokyo, Japan).
DNA extraction and direct sequencing analysis. All 73 surgicalsamples were FFPE; analysis of EGFR mutations was
performed. Tumor cells were microdissected under stereoscopic
microscopy, and DNA was extracted from tumor cells with a
DNeasy Tissue kit (Qiagen, Inc.). PCR was performed usinga primer set flanking exons 18 to 21. PCR products were run
on a gel, and the appropriate bands were cut, and then DNA
was extracted from the gel with the QIAquick Gel Extraction
kit (Qiagen, Inc.). Sequencing analysis was performed using an
ABI Prism 3100‑Avant genetic analyzer (Applied Biosystems;
Thermo Fisher Scientific, Inc., Waltham, MA, USA) according
to the manufacturer's protocol.
Determination of the efficacy of EGFR‑TKI therapy.
Definition of the efficacy of EGFR‑TKI therapy was applied
according to the Response Evaluation Criteria in Solid
Tumors (9). In the case of a partial response (PR) or complete
response (CR), changes in tumor measurements were confirmedby repeated assessments that were performed no less than
4 weeks following the fulfillment of the response criteria.
Stable disease (SD) was defined as fulfilling the SD criteria
for at least 8 weeks following the start of EGFR‑TKI therapy.
Patients receiving >4 weeks of treatment were consideredassessable, and patients who had received EGFR‑TKI therapy
for <4 weeks were considered not assessable. Response rate(RR) was defined as the percentage of PR and CR divided by
the total of the assessable patients, and disease control rate
(DCR) was the percentage of response and SD divided by the
total of the assessable patients (Table II).
Frequency of detectable mutations by the 2 IVD PCR methods.
Two widely used IVD PCR methods (Scorpion ARMS andcobas v2) were used in the present study. Scorpion‑ARMS
can detect 29 types of EGFR nucleotide mutations, andcobas v2 can detect 42 types of EGFR nucleotide mutations(Table III) (7,8). The two methods of detection have previously identified common mutations such as specific exon 19
deletions and specific exon 21 mutations including L858R.
Using the 2 methods, patients who harbored mutations that
could not be identified were confirmed, and the frequency ofdetectable EGFR mutations was calculated. The results fromthe 2 methods were compared with the predicted frequency ofdetectable mutations from the catalog of somatic mutations incancer (COSMIC) version 84 database. COSMIC is the world's
largest and most comprehensive resource for analyzing theeffects of somatic mutations in human cancers. EGFR consistsof 564 nucleotides from exon 18 to exon 21 (codons 688‑875),
and EGFR mutations include substitutions, deletions, insertions, and combinations of these (9-12). The COSMIC v84
database was used in the present study to count the types of
EGFR mutations (released on February 13, 2018).
Detection of EGFR mutations by the 2 IVD PCR methods. Owing
to the large number of clinical samples, DNA was extracted
from FFPE tumor specimens without microdissection, foranalysis by the 2 IVD PCR methods. The DNA was subjected to
Scorpion‑ARMS at SRL Inc. (Tokyo, Japan) and cobas v2 at BML
Inc. (Tokyo, Japan), along with sizing capillary electrophoresis
using MCE‑202 MultiNA (Shimazu Corporation, Kyoto, Japan)
at BML Inc. for the comprehensive detection of exon 19 deletions.
Sizing capillary electrophoresis measures the length of DNA todistinguish between wild‑type exons and exons with deletions,and covers all exon 19 deletions along the 99 nucleotides fromcodon 729 to 761 (Fig. 1A). This was performed to detect exon 19
deletions that were not identified by the 2 IVD PCR methods.
Results
EGFR mutation status and response to EGFR‑TKI therapy.
EGFR mutations were detected by direct sequencing in
35 patients (47.9% of clinical samples), including, 1 exon 18 mutation, 23 exon 19 mutations and 11 exon 21 mutations. Amongthe 35 patients with EGFR mutations who received EGFR‑TKI
therapy, 30 patients were assessable, which included 1 patientwith an exon 18 mutation, 19 patients with exon 19 mutations
and 10 patients with exon 21 mutations. The RR of the EGFR
mutations was 36.7% and the DCR was 97%. In exons 19 and 21,
including common mutations, the RR of exon 19 mutations was
42.1% and the RR of exon 21 mutations was 20% (Table II).
Frequency of mutations identified by the 2 IVD PCR methods.
In all 73 cases, there were 11 types of EGFR mutations amongthe 35 patients with an EGFR mutation, including 1 type of-478-

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Table I. Background information of the 73 patients with NSCLC.
Variable

Total n, (%)

Age, years, range (median)Sex: Male/Female
Histology: ADC/SCC/La/Pleomorphic
Smoking: Never/Former and Current
EGFR mutation

30‑92 (65.6)
45 (61.6)/28 (38.4)
59 (80.8)/9 (12.3)/4 (5.5)/1 (1.4)
29 (39.7)/44 (60.3)
35 (47.9)

NSCLC, non‑small cell lung cancer; ADC, adenocarcinoma; SCC, squamous cell carcinoma; La, large cell carcinoma; Pleomorphic, pleomorphic carcinoma; EGFR, epidermal growth factor receptor.

Table II. Information on 35 NSCLC patients with EGFR mutations receiving EGFR‑ tyrosine kinase inhibitor therapy.
Variable

Total nAssessable patients/patients with EGFR
mutations
Mutations of exon 18/19/21
Mutation types in 30 assessable patients
Mutations of exon 18/19/21
EGFR mutation RR
EGFR mutation DCR
Exon mutations
Exon 18 mutation RR
Exon 19 mutations RR
Exon 21 mutations RR

30/35
1/23/11
1/19/10
36.7% (11/30)
97% (29/30)
100% (1/1)
42.1% (8/19)
20% (2/10)

NSCLC, non-small cell lung cancer; EGFR, epidermal growth factor
receptor; RR, response rate; DCR, disease control rate.

exon 18 mutation, 9 types of exon 19 mutations and 1 type ofexon 21 mutation, as detected by direct sequencing. The exon 18
mutation was G719S, the exon 19 mutations were all deletions(9 types), including deletion and insertion compound mutations, and the exon 21 mutation was L858R. All exon 18 and 21
mutations were identified by Scorpion ARMS and cobas v2.
However, among the 9 types of exon 19 deletions, 3 types were
not identified by the 2 methods. These unidentified deletions
were the E746_S752 deletion, the T751_I759 deletion withinsertion S and the K757_L760 deletion with insertion N.
Two patients with unidentified deletions had achieved disease
control (CR and SD) via EGFR‑TKI therapy (Table IV). Of the
35 patients with EGFR mutations, the frequency of detectable
EGFR mutations by the 2 methods was 91.4% (32/35); thatof exon 19 mutations was 87.0% (20/23) and that of exon 21
mutations was 100% (11/11).
The COSMIC v84 database contains a total of 5,908 EGFR
mutations comprising 448 types. The distribution of themutations was 4.1% in exon 18, 41.3% in exon 19, 10.5% inexon 20 and 44.1% in exon 21. In the COSMIC v84 database,the predicted frequencies of detectable EGFR mutations by
Scorpion‑ARMS and cobas v2 were 85.9 and 86.9%, respectively (Fig. 2). Regarding exon 19 mutations, the distributionof deletions, including deletion and insertion compound mutations, was 94.5%, and the predicted frequencies of thesemutations by the two methods were 87.8 and 89.8%, respectively (Fig. 3A). Regarding exon 21 mutations, the distributionof L858R was 89.7%, and the predicted frequencies of the two
methods were 92.9 and 93.1%, respectively (Fig. 3B).
Detection of EGFR mutations by the 2 IVD PCR methods.
Scorpion‑ARMS and cobas v2 analyses were performed on
25 samples among the 35 patients who had EGFR mutations
that were detected by direct sequencing, which included 1
exon 18 mutation (G719S), 18 exon 19 deletions comprised of
6 different types (including 1 exon 19 deletion not identified by
the 2 methods), and 6 exon 21 mutations (L858R). All exon 18
and 21 mutations were detected by the two methods; however,both methods failed to detect exon 19 deletions in 4 patients(2 squamous cell carcinoma and 2 adenocarcinoma patients).
The undetected deletions of exon 19 from 3 samples were actually the common E746‑A750 mutation, which was expected
to be identified by the two methods. One undetected complex
with a deletion and insertion of exon 19 was T751_I759 andinsertion S (COSMIC ID 1667027), which were not identified by either of the methods. The deletion was identified in apatient with adenocarcinoma, with long SD (43.4 months) by
EGFR‑TKI therapy. Subsequent sizing capillary electrophoresis was able to detect the deletion (Fig. 1B).
Discussion
Gefitinib and erlotinib were approved as a first‑generation
EGFR‑TKI. Subsequently, afatinib and dacomitinib as asecond‑generation and osimertinib as a third‑generation
were approved. EGFR mutations are the major factors
determining the efficacy of EGFR‑TKI therapy in patientswith NSCLC (1-6). EGFR consists of 564 nucleotides fromexons 18 to 21, and EGFR mutations include substitutions,deletions, insertions, and a combination of any those (10-13).
Therefore, different types of mutations are continuously being
identified. In the COSMIC v84 database, exon 19 and 21
mutations account for the majority of EGFR mutations, andthe distribution of these mutations among each exon is similar
to previous studies (14,15) Scorpion‑ARMS and cobas v2 candetect a proportion of the EGFR mutations, including common
exon 19 deletions and the exon 21 L858R mutation. The present
results revealed that the 2 methods did not identify 3 exon 19

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Table III. Types of EGFR mutations identified by the 2 IVD PCR methods.
PCR method
Scorpion‑ARMS
Cobas v2

Exon 18

Exon 19

Exon 20

Exon 21

Total n

3
3

19
29

5
7

2
3

29
42

EGFR, epidermal growth factor receptor; IVD, in vitro diagnostics; PCR, polymerase chain reaction; Scorpion‑ARMS, Scorpion Amplification
Refractory Mutation System.

Figure 1. (A) Diagram presenting the primer and amino acid positions of exon 19, from codon 729 to 761, in sizing capillary electrophoresis. (B) Sizing capillary electrophoresis was able to detect 1 exon 19 deletion in a patient with adenocarcinoma and long stable disease (43.4 months) who received EGFR‑tyrosine
kinase inhibitor therapy, namely, the T751_I759 deletion and insertion S, which were undetected by Scorpion‑Amplification Refractory Mutation System andcobas v2. EGFR, epidermal growth factor receptor.

deletions, in which 2 deletions were adenocarcinomas and
1 deletion was squamous cell carcinoma. In squamous cell
carcinoma with EGFR mutations, a patient with a common
exon 19 deletion (E746‑A750) had achieved disease control
via EGFR TKI therapy. Regardless of the histological type oflung cancer, exon 19 deletions are important for EGFR TKI
therapy.
In the present study and COSMIC v84 database, thefrequencies of detectable EGFR mutations by the 2 methods
were similar, despite the different number of mutations
identified by the 2 methods. In particular, in regard toexon 19 mutations, Scorpion‑ARMS and cobas v2 identified
19 and 29 deletions, respectively. Mutations identified by thetwo methods include common mutations (exon 19 E746‑A750
deletion and exon 21 L858R mutation); however, there are manyother types of mutations, for example there are 99 different
types of exon 19 deletions listed in the COSMIC database v84.
In a recent clinical study, the efficacy of EGFR‑TKI therapy
in NSCLC patients with common exon 19 deletions and theexon 21 L858R mutation were analyzed (4-6). Methodologicalstudies tend to use methods with higher sensitivity for liquidbiopsy. Scorpion‑ARMS and cobas v2 are highly sensitive PCR methods for the detection of common mutations.
Cobas v2 is a recently established reliable PCR method, whichis the companion diagnostic for osimertinib (6). However,the frequency of detectable EGFR mutations and the clinicalsignificance of unidentified mutations by the 2 methods areunclear. Previous studies have demonstrated that NSCLC
patients with tumors harboring exon 19 deletions have alonger survival rate when compared with those harboring the
L858R point mutation of exon 21, following treatment with
EGFR‑TKIs, which was reported between 2006 and 2007prior to the approval of cobas v2 as an IVD (16-18). Afatinib
treatment resulted in a significant improvement in the overall
survival of NSCLC patients with exon 19 deletions, comparedwith cytotoxic chemotherapy (4). In the present study, theRR of patients with exon 19 deletions was higher than that ofexon 21 L858R. Among patients with exon 19 deletions, that-480-

ONCOLOGY LETTERS 17: 5125-5131, 2019

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Table IV. Information on 35 EGFR mutations detected by direct sequencing and the EGFR‑TKI response of the patients.
Amino acid/deletion type
Exon 18
G719S
Exon 19
E746‑A750 del
E746-S752 dela
L747‑E749 del
L747-L751 del
L747‑P753 ins S
T751‑I759 ins Sa
K757‑L760 ins Na
Exon 21
L858R
Total EGFR mutations

Nucleotide

No.

Histology ADC/SCC

Response CR/PR/SD/PD/NA

G2155A2,235‑2,249
2,236‑2,250
2,236‑2,256
2,239‑2,247
2,239-2,253
2,240‑2,254
2,240‑2,257
2,252‑2,275 T2276G
2,271‑2,279

1
23
‑
13
1
1
3
3
1
1

2/0
20/3
‑
11/2
1/0
1/0
3/0
3/0
1/0
0/1

0/1/0/0/0
2/6/10/1/4‑
0/3/7/1/2
1/0/0/0/0
1/0/0/0/0
0/2/0/0/1
0/1/2/0/0
0/0/1/0/0
0/0/0/0/1

T2573G
‑

11
35

11/0
32/3

0/2/8/0/1
2/9/18/1/5

Exon 19 deletion that was not identified by Scorpion‑ARMS and cobas v2. EGFR, epidermal growth factor receptor; TKI, tyrosine kinase
inhibitor; ADC, adenocarcinoma; SCC, squamous cell carcinoma; CR, complete response; PR, partial response; SD, stable disease; PD,
progressive disease; NA, no assessable patients; del, deletion; ins, insertion.

aFigure 2. Distribution of EGFR mutations and predicted frequencies of mutations from the Catalog Of Somatic Mutations In Cancer v84 database that weredetectable by the 2 polymerase chain reaction methods. EGFR, epidermal growth factor receptor; Scorpion‑ARMS, Scorpion‑Amplification Refractory
Mutation System.

were not identified by Scorpion‑ARMS and cobas v2, there
were patients who responded to EGFR‑TKI therapy.
Sizing capillary electrophoresis not only detected theexon 19 deletions identified by Scorpion‑ARMS and cobas v2,
but also detected the exon 19 deletion that was not identified

by the 2 methods. This was observed in a patient with adenocarcinoma for whom EGFR‑TKI therapy was effective. The
samples of 3 patients with the exon 19 E746‑750 deletion(common mutation), which was detected by sequencing wereconcluded to be wild‑type by the 2 methods. The reason for this

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Figure 3. Percentage of deletions among (A) exon 19 mutations and L858R among (B) exon 21 mutations in the Catalog Of Somatic Mutations In Cancer v84
database. Scorpion‑ARMS, Scorpion‑Amplification Refractory Mutation System.

was that the tumors are heterogeneous, comprised of a mixtureof wild‑type cells and cells with mutations, and as there was atime lag between performing sequencing and the 2 methods,
the DNA may have been damaged during this delay.
Sequencing has the ability to analyze the wholeexome. EGFR contains 564 nucleotides in exons 18 to 21(codons 688‑875), and for the majority of NSCLC patients,
sequencing of the whole exome of EGFR is difficult due tothe high cost and long time required (19,20). The frequency
of false negatives is an important factor in screening prior
to treatment, as patients may lose the opportunity to receive
EGFR‑TKI therapy. Exon 19 mutations in particular had 10%
false negatives in Scorpion‑ARMS and cobas v2, and were
expected to achieve improved overall survival via EGFR
TKIs therapy when compared with other exon mutations. In
addition, different types of exon 19 deletions have continuously increased in the COSMIC database between v84
and v87 over the 10 month time period between versions,
therefore the number of false negatives is increasing. In
conclusion, exon 19 deletions are the most important EGFR
mutations in exons 18 to 21 for EGFR‑TKI therapy, and thefrequencies of exon 19 deletions detected by Scorpion‑ARMS
and cobas v2 were less than those of exon 21 mutations with
L858R. Therefore, the results of the present study suggestthat decreasing the rate of the false negatives of exon 19

deletions via widely used PCR methods may be important
for the clinical testing of EGFR mutations.
Acknowledgements
The authors would like to thank the Department of International
Medical Communications of Tokyo Medical University(Tokyo, Japan) for their assistance with the manuscript.
Funding
No funding was received.
Availability of data and materials
The datasets regarding the clinical samples used and analyzed
during this study are available from the corresponding author
on reasonable request. The datasets analyzed in this study areavailable in the COSMIC database repository (cancer.sanger.
ac.uk/cosmic/).
Authors' contributions
EN and MS conceived and designed the study. KF obtained
the clinical samples from Tokyo Medical University Ibaraki-482-

ONCOLOGY LETTERS 17: 5125-5131, 2019

Medical Center (Ibaraki, Japan). HT and OU obtained theclinical samples from Tokyo Medical University Hachioji
Medical Center (Tokyo, Japan). YK, TO and NI obtained theclinical samples from Tokyo Medical University Hospital(Tokyo, Japan). EN, MS and TO processed and analyzed thesamples. TO, NI and FRH interpreted the data. JM and WAF
analyzed and interpreted the pathological samples. All authors
have read and approved the final manuscript.
Ethics approval and consent to participate
The present study received ethics approval from theInstitutional Review Board of Tokyo Medical University(Tokyo, Japan), and written informed consent to participate
was obtained from all of the patients.
Patient consent for publication
The present study obtained consent for publication from eachpatient.
Competing interests
The authors declare that they have no competing interests.
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Received: 13 July 2018

Revised: 7 December 2018

Accepted: 26 February 2019

DOI: 10.1002/dc.24171

ORIGINAL ARTICLE

Cytological differences between invasive and noninvasiveor minimally invasive lung adenocarcinomas diagnosed
in Japanese patients using needle biopsy specimensof pulmonary lesions ≤3 cm in diameter
Jun Matsubayashi MD, PhD1,2| Shinji Miyake CT, CTIAC2 | Yujin Kudo MD, PhD3 |Yoshihisa Shimada MD, PhD3 | Junichi Maeda MD, PhD3 | Hisashi Saji MD, PhD4 |Masatoshi Kakihana MD, PhD3 | Jinho Park MD, PhD5 | Naohiro Kajiwara MD, PhD3 |Shigeru Inoue MD, PhD6 | Kazuhiro Saito MD, PhD7 | Tatsuo Ohira MD, PhD3 |Norihiko Ikeda MD, PhD3 | Koichi Tokuuye MD, PhD7 | Toshitaka Nagao MD, PhD1,2
1

Department of Anatomic Pathology, Tokyo
Medical University, Tokyo, Japan

Background: According to the WHO classification for lung cancer, adenocarcinoma in situ (AIS)

2

and minimally invasive adenocarcinoma (MIA) have a better prognosis than invasive adenocarci-

Diagnostic Pathology Division, Tokyo Medical
University Hospital, Tokyo, Japan3

Department of Thoracic Surgery, Tokyo
Medical University, Tokyo, Japan

4

Department of Chest Surgery, St. Marianna
University School of Medicine, Kawasaki,
Japan

5

Department of Radiology, Tokyo Medical
University Hachioji Medical Center, Tokyo,
Japan

6

Department of Preventive Medicine andPublic Health, Tokyo Medical University,
Tokyo, Japan

7

Department of Radiology, Tokyo Medical
University, Tokyo, Japan
Correspondence
Jun Matsubayashi, Department of Anatomic
Pathology, Tokyo Medical University, 6-7-1
Nishishinjuku, Shinjuku-ku, Tokyo 160-0023,
Japan.
Email: jun-ma@tokyo-med.ac.jp

noma (IAD). However, detecting the foci of invasion in lung adenocarcinomas radiologicallyremains difficult. The present study examined whether or not differences in the cytological characteristics between IAD and AIS or MIA (noninvasive or minimally invasive adenocarcinomas[NMIAD]) plays a role in the differential diagnosis.
Methods: Seventy surgical resection specimens of primary lung adenocarcinoma with preoperative cytology, in which several parameters were evaluated and assessed.
Results: The histopathological diagnoses of surgical resection specimens were AIS in 8, MIA in
31, IAD in 31 including lepidic adenocarcinoma in 9, and papillary adenocarcinoma in 22.
NMIAD had a 100% 5-year recurrence-free survival (RFS), while IAD had an 82.8% 5-year RFS.
The numbers of tumor cells (at ×10 magnification in 10 fields) were 60.3 ± 40.5 in IAD and39.8 ± 28.7 in NMIAD (P = 0.0017). A univariate analysis of cytological parameters revealed significant differences in large tumor cell clusters, three-dimensional (3D) tumor cell clusters, andirregular nuclear contours between the two groups. The frequency of irregular nuclear contours
continued to be significantly different according to a multivariate analysis.
Conclusion: Large or 3D tumor cell clusters and irregular nuclear contours may be importantcytological factors for distinguishing IAD from NMIAD, with the latter being potentially more
important for distinguishing between the two groups.

1 | I N T RO D UC T I O N

health checkups and chest computed tomography (CT) now being
widely used in routine investigations. Furthermore, recent develop-

The detection of early lung carcinomas has been facilitated in Japanments in imaging modalities and the widespread use of low-dose heli-by chest radiography imaging being commonly performed in annual

cal CT for the screening of lung cancer have led to an increase in thedetection rate of pulmonary peripheral lesions.1 Noguchi et al. previ-

Jun Matsubayashi and Shinji Miyake share joint first authorship as they contributed equally to this work.

ously reported that bronchioloalveolar carcinoma (BAC), which isregarded as adenocarcinoma in situ (AIS), has a favorable prognosis

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used for commercial purposes.
© 2019 The Authors. Diagnostic Cytopathology published by Wiley Periodicals, Inc.
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with a 5-year survival rate of 100%, while adenocarcinoma with an

Tokyo Medical University Hospital. Among these patients, 70 wereinvasive component has a poor prognosis despite the small tumor

pathologically proven to have primary peripheral lung adenocarcinomas

size.2 Furthermore, according to the International Association for the≤3 cm in diameter that showed pure ground-glass opacity (GGO) or

Study of Lung Cancer/American Thoracic Society/European Respira-partly solid nodules on preoperative CT-guided fine-needle aspiration

tory Society (IASLC/ATS/ERS) classification of lung adenocarcinomas,

cytology due to the difficulty of a transbronchial approach. Any patientspatients with AIS and minimally invasive adenocarcinoma (MIA) showwith pure solid nodules on CT were excluded from this study, as werea more favorable course than those with invasive adenocarcinoma

patients with synchronous multiple adenocarcinomas.

3(IAD). MIA is defined as a small (≤3 cm in diameter), solitary adenocarcinoma with a predominantly lepidic pattern and ≤5 mm invasion in
the greatest dimension. MIA is excluded if the tumor invades the lymphatics, blood vessels, air spaces, or pleura; contains tumor necrosis;
or spreads through the air spaces.3 In 2015, the 4th edition of WHO
classification of lung adenocarcinomas, which was based on the IASLC/ATS/ERS classification, was published, emphasizing the significanceof histological subtypes for assessing the prognosis of patients. Thishas been validated in independent cohorts.4,5Previous studies have examined the relationship between the
IASLC/ATS/ERS classification of histological subtypes and radiologicalfeatures in lung adenocarcinomas.6–8 We reported that high-resolution
CT (HRCT) findings correlated with the IASLC/ATS/ERS classification
and were useful for evaluating the histological features of tumors.8
This study revealed that the consolidation/tumor ratio and consolidation size in partly solid tumors are important factors for predicting various pathological invasion factors (PIFs), that is pleural invasion,
vascular invasion, and lymphatic permeation, as well as for diagnosing
IAD. Furthermore, most purely solid tumors are considered to represent a potentially high-grade malignancy, featuring lymph node metastasis and various PIFs. A multicenter registration study showed thatthe solid tumor size on HRCT and maximum standardized uptake

2.2 | Survival analyses
Data collection and analyses were approved and the need to obtainwritten informed consent from each patient was waived by the Institutional Review Board of Tokyo Medical University. The overall survival (OS) and recurrence-free survival (RFS) were estimated using the
Kaplan-Meier method, and differences in the survival rates were
determined using a Log-rank analysis (Mantel Cox method). The OS
was defined as the time elapsed from the date of pulmonary resection to the date of death from any cause. Among patients in ourseries, the RFS was defined as the interval from the date of pulmonary
resection to the date of recurrence, lung cancer-related death, or thelast follow-up. The last follow-up observation was censored if thepatient was alive or lost to follow-up.
A univariate analysis was conducted among the different groups.
Categorical variables were analyzed using the chi-squared test. Differences between two groups were tested using the Mann-Whitney
U test. All P-values were two-sided, and P-values of <0.05 were considered to indicate a significant difference. All statistical calculations
were performed using the StatView software program for Windows,
version 5.0 (SAS Institute Inc, Cary, North Carolina).

values on positron emission tomography (PET)/CT have greater predictive value for high-grade malignancy and the prognosis in clinical stage
9

2.3 | Histological and cytological evaluations

IA lung adenocarcinoma than the whole tumor size. However, difficul-

All resected specimens were fixed in formalin and embedded in paraf-

ties remain with accurately identifying the foci of invasion in lung ade-

fin using routine methods and then stained with hematoxylin andnocarcinomas clinically due to the challenges associated witheosin. Elastica-van-Gieson stains were performed where appropriate

distinguishing central scars from the foci of invasion on CT.8 Furthermore, while active fibroblasts and a papillary growth pattern are histological hallmarks of invasion in lung adenocarcinomas,2,10 it is difficult
to detect these findings in small biopsy or cytology specimens.
We investigated the cytological differences between IAD andnoninvasive or minimally invasive adenocarcinoma (NMIAD) diagnosed in Japanese patients based on the new WHO classification
using CT-guided needle aspiration cytology specimens of peripheral
pulmonary lesions ≤3 cm in diameter to investigate whether or not
differences in the cytological characteristics between IAD and NMIAD
may contribute to the differential diagnosis. Therefore, the objectiveof the present study was to identify more readily discernible differences in the cytological characteristics of IAD and NMIAD.

to evaluate the tumor structure and vascular invasion. These cases
were all reclassified by two pathologists (J.M., T.N.) according to the
4th edition of the WHO classification based on the IASLC/ATS/ERS
classification for lung adenocarcinomas. All patients had diagnostic
preoperative CT-guided fine-needle aspiration cytology specimens
available for review. Cytology specimens were fixed in 95% ethanoland stained using the Papanicolaou method. They were then evaluated by one pathologist (J.M.) and one experienced cytotechnologist(S.M.) for the following parameters: (a) the number of tumor cells (×10magnification in 10 fields), (b) the number of stromal clusters in two
slides, (c) the presence of tumor cell clusters composed of ≥20 tumor
cells (large tumor cell clusters), (d) three-dimensional (3D) tumor cellclusters, (e) irregular nuclear contours (mainly means multinodularshaped nucleus with prominent notch), (f) nuclear pleomorphisms(mainly means nuclear-sized variety), (g) prominent nucleoli, and

2 | MATERIALS AND METHODS(h) intranuclear cytoplasmic inclusion. In the histologic and cytologic
examinations, each researcher evaluated all specimens independently

2.1 | Patient selection

and in random order. Regarding cytological parameters (a) and (b), dif-

A total of 1752 consecutive patients underwent pulmonary resection forferences in these values between AIS or MIA (NMIAD) and IAD wereprimary lung cancer between January 1997 and December 2008 at

examined using a t test. We evaluated cytological parameters (c)-

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MATSUBAYASHI ET AL.

(h) based on three criteria (score 0 [absent], score 1 [a few or slight],

TABLE 2The overall survival

and score 2 [prominent]). If the results differed between the two
observers, then one other pathologist (T.N.) evaluated these parameters, and three authors (ie, two pathologists and one cytotechnologist)
ultimately made a consensus diagnosis after carefully discussing thefindings.
The significance of differences between a score of 0 or 1 and ascore of 2 was determined using the chi-squared test. All P values
were two-sided, and P values of <0.05 were considered to indicate asignificant difference. All statistical analyses were performed using the
Statview software program (SAS Institute Inc).

3 | RESULTS
3.1 | Patient characteristics and the survival analysis
Table 1 summarizes the patient characteristics. Of the 70 cases examined, 32 (45.7%) were male, and 38 (54.3%) were female. The medianage was 67 years old (range 38-83). The median tumor size was 1.5 cm(range 0.7-3.0 cm). The histopathological diagnoses of the 70 surgicalresection specimens were AIS in 8, MIA in 31, IAD in 31 including lepidic adenocarcinoma (means lepidic-predominant adenocarcinoma) in
9, and papillary adenocarcinoma (means papillary-predominant adenocarcinoma) in 22. There were no acinar, solid, micropapillary, or mucinous adenocarcinoma cases in the IAD group in the present study. The
median clinical follow-up period was 2873 days (range: 6-5095 days).
The 5-year OS rates were 100%, 93.3%, 88.9%, 90.2%, 94.7%, andAbbreviations: 5-y OS, 5-year overall survival; AD, adenocarcinoma; AIS,
adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma; NS,
not significant.

between those with AIS + MIA and lepidic adenocarcinoma + papillaryadenocarcinoma tumors (P = 0.002 and P = 0.003, respectively), while
no significant differences were found in the RFS between patients withAIS and papillary adenocarcinoma tumors or between those with lepidic
and papillary adenocarcinoma tumors (P = 0.101 and P = 0.106, respectively; Table 3, Figure 1B).
As a significant difference was observed in the RFS between
patients with AIS + MIA and lepidic adenocarcinoma + papillary adenocarcinoma tumors, we evaluated both groups cytologically.

90.0% for AIS, MIA, lepidic adenocarcinoma, papillary adenocarcinoma,
AIS + MIA, and lepidic adenocarcinoma + papillary adenocarcinoma,

3.2 | The cytological analysis

respectively (Table 2, Figure 1A). No significant differences were

The numbers of tumor cells were 39.8 ± 28.7 in the NMIAD groupobserved in the OS between patients with AIS and papillary adenocarci-

and 60.3 ± 40.5 in the IAD group (P = 0.0017; Figure 2A). The num-noma tumors, between those with MIA and papillary adenocarcinoma

bers of stromal clusters were 11.8 ± 11.5 in the NMIAD group andtumors, between those with lepidic adenocarcinoma and papillary adeno-

13.3 ± 11.2 in the IAD group (P = 0.59; Figure 2B).

carcinoma tumors, or between patients with AIS + MIA and lepidic ade-

A univariate analysis revealed significant differences between the

nocarcinoma + papillary adenocarcinoma tumors (P = 0.192, P = 0.431,

groups in the rates of large tumor cell clusters (P = 0.0106), 3D tumor

P = 0.596, and P = 0.298, respectively; Table 2, Figure 1A). Furthermore,cell clusters (P = 0.0017), and irregular nuclear contours (P = 0.0007;

AIS, MIA, lepidic adenocarcinoma, and AIS + MIA had a 100% 5-year

Figure 3A,C). However, no significant differences were found in the fre-

RFS, while papillary adenocarcinoma and lepidic adenocarcinoma + papil-

quency of nuclear pleomorphisms (P = 0.2008), prominent nucleoli

lary adenocarcinoma had a 76.2% and 82.8% 5-year RFS, respectively(P = 0.2484),(Table 3, Figure 1B). Significant differences were observed in the RFS

Figure 3D,F). A multivariate analysis identified a significant difference

between patients with MIA and papillary adenocarcinoma tumors andbetween the groups in only one factor: the frequency of irregular nuclear

TABLE 1orintranuclear

cytoplasmic

inclusion(P = 0.8689;

contours.

Patient characteristics (n = 70)

Variable

CategorySexMenn = 32 (45.7%)

Womenn = 38 (54.3%)

4 | DI SCU SSIONMedian age (range)

67 (38-83)

A number of studies have described the cytological features of lungMedian whole tumor size (range)

1.5 cm (0.6-3.0 cm)

adenocarcinoma.11–14 Sigel et al. examined 113 fine-needle aspiration

AIS

n = 8 (11.4%)

cytology specimens of excised pathological stage I lung adenocarci-

MIA

n = 31 (44.3%)

noma.11 The surgically resected specimens were histologically graded

Lepidic AD

n = 9 (12.9%)

according to the predominant pattern. In summary, grade 1 tumors

Papillary AD

n = 22 (31.4%)

are those with a predominantly lepidic pattern, grade 2 tumors are

Histopathological diagnosis

Abbreviations: AD, adenocarcinoma; AIS, adenocarcinoma in situ; MIA,
minimally invasive adenocarcinoma.

those with predominantly acinar or papillary patterns, and grade
3 tumors are those with predominantly solid or micropapillary

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MATSUBAYASHI ET AL.

Probability of overall survival(A)

5-y OS (%)

1.0: AIS + MIA 94.7% (n=39)

0.8

: IAD

90.0% (n=31)

0.6
P = 0.298
Log-rank test

0.4
0.2
0
0

Probability of recurrence-free survival

(B)

12

24

36

60

48

5-y RFS (%)

1.0: AIS + MIA 100.0% (n=39)

0.8

: IAD
0.6

82.8% (n=31)
P = 0.003
Log-rank test

0.4
0.2
0
0

12

24

36

60

48

FIGURE 1 A, The overall survival (OS) curve for AIS + MIA (NMIAD) and lepidic and papillary adenocarcinoma (IAD). The 5-year OS rates were
94.7% and 90.0% for NMIAD and IAD, respectively (P = 0.298). B, The RFS curve for AIS + MIA (NMIAD) and lepidic and papillaryadenocarcinoma (IAD). The 5-year RFS rates were 100% and 82.8% for NMIAD and IAD, respectively (P = 0.003)

patterns. Three variables (nuclear size, chromatin pattern, and nuclear

between the cytological scoring system and histological grading, as

contours) among several cytological features showed a significant

defined by the predominant histological architectural patterns of theassociation with the histological grade and disease-free survival. Fur-

excised tumors. Rodriguez et al. examined 133 consecutive surgicalthermore, Sigel et al. proposed the establishment of a composite

resection specimens of primary lung adenocarcinoma with preopera-

original cytological scoring system based on the above three cyto-

tive cytology specimens, including samples obtained via CT-guided

logical features. By grouping the cytological scores, they stratified

needle aspiration. Sixty-six patients had diagnostic cytology specimens

the tumors into three groups (low, intermediate, and high rate ofavailable for review.12 Sufficient cellularity was noted on cytologic

DFS). They also revealed that there was a significant association

smears in 58 of 66 cases for subtyping, whereas eight cases could
not be subclassified based on their cytology because of scant cellular-

TABLE 3ity or indefinite patterns. Subtyping of adenocarcinoma was concor-

The recurrence-free survival

dant between the dominant pattern on resection and cytology
specimens in 26 cases (40%) and discordant in 32 (48%) cases. The
acinar pattern was the most common in concordant cases, whereas
discordant cases had a predominantly solid pattern. The authorsemphasized that the identification of solid and micropapillary patterns
was prognostically important but might be unreliable and difficult withcytology specimens. Furthermore, several studies have described thecytological features of lung BAC, that is, AIS in the 4th edition of
WHO classification.13,14 In 2004, Ohori and Santa Maria reported thecytological characteristics of 42 lung adenocarcinoma cases, including
6 BACs (three non-mucinous, three mucinous), 17 mixed adenocarcinoma, 6 papillary adenocarcinoma, and 12 IAD.13 The distribution oftypes of cytological specimens was as follows: fine-needle aspiration,
30; bronchial washing, 4; bronchial brushing, 4; bronchoalveolar
Abbreviations: 5-y RFS, 5-year recurrence-free survival; AD, adenocarcinoma; AIS, adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma;NS, not significant.

lavage, 2; and sputum 1. On comparison of the 6 BACs with the
12 IADs, they identified 9 statistically significant cytological features

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(A) 18039.8±28.7

biopsy (CTNB) was first reported in 197617 and has since become a60.3±40.5

widely used diagnostic tool for the management of patients with sus-

160

pected lung cancer. CTNBs are a relatively accurate method of diag-140

nosing peripheral pulmonary lesions.18–21 In the present study, all120cases evaluated were obtained using a CTNB because of their small
size and peripheral location. Therefore, all of the cytology specimens

100

were evaluated under the same conditions, morphologically. Further-80

more, the IAD and NMIAD groups evaluated in the present study con-60

tained 31 and 39 cases, respectively, both of which are greater than in40

previous cytological studies of small lung adenocarcinomas.
To our knowledge, this is the first report of the cytological differ-

200(B) 70ences between IAD and NMIAD, which had pure GGO or partly solid

AIS + MIA
11.8±11.5

IAD

nodules on preoperative CT in small peripheral pulmonary lesions, with

P = 0.017

a focus on invasion in early lung adenocarcinoma. Patients with pure13.3±11.2

solid nodules on CT were excluded from this study because in general,
tumors in patients with purely solid nodules on preoperative CT histo-

60

logically have much more prominent nuclear atypia and/or structural
atypia than those in patients with pure GGO or partly solid nodules.

50

Although there were some cases with acinar, solid, micropapillary, or

40

mucinous adenocarcinoma components, there were no acinar, solid,
micropapillary, or mucinous-predominant adenocarcinoma cases in the

30

IAD group in the present study. The reason why no mucinous adenocarcinoma cases were observed may have been because the patients

20

in our study were all Japanese and therefore included many cases of

100adenocarcinoma with EGFR mutations. The findings from the present
study suggest that several cytological parameters—namely irregular

AIS + MIA

IAD

nuclear contours, large tumor cell clusters, and 3D clusters—may be

P = 0.59

useful for distinguishing IAD from NMIAD. A multivariate analysis

A, The numbers of tumor cells (×10 magnification in
10 fields) were 39.8 ± 28.7 in the AIS + MIA (NMIAD) group and60.3 ± 40.5 in the IAD group (P = 0.017). B, The numbers of stromalclusters in two slides were 11.8 ± 11.5 in the NMIAD group and13.3 ± 11.2 in the IAD group (P = 0.591)

FIGURE 2

showed that the frequency of irregular nuclear contours was significantly higher in the IAD group than in the NMIAD group. Furthermore,
the number of tumor cells was higher in the IAD group than in the
NMIAD group, whereas no significant differences were observed in
the number of stromal clusters between the two groups. These cyto-

as the features of BAC cases: a clean background, the absence of 3D
clusters, neoplastic cells in flat sheets, the orderly arrangement of cellswith round uniform nuclei, a predominance of mucinous cells, theabsence of nuclear overlap, the absence of irregular nuclear membranes,
fine granular chromatin, and nuclear grooves. MacDonald and Yazdi
reported on 49 patients diagnosed by a fine-needle aspiration biopsy(FNAB) and/or surgical biopsy.14 Twenty-four of the patients diagnosed
with BAC by a FNAB had histologic confirmation. Surgical pathology
revealed BAC in 15 patients with a cytologic diagnosis of large-cell car-logical characteristics, which were significantly higher or more prominent in the IAD group than in the NMIAD group, appear to support
peripheral pulmonary lesions featuring pure GGO or partly solid nodules being IAD pathologically.
Adenocarcinoma is the most common histological subtype of lungcancer in Japan; it accounts for approximately 70% of all surgicallyresected lung cancers, and most cases occur as pulmonary peripherallesions.22 Patients with GGO-dominant small lung adenocarcinomas
were previously reported to have a favorable prognosis.23 Limited resection of the lung for early-stage lung cancers has recently been

cinoma or adenocarcinoma. Nine patients diagnosed with BAC by FNAB

advocated from a clinical standpoint for the preservation of the lungwere found to have adenocarcinoma histologically. One unsatisfactory

function.24 Therefore, GGO-dominant peripheral lung lesions may be

FNAB aspirate diagnosed as BAC histologically was due to a samplingcandidates for limited surgical resection.25 However, there are cur-error. A review of 15 FNAB specimens with a diagnosis of large cell car-rently no established criteria for the indication of limited resection.

cinoma or adenocarcinoma revealed cytologic features typical of BAC.

The new WHO classification clearly emphasizes the significanceIn six aspirates of these cases, additional features, such as pronouncedof the histological subtype for determining the prognosis of patients,

nuclear crowding and overlapping, variation in nuclear size, and an

and this has been validated in independent cohorts. Under the newincreased number of pleomorphic cells, interfered with the FNAB diag-classification system, AIS is defined as a preinvasive lesion, while

nosis of BAC. Nine FNABs with a diagnosis of BAC were found histo-

MIA is in a new, separate category; both are distinguished from IAD

logically to have adenocarcinoma with a focal BAC growth pattern.

because AIS and MIA have 100% disease-free survival and RFS

For small-sized peripheral lung adenocarcinoma, it is especiallydifficult to obtain a preoperative diagnosis.

15,16

A CT-guided needle

rates if the tumor is completely resected.4,26 We also revealed in
the present study that AIS and MIA had a more favorable prognosis

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MATSUBAYASHI ET AL.

(A)

(D)

(B)

(E)

(C)

(F)

(A) Large tumor cell clusters (tumor cell clusters composed of ≥20 tumor cells; P = 0.0106), (B) three-dimensional tumor cell clusters(P = 0.0017), (C) irregular nuclear contours (P = 0.0007), (D) nuclear pleomorphisms (P = 0.2008), (E) prominent nucleoli (P = 0.2484), and(F) intranuclear cytoplasmic inclusion (P = 0.8689)

FIGURE 3

than IAD; the NMIAD group had a 100% 5-year RFS, while the IADfrom the date of pulmonary resection to the date of death from anygroup had an 82.8% 5-year RFS. There was no difference in thecause, which is why the OS was lower than the RFS in several

RFS between the patients with NMIAD and those with lepidic ade-groups. Therefore, limited resection may be indicated for adenocar-

nocarcinoma tumors in our cohort, possibly because our study

cinomas classified as NMIAD, that is, AIS or MIA.

included only nine cases with lepidic adenocarcinoma. The differ-

Chest CT may be useful for detecting the foci of invasion in lungence in the RFS was significant between patients with NMIAD andtumors. However, the foci of invasion are indistinguishable from cen-

IAD tumors. In IAD tumors with recurrence, there were some cases

tral scars in lung cancer when only chest CT is performed, as NMIAD

with tiny foci of a solid (two cases) or micropapillary (one case) ade-with a central scar may have partly solid nodules on chest CT. A multi-

nocarcinoma component, which are poor prognostic factors. Of

center registration study recently showed that the solid tumor sizes

note, the RFS in our study was defined as the interval from the dateon HRCT and maximum standardized uptake values on PET/CT haveof pulmonary resection to the date of recurrence, lung cancer-

greater predictive value than the whole tumor sizes for high-grade

related death, or the last follow-up. In contrast, no significant differ-malignancy and the prognosis with clinical stage IA lung adenocarci-

ences were observed between NMIAD and IAD in the 5-yearnoma.9 Although PET-CT is useful for detecting the foci of invasion in

OS. The OS in the present study was defined as the time elapsed

lung adenocarcinomas, detecting particularly small foci can be difficult.

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Furthermore, it is not possible to diagnose AIS or MIA using biopsy
specimens alone, as central scars cannot be distinguished from thefoci of invasion in lung cancer using only preoperative biopsy specimens.14 In the present study, no significant differences were observedin the number of stroma between the IAD and NMIAD groups. This
result indicates that it is difficult to distinguish the foci of invasion in
IADs from the scars in NMIAD.
Therefore, in addition to the chest CT and PET findings, the cytological characteristics of irregular nuclear contours, large tumor cellclusters, and 3D tumor cell clusters may play a role in making moreaccurate preoperative judgment of IAD.

ACKNOWLEDGMENT
English language editing support was provided by Brian Quinn (Japan
Medical Communication).
ORCID
Jun Matsubayashi

https://orcid.org/0000-0001-7545-9332

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How to cite this article: Matsubayashi J, Miyake S, Kudo Y,
et al. Cytological differences between invasive and noninvasive or minimally invasive lung adenocarcinomas diagnosed inJapanese patients using needle biopsy specimens of pulmonary lesions ≤3 cm in diameter. Diagn Cytopathol. 2019;47:
688–694. https://doi.org/10.1002/dc.24171

ONCOLOGY LETTERS 18: 6451-6458, 2019

Epidermal growth factor receptor mutation subtype
has differential effects on adjuvant chemotherapy for resectedadenocarcinoma pathological stages II‑III
YOHEI KAWAGUCHI1*, TETSUYA OKANO1*, KENTARO IMAI1, SACHIO MAEHARA1,
JUNICHI MAEDA1, KOICHI YOSHIDA1, MASARU HAGIWARA1, MASATOSHI KAKIHANA1,NAOHIRO KAJIWARA1, TATSUO OHIRA1, JUN MATSUBAYASHI2 and NORIHIKO IKEDA1
1

Department of General Thoracic Surgery, Tokyo Medical University Hospital;
Department of Anatomic Pathology, Tokyo Medical University, Tokyo 1600023, Japan

2

Received April 18, 2019; Accepted September 25, 2019
DOI: 10.3892/ol.2019.11050
Abstract. The aim of the present study was to investigate
epidermal growth factor receptor (EGFR) mutations as aprognostic factor for postoperative patients with positive
EGFR mutations treated with postoperative platinum-based
adjuvant chemotherapy (PBAC), and whether two common
EGFR mutations exhibit different responses to PBAC. A
total of 110 patients who underwent complete surgical resection were enrolled, and overall survival (OS) and disease-freesurvival (DFS) were investigated based on EGFR mutation
status and PBAC. The 3 year OS rate in patient groups wereas follows: Patients with EGFR mutations (MT) undergoing
PBAC, 89.3%; MT patients without PBAC, 83.3%; wild-type(WT) patients with PBAC, 82.3%; and WT patients without
PBAC, 62.2%. Statistically significant differences wereobserved between WT patients based on PBAC (P=0.026).
No statistically significant differences were observed
between MT patients with PBAC and MT patients without
PBAC. On the basis of mutation subtypes, the 3 year OS rateof patient groups were as follows: Patients with in-framedeletions in exon19 (19 del) with PBAC, 92.3%; patients with
19 del without PBAC, 85.7%; patients with the point mutation

Correspondence to: Dr Tetsuya Okano, Department of General

Thoracic Surgery, Tokyo Medical University Hospital, 6-7-1
Nishishinjuku, Shinnjuku-ku, Tokyo 1600023, Japan
E-mail: tokano@tokyo-med.ac.jp*

Contributed equally

Abbreviations: CT, computed tomography; DFS, disease-freesurvival; EGFR, epidermal growth factor receptor; NSCLC, nonsmall-cell lung cancer; OS, overall survival; PBAC, platinum-based
adjuvant chemotherapy; TKI, tyrosine kinase inhibitors; WT, wild-typeKeywords: epidermal growth factor receptor mutation,
platinum-based adjuvant chemotherapy, mutation subtypes, lungadenocarcinoma

L858R inexon21 (21L858R) with PBAC, 86.7%; and patientswith 21L858R without PBAC, 81.5%; the respective 3-year
DFS rates were 53.8, 14.3, 40.2 and 26.9%. Statisticallysignificant differences were observed in the DFS rates in
19 del patients, which was dependent on PBAC (P=0.040).
EGFR mutation-positive patients exhibited a decreased
benefit from PBAC for increasing in survival rate comparedwith WT patients. It may be necessary to consider postoperative strategies based on EGFR mutations and their subtype
in the future.
Introduction
The best therapeutic modality for clinical stages I, II, andappropriate stage IIIA non-small-cell lung cancer (NSCLC)
is complete surgical resection (1,2). However, despite improvements in diagnostic and therapeutic approaches, only 60% of
patients survive 5 years after surgery (1).
To improve the survival rates, the first trial of adjuvant
chemotherapy was performed decades ago (1). Subsequently,
randomized trials reported the benefit of platinum-based
adjuvant chemotherapy (PBAC) in patients with pathologic
stages II and III NSCLC with lymph node involvement, andsubset analyses suggested a benefit in patients with large IB
tumors (3-5). However, the 5 year overall survival (OS) advantage for patients who underwent adjuvant chemotherapy wasreported to be a modest 5.4% (6). Furthermore, PBAC oftencauses adverse events. Several randomized trials of adjuvant
chemotherapy for NSCLC have reported that almost 30% of
patients experienced grade III or IV toxicity and there were0.8-2.0% of adjuvant chemotherapy-related deaths (7). Thus,
its significant toxicity may limit its use.
On the basis of the above, it is very important to adapt adjuvant chemotherapy and to identify useful factors predicting its
efficacy.
Mutations in the epidermal growth factor receptor (EGFR),
most of which have been detected in lung adenocarcinoma,
are predictors of response to EGFR tyrosine kinase inhibitors (TKIs), which have proven efficacy in the treatment ofadvanced stage NSCLC (8,9).

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Although EGFR mutations are prognostic factors in only
unresectable advanced NSCLC, there are few reports of
their utility as prognostic factors in resectable NSCLC (10).
In particular, there are no studies in the context of predictive
factors for PBAC effects.
EGFR mutations are categorized into two groups, common
mutations and minor mutations. Common mutations comprise
85% of all EGFR mutations and include two subtypes, in-framedeletions in exon19 (19 del) and the point mutation L858R inexon21 (21L858R) (11). Those two mutations are expected tohave different biological features (11,12).
Here, we investigated EGFR mutations as prognostic
factors in postoperative EGFR mutation-positive (MT) patientstreated with PBAC and whether the above two common
mutations are associated with differential responses to PBAC.
Materials and methods
Patient selection and study design. A total of 720 consecutive
patients with adenocarcinoma underwent pulmonary resection
with no evidence of residual cancer either macroscopically or
microscopically between January 2009 and December 2013 at
Tokyo Medical University Hospital. Of these, 171 patients inpathological stages II and III (per the 7th Edition of the TNM
Classification for Lung and Pleural Tumors of the Union
for International Cancer Control) were enrolled (13). We
excluded 45 patients because they had received preoperative
chemotherapy, radiotherapy, or both; because their tumors
had a mutation in EGFR exon20 (an indicator of resistance to
EGFR-TKIs), mutations in the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion gene, orother minor mutations; or because the presence of EGFR mutations could not be analyzed. We also excluded 10 patients who
had received adjuvant chemotherapy other than platinum-based
regimens such as uracil-tegafur or tegafur-gimeracil-oteracil
and six patients who received only the best supportive careincluding palliative radiotherapy for controlling pain from bonemetastasis as lung cancer recurrence and no treatments such asradiotherapy, chemotherapy, or surgical treatment after disease
recurrence. We enrolled the remaining 110 patients in the study.
The study followed a retrospective, single institutional design
and investigated EGFR mutation subtype in patients who had
undergone surgical resection of adenocarcinoma. This study
was approved by the ethics committee of the Tokyo Medical
University; the approval number is 2016-167.
Analysis of patients. We reviewed the medical records ofpatients to assess the following clinicopathological information: Age; sex; smoking history; surgical procedures; tumor
differentiation; blood vessel invasion; lymphatic permeation;
visceral pleural invasion; EGFR mutation status; PBAC
regimen; OS defined as the time elapsed from the date ofsurgery to the date of death; and disease-free survival (DFS)
defined as the time elapsed from the date of pulmonary
resection to the date of initial recurrence or death.
We divided the patients into a mutation (MT) group anda wild-type (WT) group based on their EGFR status andanalyzed their prognosis.
After surgical resection, the patients were examined at
3 month intervals for 3 years, then at 6 month intervals for the

next 2 years, and thereafter at 1 year intervals. The systemic
evaluations of patients included physical examinations, chest
roentgenograms, chest and abdominal computed tomography(CT) scans, and tumor marker measurement. Brain magnetic
resonance imaging and bone scintigraphy or positron emission
tomography/CT scanning was performed every year.
EGFR mutation analysis. All of the surgical specimens
collected were fixed in 10% formalin and embedded inparaffin. Representative sections were routinely stained withhematoxylin and eosin. Experienced pathologists reviewed
the samples to confirm that the sections contained carcinoma
cells. EGFR mutation analysis of the histology specimens was
screened using the direct sequencing method until October
2009. Direct sequencing method has a detection sensitivityof around 25%, while the cycleave polymerase chain reaction
method is around 1 to 5%. So we use cycleave polymerase
chain reaction method (after November 2009), as previously
described (14,15).
Statistical analysis. OS and DFS were estimated using the
Kaplan-Meier method. Differences in survival rates were
determined using log-rank analysis. Hazard ratios and their
95% confidence intervals were calculated using the Cox
proportional hazards model to determine independent predictors of the OS and DFS. Significance differences among thecategorized groups were compared using Chi-square tests.
P<0.05 was considered to indicate a statistically significant
difference. Statistical analyses were performed using the software package SPSS (version 24.0; SPSS, Inc.).
Results
Patient demographics. The median follow-up period for allof the patients was 35.9 months (range, 0.27-88.5 months).
Characteristics of the patients are summarized in Table I.
PBAC was carried out in 62 patients (56.4%). The most common
regimen of the PBAC comprised cisplatin and vinorelbine(41 of 62; 66.1%), and other regimens were as described inTable I. Forty-eight patients (of 110 patients; 44%) did notreceived adjuvant chemotherapy. The most common reason
was the patient's comorbidity such as renal dysfunction, etc.
The second most common reason was the patient's preference.
Another one was elder and poor performance status.
Among the 110 patients, 63 (57.2%) patients had confirmed
recurrence. EGFR mutations were detected in 50 patients(45.6%), and the most common EGFR mutation was L858R(29 of 50; 58.0%). The second most common mutation was
19 del (21 of 50; 40.2%).
The differences between patients in the PBAC groupand those in the observation group are shown in Table II.
Statistically significant differences in age were found between
the two groups (P<0.001). In the PBAC group, five patients(of 62 patients; 8%) were over 75 years old. In the observationgroup, 17 patients (of 48 patients; 35%) were over 75 years old.
There were no statistically significant differences in the otherclinicopathological factors between the groups.
Prognostic outcomes. Survival time was investigated on thebasis of EGFR mutation status and PBAC status. DFS curves
stratified on the basis of EGFR and PBAC status are shown-492-

ONCOLOGY LETTERS 18: 6451-6458, 2019

Table I. Patient characteristics (n=110).
Variables
Sex
Men
Women
Age (years)
Median age
Range
Smoking habits
Ever-smoker/unknown
Never smoker
Surgical procedure
Lobectomy
Segmentectomy
Pathological stage
II
III
Vascular invasion
Positive
Lymphatic permeation
Positive
Viscreral visceral pleural invasion
Positive
Tumor differentiation
Poor
Well/Moderate
Adjuvant chemotherapy
PBAC
None
Tumor recurrence
Recurrence
Non-recurrence
EGFR mutation status
Mutant; Exon 19/21

Wild-type
PBAC regimen
Cisplatin + Vinorelbine
Cisplatin + Gemcitabine
Cisplatin + tegafur/gimeracil/oteracil
Cisplatin + Docetaxel
Cisplatin + Pemetrexed
Carboplatin + Gemcitabine
Carboplatin + Paclitaxel
Observation

6453

Table II. Patient characteristics based on PBAC status.
Value (%)
64 (58)46 (42)

Variables66
40-88
51 (46)59 (54)109 (99)
1 (1)
52 (47)58 (53)86 (78)98 (89)47 (43)31 (28)79 (72)62 (56)48 (44)63 (57)47 (43)50 (45);21 (19)/29 (26)60 (55)41 (37)
1 (1)
1 (1)
7 (6)
1 (1)
8 (7)
3 (3)
48 (44)

Sex
Men
Women
Age, years<75≥75
Smoking habits
Ever-smoker/unknown
Never smoker
Surgical procedure
Lobectomy
Segmentectomy
Pathological stage
II
III
Vascular invasion
Present
AbsentLymphatic permeation
Present
AbsentViscreralvisceral pleuralinvasion
Present
AbsentTumor differentiation
Poor
Well/Moderate
Tumor recurrence
Recurrence
Non-recurrence
EGFR mutation status
MutantWild-type

PBAC status,n (%)-----------------------------------PBAC
None(n=62)(n=48)
32 (29)30 (27)32 (29)16 (15)57 (52)5 (1)31 (28)17 (15)34 (31)28 (25)25 (23)23 (21)61 (55)1 (1)48 (44)025 (23)37 (34)27 (25)21 (19)50 (45)12 (11)36 (33)12 (11)58 (53)4 (3)40 (36)8 (8)24 (22)38 (34)23 (21)25 (23)17 (15)45 (41)14 (13)34 (31)37 (34)25 (23)26 (23)22 (20)29 (26)33 (30)21 (19)27 (25)P-value
0.112<0.001
0.774
0.377
0.097
0.477
0.088
0.333

0.840
0.562
0.752

PBAC, platinum-based adjuvant chemotherapy; EGFR, epidermal
growth factor receptor.

EGFR, epidermal growth factor receptor; PBAC, platinum-based
adjuvant chemotherapy.

in Fig. 1A and B. The 3 year DFS rates in the groups were as
follows: MT with PBAC, 46.5%; MT without PBAC, 22.5%;

WT with PBAC, 57.1%; and WT without PBAC, 47.1%; there
were no statistically significant differences among them.
The OS curves stratified on the basis of EGFR and PBAC
status are shown in Fig. 1C and D. The 3 year OS rates in thegroups were as follows: MT with PBAC, 89.3%; MT without
PBAC, 83.3%; WT with PBAC, 82.3%; WT without PBAC,
62.2%; Fig. 1D shows statistically significant differences in
OS of WT patients with PBAC and that of WT patients without

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Figure 1. Disease-free survival curves based on (A) PBAC in EGFR MT patients and (B) PBAC in EGFR mutation WT patients. There were no significant
statistically differences between MT patients with PBAC and MT patients without PBAC. Overall survivalcurves based on (C) MT patients with PBAC and(D) WT patients with PBAC. PBAC, platinum-based adjuvant chemotherapy; EGFR, epidermal growth factor receptor; MT, mutation-positive; WT, wild-type.

PBAC (P=0.026). There were no statistically significant
differences between MT patients with PBAC and MT patients
without PBAC (P=0.503).
We also examined OS and DFS of the patients with mutants
stratified per mutational subtypes (Fig. 2). The 3 year DFS of
the patients in the stratified groups was as follows: 19 del with
PBAC, 53.8%; 19 del without PBAC, 14.3%; 21L858R with
PBAC, 40.2%; and 21L858R without PBAC, 26.9%.
The 3 year OS rates of the above groups of patients were as
follows: 19 del with PBAC, 92.3%; 19 del without PBAC, 85.7%;
21L858R with PBAC, 86.7%; and 21L858R without PBAC,
81.5%. Statistical analysis of patients with 19 del revealed that
there were statistically significant differences in their DFS
rates, dependent on PBAC being carried out (P=0.040). On theother hand, there were no statistically significant differences intheir OS rates (P=0.208). Statistical analysis of patients with
21L858R found no statistically significant differences in OS(P=0.981) or DFS (P=0.844).
There were no statistical difference about DFS in patientswith 19 del according to drugs combination of PBAC (P=0.99).
Risk factors for poor prognosis. The potential risk factors
for DFS and OS were analyzed using univariate survival

analysis based on mutational status (Table III). In MT patients,
univariate survival analysis showed that blood vessel invasion was the only risk factor associated with DFS (HR, 2.781;
CI, 1.140-6.783; P=0.025). On the other hand, there were no
significant risk factors associated with OS. In WT patients,
the potential risk factors associated with OS were visceralpleural invasion (HR, 3.326; CI, 1.202-9.207; P=0.021) and
PBAC (HR, 3.005; CI, 1.088-8.300; P=0.034). Analysis of
DFS in WT patients using univariate survival analysis showedthat smoking was the only associated risk factor (HR, 2.174;
CI, 1.046-4.518; P=0.038). Visceral pleural invasion was risk
factor for OS in univariate analysis, but not in multivariate
analysis. It might be because of small sample sizes.
Discussion
We performed the present study to clarify whether EGFR
mutation status can be considered a prognostic factor
predicting the effect of PBAC and to elucidate whether there
is a difference in the effect of adjuvant chemotherapy based on
mutational subtype.
We found that EGFR mutation status was not predictiveof OS or DFS benefit from PBAC in lung adenocarcinoma;-494-

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6455

Figure 2. DFS curves based on (A) PBAC in patients with in-frame deletions in exon19 (19del) and (B) PBAC in patients with the point mutation L858R inexon21 (21L858R). There were statistically significant differences in the DFS rates independent of PBAC in 19del patients. Overall survival curves based on(C) PBAC in patients with 19del and (D) PBAC in patients with 21L858R. DFS, disease-free survival; PBAC, platinum-based adjuvant chemotherapy.

however, in WT patients, PBAC was a significant prognostic factor for OS. This indicates that EGFR mutation
status might be considered as a predicting factor for poor
effect of PBAC and MT patients might benefit less fromadjuvant chemotherapy than WT patients would. In general,
EGFR-TKI as first-line treatment for advanced NSCLC
prolongs progression-free survival and increases theobjective response rate compared with platinum doublet
chemotherapy (8,9). Few studies focused on the prognostic
value of EGFR mutation status in patients with advanced
NSCLC with chemotherapy.
In this present analysis of OS, there were no statisticallysignificant differences between MT patients with PBAC andthose without PBAC. The reason for this outcome may be
the effectiveness of EGFR-TKIs in MT patients in general.
Prolonged survival after recurrence in both groups of MT
patients might have obscured the difference in OS between
the groups. Kudo et al reported that the presence of EGFR
mutations is a good prognostic factor in MT patients withpostoperative recurrence (10). In contrast, in WT patients,
EGFR-TKIs have not been approved for the treatment of
NSCLC except erlotinib and have less effectiveness than in
MT patients.

EGFR positive lung cancer is effective by EGFR-TKI, andthe benefits of PBAC might been diminished due to the longer
treatment period after relapse compared to the time to relapse.
Instead of PBAC in MT patients whose effect may be lesseffective, EGFR-TKI treatment as postoperative adjuvant
chemotherapy for MT patients has been investigated. Several
studies suggested a possible benefit of EGFR‑TKI as postoperative adjuvant chemotherapy for MT patients. In an analysisof 167 patients with resected stages I to IIIA NSCLC with
EGFR mutations, patients who received EGFR-TKI treatment
showed a more favorable 2 year DFS rate than an untreatedgroup (HR, 0.53; 95% CI, 0.28-1.03; P=0.06) (16). Anotherstudy, a single-arm, multi-institutional, prospective phase II
study called the SELECT trial, showed that 100 patients withresected stages IA to IIIA NSCLC and EGFR mutations
receiving adjuvant erlotinib for 2 years after standard-of-caretreatment had a 2 year DFS rate of 89% with a medianfollow-up of 3.4 years (17).
The above studies suggest the possible benefit of EGFR‑TKI
adjuvant chemotherapy in MT patients. On the other hand,a randomized, double-blind, placebo-controlled phase III
trial called the RADIANT study using erlotinib as adjuvant
EGFR-TKI analyzed a total of 278 patients and failed to

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KAWAGUCHI et al: EGFR MUTATION IMPACT ON ADJUVANT CHEMOTHERAPY FOR LUNG ADENOCARCINOMA

Table III. Univariate and Multivariate analysis of disease-free survival and overall survival.
A, All patients

Variable

Overall survival
Disease-free survival---------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------MVA
MVA
UVA ---------------------------------------------------------------------------- UVA -------------------------------------------------------------------P-value Hazard ratio
95%CI
P-value P-value Hazard ratio
95%CI
P-value

Sex
Age
Smoking habits
Pathological stage
Vascular invasion
Lymphatic premeation
Viscreral pleural invasion
Tumor differentiation
Adjuvant chemotherapy

0.471
0.118
0.093
0.186
0.034
0.403
0.021
0.020
0.030

4.872
2.000
2.260
2.519

1.073-22.122
0.04
0.915-4.373 NS (0.082)
1.040-4.911
0.039
1.143-5.550
0.022

0.233
0.130
0.032
0.216
0.084
0.854
0.036
0.709
0.073

1.695
1.681
-

1.047-2.746
1.038-2.723
-

0.032
0.035
-

B, Patients with EGFR wild-type

Variable

Overall survival
Disease-free survival---------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------MVA
MVA
UVA ---------------------------------------------------------------------------- UVA -------------------------------------------------------------------P-value Hazard ratio
95%CI
P-value P-value Hazard ratio
95%CI
P-value

Sex
Age
Smoking habits
Pathological stage
Vascular invasion
Lymphatic premeation
Viscreral pleural invasion
Tumor differentiation
Adjuvant chemotherapy

0.987
0.489
0.227
0.433
0.125
0.526
0.021
0.085
0.034

3.242
2.928

1.173-8.958
1.059-8.091

0.023
0.038

0.319
0.920
0.038
0.872
0.583
0.500
0.050
0.290
0.218

-

-

-

B, Patients with EGFR mutant

Variable

Overall survival
Disease-free survival---------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------MVA
MVA
UVA ---------------------------------------------------------------------------- UVA -------------------------------------------------------------------P-value Hazard ratio
95%CI
P-value P-value Hazard ratio
95%CI
P-value

Sex
Age
Smoking habits
Pathological stage
Vascular invasion
Lymphatic premeation
Viscreral pleural invasion
Tumor differentiation
Adjuvant chemotherapy
Mutational status

0.127
0.068
0.186
0.118
0.172
0.759
0.558
0.286
0.505
0.343

-

-

-

0.925
0.073
0.767
0.136
0.025
0.444
0.433
0.944
0.287
0.955

-

-

-

-, Not included in MVA. EGFR, epidermal growth factor receptor; NS, no statistical difference; MT, mutation-positive; UVA, uni-variate
analysis; MVA, multi‑variate analysis; CI, confidence interval.

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demonstrate a DFS benefit of erlotinib in EGFR mutation-positive patients (18).
In the analysis of subtypes, 19del patients with PBAC
showed significantly longer DFS than 19del patients without
PBAC, but 21L858R patient groups showed no statisticallysignificant difference in DFS. There are likely differences inclinical and biological features between patients with 19del
and those with L858R. It was reported that the patterns of both
EGFR amplification and EGFR autophosphorylation wereshown to differ between cell lines harboring the two mostcommon EGFR mutation types of 19del and L858R mutation (19). Several studies have reported data regarding thesetwo common EGFR mutations. In an analysis of MT patients
receiving no EGFR-TKI therapy, those with the 19 del mutation
had worse survival than those with the L858R mutation (11).
Yamashita et al reported that among NSCLC patients receiving
platinum-based chemotherapy, those with exon 19 deletion
have a longer PFS and OS. This result supported the significant
effect of PBAC in DFS rates in 19 del patient (20).
A multicenter, international, open-label, exploratory,
randomized controlled trial called the LUX-Lung7 showed adifference in OS between patients with the common EGFR
mutations (21) and suggested that 19del patients might be moresensitive to EGFR-TKIs than those with 21L858R.
On the basis of previous reports, we speculated that the
number of patients with the 19del mutation who remain at
the micro-metastasis stage after complete cancer resection
would be larger than those with the 21L858R mutation. In
terms of postoperative recurrence, EGFR-TKIs showed goodeffectiveness among 19del patients (16,21). We found a statistically significant difference in DFS but not OS between 19delpatients with PBAC and those without PBAC.
We must acknowledge three limitations in the present study.
First, the number of patients was small and data analysis may
not have been maximally effective. In future studies, we plan
to collect more data regarding surgically resected adenocarcinoma with pathologically confirmed lymph node metastasis.
Second, this study was retrospective and single institutional.
Third, we excluded patients with unknown EGFR mutation
status; even though the number of such excluded patients was
small, it may have contributed to selection bias.
In the present study, compared with patients with WT, EGFR
mutation‑positive patients showed less influence of PBAC on
improvement of survival rate. PBAC might not be necessary forMt patients with pathological stage II/III lung adenocarcinoma
On the basis of the EGFR mutation subtype, patients withthe 19 del mutation undergoing PBAC showed significantly
more favorable DFS than those without PBAC. It may therefore be necessary to consider postoperative strategy based on
the presence of EGFR mutations and their subtype in patientswith completely resected adenocarcinoma.

6457

Availability of data and materials
The datasets used and/or analyzed during the present study
are available from the corresponding author on reasonable
request.
Authors' contributions
YK, TeO and NI designed the study; YK performed theresearch, with the assistance of TeO, KI, SM, JM, KY, MH,
MK, NK TaO and JM. YK and TeO analyzed the data. YK
and TOK generated the figures and wrote the paper. TeO andNI revised the figures and critically revised the manuscriptfor important intellectual content. All authors have read andapproved the manuscript.
Ethics approval and consent to participate
The present study was approved by the ethics committee of theTokyo Medical University (approval number: 2016-167).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
References

Acknowledgements
Not applicable.
Funding
The present study was supported by AMED [grant
no. JP17ck0106295].

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Attribution-NonCommercial-NoDerivatives 4.0
International (CC BY-NC-ND 4.0) License.

Differential regulation of T-cell dependent and T-cell
independent antibody responses through arginine
methyltransferase PRMT1 in vivo
Kikumi Hata1, Noriko Yanase1, Katsuko Sudo2, Hiroshi Kiyonari3,4, Yoshiko Mukumoto4,
Junichiro Mizuguchi1 and Tadashi Yokosuka1
1 Department of Immunology, Tokyo Medical University, Japan
2 Animal Research Center, Tokyo Medical University, Japan
3 Animal Resource Development Unit, RIKEN Center for Life Science Technologies, Kobe, Japan
4 Genetic Engineering Team, RIKEN Center for Life Science Technologies, Kobe, Japan

Correspondence
K. Hata, Department of Immunology, Tokyo
Medical University, 6-1-1 Shinjuku, Shinjukuku, Tokyo 160-8402, Japan
Fax: +81 3 3341 2941
Tel: +81 3 3351 6141
E-mail: khata@tokyo-med.ac.jp(Received 11 February 2016, revised 21
March 2016, accepted 22 March 2016,
available online 8 April 2016)
doi:10.1002/1873-3468.12161
Edited by Wilfried EllmeierProtein arginine methyltransferase 1 (PRMT1), a major PRMT in mammalian cells, has been shown to play a crucial role in multiple biological functions in vitro. To explore the role of PRMT1 in B cells in vivo, we generated
B cell-specific PRMT1-deficient (Prmt1/) mice using a Cre-loxP system.
Prmt1/ mice showed a defect in B-cell development with diminished levelsof serum antibodies. Antibody responses in Prmt1/ mice were absent after
stimulation with the type 2 T cell-independent antigen NP-Ficoll but intact
after stimulation with the T cell-dependent antigen NP-OVA. Our findings
comprise the first evidence showing that PRMT1 is necessary for lymphocyte
functions in vivo.
Keywords: antibody response; arginine methylation; B cells; PRMT;
T-dependent antigen; T-independent antigen

B cell responses such as proliferation, early differentiation, antibody production and isotype switching are
initiated by the interaction between a B cell receptor(BCR) and its cognate antigen, and further modulated
by various cytokines derived from T cells in a different immune status and environment [1]. Antigeninduced engagement of BCRs initiates the signalingcascades, including protein tyrosine kinases Lyn andBruton’s tyrosine kinase (BTK), a small G-protein
Ras, and transcription factors nuclear factor of activated T-cell, nuclear factor kappa-B (NF-jB) andactivating protein-1 [2,3]. Such activation signals leadto the generation of two individual B cell subsets that
produce distinct antibodies [4,5]. Marginal zone B(MZB) cells possess the capacity to respond to thebacterial products such as repetitive immunogenic epitopes in lipopolysaccharide (LPS) or bacterial capsularpolysaccharide. These MZB cells occasionally differentiate into short-lived plasma cells producing
immunoglobulin (Ig)M or class-switched antibodies(IgG2a and IgG3) without T cell help (T cell-independent; TI) [6,7]. The widely used TI model antigens
NP-LPS(4-hydroxy-3-nitrophenylacetyl hapten coupled to
lipopolysaccharide) and NP-Ficoll are further categorized into T cell-independent type 1 (TI-1) and type 2(TI-2) antigens, respectively [8]. Immunogenic NP residues on the polysaccharide backbone efficiently crosslink the multiple NP-specific BCRs. In response toprotein antigens, follicular B (FOB) cells produce

Abbreviations
BCR, B cell receptor; BTK, Bruton’s tyrosine kinase; CFSE, carboxyfluorescein diacetate succinimidyl ester; FOB, follicular B; Ig,
immunoglobulin; IL, interleukin; LPS, lipopolysaccharide; mAb, monoclonal antibody; MZB, marginal zone B; NF-jB, nuclear factor kappa-B;
PE, phycoerythrin; PRMT, protein arginine methyltransferase; TD, T cell-dependent; TI, T cell-independent.

1200

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Role of PRMT1 in B cells

K. Hata et al.

class-switched high-affinity antibodies in collaboration
with T helper cells and this process is called ‘affinity
maturation’, occurring in germinal center [9,10].
Arginine methylation within various proteins is a
critical post-translational modification and this methylation is further involved in chromatin remodeling, signal transduction, cellular proliferation and RNA
processing [11,12]. Modification of arginine residues iscatalyzed by a series of arginine methyltransferases(PRMTs), which are divided into type I (PRMT1, 3,
4/CARM1, 6 and 8) and type II (PRMT5, PRMT7
and 9) enzymes in accordance with the production ofasymmetric and symmetric dimethylarginine, respectively [11]. Recent findings suggest that arginine
methylation plays a crucial role in the lymphocyte signaling and its function [13–16]. Coengagement of T
cell receptors with co-stimulatory receptor CD28
results in the arginine methylation of a guanine
nucleotide-exchange factor Vav1, leading to theenhanced production of interleukin (IL)-2 [16]. Amongthe arginine methyltransferases, PRMT1 is a major
isozyme in mammalian cells and is known to constitutively methylate Iga [17], which transmits signals
regulating B cell differentiation and its function by
A

Wild type allele

2

b

3

associating together with Igb and BCR [18,19]. Moreover, a general inhibitor of PRMTs prevented B cellproliferation and differentiation [20]. Although thesestudies indicate the importance of PRMT1 in a biological aspect in vitro, it is still remains to be determined
how humoral antibody responses are regulated by
PRMT1 in vivo.
Accordingly, we aimed to determine whether
PRMT1 regulates humoral antibody responses bygenerating a B cell-specific PRMT1-deficient mouse(Prmt1/) with the Cre-loxP system. The B cell-specific Prmt1/ mice demonstrated a defect in B cell
development, with diminished levels of serum IgM,
IgG2a, IgG2b and IgG3. Interestingly, however, TI
and T cell-dependent (TD) antibody responses weredifferentially regulated by PRMT1 in vivo; anti-NP
IgM or IgG3 responses to TI-2 antigen NP-Ficoll
were severely decreased in Prmt1/ mice, whereas
anti-NP IgG responses to TD antigen NP-OVA
were not changed. In the present study, we demonstrate a novel role of PRMT1 in the antibody
responses to TI and TD antigens and discuss theimportance of arginine methylation with respect to
B cell functions.

4 5

6

7 8

e
322 bpTargeting vector

DT-A

Targeted allele

2

3

2

3

a

b

Neo

4 5

Neo

4 5

d

c

6

7 8

e

470 bpB

Transitional B

PRMT1
CD19-Cre

f/f
–

f/+
+

f/f
+

FOB
f/f
–

f/+
+

MZB
f/f
+

f/f
–

f/+
+

f/f
+

C

spleen B

non-B

PRMT1
CD19-cref/f f/+ f/f– + +f/f f/+ f/f
– + +

PRMT1

Primer a+d
1.0

Primer a+c

1.0

0.1 1.0

0.8

0.6

Actin

Fig. 1. Generation of B cell-specific PRMT1-deficient mice. (A) Exons of the PRMT1 gene, loxP sites (black triangles), frt sites (white
triangles) and PCR primer annealing sites (arrows) are shown. DT-A, diphtheria toxin A fragment for negative selection; Neo, neomycinresistant cassette for positive selection. (B) CD19-Cre-mediated deletion of targeted exons flanked with loxP sites in Prmt1/ mice.
Genomic DNAs of transitional B, FOB or MZB cells from the indicated mice were amplified by PCR with the primer pair b and g in (A) for
the targeted alle or a and c in (A) for the control. (C) The amount of PRMT1 protein in Prmt1/ mice was assessed by western blotting
using anti-PRMT1 or control anti-actin as a loading control.

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Materials and methods

Japan) in combination with primers with the sequences: (a)(50 -CTTGCCATACAAGAGATCCCTG-30 ); (b) (50 -CAA
GGTTCATTTTGGTGATCTAAG-30 ); (c) (50 -CTTAGATCACCAAAATGAACCTTG -30 ); (d) (50 -GACTAGTGAG
ACGTGCTACTTC-30 ); (e) (50 -GATACTGGAACACTCAATCTAAG-30 ). The PCR products were resolved on 2%
agarose gels.

Animals
PRMT1 conditional knockout mice (accession number
CDB0853K: http://www.clst.riken.jp/arg/mutant%20mice%
20list.html) were generated as described elsewhere (http://
www.clst.riken.jp/arg/Methods.html). To generate a targeting vector, genomic fragments of the PRMT1 locus wereobtained from the RP23-373N14 BAC clone (BACPAC
Resources Center, Oakland, CA, USA). A 750 bp region
containing exons 4 and 5 of the PRMT1 gene was flanked
by loxP sites (Fig. 1A). Targeted ES clones were microinjected into ICR eight-cell stage embryos, and injected
embryos were transferred into pseudopregnant ICR
females. The resulting chimeras were bred with C57BL/6
mice, and heterozygous offspring were identified by PCR.
Mice with loxP sites flanking exons 4 and 5 of PRMT1(Prmt1f/f) were crossed with Cd19-Cre (Cd19Cre) mice todelete PRMT1 at multiple stages of B cell development.
The mice were bred and maintained under pathogen-free
conditions at Tokyo Medical University. They were handled in accordance with the guidelines of the Ethical Committee for Animal Experiments.

Western blotting
Western blotting analysis was carried out as described previously [21]. Briefly, samples were separated by SDS/
PAGE, and then transferred to poly(vinylidene difluoride)
membrane (Millipore, Darmstadt, Germany). The blots
were incubated with primary antibodies (Abs): anti-PRMT1(Millipore) and anti-actin (Sigma-Aldrich, St Louis, MO,
USA). Membrane-bound horseradish peroxidase-conjugated Abs were visualized by electrochemiluminescence(GE Healthcare, Little Chalfont, UK).

Flow cytometry
Single cell suspensions (1 9 106) were incubated with
FcBlock antibodies in staining buffer (3% FBS, 0.1%
sodium azide in PBS) for 15 min on ice, followed by incubation with a cocktail of phycoerythrin (PE), FITC, APC,PerCP, Pacific blue or PE-Cy7 conjugated antibodies (BDPharmingen, San Diego, CA, USA; eBioscience, Carlsbad,
CA, USA; BioLegend, San Diego, CA, USA). The Abs
used were: CD3, CD4, CD5, CD8, CD11b, CD23, CD69,
CD80, CD86, IgM, IgG1, IgG3, IgE, B220, MHC class II
and AA4.1. Cells were analyzed on FACSCanto II flow
cytometers (Nippon Becton Dickinson, Tokyo, Japan). The
data were analyzed using FLOWJO (Tomy Digital Biology,
Tokyo, Japan).

Genotyping and genomic PCR
Genotyping was performed by PCR using genomic DNA
extracted from tail tissue (Fig. S1). Genomic PCR were
performed using genomic DNA extracted from purified
pro-B (IgM�B220+c-kit+), pre-B (IgM�B220+CD25+),
immature B (IgM+B220mid), recirculating B (IgM+B220hi),transitional B (B220+AA4.1+), FOB (B220+AA4.1�
CD23+) and MZB (B220+AA4.1�IgM+CD23�) cells.
PCR reactions utilized Taq polymerase (Takara, Kyoto,103

P = 0.008

P = 0.113

P = 0.059

P = 0.029

P = 0.012

P = 0.015

Cont.
Prmt1–/–

Serum Ig (μg·mL–1)

102

101

100

10–1

1202

IgM

IgA

IgG1

IgG2a

IgG2b

IgG3

Fig. 2. Serum Ig levels in Prmt1�/� orcontrol mice. Concentrations of serum Igs
were determined by ELISA in Prmt1�/�and control mice. Data are shown as themean � SD (n = 6). P-values were
calculated using Student’s t-test.

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Immunization and serum analysis
Prmt1/ and control mice were immunized with 100 lg of
NP-Ficoll in PBS or 100 lg of NP-OVA in alum, and the
OVA-primed mice were boosted with 50 lg of NP-OVA in
PBS on day 28. Sera were harvested on the indicated days,
followed by an assay for an NP-specific antibody by
ELISA.

ELISA
ELISA was performed in accordance with a modified procedure, as described previously [22]. Briefly, ELISA plates
were coated with 10 lgmL1 NP-BSA (for specific Ig)
and anti-mouse Igs (for total IgM, IgG subclass, IgA andIgE) in a total volume of 50 lL. After overnight incubation at 4 °C, the supernatants were discarded, and theplates were washed. The plates were pre-incubated with ablocking buffer (2% BSA and 0.05% Tween 20 in PBS)
at room temperature for 1 h. After washing, samples wereadded to the wells, and the plates were incubated at room
temeperature for 2 h. After several washes, alkaline phosphatase-labeled IgG Abs (Southern Biotech, Birmingham,
AL, USA) specific for mouse IgM, IgG1, IgG2a, IgG2b,
IgG3, IgA or IgE was added, and then the plates wereincubated at room temperature for 1.5 h. After several
washes, color was developed by incubating with phosphatase substrate p-nitrophenyl phosphate (Sigma-Aldrich)for 30 min.

Cell purification and in vitro cell culture
Splenic B cells were purified from erythrocyte-lysed singlecell suspensions of splenocytes by negative selection using
AutoMACS (Miltenyi Biotec, Bergisch Gladbach, Germany), in accordance with the manufacturer’s instructions.

IgM-

IgM-

pro-B

pro-B

2.9

2.7

IgM-

c-kit

IgM

Cell number (×106)

36

pre-B

pre-B

18.8

10.5

B220

Prmt1–/–

cont.

B220

8.4

21.7
7.8

5.5

12.9
4.5

Recirculating B
Immature B

Cell number (×106)

CD25

B220

pro-B
(c-kit+B220+)
4

*

18

pre-B
(CD25+B220+)
*
18

n.s.

Cell number (×105)

IgM-

Total

B

2

0

0

pro-pre-B(IgM-B220mid)

immature B(IgM+B220mid)

recirculating B(IgM+B220hi)

30

14

20

*

15

0

7

0

***

cont.
Prmt1–/–

90

Cell number (×105)

Prmt1–/–

Cell number (×105)

cont.

Cell number (×105)

A

***

10

0

Pro-pre-B
IgM

Fig. 3. Impairment of B cell development in Prmt1/ mice. (A, B) Bone marrow cells from the indicated mice were analyzed byfluorescence-activated cell sorting (FACS) in (A). Numbers of total, pro-B (B220+IgMc-Kit+), pre-B (B220+IgMCD25+), pro-pre-B(B220mid IgM), immature B (B220mid IgM+) or recirculating B (B220high IgM+) cells in the bone marrow from Prmt1/ or control mice in(A) are shown in the graphs in (B). (C, D) Splenocytes from the indicated mice were analyzed by FACS in (C). Numbers of transitional type 1(B220+AA4.1+IgM+CD23), transitional type 2 (B220+AA4.1+IgM+CD23+), transitional type 3 (B220+AA4.1+IgMlow CD23+), FOB(B220+AA4.1CD23+) or MZB (B220+IgM+AA4.1CD23) cells in spleen from Prmt1/ or control mice in (C) are shown in the graphs in(D). (E, F) Peritoneal cells from the indicated mice were analyzed by FACS in (E). Numbers of B1 (B220+CD23) or B2 (B220+CD23+) cells
in the peritoneal cavity from Prmt1/ or control mice in (E) are shown in the graphs in (F). The numbers indicate the percentage of thegated populations of the FACS plots. A representative of three independent experiments is shown (A, C, E). Data from three mice in the
independent experiments are shown as the mean  SD (B, D, F). *P < 0.05; **P < 0.01; ***P < 0.001 (Mann–Whitney U-test).
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For isolation of FOB and MZB cell subsets, splenocytes
were preincubated with FcBlock antibodies and stainedwith B220-FITC, anti-IgM-APC, AA4.1-PE/Cy7 and antiCD23-PE. FOB (B220+AA4.1CD23+) and MZB(B220+AA4.1IgM+CD23) cells were sorted by a FACSAria (BD). B1 cells (B220+CD23) derived from peritoneal cavity of control or Prmt1/ mice were sorted
using the antibodies: B220-APC and anti-CD23-PE.
Splenic B cells were cultured with 10 lgmL1 anti-IgM,
10 lgmL1 anti-CD40 or 20 lgmL1 LPS for 24 h, followed by assays for expression of CD69, CD80, CD86 andMHC class II. FOB, MZB and B1 cells were labeled withcarboxyfluorescein diacetate succinimidyl ester (CFSE)(Molecular Probes, Carlsbad, CA, USA) in accordancewith the manufacturer’s instructions. FOB cells were cultured with 10 lgmL1 rIL-4 and 10 lgmL1 anti-CD40
and MZB and B1 cells were cultured with 20 lgmL1
LPS.

Statistical analysis
Data are expressed as the mean  SD for each group.
Control and Prmt1/ groups were compared using an
unpaired two-tailed Student’s t-test. When counting theB220+AA4.1+

D

T2(IgM+CD23+)

T1(IgM+CD23–)

Prmt1–/–

cont.

T1 T2

IgM

22

45

B220+AA4.1+
T1 T2
12

B220

57

30

28

T3

9

0

T3

3.9 94.2

FOB(CD23+)
30

MZB

MZB
FOB

FOB
CD23

Cell number (×106)

IgM

B220+AA4
AA4.1
1-

6.8 90.8

25

15

B2

16.8

34.7
B1

B1

34.2

16.1

B220

25

0

cont.
Prmt1–/–

***

10

0

B2
(B220+CD23+)
50

Cell number (×104)

B2

Cell number (×104)

CD23

Prmt1–/–

0

20

*

B1
(B220+CD23–)
**
50

F

cont.

20

MZB
(IgM+CD23– )

0

E

***

B220+AA4.1–

CD23
AA4.1
1B220+AA4

40

0

Cell number (×105)

B220+AA4.1+

n.s.

50

***

Cell number (×105)

FOB and MZBCell number (×105)

AA4.1

18

T3
(IgMlowCD23+)

Cell number (×105)

C

*

cont.
Prmt1–/–

25

0

Fig. 3. Continued

1204

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Low levels of serum Igs in Prmt1/ mice

number of B cells, the Mann–Whitney U-test was used todetermine statistical significance.

For analysis of the isotypes of antibodies in Prmt1/
mice, concentrations of serum Igs in Prmt1/ orcontrol mice were determined by ELISA. The levelsof total IgM, IgG2a, IgG2b or IgG3 in Prmt1/
mice were slightly reduced, whereas those of IgA or
IgG1 were equal to that in control mice (Fig. 2).
These results indicate that a partial and isoformdependent reduction in serum Igs was detected in
Prmt1/ mice.

Results
Generation of B cell-specific Prmt1/ mice
To analyze the physiological roles of PRMT1 in vivo,
we specifically deleted the PRMT1 gene from the B
cell lineages. To establish the B cell-specific PRMT1deficient mice (Prmt1/), we generated a mousewhose PRMT1 gene locus was flanked by loxP sites(Prmt1f/f) (Fig. 1A) and crossed the Prmt1f/f with a
Cd19-Cre mouse, which expresses Cre recombinase
under the control of the CD19 promoter [23]. B cellspecific deletion of RRMT1 was confirmed by genomic
PCR (Fig. 1B and Fig. S1) and by western blotting(Fig. 1C). The protein of PRMT1 was reduced but
slightly expressed in Prmt1/ B cells (Fig. 1C). Therefore, we purified the transitional B, FOB and MZBcells from the spleen and the recirculating B cells fromthe bone marrow and examined PRMT1 both in the
genome (Fig. 1B and Fig. S2B) and protein(Fig. S2A). The target exons were completely deleted
in the genomic DNA of every purified B cell subset
from Prmt1/ mice (Fig. 1B and Fig. S2B) and the
PRMT1 protein was completely repressed at least in
the recirculating B cells of Prmt1/ mice (Fig. S2A).
These results suggest that PRMT1 is specificallyreduced in Prmt1/ B cells.

Altered B cell development in Prmt1/ mice
To clarify the mechanisms of the Ig reduction in
Prmt1/ mice, we analyzed the B cell subsets in thelymphoid organs by flow cytometry. The number ofthe splenic B cells from Prmt1/ mice was reduced
compared to that of control mice, whereas the numberof splenic T cells was intact (Fig. S3). Among the B
cell subsets, pre-B, immature B and recirculating B
cells in the bone marrow from Prmt1/ mice werereduced in number by 54%, 68% and 79%, respectively, compared to that from control mice (Fig. 3A,
B). Similarly, the numbers of T1, T3, FOB and MZBcells in a spleen were decreased by 56%, 47%, 22%
and 54%, respectively (Fig. 3C,D). The peritoneal B1
and B2 cells from Prmt1/ mice were markedly
decreased by 74% and 51%, respectively (Fig. 3E,F).
These results suggest that B cell development in

IgM

IgG3
4.0

4.0
***

***

***

***

**

14

21

28

Cont.
Prmt1–/–

2.0

2.0

**
0
0

B

***
***

0

Absorbance (405 nm)

Fig. 4. Diminished antibody response to
TI-antigens but not to TD-antigens inPrmt1/ mice. (A, B) Prmt1/ andcontrol mice were immunized with 100 lg
of NP-Ficoll in PBS (A) or 100 lg of NPOVA in alum (B). Then, NP-OVA-primed
mice were boosted with 50 lg of NP-OVA
in PBS on day 28 after the priming.
Peripheral blood from these mice weretaken every 7 days and analyzed for antiNP-specific IgM and IgG3 (NP-Ficoll) or
IgM and IgGs (NP-OVA) in murine serumby ELISA. Data are shown as themean  SD (n > 5). Experiments were
performed three times, with essentiallysimilar results. **P < 0.01; ***P < 0.001(Student’s t-test).

Absorbance (405 nm)

A

7

14

21

28

IgM

3.0

7

IgG

3.6

1.5

0

0

1.8

0

7

14

21

28

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0

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% of max% of max% of max% of max% of maxClass II

% of max% of max% of max

CD86% of max% of max% of max% of max

LPS% of max

α-CD40% of maxα-IgM% of max

MediumCD80% of max

CD69

A

Fig. 5. Immunoglobulin production and activation of Prmt1/ B cells in vitro. (A) Total B cells are isolated from Prmt1/ or control mice,stimulated with 10 lgmL1 anti-IgM, 10 lgmL1 anti-CD40 mAb or 20 lgmL1 LPS in vitro for 1 day, and analyzed for the expression ofactivation markers, CD69, CD80, CD86 and class II molecules, using a flow cytometer. Isotype control staining is indicated by a solid line
(control) or a dotted line (Prmt1/). Specific Abs staining shown at the top is indicated by a filled solid line (control) or a filled dotted line
(Prmt1/). (B) CFSE-labeled FOB, MZB and B1 cells were stimulated as described in the Materials and methods and then examined for cell
division and class switch recombination by flow cytometry. Histograms show cell division profiles of CFSE-labeled cells. Two-color flow
cytometric profiles show IgG1, IgE and IgG3 expression of each cell division. Dashed lines show the position of the cell division peaks forreference. (C) The percentage of cells in each cell division is indicated in the upper panels. The percentage of class switched cells in eachcell division is indicated in the lower panels. (D) FOB cells were stimulated with 10 lgmL1 rIL-4 and 10 lgmL1 anti-CD40. MZB and B1
cells were stimulated with 20 lgmL1 LPS. On 7 days after stimulation, culture supernatants were assayed for IgG1, IgE or IgG3. A
representative of three independent experiments is shown (A–C). Data from three mice in the independent experiments are shown as themean  SD (D). *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t-test).

Prmt1/ mice is substantially blocked in the bonemarrow, as well as in peripheral lymphoid organs.
Antibody responses in Prmt1/ mice after
stimulation with NP-Ficoll and NP-OVA in vivo
To examine the role of PRMT1 in antibody production in vivo, Prmt1/ or control mice were immunizedwith 100 lg of NP-Ficoll (TI-2 antigen) in PBS or
100 lg of NP-OVA (TD antigen) in alum. The
NP-OVA-primed mice were boosted with 50 lg of
NP-OVA in PBS, and then both NP-Ficoll- and
NP-OVA-primed mice were followed by assays foranti-NP antibody production. Prmt1/ mice showeda markedly reduced production of anti-NP IgM and
IgG3 after NP-Ficoll administration compared to con1206

trol mice (Fig. 4A). However, the antibody responses
of anti-NP IgGs to NP-OVA were unaltered in
Prmt1/ mice (Fig. 4B), suggesting that PRMT1 differentially regulates the TI or TD antibody responses.
Activation and antibody production of Prmt1/
B cells in vitro
To determine the requirement of PRMT1 in B cell
activation, the Prmt1/ and control B cells were
stimulated with 10 lgmL1 anti-IgM, 10 lgmL1
anti-CD40 or 20 lgmL1 LPS for 1 day, followed byassays for the expression of activation markers such as
CD69, CD80, CD86 and MHC class II molecules.
These four activation markers were up-regulated byany stimulation in both Prmt1/ and control B cells

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Role of PRMT1 in B cells

K. Hata et al.

FOB
Day 5
IgG1

FOB
Day 5
IgE

MZB
Day 4
IgG3

B1
Day 4
IgG3

Diviision number

Diviision number>87 6 5 4 3 2 1 0>8 7 6 5 4 3 2 1 0>8 7 6 5 4 3 2 1 0

Cell counts

Diviision number>8 7 6 5 4 3 2 1 0

Cell counts

Diviision number
Cell counts

Cell counts

B

IgG3

IgG3

IgE

IgG1

cont.

CFSE

CFSE

CFSE

Diviision number

Diviision number

Diviision number

Diviision number>8 7 6 5 4 3 2 1 0>876 5 4 3 2 1 0>8 76
6 54 3 2 1 0>8 7 6 5 4 32 1 0
Cell counts

Cell counts

Cell counts

CFSE

CFSE

IgG3

IgG3

IgE

IgG1

Prmt1–/–

CFSE

CFSE

CFSE

Fig. 5. Continued(Fig. 5A). These results suggest that PRMT1 is dispensable for early B cell activation after stimulation
with anti-IgM, anti-CD40 or LPS.
Previous studies have demonstrated that FOB cells
mount antibody responses to TD antigen, whereas
MZB or B1 cells contribute to TI antigen responses[6–10]. The down-regulation of the antibody production
to TI antigen may reflect a decrease in the number of
MZB or B1 cells. On the other hand, Prmt1/ mice
showed no significant deficiencies in antibody production in response to TD antigen despite a reduction in
the number of FOB cells. Therefore, we isolated
FOB, MZB and B1 cells and analyzed the correlation
between proliferation and antibody production in vitro
using CFSE and antibody for Ig isotypes [24,25]. No
significant difference was observed in the division-based
switch rate to IgG1 between the control and Prmt1/
FOB cells (Fig. 5B,C); however, IgG1 production
was significantly increased in Prmt1/ FOB cells(Fig. 5D). Prmt1/ FOB cells proliferated more vigorously and produced more IgG compared to control
FOB cells, which could account for the augmentationof the IgG1 production in Prmt1/ FOB cells stimulated in vitro (Fig. 5B–D). By contrast to IgG1, IgG3
production in Prmt1/ MZB and B1 cells was lesscompared to control cells when stimulated in vitro(Fig. 5D). Although the proliferation and IgG3 isotype switching were not affected by PRMT1 deficiency in both MZB and B1 cells (Fig. 5B,C), we
noted a tendency for Prmt1/ MZB and B1 cells to
die after in vitro stimulation (data not shown), possibly leading to the low production of IgG3 by
Prmt1/ MZB and B1 cells. Taken together, theseresults indicate that the enhancement of IgG1 production by Prmt1/ FOB cells could compensate for the
reduction in the number of FOB cells. This could
explain the normal concentration of serum IgG1 in
Prmt1/ mice immunized by TD antigen in vivo
(Fig. 4B).

Discussion
Arginine methylation plays a crucial role in various cell
types, including immune cells [11,13,14]. In T cell activa-

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Role of PRMT1 in B cells

K. Hata et al.

% Total

B1
Day 4
IgG3

% IgG3+

Number of cell divisions

cont.
Prmt1–/–

Number of cell divisions

cont.
Prmt1–/–

Number of cell divisions

Number of cell divisions

Number of cell divisions

Number of cell divisions

FOB

FOB
***

MZB

B1

600

0

1000

1200

600

0

0

280

*
IgG3 (ng·mL–1)

2000

**

IgG3 (ng·mL–1)

1200

IgE (ng·mL–1)

D

IgG1 (ng·mL–1)

Number of cell divisions% IgE+% IgG1+

Number of cell divisions

MZB
Day 4
IgG3

% IgG3+% Total% Total

FOB
Day 5
IgE
% Total

FOB
Day 5
IgG1

C

*
cont.
Prmt1–/–

140

0Fig. 5. Continued

tion machinery, the engagement of T cell receptors
together with co-stimulatory receptor CD28 has beenreported to induce arginine methylation, resulting inenhanced IL-2 production in vitro [16]. We recently
demonstrated that PRMT1 also plays a crucial role in
BCR signaling in a B lymphoma cell line [21]. Moreover,a general inhibitor of arginine methylation, adenosine20 ,30 -dialdehyde, prevented the IgG1 production after
stimulation with LPS, IL-4 or CD40 ligand [20]. To clarify the contribution of PRMT1 in B cell responses
in vivo, we established B cell-specific PRMT1-deficient
mice as a result of embryonic lethality by the targeted
disruption of PRMT1 [26]. Prmt1�/� mice demonstrated
a developmental defect in B cell lineages, with reduced
serum Igs and impaired antibody production to the TI-2
antigen NP-Ficoll but not to the TD antigen NP-OVA.
Prmt1f/f mice were crossed with Cd19-Cre mice [23]because CD19 is expressed from pro-B cell stage
throughout B cell development [27]. We confirmed theprevious finding that PRMT1-mediated arginine
methylation regulates the development from pre-B to
immature B cells [17] and further showed that peripheral B cell development was also blocked in Prmt1�/�
1208

mice. The serum Ig levels, including IgM, IgG2a/
IgG2b and IgG3, were diminished in Prmt1�/� mice.
PRMT1 protein was selectively and severely reducedin B cells but not in non-B cells from Prmt1�/� mice,suggesting that PRMT1 is required for proper B cell
development and its functions.
The expression of PRMT1 protein is higher in MZBthan in FOB cells [17]. MZB and B1 cells play animportant role in the early phase of the TI antibody
response, whereas FOB cells are the main players in thelate TD antibody response, such as the germinal center
reaction [28]. In the present study, Prmt1�/� mice
showed a lower antibody response to TI antigen but anormal reponse to TD antigen. This reduction in TI
antigen response in Prmt1�/� mice is possibly a resultof both the decrease in the number of Prmt1�/� MZB
and B1 cells responding to TI antigens and the lower
antibody productivity of individual Prmt1�/� MZB andB1 cells. In the TD immune response, increasing antibody production by each Prmt1�/� FOB cell appears tocompensate for the decrease in the number of Prmt1�/�
FOB cells responding to TD antigens. This may resultin the normal antibody response to TD antigens in

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Role of PRMT1 in B cells

K. Hata et al.

Prmt1/ mice. Taken together, these results indicatethat PRMT1 is required for the TI antibody response
but not for the TD antibody response.
Similar to the X-linked immunodeficient mousemodel [29–31], Prmt1f/f-Cd19-Cre mice appeared tohave several defects in B cell development and function, including the maintainance of serum Ig level,
and also the TI-2 antibody response but not the TD
response [29–31]. X-linked immunodeficient mice harbour the R28C mutation within the gene encoding
BTK [29], which means a functionally inactive form.
Stimulation of MZB cells with TI-2 antigens introduces the activation of an alternative NF-jB2/p100
signalling pathway [4], whereas the TD antibody
response requires NF-jB activation [5]. Because BTK
couples BCR to the NF-jB pathway, it is possiblethat the BTK-NF-jB axis is involved in the B cell
response mediated by PRMT1 upon antigen stimulation. The molecules that could be targeted by
PRMT1 for arginine methylation and the regulationof protein function remain to be investigated infuture studies.
In conclusion, the present study, using B cell-specificPrmt1/ mice, is the first to demonstrate that thearginine methyltransferase PRMT1 plays a crucial rolein lymphocyte development, proliferation and differentiation in vivo.

Acknowledgements
This work was supported by MEXT KAKENHI
Grant Number 24590286.

Author contributions
KH designed and performed most of the experiments,
analyzed the data, and wrote the manuscript. NY performed the experiments. KS and YM established the
PRMT1 conditional knockout mice. SK generated andmaintained the mice. JM and TY wrote the manuscript and supervised the project.

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Supporting information
Additional Supporting Information may be foundonline in the supporting information tab for this article:
Fig. S1. Genotyping of control, Prmt1f/+ and Prmt1f/f
mice. Genomic DNA prepared from tail of indicatedmice was amplified by PCR with the primer pair b andd in Fig. 1A for floxed allele (470 bp) or b and e forwild-type allele (322 bp).
Fig. S2. Characterization of the various stages of B
cells in the bone marrow of control and Prmt1f/f mice.
(A) The expression of PRMT1 was detected by western blotting with cell lysates of sorted pro-, pre-,immature and recirculating B cells from control andPrmt1/ mice. The expression of actin was used as aloading control. (B) Genomic DNAs of pro-, pre-,immature and recirculating B cells from the indicatedmice were amplified by PCR with the primer pair b
and g (Fig. 1A) for the targeted allele or a and c(Fig. 1A) for the control.
Fig. S3. Reduction of number of spleen B but not T
cells in Prmt1/ mice. Spleen cells from the indicatedmice were analyzed by fluorescence-activated cell sorting (FACS) in (A). Numbers of total splenocytes or
splenic B cells (B220+CD3) and T cells(B220CD3+) from Prmt1/ or control mice in (A)are shown in the graphs (B). The numbers indicate thepercentage of the gated populations of the FACS
plots. A representative of three independent experiments is shown in (A). Data from three mice in the
independent experiments are shown as the mean  SD(B). *P < 0.05; **P < 0.01; ***P < 0.001 (Mann–
Whitney U-test).
Fig. S4. CFSE-labeled FOB, MZB and B1 cells were
stimulated as shown in Fig. 5 and then examined for cell
division and staining with isotype control antibodies.

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Chapter 34

The Multifaceted Role of PD-1 in Healthand Disease
Mohamed El Sherif Gadelhaq Badr, Kikumi Hata, Masae Furuhata,
Hiroko Toyota, and Tadashi Yokosuka

Abstract Properly functioning T cells should clear invading pathogens and tumors
without causing damage to the host. Programmed cell death-1 (PD-1) is a negative
costimulatory receptor that is expressed on an effector T cell to regulate its
functions; otherwise, uncontrolled activation of T cells might cause collateral tissue
damage and autoimmune diseases. However, such a beneficial regulatory tool has
been hijacked by several pathogens and tumors to promote chronic infection andevade antitumor immunity, respectively. PD-1 ligands are expressed by a variety of
haematopoietic and non-haematopoietic cells all over the body under steady state
and their expression is further modulated within different pathological conditions,suggesting that PD-1 is an immune checkpoint that is utilised by the body toregulate T-cell functions in different contexts. Furthermore, blocking PD-1 hasrecently received attention as a promising therapeutic approach for the treatment ofcancer and chronic viral infections. Herein, we shed some light on the current
knowledge of PD-1 biology and role in its health and disease.
Keywords Programmed cell death 1 • PD-1 • Immune checkpoint • Tumor
immunotherapy • T-cell signaling • T-cell receptor • Immunological synapse •Microcluster • Imaging • Regulatory T cell

M.E.S.G. Badr
Laboratory for Cell Signaling, Center for Integrative Medical Sciences, RIKEN Yokohama
Institute, Kanagawa 230-0045, JapanWPI Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, JapanK. Hata • M. Furuhata • H. Toyota
Department of Immunology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo
160-8402, JapanT. Yokosuka (*)
Department of Immunology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo
160-8402, Japan
PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012, Japane-mail: yokosuka-ths@umin.ac.jp© Springer Japan 2016
M. Miyasaka, K. Takatsu (eds.), Chronic Inflammation,
DOI 10.1007/978-4-431-56068-5_34
yokosuka-ths@umin.ac.jp

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34.1

M.E.S.G. Badr et al.

Introduction

Inflammation is one of the biological responses to restore body homeostasis thatwas disrupted by insult from the outer world such as infection with pathogenic
invaders such as bacteria, fungi, and viruses and physical injuries. However,uncontrolled or exaggerated inflammatory responses can backfire on the bodycausing allergic or devastating autoimmune diseases. An inflammatory response
is launched mainly by innate leukocytes, including neutrophils, which sense
infecting pathogens and tissue disruption and then secrete various chemical mediators in response. On the contrary, a prolonged inflammatory response, called‘chronic inflammation’, is mediated mainly by acquired immunity through theantibody production by B cells and various helper functions by T cells. Because
T cells are playing the role of the maestro in coordinating the orchestra of theimmune response symphony, it should be quite important to unveil the mechanismsof T-cell activation regulation to reach the ultimate goal of bringing chronic
inflammation under control.
T cells are activated upon recognition of their cognate antigen peptide loaded ona major histocompatibility complex (MHC) and the functional characteristics of anactivated T cell are further modified by various signals through costimulatory
receptors and cytokines within the surrounding environment. T cells express several
costimulatory receptors, either with positive or negative functions, the expressionof which is changing according to the activation status of a T cell. On the other
hand, the ligands for those costimulatory receptors are expressed on various antigen
presenting cell (APC) subsets and nonhaematopoietic cells all over the body; andthe types and the expression levels of the ligands also change according to the
inflammation status of the surrounding microenvironment within the tissue. Therefore, T cells, with help from costimulatory receptors, sense the changes within theirenvironment to respond with the proper functional outputs. In this review, we
discuss the regulatory functions of the negative costimulatory receptor,
programmed cell death-1 (PD-1; CD279), which has become recently appreciatedas an important ‘immune checkpoint’ molecule.

34.2

Characteristics of PD-1 and Its Ligands

T cell costimulatory receptors contain two receptor superfamilies (SFs), immunoglobulin (Ig) SF and tumor necrosis factor (TNF) SF. The IgSF is further divided
into two functionally opposing groups: activating IgSF receptors, for example,
CD28 and inducible T-cell costimulator (ICOS, CD278) and inhibitory IgSF
receptors, for example, cytotoxic T-lymphocyte associated protein 4 (CTLA-4 orCD152), PD-1, and B and T lymphocyte attenuator (BTLA, CD272). PD-1 wasoriginally discovered as an apoptosis-related molecule and characterised as a type I
membrane protein of 288 amino acids; composed of an extracellular Ig-likeyokosuka-ths@umin.ac.jp

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34

The Multifaceted Role of PD-1 in Health and Disease443

variable domain, transmembrane domain (27 amino acids), and a cytoplasmic(94 amino acids; Ishida et al. 1992). Similar to CTLA-4, PD-1 is expressed in a T
cell after activation; but, because PD-1 possesses neither a membrane-proximal
cysteine residue as in CD28 nor a clathrin–adaptor protein complex 2 (AP-2)
binding domain as in CTLA-4, therefore, PD-1 exists on the plasma membrane asa monomeric receptor (Fig. 34.1, left). There are two known ligands for PD-1: PD-1
ligand-1 (PD-L1; B7-H1; CD274; Freeman et al. 2000) and PD-L2 (B7-DC;
CD273; Latchman et al. 2001; Tseng et al. 2001). Both ligands belong to the B7
family, which is characterised by bearing one IgV and one IgC domains in the

Activated effctor T cell
PD-1 aPD-L1
c PD-1PD-L1CTLA-4

CD80

PD-1

CD80
PD-L2

e
PD-1

e
CD86

Antigenpresenting
cell

PD-L1

c

d

MHC

PD-L2

PD-L1

CD86

ICOSL

CD80

MHC

e

PD-L1

CTLA-4

TCR

PD-1

ICOS

CTLA-4CD28

d CTLA-4beTreg cell

CD80

CD80
TCR

Naive T cell

Primary
T cellFig. 34.1 The B7-CD28 family-dependent crosstalk between APCs and T cells. The diagramshows the B7-CD28 costimulatory or coinhibitory receptors and their ligands’ dependent interaction between a naı̈ve T cell (green), an activated effector T cell (orange), a regulatory T cell (blue),and an antigen-presenting cell. TCR, costimulatory (red) and coinhibitory receptors (blue),occasionally, through binding to their ligands convey positive and negative signaling, respectively.
Treg cells suppress effector T-cell functions and also promote conversion of effector T cells into
iTreg cells through the interaction between PD-1 and PD-L1 (a). The PD-1–PD-L1 interaction
suppresses T-cell functions and promotes iTreg cell differentiation (b). The sustained PD-1
signaling maintains the Foxp3 expression, suppressive functions, and survival of Treg cells (c).
Signaling through CTLA-4 enhances the Treg-cell–suppressive functions (d). Crosslinking of
CD80/CD86 by CD28 or CTLA-4 induces either immune enhancing or suppressive outcomes inantigen-presenting cells (APCs), respectively. Treg cells strip off CD80/CD86, the ligands for
CD28, from the surface of APCs via CTLA-4, a phenomenon known as trogocytosis (e)yokosuka-ths@umin.ac.jp

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M.E.S.G. Badr et al.

extracellular region. PD-L1 consists of 290 amino acids, with a 30 amino acidtransmembrane region, and is expressed on a variety of cells of both haematopoietic
and nonhaematopoietic origins. PD-L1 was also reported to bind the CD28 ligand,CD80, with introducing bidirectional negative signals (Butte et al. 2007). In
contrast, PD-L2 is inducibly expressed on professional APCs after their activationand is known to have triple higher affinity for PD-1 than PD-L1 (Fig. 34.1, bottom).
Although human PD-L2 consist of 273 amino acids with a 30 amino acid cytoplasmic tail and the murine counterpart consists of 274 amino acids with only a 4 aminoacid cytoplasmic tail, PD-L2 is speculated to induce functional signaling to dendritic cells (DCs), because the activation or survival signals in dendritic cells wereaugmented by treatment with the anti-PD-L2 monoclonal antibody, sHIgM12,
originally isolated from the primary (Waldenstorm’s) macroglobulinemia patients(Nguyen et al. 2002).

34.3

Expression Regulation of PD-1 and Its Ligands

PD-1 is expressed on various types of cells, such as activated T and B cells, humannaı̈ve B cells, exhausted T cells, regulatory T (Treg) cells, follicular helper T (Tfh)cells, natural killer T (NKT) cells, activated CD11c+ myeloid DCs (mDCs), andmonocytes, but not on macrophages and plasmacytoid DCs (pDCs). In vitro
experiments have shown that anti-CD3 plus anti-CD28 and anti-IgM plus antiCD40 induce the expression of PD-1 on T and B cells, respectively; and theexpression level was proportional to the strength and duration of the stimulation.
PD-1 is expressed in T cells within 2–6 h following TCR stimulation; earlier than
CTLA-4 and BTLA or than the first cell division. Because PD-1 expression on T
cells is sustained for more than one week, starting from the initial activation ofnaı̈ve T cells till the differentiation into effector T cells, therefore, it is considerable
that PD-1 is modulating all the developmental stages of memory T cells (Chikuma
et al. 2009). PD-1 expression in T cells is further enhanced by stimulation with type
I interferon (IFN). In fact, the promoter region of PD-1 contains a binding region for
the transcription factor interferon regulatory factor 9 (IRF9) which is activateddownstream of type I IFN receptor (Terawaki et al. 2011) as well as a binding
region for the transcription factor nuclear factor of activated T cells, cytoplasmic
1 (NFATc1) which is activated downstream of TCR (Oestreich et al. 2008). Moreover, chronic viral infection has been shown to play a role in regulating themethylation status of the CpG islands within the promoter region of PD-1 (Youngblood et al. 2011). Independent of TCR activation, the common gamma chain (cγ)
cytokines; IL-2, IL-7, IL-15, and IL-21 have been reported to induce the expressionof PD-1 in T cells (Kinter et al. 2008). On the other hand, Listeria infection induced
the expression of PD-1 in mDCs through the activation of Toll-like receptor
2 (TLR2), TLR3, TLR4, and NOD-like receptors. It is reported that estrogenenhances the expression of PD-1 on Treg cells, macrophages, B cells, and mDCs

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The Multifaceted Role of PD-1 in Health and Disease445

and this inhibitory machinery contributes to the immune suppression during pregnancy (Polanczyk et al. 2007).
PD-L1 is constitutively expressed not only by immune cells, such as T cells, B
cells, macrophages, m/pDCs, and bone marrow mast cells, but also by tissueconstructing cells, such as endothelial cells, fibroblastic reticular cells, gut epithelial cells, pancreatic islet cells, astrocytes, central neurons, placental trophoblasts,
retinal pigmented epithelial cells, and optic nerve cells. The expression of PD-L1 isenhanced upon stimulation with type I IFNs, IFN-γ, and TNF-α consistent with thepresence of the IRF1 and signal transducer and activator of transcription 3 (STAT3)
binding domains in its promoter region. T and B cells also express more PD-L1
upon stimulation with cγ cytokines whereas DCs and macrophages upregulate
PD-L1 upon stimulation with granulocyte/macrophage-colony stimulating factor(GM-CSF) and IL-4. PD-L1 is also expressed by many cancer cells; furthermore,
PD-L1 is known to be a target for the transcription factor Hypoxia-inducible factor1α (Hif-1α), therefore an hypoxic tumor microenvironment can further augment theexpression of PD-L1 (Noman et al. 2014). It was also reported that multiple
myeloma cells upregulate the expression of PD-L1 upon stimulation with IFN-γ
and TLR ligands via myeloid differentiation primary response (MyD88)–TNF
receptor associated factor 6 (TRAF6)–mitogen-activated protein kinase (MAPK)/
extracellular signal-regulated kinase (Erk) kinase (MEK) pathway (Liu et al. 2007).
As mentioned previously, PD-L2 is expressed by m/pDCs, macrophages, B-1
cells, and bone marrow mast cells and its expression is further enhanced bystimulation with several cytokines, for example, GM-CSF, IFN-γ, IL-4, cγ cytokines, and TNFα. Therefore, it is plausible that the increased expression of IFNs
and other cytokines, responsible for the maintenance of activated and memory T
cells especially in the case of chronic infection and inflammation, induce theexpression of PD-1 and its ligands PD-L1/2; the interaction between them transduces bidirectional suppressive signals that induce tolerance and suppress that
excessive activation of immune system and ultimately lead to chronic inflammation

34.4

PD-1 Signaling Machinery

The cytoplasmic domain of PD-1 contains two tyrosine-based signaling motifs: an
immunoreceptor tyrosine-based switch motif (ITSM, TxYxxL/I) and an
immunoreceptor tyrosine-based inhibitory motif (ITIM, S/I/LxYxxI/V/L). Uponcrosslinking of PD-1, the tyrosine residues get phosphorylated and consequently
they act as docking sites to recruit the Src homology 2 (SH2) domain-containing
tyrosine phosphatase 2 (SHP-2) and SHP-1 which subsequently dephosphorylate
the signalling molecules downstream of TCR resulting in the diminished activationof the Erk and Akt signaling pathways. In fact, it was shown that PD-1 suppressed
Erk activation in human T cells which led to decreased transcription of the ubiquitin
ligase SKP2 that is responsible for the degradation of the inhibitors of cycling-yokosuka-ths@umin.ac.jp

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dependent kinases (Cdks), ending up blocking cell-cycle progression at G1 thephase (Patsoukis et al. 2012).
Although the kinases important for the phosphorylation of the cytoplasmic
domain of PD-1 have not been definitively shown, the pull-down experiments,using the ITSM/ITIM motifs as bait followed by mass spectrometry, havesuggested that Lck and C-terminal Src kinase (Csk) could be the responsible
kinases (Sheppard et al. 2004). The phosphorylated ITSM motif recruits both
SHP-1 and SHP-2 whereas the phosphorylated ITIM motif recruits only SHP-2
and mutation experiments of the tyrosine residues have suggested that the suppressive functions of PD-1 are mainly attributed to the ITSM motif (Chemnitz
et al. 2004). In T cells, SHP-2 and possibly SHP-1 associate with both the ITSM
and ITIM motifs and dephosphorylate the CD3ζ-chain, zeta-associated protein of
70kD (Zap70), and protein kinase C θ (PKCθ) (Sheppard et al. 2004). In B cells,
only SHP-2 is recruited to the PD-1 cytoplasmic tail and dephosphorylate Igβ-chain, spleen tyrosine kinase (Syk), Erk, and phospholipase Cγ2 (PLCγ2) tosuppress BCR signaling (Okazaki et al. 2001).
Formation of an immunological synapse is required for the antigen recognition
and activation of T cells, when T cells contact with APCs bearing their cognate
antigens. The immunological synapse is a concentric, bull’s eye-like, structure
generated at the interface between a T cell and an APC, where various immune
receptors including TCRs, coreceptors CD4 and CD8, costimulatory receptors, andtheir signaling transducers are gathering together for the efficient signal transduction and are surrounded by adhesion molecules such as lymphocyte functionassociated antigen-1 (LFA-1), intercellular adhesion molecule-1 (ICAM-1), verylate antigen-4 (VLA-4), and vascular cell adhesion molecule-1 (VCAM-1) to keep astable conjugation between a T cell and an APC. We discovered that the immunological synapse is constructed by a number of more tiny signaling units that we
named ‘TCR microclusters’ (Yokosuka et al. 2005; Fig. 34.2, left). Our imaging
analysis has shown that a TCR microcluster is composed of a few decades of TCRs
and that PD-1 is recruited to a TCR microcluster upon binding to PD-L1, where its
ITSM/ITIM motifs get phosphorylated and subsequently recruit SHP-2 (Yokosuka
et al. 2012). In fact, the SHP-2 substrates, such as CD3ζ, Zap70, and PKCθ, are also
localised within the TCR microclusters suggesting that PD-1-mediated dephosphorylation reactions and regulation of T-cell activation primarily take place within
the microclusters. Interestingly, SHP-2 which is recruited to the phosphorylated
ITSM/ITIM in the PD-1 cytoplasmic tail could dephosphorylate not only the SHP-2
substrates but also PD-1 itself and subsequently SHP-2 dissociates from the PD-1TCR microclusters (Fig. 34.2, right). Although the negative costimulatory receptor
CTLA-4 also has a cytoplasmic ITIM-like motif that is believed to recruit SHP-2
and SHP-1, its suppressive mechanism is different from PD-1. Whereas PD-1
dephosphorylates phosphatidylinositol-3-kinase (PI3K) which is an upstream regulator of Akt, CTLA-4 recruits the protein phosphatase 2A (PP2A) which directly
dephosphorylates Akt (Parry et al. 2005). However, our imaging studies demonstrate that the mechanism of CTLA-4–mediated suppression is mainly accomplished by the competition with CD28 for binding to the ligand CD80/CD86

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Immunological synapse
T cellMHC+
peptide
APC

APC

447

PD-L1
PD-L2
CD4

TCR
T cell

CD28

PD-1
Lck

CD3z

CD80
CD86

SHP-2

PKCq

Zap70
PLCg1

Vav

Erk

TCR microclusters

NFAT

MAPK

Cytoskeleton

NF-kB

Fig. 34.2 PD-1-mediated inhibition of TCR signaling at microclusters. A T cell and an APC
form the immunological synapse at the interface between the two cells, which is initiallyconstructed by tiny clusters of TCRs with their downstream signaling molecules ‘TCR
microclusters’. At TCR microclusters, the engagement of TCRs and CD28 triggers several
downstream kinases (orange arrow) to activate distinct but synergistic signaling cascades thatmodify the T cells’ cytoskeleton, transcriptional, and functional program. PD-1 also translocates at
TCR microclusters via binding to PD-L1/2 and specifically recruits SHP-2, which dephosphorylates various signaling molecules downstream of TCRs (blue dotted line)

which is critical for NF-kB signaling, whereas PD-1 is suppressing the proximal
TCR downstream signaling via recruiting SHP-2.

34.5

PD-1 and Thymic T-Cell Development

The expression of PD-1 is detected on CD4 CD8 double negative (DN) αβ T
cells and γδ T cells in the thymus. PD-1 expression starts from CD44+ CD25 DN
II stage, peaks at the CD44 CD25 DN IV stage with the termination of the
β-chain rearrangement, and then ceases at the double positive (DP) stage(Nishimura et al. 1996). PD-L1 is expressed in both thymic cortical epithelial
cells and thymocytes and PD-L2 is in the thymic medulla. TCR transgenic mice,
2C and H-Y, on PD-1 deficient background showed an increased number of DP
thymocytes and decreased number of mature CD8+ single positive T cells

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indicating that PD-1 in the thymocytes adjust the TCR signaling threshold toregulate β-selection negatively, modify positive selection, and ultimately select a
TCR repertoire with reduced autoreactivity (Nishimura et al. 2000). Indeed, microarray analysis of NOD mice, that spontaneously develop type I diabetes, suggested
that PD-1 expression in the thymus could be involved in the induction of tolerance(Zucchelli et al. 2005).

34.6

Multiple Functions of PD-1 in Regulatory T Cells

Inducible Treg (iTreg) cells could be developed from naı̈ve T cells in vitro throughthe TCR stimulation with anti-TCR/CD3 in the presence of tumor growth factor-β(TGF-β). Recently, the addition of the PD-L1-immunoglobulin (Ig) fusion proteinsis shown to enhance the development of iTreg cells further in the same in vitro
culture system and similarly Cd274/ (PD-L1-deficient) APCs are less efficient in
inducing the iTreg cell differentiation (Francisco et al. 2009). The induction of
iTreg cells is enhanced by a mechanistic target of rapamycin (mTOR) inhibitor,
Rapamycin; similarly, PD-L1-Ig could suppress the Akt-mTOR activation andinduce the PTEN enzymatic activity to upregulate Foxp3 expression and sustain
the suppressor functions of iTreg cells (Francisco et al. 2009). In contrast, because
the expression of PD-L1 is dependent on PTEN, the PD-1–mediated suppression of
PTEN itself interferes with the iTreg cell development. The expression of PD-L1 on
APCs or tissue stromal cells is also enhanced under inflammatory conditions orwithin the tumor microenvironment. This alteration of PD-L1 expression induces
the differentiation of naı̈ve or effector T cells into iTreg cells to sustain chronic
infection and preserve peripheral immune tolerance (Fig. 34.1, right). Not only
iTreg cells but also natural Treg (nTreg) cells express both PD-1 and PD-L1. Treg
cells expressing PD-L1 suppress other effector T cells expressing PD-1 by the
PD-L1–PD-1 interaction and furthermore promote conversion of effector T cells
into iTreg cells. Treg cells also suppress the expression of CD80/CD86 on DCs
expressing PD-1 by the PD-L1–PD-1 interaction (Yao et al. 2009). One of the splicevariants of PD-1, the soluble PD-1, is reported to induce CD4+ T cells and DCs to
produce the inhibitory cytokine, IL-10, without indoleamine 2,3 dioxygenase (IDO)
production (Kuipers et al. 2006).

34.7

PD-1-Deficient Mice Develop Spontaneous
Autoimmune Diseases

Pdcd1/ mice were originally generated on the 129 genetic background and thesemice have shown augmented antibody production upon immunisation with foreign
antigens. However, by backcrossing Pdcd1/ mice to the B6 genetic background,yokosuka-ths@umin.ac.jp

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The Multifaceted Role of PD-1 in Health and Disease

449

they started to develop symptoms of lupus-like autoimmune disease, for example,
splenomegaly, autoantibody production, glomerulonephritis, and lupus-like proliferative arthritis as they exceeded 6 months of age (Nishimura et al. 1999). In
contrast, Pdcd1/ mice on the BALB/c genetic background developed dilated
cardiomyopathy (DCM) and autoimmune gastritis (Nishimura et al. 2001). In fact,that was the first experimental model to show that DCM could have an autoimmune
basis; later on, it was found that deletion of the negative costimulatory receptor
lymphocyte activation gene 3 (LAG-3) further exacerbated the pathology of thedisease in Pdcd1/ mice (Okazaki et al. 2011). Diabetes with early onset andseverity is shown by the NOD mice crossed with Pdcd1/ (Wang et al. 2005) orinoculation with anti-PD-1 or PD-L1. Furthermore, fatal autoimmune myocarditis
was developed by the autoimmune-prone mice, MRL, crossed with Pdcd1/ mice
(Wang et al. 2010). Focusing on the differences in function among negative
costimulatory receptors, Ctla4/ mice, irrespective of the genetic background,showed infiltration of the inflammatory cells, mainly T cells, into the entire bodywithout a clear specific tissue tropism and died within a few weeks after birth; incontrast, Pdcd1/ mice demonstrate the tissue-specific autoimmune diseases in amuch later phase. Generally, CTLA-4 suppresses T-cell activation via both cell
intrinsic and extrinsic mechanisms, whereas PD-1 inhibitory effect is mediatedmainly by extrinsic factors. The study with bone marrow chimera from wild-type
and receptor-deficient mice demonstrates that autoimmune diseases are developingin the wild type–Ctla4/ chimeras, not in the wild type–Pdcd1/ chimeras.

34.8

PD-1 Polymorphism and Diseases

The first report to correlate between single-nucleotide polymorphisms (SNPs) in
PD-1 and autoimmune diseases was about a group of systemic erythematous lupus(SLE) patients in Europe and Mexico. These SLE patients demonstrated SNPs in
the runt-related transcription factor 1 (Runx1)-binding region in the enhancer
element of PD-1 (Prokunina et al. 2002). The same SNPs were also identified in
type I diabetes patients in Denmark, multiple sclerosis in Germany, arthritis inAustralia, Basedow disease in Germany, Kawasaki disease in Korea, and ankylosing spondylitis in China. In addition, SNPs in PD-L1 and PD-L2 are reported in
Basedow disease patients in Japan and SLE patients in Taiwan, respectively.

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34.9

M.E.S.G. Badr et al.

Control of Inflammation During Viral Infection
by PD-1

Inhibition of clearance of viral infection by PD-1 was first demonstrated in a murine
adenovirus-induced hepatitis model (Iwai et al. 2003). Pdcd1/ mice showedfaster clearance of adenovirus but more severe destruction of liver tissues. Therefore, PD-1, rather than promoting viral infection by its immunosuppressive functions, prevents tissue damage by infiltrating immune cells and cytokines during theacute phase of infection and consequently promotes chronic viral infections. In fact,
the lymphocytic choriomeningitis virus (LCMV) Armstrong strain, an acute infectious strain, was similarly cleared from WT and Pdcd1/ mice. However, upon
infection with clone 13, a chronic infectious strain, the virus antigen-specific PD-1hi
CD8+ T cells were rendered unresponsive or so-called anaergic, a phenomenon thatwas called T-cell exhaustion (Barber et al. 2006). Clone 13-induced exhausted
T cells did not terminally lose cytotoxicity, because these cells recover cytotoxicity
upon treatment with anti-PD-1 or anti-PD-L1. In contrast, Cd274/ (PD-L1–
deficient) mice that were infected by Clone 13 died of liver failure caused byexcessive T-cell responses and resistance to exhaustion. Virus-specific PD-1hi CD8+
T cells in HIV-infected individuals, as an example of chronic viral infection inhumans, showed exhausted phenotypes and blocking PD-L1 could restore thefunction of those exhausted cells (Day et al. 2006) (Trautmann et al. 2006). In
chronic hepatitis B or C virus infections, virus-specific CD8+ T cells express PD-1
and both CD14+ monocytes and mDCs are shown to upregulate PD-L1. Other thanviral infections, the PD-1–PD-L1 axis can promote for the induction of chronic
inflammation during bacterial or helminthic infections; Helicobacter pylori infection enhanced the expression of PD-L1 in the gastric mucosal epithelium; Taenia
crassiceps infection induced upregulation of PD-L1/PD-L2 and PD-1 in macrophages and CD4+ T cells, respectively; Schistosoma mansoni infection induced
anaergy in T cells via upregulation of PD-L1 in macrophages (Smith et al. 2004).
Therefore, breaking the PD-1–PD-L1 axis, to restore the functions of exhausted
pathogen-specific T cells, is a promising approach for the treatment of chronic
infections.

34.10

Blocking PD-1 Signaling as a Cancer Therapy

The first evidence for a role of PD-1–PD-L interaction in tumor immune evasion
came from the study of a murine mastocytoma cell line, P815, transgenicallyoverexpressing PD-L1, which showed resistance to the cytolytic activity of cytotoxic CD8+ T cells (Iwai et al. 2002). Moreover, the administration of anti-PD-L1
not only decreased the tumor growth but also suppressed its metastasis resulting in ahigher survival rate. In fact, PD-L1 is expressed in the human lung, colon, andovarian cancers and in malignant melanoma. The expression of PD-L1 on tumor

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The Multifaceted Role of PD-1 in Health and Disease451

cells is further up-regulated by IFN-γ and PD-L1 could itself suppress tumorspecific CD8+ T cells and induce their apoptosis through FasL and IL-10 (Donget al. 2002). Recently, other types of tumor cells bearing PD-L1 were also reported,
such as breast cancer, bladder cancer, hepatocellular carcinoma, salivary gland
carcinoma, gastric cancer, glioma, and thyroid cancer. From a clinical viewpoint,
PD-L1 expression is correlated with the poor prognosis of kidney, ovarian, bladder,breast, gastric, esophageal, and pancreatic cancers (Thompson et al. 2004). Simultaneous expression of PD-1 and PD-L1 was also observed in lymphoid tumors suchas multiple myeloma and Hodgkin’s lymphoma. Furthermore, PD-L1 is expressedby monocyte-derived mDCs residing within the tumor draining lymph nodes andblocking the PD-1–PD-L1 interaction enhances DC-mediated autologous tumorspecific T-cell activation and antitumor immunity (Curiel et al. 2003).
In appreciation for the role of the PD-1–PD-L1 axis in tumor immune evasion,
the humanised anti-PD-1 monoclonal antibody (Nivolumab) was approved in lastyear in Japan for the treatment of malignant melanoma. In a phase I clinical trial in
the United States, anti-PD-1 blocking antibody has shown efficacy in the treatmentof recurrent solid tumors (Brahmer et al. 2010). In another large-scale phase I
clinical trial, the cumulative response rates were 28 % in melanoma, 18 % in
non-small cell lung cancer, and 27 % in renal cell cancer patients (Topalian
et al. 2012). On the other hand, a phase I clinical trial for anti-PD-L1 conducted
on 207 patients with advanced cancers, the cumulative response rates were 17 % inmalignant melanoma, 10 % in non-small cell lung cancer, 12 % in renal cell cancer,
and 6 % in ovarian cancer patients, which is an historically remarkable outcome(Brahmer et al. 2012). The combination therapy of anti-CTLA-4 (Ipilimumab) andanti-PD-1 was reported to double the response rate, compared to monotherapy, in aphase I clinical trial for the treatment of advanced melanoma (Wolchok et al. 2013).
Therefore, targeting the PD-1–PD-L1 axis is expected to introduce a breakthroughin cancer treatment when applied in combination with chemotherapy, radiotherapy,
and surgical interventions.

34.11

PD-1 and Autoimmune Diseases

As mentioned earlier, the PD-1–PD-L1/2 binding not only controls the functions ofT cells and APCs but also contributes to the differentiation of iTreg cells that are
induced by the endothelial and epithelial cells in the peripheral tissues. Therefore,
the dysfunction of PD-1 both directly or indirectly, through induction of iTreg cells,
leads to uncontrolled functions of effector T cells which end up in autoimmune
diseases. On the other side, when the autoimmune diseases develop from othercauses, the chronic inflammation within the disease microenvironment enhances
the expression of PD-L1 and iTreg cell differentiation.
It was experimentally shown that the deficiency of PD-1 increases the severity oftype I diabetes in NOD mice. Although the relationship between PD-1 and theyokosuka-ths@umin.ac.jp

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destruction of β cells of the pancreatic islets was not definitively shown in humans,
the number of Treg cells and their suppressive functions in pancreatic islets wasmarkedly reduced in type I diabetic patients (Kukreja et al. 2002). The inhibitory
function of Treg cells in the peripheral blood of type I diabetic patients appears to
be normal but it is unsure that the same is true for Treg cells residing within the
microenvironment of the pancreatic islets. In mice, pancreatic island β cells are
expressing PD-L1, thereby directly suppressing the cytolytic activity of effector
CD8+ T cells and possibly inducing the differentiation of CD4+ T cells into iTreg
cells. Moreover, it seems that PD-L1 expressed by the pancreatic epithelial andvascular endothelial cells has a similar suppressive function. Therefore, PD-1 exerts
its inhibitory activity on T cells from different angles. Using two-photon microscopy, it was shown that antibody blockade of PD-L1 resulted in decreased motilityof tolerant islet-antigen–specific CD4+ T cells within the pancreatic island concomitant with breaking the tolerance and induction of autoimmune diabetes (Fifeet al. 2009). Functions and differentiation of effector T cells could be cooperatively
determined by not only the direct signaling through the PD-1–PD-L1 binding but
also the duration of TCR signaling and the cytokine concentration within the
microenvironment.
Immunosuppressive mechanisms by PD-1 are also believed to control thedevelopment of multiple sclerosis. PD-1 and PD-L1 are widely expressed byvarious types of cells within the central nervous system (CNS); furthermore, retinal
ganglion cells not only express PD-1 but also upregulate the PD-L1/2 expression
under inflammatory conditions. Astrocytes and brain vascular endothelial cells
express PD-L1. Microglia, the antigen-presenting cells within the CNS, also
express PD-L1 upon stimulation with IFN-γ. In experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis, PD-1 and PD-L1/2 areexpressed by the inflammatory cells infiltrating within the cerebrospinal fluid.
Furthermore, the deficiency of PD-1 or the antibody blockade of the PD-1–PD-L1
interaction leads to more severe EAE with an earlier onset, suggesting that PD-1
plays a critical role in limiting inflammation within CNS. Bone marrow chimera
experiments have further shown that the expression of PD-1 on macrophages andother myeloid cells is as important as its expression on lymphocytes for the control
of EAE (Rui et al. 2013). Under inflammatory conditions, nerve cells themselves
express PD-1 and TGF-β; those induce the differentiation of the infiltrating encephalitogenic effector T cells into iTreg cells, promoting the relief of inflammation(Liu et al. 2006).
In inflammatory bowel disease (IBD) patients, PD-L1 is highly expressed byintestinal epithelial cells suggesting that intestinal mucosal epithelia could contribute to the tolerance induction via PD-1 (Nakazawa et al. 2004). CD103+ DCs within
the gut-associated lymphoid tissue (GALT) could induce oral tolerance via the
induction of iTreg cell differentiation. Additionally, iTreg cells could also be
induced by PD-L1–expressing intestinal epithelial cells. Inflammation-related cytokines such as IL-12, IL-17, IL-21, IL-23, and IFN-γ were locally concentrated in the
microenvironments under the IBD conditions and induce T-cells differentiation
into Th17 cells, the major T-helper subset responsible for IBD. Although theyokosuka-ths@umin.ac.jp

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number of Foxp3+ cells is increased in the gut, Th17 cells drastically increase in
number compared to Treg cells and consequently the decreasing Treg/Th17 ratio
results in IBD. The murine IBD model, in which CD4+ CD45RBhi T cells weretransferred into severe combined immunodeficient (SCID) mice, demonstrated that
not only iTreg cells but also CD4+ CD25 PD-1+ Foxp3+ effector T cells contributeto the control of inflammation; indeed, the pathogenic CD4+ CD45RBhi T cells that
expressed PD-1 became nonpathogenic after several rounds of sequential transfer
into SCID mice and finally could suppress colitis upon cotransfer with colitogenic
CD4+ T cells (Totsuka et al. 2008)

34.12

Closing Remarks

Although PD-1 was originally cloned as an apoptosis-related molecule, now its
negative costimulatory function has become very clear. Biological characteristics
of PD-1 are unique compared to the other IgSF costimulatory molecules; first, its
ligand expression pattern is complicated as it is not only restricted to immune cells
but rather expressed by several cell types all over the body. The expression of PD-1
and its ligands are controlled by various environmental cues and also both thereceptor and their ligands transduce inhibitory signaling as counterreceptors.
Nonhaematopoietic cells expressing PD-1 ligands can regulate T-cell activation
through an MHC Class II-independent fashion. Second, the receptor PD-1 and theligands PD-L1/2 are simultaneously expressed on the same cell forming a cell
intrinsic inhibitory machinery that might also regulate the developmental or functional outputs in concert with the extrinsic machinery. Collectively, PD-1 was
unequivocally proved to be a superior target molecule for cancer and chronic
inflammatory disease therapy, probably because its immune-suppressive functions
are mediated at multiple stages of T-cell life and via crosstalk between T cells,
haematopoietic cells, and even almost all types of cells within the body.

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Micro–adhesion rings surrounding TCR microclusters are
essential for T cell activation
Akiko Hashimoto-Tane,1 Machie Sakuma,1 Hiroshi Ike,1,3 Tadashi Yokosuka,1 Yayoi Kimura,4Osamu Ohara,2,5 and Takashi Saito1,3
1

Laboratory for Cell Signaling and 2Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan
Laboratory for Cell Signaling, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan
4
Advanced Medical Research Center, Yokohama City University, Yokohama, Kanagawa 236-0004, Japan
5
Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan3

T he immunological synapse (IS) formed at the interface between T cells and antigen-presenting cells represents a hallmark ofinitiation of acquired immunity. T cell activation is initiated at T cell receptor (TCR) microclusters (MCs), in which TCRs andsignaling molecules assemble at the interface before IS formation. We found that each TCR-MC was transiently bordered by aring structure made of integrin and focal adhesion molecules in the early phase of activation, which is similar in structure to
the IS in microscale. The micro–adhesion ring is composed of LFA-1, focal adhesion molecules paxillin and Pyk2, and myosin II(MyoII) and is supported by F-actin core and MyoII activity through LFA-1 outside-in signals. The formation of the micro–
adhesion ring was transient but especially sustained upon weak TCR stimulation to recruit linker for activation of T cells (LAT)and SLP76. Perturbation of the micro–adhesion ring induced impairment of TCR-MC development and resulted in impaired
cellular signaling and cell functions. Thus, the synapse-like structure composed of the core TCR-MC and surrounding micro–
adhesion ring is a critical structure for initial T cell activation through integrin outside-in signals.
When T cells recognize an antigen (Ag) MHC on APCs
by their TCRs, TCR microclusters (MCs) are generated
at the interface and recruit kinases and adapters to initiate activation signals. The TCR-MCs move to the centerof the interface to form the central supramolecular activation cluster (SMAC [cSMAC]), generating the bull’s
eye–type immunological synapse (IS; Bunnell et al., 2002;
Campi et al., 2005; Yokosuka et al., 2005; Saito and Yokosuka, 2006; Hashimoto-Tane et al., 2011). Because mostmembrane-proximal signaling molecules critical for T cell
activation, such as ZAP70 (ζ-chain–associated kinase protein 70 kD), LAT (linker for activation of T cells), SLP76(SH2 domain–containing leukocyte protein of 76 kD),
PLC-γ (phospholipase Cγ), etc., are recruited to TCRMCs, the quality and quantity of the activation depends
on regulation of these structures (Yokosuka et al., 2005;
Čemerski et al., 2007). However, how the signaling complexes translate signal strength to lead to different outcomesis still largely unknown.
Correspondence to Takashi Saito: takashi.saito@riken.jp; or Akiko Hashimoto-Tane:
akiko.tane@riken.jp
T. Yokosuka’s present address is Dept. of Immunology, Tokyo Medical University, Tokyo
160-8402, Japan.
Abbreviations used: Ab, antibody; ADAP, adhesion- and degranulation-promoting adapter protein; Ag, antigen; Arp2/3, actin-related protein 2/3; cSMAC, central
SMAC; dKD, double knockdown; Erk, extracellular signal–regulated kinase; ICAM, intercellular adhesion molecule; IS, immunological synapse; LAT, linker for activation of
T cells; MC, microcluster; MCC, moth cytochrome C; MTOC, microtubule-organizing
center; SMAC, supramolecular activation cluster; tg, transgenic; TIRF, total internalreflection fluorescence; WASP, Wiskott-Aldrich syndrome family protein.

T cell activation primarily requires integrin-mediatedconjugate formation between T cells and APCs. Activationof LFA-1, the major integrin expressed on T cells (Dustin etal., 1997), is triggered by the inside-out signal from TCR or
chemokine stimulation. A further change into the high-affinity conformation of LFA-1 is mediated by the outside-in signal upon binding to its ligand intercellular adhesion molecule
1 (ICAM-1; Springer and Dustin, 2012). LFA-1–deficient(KO) mice have reduced sensitivity to Ag (Perez et al., 2003;
Wang et al., 2008), although the in vivo responses of the KO
mice differ depending on the experimental systems (Schmits
et al., 1996; Eskan et al., 2012). In the context of the IS, LFA-1
and its ligand ICAM-1 accumulate outside of the cSMAC
as the peripheral SMAC (Monks et al., 1998; Grakoui et al.,
1999; Krummel et al., 2000). Recently, it was revealed that all
three LFA-1 activation states, low-, medium-, and high-affinity forms, are present at the peripheral SMAC (Comrie et al.,2015). The integrin-mediated outside-in signal induces activation of kinases and clustering of SLP76 (Perez et al., 2003;
Baker et al., 2009). However, how the LFA-1–mediated outside-in signal contributes to T cell activation remains unclear.
In general, integrin signals activate focal adhesion molecules (Mitra et al., 2005). The major components, talin, paxillin (Pxn), and tyrosine kinase Pyk2, have been observed at
the IS (Monks et al., 1998; Sancho et al., 2002; Robertson© 2016 Hashimoto-Tane et al. This article is distributed under the terms of an Attribution–Noncommercial–
Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–
Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/
by-nc-sa/3.0/).

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The Journal of Experimental Medicine

Ar ticle

RESULTS
TCR-MCs are transiently bordered by
focal adhesion molecules
To analyze the contribution of the LFA-1–mediated outside-in signal in the IS formation and T cell activation, we
analyzed LFA-1–related molecules using a planar bilayer
containing moth cytochrome C (MCC)–loaded MHC class
II I-Ek and ICAM-1. T cells from AND-TCR transgenic(tg) mice were transduced with GFP-labeled molecules and1610

stained with Dylight549 (Dy549)-labeled anti–TCR-β (H57)
Fab to visualize the TCR. The images of fluorescence at theinterface between the T cell and the bilayer were collectedby total internal reflection fluorescence (TIRF) microscopy.
Staining of LFA-1 and TCR on fixed T cells revealed
that LFA-1 formed small rings surrounding TCR-MCs(Fig. 1 A). GFP-labeled Pxn (Pxn-GFP) in live T cells showed
clearer rings surrounding TCR-MCs (Fig. 1 B, left).The fluorescent intensity profiles of Pxn-GFP and TCRs showed thatthe fluorescent peaks did not overlap and distributed in analternating way (Fig. 1 B, middle). The nonoverlapping feature was confirmed by plotting normalized pixel intensities of
Pxn-GFP versus TCRs showing low correlation (Fig. 1 B and
Fig. 2 C, right). The distribution and dynamics of the ringsof Halo tag–labeled Pxn (Pxn-Halo) were almost identicalto focal adhesion kinase Pyk2-GFP (Fig. 1 C). The fluorescent intensity profiles of Pyk2-GFP and Pxn-Halo showed
their colocalization (Fig. 1 C, middle).The plot of normalized
pixel intensities of Pyk2-GFP versus Pxn-Halo revealed ahighly positive correlation (Figs. 1 C and 2 C, right). Stainingof vinculin on fixed T cells as an endogenous focal adhesion
molecule also showed similar rings (Fig. 1 D).Therefore, both
integrin and focal adhesion molecules formed the micro–
adhesion ring surrounding TCR-MCs. The criteria of the
micro–adhesion ring are shown in Fig. 1 F. The concentricarrangement of TCRs and micro–adhesion rings resembles
mature ISs in microscale.
Analysis of sequential images of TCR and Pyk2-GFP
at the adhesion area revealed that the formation of micro–
adhesion rings is transient (Fig. 1 E and Video 1). The generation of micro–adhesion rings began just after the initial
TCR-MC formation, and their numbers increased with increasing TCR-MCs. However, these structures disappeared
before cSMAC formation. The images of single micro–adhesion rings chronologically depicted and an array illustrating
the duration of individual TCR-MCs and micro–adhesion
rings showed that the micro–adhesion ring was formed justafter TCR-MC generation at a very early phase of activation
but soon disappeared (Fig. 1 G).
Synapse-like structures at the interface of T cells and APCs
We confirmed these synapse-like structures in microscale atthe interface of T cells and APCs (Fig. 2). T cells expressing SLP76-GFP were incubated with Ag-pulsed B cells andstained with anti–LFA-1 antibody (Ab).The x–y dimensional
analysis at the interface revealed complex distributions of
SLP76-GFP and LFA-1 (Fig. 2, A and B). In SLP76-GFP–
positive cells conjugated with APCs, 45.2 ± 18.2% of thecells showed SLP76-GFP clusters and LFA-1 staining orderedalternately at the interface. The T cells expressing SLP76GFP without clustering would have ceased the formationof SLP76 clusters because the SLP76 cluster is generated
transiently at the very early activation stage (see Fig. 3 G).
The number of SLP76 clusters between LFA-1 staining wasevaluated. From the fluorescent intensity profiles shown in
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and Ostergaard, 2011). Pxn and Pyk2 have also been observedin the microtubule-organizing center (MTOC) and possibly
participate in the MTOC relocation and killing function incytotoxic T cells and NK cells (Herreros et al., 2000; Sancho et al., 2000, 2002; Robertson et al., 2005; Robertson andOstergaard, 2011). Pyk2 was shown to be involved in CD28
signaling, integrin co-stimulation, and the development ofshort-lived CD8 effector T cells (van Seventer et al., 1998;
Tsuchida et al., 1999; Beinke et al., 2010). However, the molecular basis of the function of focal adhesion molecules in
the initial TCR signaling has not yet been elucidated.
Signals from both TCRs and integrin trigger generationof filamentous actin (F-actin) polymer through activation of
Wiskott-Aldrich syndrome family proteins (WASPs, WASP
family verprolin-homologous proteins [WAVE], etc.) andactin-related protein 2/3 (Arp2/3) complexes (Dustin andCooper, 2000; Barda-Saad et al., 2005). During IS formation,it has been shown that F-actin polymerization is induced
initially at TCR-MCs and particularly supports the recruitment of PLC-γ (Kumari et al., 2015). In later phases, F-actin
formed in the marginal area is critical for cell adhesion andlamellipodia formation. F-actin is also essential for promoting
the initial movement of newly generated TCR-MCs throughactin retrograde flow (Barda-Saad et al., 2005; Campi et al.,2005; Yu et al., 2010). The actin-binding protein nonmuscle
myosin II (MyoII) is one of the major players of F-actin–
dependent functions. Although its role in T cell function
was controversial, T cell motility, assembly and movementof TCR-MCs, and MTOC relocation are regulated by the
MyoII-mediated contractility of actin (Jacobelli et al., 2004;
Ilani et al., 2009; Liu et al., 2013).
In this study, we discovered a novel structure of adhesion
molecules surrounding TCR-MCs that is required for T cell
activation, particularly for facilitating activation under weakstimulation. The structure was similar to the IS, although thesize was almost 10 times smaller. This ring structure includes
LFA-1, Pxn, Pyk2, vinculin, and MyoII, which are supportedby F-actin and MyoII activity through LFA-1 outside-in signals.These micro–adhesion rings supported TCR-MC development, especially LAT and SLP76 clustering. Disruption of
the micro–adhesion ring by down-regulation of both Pxn
and Pyk2 resulted in the impairment of TCR-MC formation, phosphorylation of signal molecules, and IL-2 production. These results indicate that the micro–adhesion ring isessential for T cell activation.

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Figure 1. TCR-MCs in the early phase of activation are bordered with focal adhesion molecules. (A) T cells were fixed on the planar bilayer, stainedwith anti–LFA-1–Dy549 and anti-TCR–Alexa Flour 647 Abs, and analyzed with a confocal microscope. Representative images of 58 cells are shown. (B) T
cells expressing Pxn-GFP were stained with H57(Fab)-Dy549 (TCR) and analyzed on the planar bilayer by TIRF microscopy. The images at the contact area
are shown (top left). For better visualization, the image in the boxed area is shown at higher magnification (bottom left). Representative images of 24 cells
are shown. The fluorescent intensity profiles of Pxn-GFP and TCRs between the white arrows are plotted (middle). The pixel intensities of Pxn-GFP and TCRs
from 188 pixels in the central area as indicated by the arrows were normalized to their median values and plotted (right). a.u., arbitrary units. (C) T cells

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Apparent formation of micro–adhesion rings and SLP76
clusters by weak stimulation
For quantitative analysis, the number of micro–adhesion
rings was counted manually using the criterion explainedin Fig. 1 F, whereas the number of TCR-MCs per cell wascounted automatically using IMARIS software (BitPlane).
First, we analyzed Ag dose dependency of micro–adhesion
ring formation (Fig. 3 A and Video 1). The numbers of both
micro–adhesion rings and TCR-MCs increased as cellularadhesion progressed, but the number of micro–adhesion ringsat the higher doses of Ag (1 and 10 µM) decreased rapidly.
In contrast, with lower doses (10 and 100 nM), micro–adhesion rings were generated and sustained for a longer period.
Interestingly, whereas the number of TCR-MCs at 10 nM
Ag was noticeably reduced, the number of micro–adhesion
rings at 10 nM Ag was similar to that of 100 nM Ag. Because the adhesion area was also affected by the strength of
TCR stimulation, we evaluated the densities of TCR-MCs
and micro–adhesion rings upon stimulation with various Ag
doses. The numbers of TCR-MCs or micro–adhesion ringsat the peaks were divided by area (square micrometers) andplotted as the density (Fig. 3 B). Interestingly, the densities of
TCR-MCs increased as the Ag dose increased, whereas thoseof micro–adhesion rings were constant except for those withno Ag. Without Ag, 17.4% of added cells showed transientadhesion, but they moved quickly on the bilayer. Consequently, only 1.6% of cells stopped and made a small num-

ber of TCR-MCs, which could represent preexisting MCs
formed in the absence of Ag as reported previously (Fig. 3,
A and C; Crites et al., 2014). Although we found that someof the preexisting MCs were surrounded by micro–adhesion
rings (Fig. 3 A), they failed to recruit phosphorylated SLP76(Fig. 3 D), suggesting that the micro–adhesion rings might
not support delivering activation signals in the preexistingMCs. Considering that 10 nM is the minimum Ag dose for
T cell stimulation based on the capacity to induce sustained
calcium responses and produce IL-2 under this experimental
condition (Fig. 3 E), these data suggest that the micro–adhesion ring plays a critical role in supporting T cell activation,
particularly upon weak stimulation with low doses of Ag.
We next analyzed the Ag dose dependency in the clustering of ZAP70, LAT, and SLP-76 at TCR-MCs. With
1 µM Ag, all of the ZAP70-GFP, LAT-GFP, and SLP76-GFP
formed clear clusters at the TCR-MCs and moved toward
the center of the interface with TCR-MCs as previously reported (Fig. 3 F, left; and Video 2; Yokosuka et al., 2005). In
contrast, upon stimulation with 10 nM Ag, LAT-GFP and
SLP76-GFP formed significant numbers of distinct, clear
clusters, but ZAP70-GFP showed only a small number ofdiffuse, unclear clusters (Fig. 3 F, right). The kinetics of thecluster number per cell was examined (Fig. 3 G).The number
of ZAP70-GFP clusters by stimulation with 1 µM Ag initially
increased and then decreased gradually and was comparablewith that of TCR-MCs, but the number with 10 nM Ag was
dramatically reduced. In the case of LAT-GFP, the number ofclusters was quite similarly induced to that of ZAP70 upon
stimulation with 1 µM Ag, but the number of LAT clusters bystimulation with 10 nM Ag was similar to those with 1 µM
Ag. The number of SLP76 clusters by stimulation with 1 µM
Ag also initially increased and then decreased more rapidly
than those of TCR-MCs, and the number of SLP76 clusters
by stimulation with 10 nM Ag was similar to those with 1 µM
Ag. The kinetics of cluster intensities showed the difference
between ZAP70, LAT, and SLP76-GFP clusters more clearly(Fig. 3 H). With 1 µM Ag, the intensities of TCR-MCs
and ZAP70-GFP clusters gradually increased, whereas the
SLP76-GFP clusters rapidly disappeared, and LAT-GFP clusters showed the intermediate pattern of ZAP70 and SLP76.
TCR-MCs and ZAP70-GFP clusters upon stimulation with
10 nM Ag had much lower intensities than those with 1 µM
Ag, whereas SLP76-GFP clusters showed similar intensities
throughout the period, and LAT again showed the interme-

expressing both Pyk2-GFP and Pxn-Halo were examined. The representative images of five cells are shown. The fluorescent intensity profiles of Pyk2-GFP
and Pxn-Halo between the white arrows are plotted (middle). The pixel intensities of Pyk2-GFP and Pxn-Halo from 188 pixels analyzed the same as in B
are plotted (right). (D) Fixed T cells were stained with anti-vinculin–FITC and anti-CD3–Alexa Fluor 647 mAbs and analyzed as in A. Representative imagesof 58 cells are shown. (E) T cells expressing Pyk2-GFP were similarly analyzed. Sequential images of Pyk2-GFP and TCR are shown in chronological order.
Representative cells of 23 cells were selected. (F) Criterion for counting micro–adhesion ring is shown for Pyk2 and TCR images. TCR-MCs surrounded bymore than half of their periphery with Pyk2 were counted. In this example, the three images from the top are positive, and the one on the bottom is negative. (G) The images of each individual TCR-MC and Pyk2-GFP ring from the cell shown in E (left) and an array illustrating the duration of 43 TCR-MCs andmicro–adhesion rings (right) are shown. Red bars indicate the total lifetime of each TCR-MC, and green bars show the period when it was bordered by the
Pyk2-GFP ring indicative of micro–adhesion ring formation. Time 0 means the time point of observed cluster formation. Bars: (A–E) 5 µm; (F and G) 1.5 µm.
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Fig. 2 C (left), the number of SLP76 clusters was estimatedas 4.8 ± 2.3 on a diameter and 18.6 ± 9.18 per contact area.
The plot of normalized pixel intensities of SLP76-GFP versus
LFA-1 from representative cells (middle) was also shown. The
means of correlation coefficients of SLP-GFP and LFA-1
from 10 cells were plotted in Fig. 2 C (right). These dataclearly showed that SLP76 and LFA-1 were nonoverlapped.
As a colocalization positive control, the higher correlation coefficient of Pyk2-GFP and Pxn-Halo was plotted (Fig. 1 C).
The lower correlation coefficient of TCR and Pxn-GFP was
also plotted for confirmation of similar value from different
experimental conditions (Fig. 1 B). We also reconstituted
the x–z dimension image and confirmed that SLP76-GFP
clusters were surrounded by LFA-1 (Fig. 2 D). These dataclearly show that the TCR-MCs in the early phase of activation induce the transient formation of synapse-like structureswith micro–adhesion rings.

diate pattern of ZAP70 and SLP76. In this analysis, only theclusters generated within 1 min after attachment were analyzed because the clusters may have fused to each other later.
We detected a similar difference in the cluster formation
between ZAP70 and SLP76 when using lower affinity Ag (avariant MCC K99A peptide) or T cells expressing low-affinity TCR (5c.c7-TCR tg T cells; unpublished data). These results demonstrate that ZAP70 clusters depend on the strengthof TCR stimulation, whereas SLP76 clusters are relatively
independent of TCR signal strength, and LAT clusters areformed in both a TCR signal–dependent and –independent
fashion. TCR signal–independent LAT clustering and SLP76
clustering were similar to micro–adhesion ring formation.
Consistently, almost all of the LAT clusters and SLP76 clusters
were at the center of the micro–adhesion ring like small ISs
upon 10 nM Ag stimulation (Fig. 3 I).
Formation of micro–adhesion ring depends on LFA-1–
mediated outside-in signaling
It has been shown that SLP76 clusters are induced not only
by TCR stimulation, but also by the LFA-1–mediated outside-in signal (Baker et al., 2009). Therefore, we modulated
the strength of the outside-in signal by changing the concentration of the ligand ICAM-1 on the planar bilayer (Fig. 4 A).
The concentration of 200/µm2 of ICAM-1 was used throughout the study because it is the equivalent level to that on theactivated dendritic cells. When the ICAM-1 concentration
was decreased to 20/µm2, the number of micro–adhesion
rings was reduced to half, whereas that of TCR-MCs was
not changed. With 2/µm2 of ICAM-1, the micro–adhesion
rings were not induced at all, whereas TCR-MCs were stilldetected, suggesting that the generation of micro–adhesion
rings strongly depends on the LFA-1 outside-in signal. The
densities of micro–adhesion rings (the number per area) at
the peak also decreased by reducing ICAM-1 concentration,
whereas those of TCR-MCs were not significantly changed(Fig. 4 B). The number and density of SLP76 clusters by theoutside-in signal showed similar results as the micro–adhesion
rings (Fig. 4, A and B). Therefore, SLP76 clusters upon stimulation with low-dose Ag would be supported by the micro–
adhesion ring. The intensity of TCR-MCs slightly increased
with 2/µm2 of ICAM-1, probably because of the excessive
accumulation of TCRs through reduced integrin activation(Fig. 4, C and E). Because the majority of the cells did notadhere to the bilayer with 2/µm2 of ICAM-1 and only a few
cells were adherent and stopped, it may not represent a physiological condition (Fig. 4 D). Nevertheless, even under suchconditions, SLP76-GFP clusters with 2/µm2 of ICAM-1 hadsignificantly lower intensity (Fig. 4 B). These data indicatethat micro–adhesion rings depend on LFA-1–mediated outside-in signaling and that SLP76 clusters were predominantly
supported by this structure.
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Figure 2. Micro–adhesion rings at the interface between T cells and APCs. (A–D) T cells expressing SLP76-GFP were incubated with 1 µM Ag–loaded
activated B cells for 5 min and fixed. The images of SLP76-GFP and anti–LFA-1–Dy549 staining are shown. (A) The xyz dimensions are indicated in the
diagram (left). The representative images of 14 cells in the x–y dimension are shown (right). DIC, differential interference contrast. (B) The region in the box
in A with a higher magnification is shown. (C) The fluorescence intensity profiles of LFA-1 and SLP76-GFP in between the white arrows in B were plotted(left). The pixel intensities of SLP76-GFP and LFA from 189 pixels were normalized to their median values and plotted (middle). The mean of the correlation
coefficients of SLP76-GFP and LFA from 10 T cells conjugated with APC was plotted (right). The correlation coefficients of Pyk2-GFP and Pxn-Halo and TCR
and Pxn-GFP were also evaluated from the cells analyzed on the planer bilayer shown in Fig. 1 (B and C). Data are the mean from five cells ± SD. *, P < 0.01.
a.u., arbitrary units. (D) The reconstituted x–z images in the box in B are shown. Bars, 3 µm.

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Figure 3. Apparent formation of micro–adhesion rings and LAT and SLP76 clusters at weak TCR stimulation. (A) T cells expressing Pyk2-GFP were
examined on the planar bilayers. The concentration of loading Ag peptide was changed from 10 nM to 10 µM, and Pyk2-GFP rings and TCR-MCs per cellwere counted. (B) Concentrations (conc.) of TCR-MCs and Pyk2-rings at the time point of maximum number for each in A are plotted. (A and B) Data are
the mean from 12–24 cells for each condition ± SD. (C) The rates of cells that adhered and moved (shaded bar) or adhered and stopped (unshaded bar) areplotted. Data are the mean from 505–773 cells for each condition ± SD. (D) The number of phosphorylated SLP76 (pSLP) clusters per fixed T cells on theplanar bilayers was counted. Data are the mean from 10–24 cells for each condition ± SD. (E, left) T cells were loaded with Fluo-4 and Fura-red and analyzed
on the planar bilayers. The ratios of fluorescence intensities of Fluo-4 versus Fura-red were plotted over time. Data are the mean from 64–174 cells foreach condition ± SD. (right) T cells were stimulated with the glass beads coated with planar bilayer that was loaded with Ag peptides. IL-2 in the culture

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Furthermore, pretreatment of T cells with Arp2/3 complex inhibitor CK666 (CK) was also used to inhibit F-actin.
The T cells treated with 30 µM CK for 30 min dramaticallyreduced the number of Pyk2-GFP rings (Fig. 5 C), whereas
the significant number of TCR-MCs remained, and the intensity of TCR-MCs decreased (Fig. 5 D).
These data clearly show that micro–adhesion rings,
LAT clusters, and SLP76 clusters are dependent on F-actin,
whereas TCR-MCs and ZAP70 clusters are relatively independent. The components of the synapse-like structure wereclearly divided into two types: F-actin–dependent clustering(LAT, SLP76, micro–adhesion ring, etc.) and independent
clustering (TCR, ZAP70, etc.).
Micro–adhesion ring formation depends on MyoII activity
Because the micro–adhesion ring is tightly associated andregulated by F-actin, we next examined the involvement of
MyoII as an F-actin–related effector molecule for micro–adhesion ring function. T cells expressing Pxn-Halo or SLP76Halo were loaded with tetramethylrhodamine (TMR) ligand
and treated with 50 µM blebbistatin (Blebb), a MyoII inhibitor, for 3 h before micro–adhesion ring formation. Pretreatment with Blebb reduced Pxn-Halo rings and SLP76-Haloclusters, although it significantly retained the numbers anddensities of TCR-MCs (Fig. 6 A), suggesting that MyoII activity is strongly related to the micro–adhesion ring formation. The kinetics of the cluster intensities showed that the
TCR-MCs were slightly reduced and SLP76-Halo clusters
were reduced and shortened (Fig. 6 B). Imaging analysis byusing anti-MyoII Ab revealed that MyoII was colocalizedwith LFA-1 surrounding TCR-MCs (Fig. 6 C).
Therefore, these results suggest that MyoII and its activity are essential for the formation of synapse-like structures,
especially for the recruitment of focal adhesion molecules.
The impaired micro–adhesion ring would reduce the sizeof TCR-MCs and F-actin clusters and would result in defects in SLP76 clustering.
Critical role of the micro–adhesion ringfor generation of TCR-MCs
To investigate the importance of the micro–adhesion rings
for TCR-MCs and T cell activation, RNA interferencewith shRNA for focal adhesion molecules was used. T
cells were retrovirally transduced with shRNA for control(shCtrl), Pxn (shPxn), and Pyk2 (shPyk2). Down-regulation of Pxn and Pyk2 expression was confirmed by Western
blotting (Fig. 7 A). The shCtrl-, shPxn-, and shPyk2-trans-

supernatants from 105 cells at 24 h was measured by ELISA. Data are representative of three independent experiments. (F) T cells expressing ZAP70-GFP,
LAT-GFP, or SLP76-GFP were analyzed on the planer bilayer. Representative images of 20–36 cells at the maximum cell spreading time are shown. (G) The
cluster numbers of ZAP70-GFP, LAT-GFP, and SLP76-GFP per cell are plotted over time. Data are the mean from 20–36 cells ± SD. (H) The mean intensities
of individual clusters generated during the initial 1 min in G are plotted per time. Data are the mean from 163–272 clusters ± SD. *, P < 0.01. a.u., arbitrary
units. (I) T cells expressing both ZAP70-Halo and Pxn-GFP (top), LAT-Halo and Pyk2-GFP (middle), or SLP76-Halo and Pxn-GFP (bottom) were examined on
the planar bilayers with 10 nM Ag. Representative images of 16–36 cells at the time of maximum cell spreading are shown. Bars, 5 µm.
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F-actin is essential for micro–adhesion
rings and SLP76 clusters
It was shown that adhesion- and degranulation-promoting
adapter protein (ADAP) regulates integrin-mediated celladhesion from both inside-out and outside-in signals bystabilizing the recruitment of WASP to SLP76 for actin rearrangement (Peterson et al., 2001; Baker et al., 2009; Pauker
et al., 2011). We confirmed that the SLP76 cluster in T cells
from ADAP KO mice was severely reduced in intensity but
was intact in number, which was probably supported by intact
F-actin clusters (unpublished data). Therefore, we analyzed
the contribution of F-actin on micro–adhesion rings.
F-actin distribution was analyzed by using the F-actin–binding peptide Lifeact. Lifeact-GFP was accumulated
at TCR-MCs and in the peripheral lamellipodia area upon
stimulation with 1 µM Ag, and they showed distinct, clear
clusters, even with 10 nM Ag stimulation, which were colocalized with SLP76-GFP clusters and surrounded by Pxn-Halorings (Fig. 5, A and B; and Video 3). Other F-actin–related
molecules, WASP (Fig. 5, A and B) and the Arp2/3 complex(not depicted), were similarly detected at TCR-MCs upon
stimulation with 10 nM Ag.
To confirm the importance of F-actin for the synapse-like structure, T cells were acutely treated with cytochalasin D (CytD), which promptly inhibits actin polymerization.
CytD was added at the point of maximum spreading, andthe time course of micro–adhesion rings and other cluster
numbers are shown in Fig. 5 E. Inhibition of actin polymerization was confirmed by the rapid disappearance of Lifeact-GFP clusters after CytD addition (Fig. 5 E, bottom right).
TCR-MCs and ZAP70-GFP clusters upon stimulation with
1 µM Ag were not changed by the addition of CytD andremained. These data were consistent with the previous study
by Varma et al. (2006) that showed new generation of TCRMCs was inhibited by F-actin inhibitor, whereas they wereresistant to F-actin inhibitor after generation. The adhesionarea was also comparable in CytD-treated T cells because
CytD was added after the maximum expansion of the T cells(Fig. 5 G). In contrast, SLP76-GFP clusters and LAT-GFP
clusters upon stimulation with 1 µM Ag decreased much
more rapidly than the control by the addition of CytD. 10
nM Ag was used for Pyk2-GFP because they showed more
prolonged micro–adhesion ring formation than with 1 µM
Ag. Pyk2-GFP rings and SLP76-GFP clusters upon 10 nM
Ag stimulation rapidly disappeared by CytD treatment similar
to Lifeact-GFP. The images after the addition of CytD are
shown in Fig. 5 F and Video 4.

duced cells on planar bilayers showed no significant difference in TCR-MC numbers upon stimulation with 1 µM Ag(Fig. 7 B). However, the cells cotransduced with shRNAs
for Pxn and Pyk2 (double knockdown [dKD] cells) showed
an increased number of TCR-MCs and impaired cSMAC
formation (Fig. 7, B and C; and Video 5). dKD cells became more mobile on the planar membrane by stimulation
with 1 µM Ag than shCtrl cells (Fig. 7 D). This unstablecontact of dKD cells would be the reason for the impaired
cSMAC formation. When T cells were stimulated with 10
nM Ag, the numbers of TCR-MCs in shPxn and dKD cells
were dramatically reduced compared with that in shCtrl cells(Fig. 7 B and Video 5). The reduction in numbers appeared
to depend on the size of the adhesion area because the density of clusters was not changed, suggesting that the effectmight be based on dysregulation of focal adhesion (Fig. 7 E).
Indeed, as expected, in the dKD cells, the number and sizeof each micro–adhesion ring detected by vinculin staining
were decreased (Fig. 7 F).
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Analyses of individual clusters revealed that the intensities of TCR-MCs in the dKD cells significantly decreased
with both 1 µM and 10 nM Ag (Fig. 7 G). The Lifeact-GFP
intensity in the dKD cells was also significantly decreased(Fig. 7 H). The phosphorylation status was analyzed at TCRMCs by Ab staining. The numbers and intensities of the clusters of phospho-CD3ζ and phospho-SLP76 were decreasedin the dKD cells (Fig. 7 I). These data indicate that the development of individual TCR-MCs and the signaling through
TCR-MCs including phosphorylation and recruitment of
SLP76 are supported by the micro–adhesion ring throughregulation of cell adhesion.
Impaired T cell functions by down-modulation of micro–
adhesion ring components
To analyze the function of the synapse-like structure, dKD
cells were stimulated with Ag peptide/APC, and T cell responses were analyzed. Upon interaction with APC, the frequency of cell adhesion and cSMAC formation was reduced
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Figure 4. Micro–adhesion ring formation is dependent on integrin outside-in signal. (A) T cells expressing Pyk2-GFP or SLP76-GFP were examinedon the planar bilayers containing 200, 20, or 2/µm2 of ICAM-1. The numbers of TCR-MCs, Pyk2-GFP rings, and SLP76-GFP clusters per cell are plotted. Data
are the mean from 16–29 cells for each condition ± SD. (B) The mean concentrations of Pyk2-GFP rings, TCR-MCs, and SLP76 clusters at the peak of numbersof TCR-MCs in A ± SD are plotted. *, P < 0.05; **, P < 0.01. (C) The mean intensities of individual TCR and SLP76-GFP clusters generated during the initial 1
min are plotted over time. Data are the mean from 298–447 clusters ± SD. *, P < 0.01. a.u., arbitrary units. (D) The rates of cells that adhered and stopped
on the planar bilayer with each concentration of Ag peptides are plotted. Data are mean ± SD from 443–752 cells. **, P < 0.01. (E) Representative images of
Pyk2-GFP, TCR, SLP76-GFP, and Lifeact-GFP on the planar bilayers containing 2/µm2 ICAM-1 are shown. Bar, 5 µm.

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Figure 5. Dependency of synapse-like structures on F-actin. (A) T cells expressing Lifeact-GFP, Lifeact-GFP and SLP76-Halo, WASP-GFP, or Lifeact-GFP
and Pxn-Halo were examined with 1 µM or 10 nM Ag. Representative images of 8–32 cells are shown. (B) The fluorescence intensity profiles of Lifeact-GFP(green) and TCR (red; top), Lifeact-GFP (green) and SLP76-Halo (red; middle), and WASP-GFP (green) and TCR (red; bottom) in between each pair of whitearrows in A are plotted. (C) T cells expressing Pyk2-GFP were pretreated with 30 µM CK666 for 30 min and examined. The numbers of Pyk2 rings and TCRMCs with (black line) and without (gray line) CK666 treatment are plotted per time. Data are the mean from 14 or 16 cells ± SD. Ctrl, control. (D) The mean
intensities of individual TCR clusters generated during the initial 1 min in F are plotted. Data are the mean from 1,309–1,994 clusters ± SD. *, P < 0.01. (E)When the T cells expressing Pyk2-GFP, SLP76-GFP, ZAP70-GFP, LAT-GFP, or Lifeact-GFP had expanded on the planar bilayers with 10 nM or 1 µM Ag, CytD
was added at a final concentration of 10 µM. The numbers of rings or clusters for each molecule with (black line) and without (gray line) CytD treatment

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DISCUSSION
In this study, we describe the discovery of a specific structure of micro–adhesion rings just around TCR-MCs, whichis required for early T cell activation (Fig. 8 F). The micro–
adhesion ring is composed of integrin, focal adhesion molecules, and MyoII and surrounds each TCR-MC. Consideringthat the mature IS is composed of centralized TCRs andsurrounding LFA-1, this structure resembles the IS in microscale, which we may call a microsynapse.The formation of
the synapse-like structure is transient during the early stage ofT cell activation and depends on LFA-1–mediated outside-in
signals, F-actin, and MyoII activity. The micro–adhesion ringplays a critical role in facilitating adhesion, TCR clustering,and F-actin cluster formation to recruit LAT and SLP76 andgenerating TCR signals. Such function of the synapse-likestructure to amplify and support TCR activation was particularly obvious upon weak stimulation with low doses of Ag.
It has been previously reported that LFA-1 induces dot
clusters separately from TCR-MCs during the initial phaseof T cell activation (Kaizuka et al., 2007). In the present
study, we showed that LFA-1 was transiently accumulated
to form a ring structure surrounding TCR-MCs. The ring
structure was more clearly observed by Pxn-GFP or Pyk2GFP than by CD11a-GFP (unpublished data), probably because of the abundant expression of CD11a-GFP on the cellsurface. Indeed, more LFA-1 rings were detected by anti–
LFA-1 staining than CD11a-GFP overexpression. Therefore,
the number of the micro–adhesion rings identified by thetransfected Pxn-GFP or Pyk2-GFP might be an underestimate of the total number.
The micro–adhesion ring has some similarities to thepodosome/invadosome. The podosome is a cylindrical structure formed in a vertical direction on the ventral cell surface
to support invasion of cancer cells or dendritic cells (Murphy
and Courtneidge, 2011). The similarities include molecularcomponents, size, dependency on receptor stimulation, outside-in signaling, MyoII activity, and structure with an F-actin
core surrounded by adhesion molecules. (Collin et al., 2008;
Albiges-Rizo et al., 2009; van den Dries et al., 2013). There
has been a study describing a podosome-like protrusion at theinterface between T cells and epithelial cells or APCs (Sage etal., 2012).This structure contained TCR, F-actin, and ZAP70,
and the authors suggested its role in invading the carbohydrate forest on APC to find the peptide–MHC complex on
the cell surface. Because the micro–adhesion rings are formed
transiently during the early stage of T cell activation and are
involved in the initial cell adhesion and activation, they may
be similar to the podosome-like structure. Although we failed
to detect such a small protrusion in T cells on the planar bilayer system using electron microscopy in our previous study(Hashimoto-Tane et al., 2011), the structural characterizationof the observed synapse-like structure remains to be analyzed.
Because there is no means of specific disruption of the
micro–adhesion ring to help determine its function, we analyzed T cell function after down-regulating two componentsof the micro–adhesion ring, Pxn and Pyk2. Consistent with aprevious study of Pyk2 KO mice (Beinke et al., 2010), shPyk2

are plotted per time. Arrows indicate the kinetics of CytD addition. Data are the mean from 12–18 cells ± SD. (F) Representative images of TCR, ZAP70-GFP,
LAT-GFP, SLP76-GFP, and Lifeact-GFP at 90 s after the addition of CytD are shown. (G) The mean contact area with and without CytD treatment is plotted
over time. Data are the mean from 16 cells ± SD. a.u., arbitrary units. Bars, 5 µm.
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in dKD cells (Fig. 8 A and Video 6). Consequently, flow
cytometric analysis showed that phosphorylation of SLP76
and extracellular signal–regulated kinase (Erk [pErk]) upon
stimulation with Ag peptide/APC was reduced in dKD cells(Fig. 8 B). IL-2 production upon stimulation with Ag peptide/APC was significantly reduced in dKD cells, whereas
there was no difference even in dKD cells when stimulated
by immobilized anti-CD3/CD28 Abs. Unlike molecular assembly and phosphorylation as early signaling, because IL-2
production is a late response, the reduction might be affectedby reduced Pyk2/Pxn during a long period in addition todefective micro–adhesion rings (Fig. 8 C).
To determine the functional importance of the synapse-like structure further, we tried to dissect TCR and
LFA-1 stimulation by using lipid bilayer–coated glass beads.
We used three types of lipid bilayer–coated beads: only I-Ek–
coated beads (Ek), both I-Ek– and ICAM-1–coated beads ([Ek+ ICAM]), and a mixture of I-Ek beads and ICAM-1 beads([Ek] + [ICAM]; Fig. 8 D, top). shRNA-treated T cells were
stimulated by these glass beads with 1 µM Ag peptides, andthe level of pErk as the representative activation signal was
analyzed (Fig. 8 D). shCtrl cells stimulated with [Ek + ICAM]
showed an increased level of pErk compared with those stimulated with Ek, demonstrating that ICAM-1 enhances pErk.
In contrast, in shCtrl cells stimulated with [Ek] + [ICAM]
separately, the level of pErk was almost comparable with thosestimulated with Ek alone, suggesting that the ICAM-1 stimulation apart from I-Ek had little effect on T cell activation.
The significance of the close proximity between I-Ek andICAM-1 for T cell activation is consistent with and supports
our idea that the synapse-like structure micro–adhesion ringsurrounding TCR-MCs is critical for initial TCR signals.
In contrast, the pErk induction upon stimulation with [Ek+ ICAM] was much lower in dKD cells than that in shCtrl
cells and was not significantly changed from that with [Ek] +[ICAM] (Fig. 8 D).The proportion of pErk-positive cells wascounted, and the statistical results were plotted in Fig. 8 E(left). The same results were obtained in nontransfected CD4
T cells (Fig. 8 E, right). These results suggest that dKD cells
could not be fully activated by [Ek + ICAM], probably because of the lack of close proximity of TCR and LFA-1 by thedefect in micro–adhesion ring formation around TCR-MCs.

cells showed no structural and functional impairment. In contrast, the Pxn KO mice resulted in early lethality. It may havea role in growth and/or survival of cells, which is consistent
with our data that a considerable proportion of shPxn cells
failed to grow. Whereas the shPxn T cells had no detectabledefects in adhesion or IS formation upon strong stimulation
with 1 µM Ag, they showed some defects in cell adhesion andformation of TCR-MCs upon weak stimulation with 10 nM
Ag. In contrast, the dKD cells exhibited severely impaired
regulation of adhesion, TCR-MC formation, phosphorylation of signaling molecules, and cytokine production. Therefore, it is likely that the function of the micro–adhesion ringprimarily depends on Pxn rather than Pyk2, and synergistic
effects were observed by double deficiency of both Pxn andPyk2 for the early stage of T cell activation.
The reduced size of TCR-MCs and impaired TCR proximal signaling were caused by the alteration of micro–adhesion

rings. In addition, the defect in cell adhesion in dKD cells upon
weak stimulation was also explained by impaired micro–adhesion ring formation because usually adhesion starts from thepoint of the initial TCR-MC and the generation and maturation of TCR-MCs depends on adhesion rings. In contrast, theincreased adhesion area in dKD cells upon strong stimulation
may reflect the effects from the general function of focal adhesions; stable adhesion in sessile cells and active cells move byrecycling in moving cells (Webb et al., 2004). The dKD cells
upon strong stimulation could induce adhesion but also failed
to stop cells with narrowing down the adhesion area. The reduced IL-2 production could be mainly caused by the defective
initial activation signal but may also be affected by the lack of
Pxn or Pyk2 later during the 24-h culture period.The reduced
sensitivity of T cell activation in the dKD cells was similarly
observed in LFA-1–deficient T cells (Wang et al., 2008), whichis consistent with impaired LFA-1 signaling in the dKD cells.
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Figure 6. Micro–adhesion ring formation isdependent on MyoII activity. (A–C) T cells expressing Pxn-Halo or SLP76-Halo were pretreatedwith 50 µM Blebb for 3 h and examined on theplanar bilayers. (A) The numbers of clusters with
(black line) and without (gray line) Blebb treatment are plotted over time. Data are the meanfrom 12–16 cells ± SD (left and middle). The mean
concentration of TCR-MCs at the peak of cluster
numbers with (black bar) and without (gray bar)
Blebb treatment is plotted (right). Ctrl, control. (B)The mean intensities of individual clusters of TCRs
or SLP76 generated during the initial 1 min with
(black line) and without (gray line) Blebb treatment
analyzed in A are plotted. Data are the mean from
416–540 clusters ± SD. *, P < 0.01. a.u., arbitrary
units. (C) T cells were fixed at 2 min and stainedwith anti–MyoII-FITC, anti–LFA-1 (CD11a)–Dy549,
and anti-CD3(TCR)–Alexa Fluor 647 (top). For better visualization, the images in the boxed areas areshown at a higher magnification (middle). Representative images of 12 cells are shown. The fluorescence intensity profiles of MyoII-FITC (green)and LFA-1 (CD11a)–Dy549 in between the whitearrows in the top image are plotted (bottom left).
The percentages of MyoII cluster area colocalizedwith LFA-1 or with TCRs as a control to the total
MyoII cluster area were plotted (bottom right).
Bars, 3 µm. Data are the mean from 12 cells ± SD.
*, P < 0.01.

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Figure 7. Critical role of the micro–adhesion ring for TCR-MC development. (A–I) T cells transduced with shCtrl, shPxn, shPyk2, or shPxn + shPyk2(dKD) were examined on the planer bilayer. (A) The expression levels of Pxn and Pyk2 with shCtrl, shPxn, shPyk2, or dKD cells were examined by Western
blotting. Representative data from three independent experiments are shown. (B) The numbers of TCR-MCs per cell in shCtrl, shPxn, shPyk2, or dKD cells
on 1 µM or 10 nM Ag are plotted. Data are the mean from 18–38 cells ± SD. (C) The rates of cells with cSMACs at 10 min on 1 µM Ag–loaded bilayer wereplotted (left). Data are the mean from 87–132 cells ± SD. Representative images are shown (right). Bar, 5 µm. (D) The trajectories of movements of shCtrl
or dKD cells on 1 µM Ag for 10 min are shown. 16 cells for each condition were analyzed. (E) The concentrations of TCR-MCs in the cells analyzed in B are
plotted. Data are the mean from 18–38 cells ± SD. (F) The shCtrl or dKD cells were fixed at 1.5 min and stained with anti-vinculin–FITC mAb. Histograms ofthe numbers of vinculin rings per cell and the diameters of each ring in shCtrl and dKD cells are shown. Data are from 24 cells for each condition. (G) The
mean intensities of individual TCR-MCs in the cells in B are plotted over time. Data are the mean from 526–1,774 clusters ± SD. a.u., arbitrary units. (H) The
intensities of individual Lifeact-GFP clusters in shCtrl and dKD cells with 1 µM Ag are shown. Data are the mean of 641 and 635 clusters ± SD, respectively.
(I) The mean number of clusters per cell and individual intensities of phospho–CD3-ζ clusters and phospho-SLP76 (pSLP76) clusters in shCtrl (unshadedbars) or dKD (shaded bars) cells is depicted. Data are the mean from 1,098–1,438 clusters ± SD. *, P < 0.01.

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Figure 8. Impaired T cell functions after down modulation of focal adhesion molecules. (A) Representative images of the TCR (H57-Dy549) on
shCtrl or shPxn + shPyk2 (dKD) cells stimulated with 1 µM Ag–loaded activated B cells are displayed (left). The frequencies of adhered and stopped cells
upon interaction with APCs and the rate of the cells with cSMACs after 10 min in the adhered and stopped cells are plotted (right). Data are the mean from59 or 65 cells ± SD. *, P < 0.05. DIC, differential interference contrast. (B) The shCtrl and dKD cells incubated with 1 µM Ag–loaded activated B cells for 10
min were fixed and analyzed for the phosphorylation levels of SLP76 and Erk by flow cytometry. Representative results from four experiments are shown.
pSLP, phosphorylated SLP76. (C) The shCtrl, shPxn, shPyk2, and dKD cells were incubated with irradiated spleen cells at the indicated dose of Ag. IL-2 in theculture supernatant after 24 h was measured by ELISA. Representative results from three experiments are shown. Data are mean ± SD. *, P < 0.01. (D) T cellstimulation with lipid bilayer–coated beads. Three types of lipid bilayer–coated beads were used: I-Ek coated, both I-Ek and ICAM-1 coated, and a mixtureof I-Ek and ICAM-1 coated. The shCtrl and dKD cells were stimulated with the glass beads with 1 µM Ag peptides for 10 min and analyzed for phosphorylation levels of Erk as in C. (E) The nontransfected cells were activated with ICAM-1, Ek, [Ek + ICAM], or [Ek] + [ICAM]. The proportions of pErk-positive cells
from shRNA-treated cells in D and nontransfected cells are plotted. Data are the mean of four experiments ± SD. *, P < 0.05; **, P < 0.01. (F) Diagram of
micro–adhesion rings and ISs (pSMAC, peripheral SMAC; left). When a T cell recognizes an Ag on an APC, TCR-MCs are generated at the interface and initiate
activation signals. Then, TCR-MCs move to form cSMACs and generate a typical IS. The TCR-MCs in initial phase were surrounded by a micro–adhesion ring;

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its importance in T cell activation. The adhesion-mediatedpositive regulation of TCR activation signals was clearly visualized. Such regulation of adhesion and the cytoskeleton
will become more important in understanding the signaling
and function of T cells.
MATERIALS AND METHODS
Mice. AND-TCR tg mice were provided by S.M. Hedrick(University of California, San Diego, San Diego, CA) bycourtesy of R.N. Germain (National Institutes of Health),
and 5c.c7 tg mice were provided by M.M. Davis (Stanford
University, Stanford, CA). ADAP-deficient mice were provided by G. Koretzky (Weill Cornell Medical College, New
York, NY). C57BL/6 and B10.BR mice were purchasedfrom CLEA Japan, Inc. and Japan SLC Inc., respectively. All
mice were maintained in our animal facility in specific pathogen-free conditions and treated in accordance with the ethical guidance of the RIKEN Yokohama Institute.
Cell culture. T cells from the spleen and lymph node of
AND-tg mice were isolated and purified by using CD4(L3T4) MicroBeads (Miltenyi Biotec). The purified T cells
were stimulated with irradiated spleen cells from B10.BR
mice pulsed with 3 µM MCC mutant (MCC(88-103)
K99A) peptide (MCC(K99A)) in RPMI 1640 (Sigma-Aldrich) with 10% FBS and 1% penicillin–streptomycin (Invitrogen). The next day, the genes of interest were transduced
using a retrovirus-mediated gene transfer system. On day 4,
the infected cells were sorted using a flow cytometer (FACS
Aria; BD). Fluorescent imaging experiments were performed on the fifth or sixth day of culture. For shRNA
treatment, the cells were restimulated with irradiated spleen
and MCC(K99A) on the fifth or sixth day, transduced again
with virus supernatants on the sixth or seventh day, and subjected to imaging and functional analysis on the 10th
to 12th day of culture.
Construction and transduction. All the expression vectors
were made by PCR subcloning. The retrovirus vector pMXs
(provided by T. Kitamura, Tokyo University, Tokyo, Japan)
were used for expression of fluorescent-labeled molecules.
ZAP70, SLP76, ADAP, WASP, p16, Arp3, CapZ, Pyk2, Pxn,
and CD11a were cloned from complementary DNA of activated AND-tg T cells. GFP was derived from plasmid enhanced GFP–N1 (BD), HaloTag from HaloTag pHT2 vector

this structure resembles mature ISs as the synapse-like structure in microscale. Our results revealed the following scenario. The integrin outside-in signal–
induced micro–adhesion ring formation was composed of LFA-1, Pxn (Pax), Pyk2, vinculin, and MyoII. The micro–adhesion ring enhanced F-actin formation
and TCR clustering, especially under weak TCR stimulation. (Middle) When F-actin was acutely broken by CytD treatment after TCR-MC generation, TCR-MCs
and ZAP70 clusters remained, but the micro–adhesion ring and LAT and SLP76 clusters disappeared. However, when F-actin formation was inhibited before
TCR-MC generation by CK666 treatment, the generation of both TCR-MCs and micro–adhesion rings was inhibited. Therefore, F-actin is important for bothgeneration and maintenance of the TCR-MC signaling. (Right) In the dKD (shPxn + shPyk2) cells, the decrease of focal adhesion molecules caused defects
in micro–adhesion ring formation. Consequently, small TCR-MCs and F-actin clusters were formed, and T cell activation such as phosphorylation of SLP76
and Erk and IL-2 production was impaired. (A and D) Bars, 5 µm.
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The colocalization of Pxn and Pyk2 suggested their cooperation at the micro–adhesion ring. According to previous
studies (Rodríguez-Fernández et al., 1999; Herreros et al.,2000), it is possible that the cooperation of Pxn and Pyk2 at
the MTOC regulates T cell polarization and activation. Indeed, we confirmed reduced efficiency of MTOC translocation in the dKD cells (unpublished data). However, the defects
in MTOC translocation could also be explained by the defects in the initial T cell activation. Because the defective processes such as TCR-MC generation and phosphorylation of
SLP76 clusters were very early membrane-proximal events,
we suggest that the appropriate spatiotemporal generation of
micro–adhesion rings with Pxn and Pyk2 is critical for inducing integrin outside-in signals required for T cell activation.
The discovery of the TCR-MC as the responsible
structure and the site for inducing initial TCR activation
signals revealed that TCR, ZAP-70, LAT, and SLP76 formed
a TCR-MC, and their assembly and dynamics upon Ag
stimulation were thought to be stoichiometric. However,in the present study, we showed that although the dynamics of TCR and ZAP-70 were almost identical, LAT and
SLP76 behaved differently in terms of the kinetics of cluster
numbers and intensities and the effects of an actin inhibitor,
particularly upon weak TCR stimulation. Interestingly, thetransient recruitment of SLP76 to TCRs in the very earlyphase of activation had also been shown in recent proteome
analysis (Roncagalli et al., 2014). Whereas cluster formationof TCRs and ZAP70 was dependent on the strength of theactivation signal, LAT and SLP76 clusters were formed relatively independently of signal strength. The observed difference in the dynamics of the LAT and SLP76 clustering from
TCRs and ZAP70 was caused by its dependency on theformation of the micro–adhesion ring through LFA-1–mediated outside-in signals and F-actin. Recently, WASP-mediated F-actin–dependent clustering of phospho–PLC-γ
was reported (Kumari et al., 2015). The F-actin supporting
phospho–PLC-γ as actin foci is similar to the F-actin localized at the core of the synapse-like structure that supports
SLP76 clusters and the micro–adhesion ring. Because thecharacteristics of the phospho–PLC-γ clusters were mostly
consistent with LFA-1–mediated SLP76 clusters, it is likelythat the PLC-γ clusters would also be under the regulationof the micro–adhesion ring.
In this study, we found the synapse-like structure
micro–adhesion ring surrounding TCR-MCs and showedAbs. For live imaging of TCR, the Fab fragment of anti–
TCR-β Ab (H57) was labeled with Dylight549. anti-Pxn
Ab (BD610051) and anti-Pyk2 Ab (D-1; sc-365201) wereused for Western blotting. For immunohistochemistry, anti–CD3-ε Ab labeled with Alexa Flour 647 (BD557889), anti-vinculin Ab (F7053; Sigma-Aldrich), anti–LFA-1 Ab(H155-78; BioLegend) labeled with Dylight 549, and anti-MyoII Ab labeled with FITC (orb4013) were used. For
FACS analysis, anti–phospho-Erk Ab (Phosflow 812593;
BD) and anti–phospho-SLP76 Ab labeled with APCs (Phosflow 558438; BD) were used.
Planar bilayer system. Glycophosphatidylinositol (GPI)-anchored mouse MHC class II I-Ek and ICAM-1 were purified
as previously described (Grakoui et al., 1999). The purified
I-Ek and ICAM-1 incorporated in dioleoylphosphatidylcholine liposomes (Avanti Polar Lipids, Inc.) were applied tocover glasses (thickness of 0.12–0.17 mm; Matsunami) or glass
beads (4.86-µm sphere glass) to make the lipid bilayer containing 200 molecules of I-Ek and ICAM-1/µm2 and incubated with Ag peptides (Yokosuka et al., 2005).
In this system, when 10 µM MCC(88-103) peptide wasincubated at 37°C for 12 h, 8.9 ± 5.1% of total I-Ek was
detected as peptide binding I-Ek (I-Ek + MCC; mean fromthree experiments) by staining with anti–I-Ek + MCC mAb
D4 (Grakoui et al., 1999). Therefore, the concentration of
I-Ek + MCC was 17.8/µm2 for 10 µM. When we considered the general formula for affinity evaluation (Kd = [I-Ek]kfree × [MCC]free/[I-E + MCC]) and the published Kd valueof 0.54 µM, which was measured by using soluble I-Ek and
MCC (Reay et al., 1992), the I-Ek + MCC concentrationsfor 1 µM, 100 nM, and 10 nM MCC were estimated to be
11.3, 2.94, and 0.34 µm2, respectively.
Hepes-buffered saline containing 1% BSA, 1 mM
MgCl2, and 1 mM CaCl2 was used for the assay. For immunohistochemistry, cells were placed on a planar bilayer for theindicated periods and then fixed with 2% paraformaldehyde
at 37°C for 10 min. For FACS analysis, cells were spun downwith the glass beads and incubated for 10–15 min and fixedwith 2% paraformaldehyde at 37°C for 10 min. The cells

were permeabilized in 0.05% digitonin or 0.1% Triton X-100
and stained with Abs in PBS containing 5% BSA. For calcium
measurement, cells were incubated with 5 µM Fluo-4 AM
and 10 µM Fura-red AM in 0.5% BSA containing PBS for 20
min at room temperature.
Fluorescence microscopy. A TIRF system (Olympus) was
configured on a microscope (IX81). TIRF objective lenses(UAPON 100×, NA 1.49, oil; Olympus), dual excitation lasers, diode-pumped solid-state 488-nm and 561-nm laser systems (85BCD020 and 85YCA020; Melles Griot), and acharge-coupled device camera (ORCA flash4.0; Hamamatsu
Photonics) were used. Dual-color videos were taken every 5 s
for 10 min using Metamorph software (Molecular Devices).
The images of the interaction between T cells and activated B
cells that had been stimulated with LPS for 24 h were taken
every 10 s using the IX81 microscope with an oil objective
lens (Plan APO N 60×, NA 1.45; Olympus) under illumination with a Hg lamp.The intracellular calcium concentrations
were also measured by using the IX81 microscope with Hg
lamp illumination and an oil objective lens (UPlanAPO 40×,NA 1.00; Olympus). A microscope (TCS SP5; Leica Biosystems) with an oil immersion objective (HCX PL APO 100×,NA 1.44; Leica Biosystems) was used for fixed and stained
samples. For detecting micro–adhesion rings formed in thecell–cell interaction, a hybrid detector (HyD; Leica Biosystems) was used to improve image quality.
Image analysis. IMARIS software (BitPlane) was used forcounting and evaluation of fluorescent clusters. In this study,
all the images were normalized using Fiji as follows: subtracted numerically until the mean background count became 10 and multiplied to adjust the count in the noncluster
area of the cell to be 100. The criteria for a cluster was thatthe diameter was <5 µm, intensities at the center were >105–
120 counts, and the summation of the fluorescence was>1,600–2,400 counts.The parameters were adjusted depending on the molecules being analyzed. The movements of theclusters were tracked automatically with an algorithm ofBrownian movement, and the maximum gap distance in
one frame was set as 5 µm.
Online supplemental material. Video 1 shows TCR-MCs
transiently bordered by Pyk2 in the initial phase of activation.
Video 2 shows different behaviors of ZAP70 and SLP76
clusters upon weak stimulation. Video 3 shows F-actin
transiently accumulated at TCR-MCs in the initial phase ofactivation. Video 4 shows disappearance of SLP76 clusters
and remaining TCR-MCs and ZAP70 clusters by CytD
treatment. Video 5 shows the unstable contacts and impaired
IS formation of dKD cells on the planer bilayer. Video 6
shows the unstable contacts and impaired IS formation ofdKD cells upon interaction with APCs. Online supplemental
material is available at http://www.jem.org/cgi/content/full/jem.20151088/DC1.
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(Promega), and Lifeact from Lifeact-TagRFP2 pCMV (ibidi).
The pMSCV-LTRmiR30-PIG(LMP) retrovirus vector (GEHealthcare) was used for shRNA expression. We used several
different markers to detect doubly transduced cells. In addition to GFP, CD8 without the cytoplasmic region was used
for shPxn or shCtrl, and rat CD2 without the cytoplasmic
region was used for shPyk2 by inserting them after the internal ribosomal entry site. The sequences used were shPxn, 5′CCCCTGGTGAAAGAGAAGCCAA-3′; shPyk2, 5′-AAG
AAGTAGTTCTTAACCGCAT-3′; and shCtrl, 5′-GCA
CGGGGAAGGAGCGGGAA-3′. To produce pseudovirus,
the recombinant plasmids were transfected into Phoenix
packaging cells (G. Nolan, Stanford University, Stanford, CA)
using Lipofectamine 2000 (Invitrogen).

ACKNOWLEDGMENTS

Submitted: 2 July 2015
Accepted: 13 May 2016

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We thank K. Shiroguchi for helpful discussion, M. Unno, W. Kobayashi, and N. Suzuki
for technical support, and M. Yoshioka and H. Yamaguchi for secretarial assistance.
This work was supported by the Japan Society for the Promotion of Science KAK
ENHI with Grants-in-Aid for Scientific Research to T. Saito ([S] JP24229004) and
A. Hashimoto-Tane ([C] JP10415226) and partly by the Takeda Science Foundation(F1206042), Novartis Foundation, and Hayashi Memorial Foundation for Female
Natural Scientists.
The authors declare no competing financial interests.
Author contributions: A. Hashimoto-Tane and T. Saito designed the studies.
A. Hashimoto-Tane, M. Sakuma, and H. Ike performed experiments. T. Yokosuka provided advice and reagents. Y. Kimura and O. Ohara helped with molecular cloning.
A. Hashimoto-Tane and T. Saito wrote the manuscript.

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A novel c-Src recruitment pathway from the cytosol to
focal adhesions
Hiroaki Machiyama1, Tomoyuki Yamaguchi1, Tomonobu M. Watanabe1,2 and Hideaki Fujita1,2
1 WPI, Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan
2 Quantitative Biology Center, Riken, Suita, Osaka, Japan

Correspondence
H. Fujita, Quantitative Biology Center,
Riken, OLABB, Osaka University, 6-2-3,
Furuedai, Suita, Osaka 565-0874, Japan
Fax: +81 6 6155 0112
Tel: +81 70 6804 7335
E-mail: hideaki.fujita@riken.jp(Received 9 March 2017, revised 27 April
2017, accepted 15 May 2017, available
online 10 June 2017)
doi:10.1002/1873-3468.12696
Edited by Lukas Alfons Huber

The role of myristoylation in the localization and catalytic activity of Src at
focal adhesions was investigated by live-cell imaging and site-directed mutagenesis. Although the majority of activated Src molecules are localized at focal
adhesions, it is unclear how activated Src molecules are recruited to focal
adhesions. Because Src is activated at the cell membrane, translocation of Src
to cell membranes is considered to be essential for its recruitment to focal
adhesions. Membrane-targeting-deficient Src mutant SrcG2A localizes at focal
adhesions, indicating direct recruitment of Src from cytosol to focal adhesions.
Furthermore, directly recruited Src molecules are shown to enhance paxillin
dynamics at focal adhesions. These results reveal that the regulation of Src
activation and translocation is more complex than previously suggested.
Keywords: focal adhesion; FRAP; mechanobiology; mechanotransduction;
TIRF

Both intracellular and extracellular mechanical environments influence not only cell morphology but also
cell behavior and fate, such as migration, proliferation,and differentiation [1–4]. Individual cells can sense theirsurrounding mechanical stimuli and convert this information to intracellular biochemical signals, a mechanism termed mechanotransduction. Focal adhesions
provide a platform for mechanotransduction at theinterface between cells and the extracellular matrix(ECM). Interaction between ECM and the ECM receptor integrin enhances intracellular phosphorylation signals at focal adhesions, and subsequently alters focal
adhesion dynamics, cell-generated forces and survival
signals. Nonreceptor tyrosine kinase Src plays pivotal
roles in integrin-mediated cellular signals and many
focal adhesion proteins have Src substrate domains.
Therefore, the localization and catalytic activity of Src
markedly affects cell mechanotransduction.
The conformation of Src is known to be essential
for its kinase activity. Src molecules localized in thecytosol have closed conformation, corresponding to acatalytic inactive state [5]. Phosphorylation of the tyrosine residue at 530 (i.e. referring to the residue inhuman Src) retains the closed conformation of Src bybinding to the Src-homology 2 (SH2) domain [5]. After
dephosphorylation of tyrosine 530, autophosphorylation at tyrosine 419 in the tyrosine kinase domain isrequired for full activation of Src [6,7]. When Src isfully activated, molecules exhibit the open conformation and SH2 and Src-homology 3 (SH3) domains are
exposed. Binding between Src and its substrates
through the SH2 and/or SH3 domains then triggers
Src to serve as a kinase. Although the majority of activated Src molecules are localized at focal adhesions[8], little is known about how activated Src molecules
are recruited.
Translocation of Src to the cell membrane isachieved by myristoylation of the Src N-terminal
domain [9]. Membrane targeting of Src is independentof its kinase activity, although the kinase activation isAbbreviations
ECM, extracellular matrix; FRAP, fluorescence recovery after photobleaching; SH2, Src-homology 2; SH3, Src-homology 3; TIRF, total internal reflection fluorescence.

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presumably conducted at the cell membrane. Therefore, it is hypothesized that, subsequent to membranetargeting and activation, activated Src molecules enter
focal adhesions. Although the G2A mutation of Src
prevents membrane binding as a result of a lack of Nterminal myristoylation of Src [9], the SrcG2A mutant
has been detected in the membrane fraction whenassessed by membrane-cytosol separation method [10].
Therefore, it is assumed that Src molecules directlytranslocate from the cytosol to focal adhesions.
In the present study, we analyzed the effects of
SrcG2A mutation on the function and localization of
Src. We detected the localization of SrcG2A mutants
at focal adhesions by fluorescent microscopy. Expression of SrcG2A enhanced the exchange rate of the
focal adhesion-associated protein paxillin. The results
indicate that there is a novel pathway of Src from thecytosol to focal adhesions without cell membrane targeting, and direct recruitment of Src to focal adhesions
and membrane targeting via N-terminal myristoylation
are required for mechanotransduction at the focal
adhesions.

Microscopy

Materials and methods

To visualize the dynamics of paxillin at focal adhesions, we
carried out fluorescence recovery after photobleaching(FRAP) experiments under TIRF illumination, as described
previously [13]. Before photobleaching, the pre-bleachimages of mCherry-tagged paxillin were acquired every 1 s
for 10 s, using a 561 nm laser set at 5% of the maximum
power. Photobleaching of mCherry was conducted for 10 susing a 561 nm laser at maximum power. Fluorescencerecovery images were acquired every 1 s for 2 min using a
561 nm laser set at 5% of the maximum power. TIRFFRAP analysis was conducted as described previously [13].
Photobleaching as a result of continuous TIRF illumination during recording was corrected by correction curves
calculated from the intensity reduction time course in the
pre-bleach step.

DNA constructs
To construct the expression vectors for Venus, SrcWTVenus and SrcG2A-Venus, the cDNAs of SrcWT, SrcG2A
and Venus were amplified by PCR and cloned into the
pcDNA3.1 vector (Thermo Fisher Scientific, Waltham,
MA, USA). A flexible (GGGGS)3 linker [11] was used
between Src and Venus so as not to disturb the Src structure and function. SrcG2A/K298M-Venus and SrcG2A/
Y530F-Venus were generated by introducing point mutations using SrcG2A-Venus as a template. The expression
vector for mCherry-tagged paxillin was constructed by
PCR using the pcDNA3.1 vector as a parent vector.

Cell culture
HeLa (RRID:CVCL_0030) and SYF (RRID:CVCL_6461)
cells were cultured in Dulbecco’s modifiwed Eagle’s medium (Sigma-Aldrich, St Louis, MO, USA) containing 10%
FBS (Thermo Fisher Scientific) supplemented with 1%
penicillin/streptomycin (Sigma-Aldrich) at 37 °C in 5%
CO2. Transfection of plasmid DNA was carried out using
FuGENE6 HD (Promega, Madison, WI, USA) in accordance with the manufacturer’s instructions. The transfected
cells were transferred to collagen-coated glass dishes (AGC
Techno Glass, Shizuoka, Japan) 24 h before microscopic
observation. FAK inhibitor RF-573228 (Cayman Chemical,
Ann Arbor, MI, USA) was applied 1 h before observation.

Total internal reflection fluorescence (TIRF) images wereacquired under a conventional inverted microscope (Ti-U;
Nikon, Tokyo, Japan) equipped with an EMCCD camera
with a 512 9 512-pixel chip (iXon3; Andor Technology,
Belfast, UK) and fiber-coupled 488 and 561 nm lasers to
excite Venus and mCherry, respectively. NIS-elements software (RRID:SCR_014329; Nikon) was used for image
acquisition and IMAGEJ software (RRID:SCR_003070;
National Institutes of Health, Bethesda, MD, USA) wasused for image processing.

Quantification of Src localization at focal
adhesions
Acquired images of Venus and mCherry were cropped to3 9 3 lm2, including a few focal adhesions. To quantify
the correlation between the distributions of Venus-tagged
Src and mCherry-tagged paxillin, we calculated Pearson’s
coefficient for the two acquired images [8,12].

TIRF-fluorescence recovery after photobleaching(FRAP) experiment

Results and Discussion
A novel recruitment pathway of Src
Myristoylation of Src N-terminal domain promotes
Src translocation and binding to the cell membrane[9]. After activation at the cell membrane, Src works
as a kinase by binding with its substrate located at thecell membrane and focal adhesions [14,15]. Because
replacement of glycine 2 with alanine in the SrcG2A
mutants is known to hamper membrane targeting [9],it is assumed that recruitment to the cell membrane
and focal adhesions would decrease. In the present

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study, we examined the effect of SrcG2A mutation on
the activity and localization of Src. To visualize theeffect of SrcG2A mutation on translocation to the cell
membrane, we constructed Venus, a modified yellow
fluorescent protein [16], Venus-tagged wild-type Src(SrcWT) and SrcG2A mutant (Fig. 1A). These constructs were co-transfected with mCherry-tagged paxillin, used as focal adhesion markers, in HeLa cells,
and observed by TIRF microscopy. TIRF microscopy
enables the visualization of fluorophores within 100–
150 nm from the coverslip surface, which includes
basal cell membrane and focal adhesions, at the sametime as excluding the majority of the cytosol region[17]. When HeLa cells were observed as co-expressing
paxillin-mCherry and Venus or SrcWT-Venus, clear
co-localization with paxillin-mCherry was not detected(Fig. 1B). By contrast, SrcG2A mutant showed localization at focal adhesions (Fig. 1B). Because localization of SrcG2A mutant at focal adhesion wasobserved in HeLa cells expressing SrcG2A-Venus without paxillin-mCherry (Fig. S1), we confirmed thatoverexpression of paxillin did not affect the localization of SrcG2A. To quantify the observations, wecropped TIRF images to 3 9 3 lm2 at the same time
as including a few focal adhesions at the cell peripheral
region. The colocalization index between Venus andmCherry images was calculated as reported previously[8,12]. SrcG2A exhibited a significantly higher colocalization index than SrcWT and Venus (Fig. 1C), indicating that SrcG2A molecules directly translocate fromthe cytosol to focal adhesions, bypassing the cell membrane. Although the recruitment pathway of Src from
cytosol to focal adhesions is not completely understood, membrane targeting is considered essential fortranslocation. This is attributed to the inactive state ofSrc molecules localized in the cytosol [5] and the regulation of Src activation at the cell membrane [18].
However, a previous study has shown that Src molecules can be detected in the membrane fraction, whenanalyzed by membrane-cytosol separation assay [10];these Src molecules were possibly obtained from focal
adhesions. Therefore, we suggest a novel pathway fordirect recruitment of Src from the cytosol to focal
adhesions.
Src kinase activity enhances the directly
recruitment of Src to focal adhesion throughinteraction with FAK
To examine whether Src kinase activity require thetranslocation of SrcG2A molecules to focal adhesion,
we made myristoylation defective and kinase deadmutant SrcG2A/K298M and myristoylation defective
and constitutive active mutant SrcG2A/Y530F. The
mutants were transfected to HeLa cells with paxillinmCherry and observed using TIRF microscopy.
SrcG2A/Y530F molecules accumulates at focal adhesion and the colocalization index of SrcG2A/Y530F
was significantly higher than that of SrcG2A (Fig. 2A,
B). On the other hand, SrcG2A/K298M uniformly distributed at the cell membrane with slight accumulation
at focal adhesion (Fig. 2A). The colocalization index
of SrcG2A/K298M was comparable to that of SrcG2A(Fig. 2B). Therefore, the results indicate that catalytic
activation of Src is essential for residing at focal adhesion, although Src kinase activity does not appear to
be required for the recruitment of unmyristoylated Src
at focal adhesion.

Fig. 1. Myristoylation-deficient mutant
SrcG2A is localized at focal adhesions. (A)Schematic presentation of Src variants
used in the present study. Venus
fluorescent protein fused to C-terminus of
Src. G2A mutation in Src highly prevents
the translocation of Src from cytosol tocell membrane. (B) HeLa cells expressing
Src variants (left) and mCherry-tagged
paxillin (center) were viewed under a TIRF
microscope. The merged images of Src
and paxillin are shown on the right. Scalebar = 5 lm. (C) Box plot of thecolocalization index of Venus-tagged Src
variants and mCherry-tagged paxillin in
HeLa cells. Statistical analyses were
performed by a t-test among Src variants(n > 50, **P < 0.01).

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Fig. 2. Src kinase activity and FAKinteraction are essential for the localizationof SrcG2A at focal adhesion. (A,C) HeLa
cells expressing Src variants (left) andmCherry-tagged paxillin (center) wereviewed under a TIRF microscope. The
merged images of Src and paxillin are
shown on the right. Scale bar = 10 lm.
(B,D) Box plot of the colocalization index
of Venus-tagged Src variants and mCherrytagged paxillin in HeLa cells. Statisticalanalyses were performed by a t-test
between SrcG2A and other Src variants in(B) and in the presence and absence of
RF-573228 in (D) (n > 50, **P < 0.01).

We next examined whether focal adhesion kinase
FAK promote the translocation of SrcG2A to focal
adhesion because integrin-mediated FAK signal
induces FAK-Src interaction and involves the subsequent signaling pathway [19]. We applied an FAK
inhibitor 10 lM RF-573228 to HeLa cells expressing
SrcG2A-Venus and paxillin-mCherry for 1 h andobserved with TIRF microscopy. Localization of
SrcG2A at focal adhesion was diminished in the presence of the inhibitor. The colocalization index of
SrcG2A in the presence of the inhibitor was significantly lower than that in the absence of the inhibitor(Fig. 2C,D). Because RF-573228 inhibits autophosphorylation at FAK Tyr397, which is the site for binding with Src SH2 domain, we suggest that therecruitment of Src to focal adhesion without membrane targeting is likely to be mediated by Src-FAK
interaction.
Function of directly recruited Src molecules in
focal adhesion protein dynamics
Because integrin-mediated Src kinase activity enhances
the activity of focal adhesion proteins and their ability
to facilitate cell migration [20,21], we performed
TIRF-FRAP experiments to monitor the exchange of
focal adhesion protein paxillin at focal adhesions.
FRAP method is a powerful tool to monitor protein
dynamics at the region of interest [22]. To avoid theeffect of the endogenous Src molecules in paxillin
dynamics, SYF cells were used in which the major Src
family kinases Src, Yes and Fyn are knocked out [23].
SYF cells coexpressing mCherry-paxillin and Venus,
SrcWT-Venus, or SrcG2A-Venus, were generated andutilized in TIRF-FRAP experiments (Fig. 3A). SrcWTand SrcG2A expressing SYF cells showed fluorescencerecovery at the focal adhesions, whereas fluorescencerecovery was greatly reduced in SYF cells not expressing Src (Fig. 3B). The results indicate that both
SrcWT and SrcG2A can promote focal adhesion
dynamics. For further analysis, the fluorescence recovery curves were fitted by:FðtÞ ¼ M½1 � exp ð�t=sÞ�ð1Þ

where F(t) represents the mean fluorescence intensity
value of mCherry-paxillin at individual focal adhesions, normalized against the mean fluorescence intensity before photobleaching. The mobile fraction (M)and time constant (s) were used as fitting parameters.
The half-recovery time T1/2 was calculated using:T1=2 ¼ lnð2Þ � sÞ

ð2Þ

Because fitting scores of Venus-expressing SYF cells
were too low as a result of small fluorescence recovery,
we ignored those fitting results. The M value ofVenus-SrcG2A-expressing SYF cells was significantly
lower than that of Venus-SrcWT-expressing SYF cells

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(Fig. 3C). By contrast, the T1/2 values were comparable between SrcWT and SrcG2A (Fig. 3D). Because
lower M values indicate stable residence of paxillin
molecules at the focal adhesions and T1/2 values represent the speed at which paxillin molecules exchange
between focal adhesions and the cytosol, these results
indicate that individual catalytic activity of SrcG2A
molecules is similar to that of SrcWT molecules. However, as a result of the elimination of membrane targeting, the number of activated SrcG2A molecules at
focal adhesions was expected to be smaller than thatof activated SrcWT molecules. In the present study,
we showed the accumulation of SrcG2A at focal adhesion and also its contribution for focal adhesion
dynamics. Additionally, we confirmed that SrcG2A
was recruited to other types of adhesion sites (i.e. focal
contact and fibrillar adhesion) and that thecontribution of SrcG2A for adhesion dynamics wascomparable among three types of adhesion site
(Fig. S2).
Model of Src translocation between cytosol, cell
membrane, and focal adhesions
A novel recruitment pathway of Src from the cytosol to
focal adhesions was shown, bypassing the cell membrane through the myristoylation of the Src N-terminal(Fig. 1). Although the total catalytic activity of SrcG2A
molecules is lower than that of SrcWT molecules [24],the catalytic activity of individual SrcWT and SrcG2A
molecules at focal adhesion was comparable (Fig. 3).
However, because the majority of Src molecules localized at cytosol are in an inactivated state, it is unknown
how directly recruited Src molecules from cytosol are

Fig. 3. Expression of SrcWT and SrcG2A facilitates the exchange of paxillin at focal adhesions. (A) Representative pseudocolor images of
TIRF-FRAP experiments in SYF cells expressing Venus-tagged Src variants and mCherry-tagged paxillin. Photobeaching of mCherry
fluorescence was carried out at time 0. Scale bar = 1 lm. (B) Mean fluorescence intensities of mCherry within individual focal adhesions
were plotted against time. Mobile fraction (C) and half-recovery time (D) were obtained using Eqns (1,2). All statistical analyses were
performed between SrcWT and SrcG2A (t-test, n > 30, **P < 0.01).

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Author contributions
HM, TY and HF designed the experiments. HM performed the experiments. HM and HF analyzed data.
HM, TMW and HF wrote the paper.

References

Fig. 4. Schematic translocation model of Src. Myristoylation of Src
N-terminal domain facilitates translocation from cytosol to cell
membrane. Translocated Src molecules are activated at the cell
membrane and move to the focal adhesion. Directly recruited Src
molecules move to the cell membrane, where they are activated,
and then return to the focal adhesions. Dotted lines indicate
hypothetical translocation of Src.

activated. Indeed, SrcG2A molecules in COS-1 cells
showed clear catalytic activation by western blotting
analysis [10]. Furthermore, Hamadi et al. [25] showedthat Src is translocated from focal adhesions to membrane ruffles. Therefore, we hypothesize that directly
recruited Src molecules at focal adhesions must translocate to the cell membrane, be catalytically activated andsubsequently return to the focal adhesions (Fig. 4).
Because the SrcG2A mutation prevents prolonged residence of Src at the cell membrane, many activatedSrcG2A molecules quickly dissociate as a result of their
low affinity with the cell membrane and are unable toreturn to the focal adhesions (Fig. 4). Src kinase activity
and binding with FAK promote the localization of Src
at focal adhesion (Fig. 4). In conclusion, Src molecules
are able to translocate directly from the cytosol to focal
adhesions without binding to the cell membrane.
Directly recruited Src molecules can enhance focal
adhesion dynamics. In addition to the Src translocation
pathway from the focal adhesions to cell membrane,
clarification of the relationship between catalytic activity and Src translocation is important with respect tounderstanding the integrin-mediated mechanotransduction mechanism in detail.

Acknowledgements
The work was supported by JSPS KAKENHI Grant
Number JP15K19128 to HM, JP24570191 to HF.

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Vines RR and Parsons JT (1994) Autophosphorylationof the focal adhesion kinase, pp125FAK, directs SH2dependent binding of pp60src. Mol Cell Biol 14, 1680–
1688.
20 Ballestrem C, Erez N, Kirchner J, Kam Z, Bershadsky
A and Geiger B (2006) Molecular mapping of tyrosinephosphorylated proteins in focal adhesions using
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21 Huttenlocher A and Horwitz AR (2011) Integrins incell migration. Cold Spring Harb Perspect Biol 3,a005074.
22 Fritzsche M and Charras G (2015) Dissecting protein
reaction dynamics in living cells by fluorescencerecovery after photobleaching. Nat Protoc 10, 660–680.
23 Klinghoffer RA, Sachsenmaier C, Cooper JA andSoriano P (1999) Src family kinases are required for
integrin but not PDGFR signal transduction. EMBO J
18, 2459–2471.
24 Li L, Hisamoto K, Kim KH, Haynes MP, Bauer PM,
Sanjay A, Collinge M, Baron R, Sessa WC and Bender
JR (2007) Variant estrogen receptor-c-Src molecularinterdependence and c-Src structural requirements forendothelial NO synthase activation. Proc Natl Acad Sci
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25 Hamadi A, Deramaudt TB, Takeda K and Ronde P(2009) Src activation and translocation from focal
adhesions to membrane ruffles contribute to formationof new adhesion sites. Cell Mol Life Sci 66, 324–338.

Supporting information
Additional Supporting Information may be foundonline in the supporting information tab for this article:
Fig. S1. TIRF images of HeLa cells expressing Venus,
SrcWT-Venus and SrcG2A-Venus.
Fig. S2. TIRF images of HeLa cells expressing Venustagged Src variants and mCherry-tagged paxillin, as
well as mean fluorescence intensities of mCherry withineach type of adhesion site plotted against time.

FEBS Letters 591 (2017) 1940–1946 ª 2017 Federation of European Biochemical Societies-550-

Vol. 14, pp. 119–125 (2017)
doi: 10.2142/biophysico.14.0_119

Biophysics and Physicobiology
https://www.jstage.jst.go.jp/browse/biophysico/Regular Article

The use of a genetically encoded molecular crowding sensor
in various biological phenomena
Hiroaki Machiyama1,2*, Takamitsu J. Morikawa1*, Kazuko Okamoto1, Tomonobu M. Watanabe1
and Hideaki Fujita1,3Quantitative Biology Center, RIKEN, Suita, Osaka 565-0874, Japan
Department of Immunology, Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402, Japan
3
Waseda Bioscience Research Institute in Singapore (WABIOS), Helios, Singapore 138667, Republic of Singapore
12Received May 30, 2017; accepted July 24, 2017

We evaluated usability of a previously developed genetically encoded molecular crowding sensor in various biological phenomena. Molecular crowding refers to intracellular regions that are occupied more by proteins andnucleotides than by water molecules and is thought tohave a strong effect on protein function. To evaluate
intracellular molecular crowding, usually the diffusion
coefficient of a probe is used because it is related tomobility of the surrounding molecular crowding agents.
Recently, genetically encoded molecular crowding sensors
based on Förster resonance energy transfer were reported.
In the present study, to evaluate the usability of a genetically encoded molecular crowding sensor, molecular
crowding was monitored during several biological events.
Changes in molecular crowding during stem cell differentiation, cell division, and focal adhesion development* These authors contributed equally to this work.
Corresponding authors: Tomonobu M. Watanabe, Quantitative Biology
Center, RIKEN, OLABB, Osaka University, 6-2-3, Furuedai, Suita,
Osaka 565-0874, Japan. e-mail: tomowatanabe@riken.jp; Hideaki
Fujita, Quantitative Biology Center, RIKEN, OLABB, Osaka University, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan. e-mail: hideaki.
fujita@riken.jp

and difference in molecular crowding in filopodia locations were examined. The results show usefulness of thegenetically encoded molecular crowding sensor for understanding the biological phenomena relating to molecular
crowding.
Key words: histone, chromatin packing, filopodia,
focal adhesion, FRET
Cells are densely packed with macromolecules such asproteins, lipids, and nucleic acids [1]. Protein concentrationin cells is estimated to be 300–400 mg/mL, however, thishigh protein concentration is difficult to achieve in in vitro
studies [2,3]. These crowded conditions influence many
important characteristics of cell physiology, such as reaction
rates, protein stability, or assembly/disassembly of supramolecular complex such as polymerization/de-polymerizationof actin filament or microtubule. For example, molecularly
crowded conditions slow down diffusion of molecules thus
decreasing the reaction rate; however, the effect of confinement increases the reaction rate [4,5]. It has been reported
that protein folding is more stable under molecularly crowded◄ Significance ►Cells are densely packed with various macro-molecules with water molecules filling the gaps, a phenomenon known as molecular crowding. There
has been an increasing interest for molecular crowding as it was shown to influence many biological reactions within cells. In this paper, molecular
crowding was monitored during several biological events using a genetically encoded molecular crowding sensor we previously developed.
Changes in molecular crowding during stem cell differentiation, cell division, and focal adhesion development and difference in molecular crowding among various filopodia locations were examined. Our results show the usefulness of the genetically encoded molecular crowding sensor forunderstanding the biological phenomena relating to molecular crowding.
©2017 THE BIOPHYSICAL SOCIETY OF JAPAN

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Vol. 14conditions than under dilute conditions [6,7]. Actin filament
polymerization is known to be enhanced under crowded
conditions because of the excluded-volume effect [8]. These
examples show the importance of molecular crowding in
biological phenomena. Furthermore, a recent study revealed
that local molecular crowding, for example, at cell adhesion
sites or on microtubules, influences behavior of the related
proteins [9,10]. Thus, it is important to know the molecular
crowding distribution within the cell to correctly interpret
biological observations.
Because molecular crowding is directly related to viscosity, molecular crowding in the cell can be briefly estimatedby the measurement of the diffusion constant of a probe[11,12]. There are other methods for estimation of molecular crowding in the cell. Silver nanoclusters synthesized inpoly(methacrylic acid) are a good candidate for a molecular
crowding sensor [13]. Digitally recorded interference microscopy with automatic phase shifting can estimate protein
concentration in a live cell [14]. Raman microscopy is also
capable of assessing protein concentration in the cell [15].
Recently, genetically encoded Förster resonance energy transfer (FRET)-based molecular crowding sensors were developed, which enable direct measurement of macromolecular
crowding in live cells [16,17]. By means of these geneticallyencoded molecular crowding sensors, a change in molecular
crowding during a cell volume change has been evaluated inthese reports, but practical applications of the molecular
crowding sensor have not been addressed.
To assess the possibility of a genetically encoded crowding sensor, here, we created various fusion proteins with ourpreviously developed genetically encoded molecular crowding sensor GimRET (glycine inserted mutant FRET) [17],and assessed the change in molecular crowding under different conditions or at different locations. Changes in molecular crowding during stem cell differentiation, in the cell
nucleus during cell division, during focal adhesion development, and at filopodia locations were evaluated in this study.
The results show the ability of GimRET to reveal new biological insights into molecular crowding.

Materials and Methods
DNA constructs
GimRET was constructed as reported previously [17]. To
generate transient expression vectors for H2B-GimRET and
vinculin-GimRET, cDNAs of histone 2B (H2B) and vinculin were amplified by PCR with sense primers specific for
the HindIII and NheI sites and reverse primers specific for
the KpnI and NheI sites. The PCR products were ligated intoa mammalian expression vector for GimRET between the
HindIII and KpnI sites or into the NheI site and then transformed into Escherichia coli DH5α cells. To generate transient expression vectors for myosin-X-GimRET, cDNAs of
GimRET were amplified by PCR using sense and reverse
primers containing the Nhe I sites. The PCR products wereligated into a mammalian expression vector for myosin-X
between the Nhe I/Nhe I sites and then transformed into
DH5α. For creation of cells stably expressing the reporters,
the GimRET fusion constructs were subcloned into the lentiviral expression vector pCDH-CMV-EF1-Puro (CD510B-1,
System Biosciences). Oct3/4 reporter was generated byreplacing RFP to mKate2 in Mouse Oct4 reporter plasmid pRedZeroTM –Oct4 Differentiation Reporter Virus(SR10045VA, System Biosciences). Lentiviral infection was
carried out according to the manufacturer’s protocol.
Cell culture
Mouse embryonic stem (mES) cells (E14Tg2a) were maintained in high-glucose Dulbecco’s modified Eagle’s medium(DMEM) containing 10% of fetal bovine serum (FBS), 1%
of a penicillin/streptomycin solution (Sigma-Aldrich), 1%
of GlutaMAX-1 (Gibco), 1% of a solution of nonessential
amino acids (Gibco), 1% of a solution of nucleosides (Millipore), 1% of a solution of sodium pyruvate (Sigma-Aldrich),
0.1% of 2-mercaptoethanol (Sigma-Aldrich), and 0.1% ofleukemia inhibitory factor (LIF) (Nacalai USA Inc.). The
mES cells were cultured in 0.1% gelatin-coated 10-cm dishes(Matsunami), and the medium was refreshed daily. To visualize the pluripotency of mES cells, Oct3/4 reporter wastransfected to mES cells, i.e., undifferentiated cells show
mKate2 fluorescence. Differentiation of mES cells was
induced by removal of LIF from the medium. HeLa cells and
COS7 cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% of FBS (Thermo Fisher Scientific) and
1% of penicillin/streptomycin (Sigma-Aldrich).
Microscopic observation
The microscopy setup consisted of an epifluorescencemicroscope (Ti-E, Nikon), an objective lens (40× CFI Plan
Apo Lambda, 0.95 NA, Nikon), a relay optics box for dualcolor imaging (GA03; G-Angstrom, Japan), and an electron
multiplier-type CCD camera (EM-CCD, iXon DV887 or
DU897; Andor Technology PLC, UK). A slit was placed at
the imaging surface of the microscope in the relay optics. A
dichroic mirror (FF458-Di02, Semrock) located just outside
the slit split the optical pathway after the imaging surface
into two pathways for cyan (CFP) and yellow (YFP1G) fluorescence. The two pathways converged on the acceptancesurface of the EM-CCD camera side by side. Band-pass filters were set for each pathway (467–499 nm for CFP and
510–560 nm for YFP). Multiphoton fluorescence recovery
after photobleaching (MP-FRAP) experiments were conducted as described elsewhere [17]. Image analyses werecarried out in the ImageJ software (NIH, USA).

Results and Discussion
Molecular crowding in pluripotent cells
We first transfected GimRET into mES cells to test
whether molecular crowding changes during cell differen--552-

Machiyama et al.: Use of molecular crowding sensor

tiation because molecular crowding in the nucleus, e.g.,chromatin packing, is thought to influence pluripotency [18].
GimRET-transfected mES cells in the presence of LIF and 3,
6, or 10 days after withdrawal of LIF were visualized using
a multiphoton microscope. Intensity ratio of the CFP channel to YFP1G channel in each pixel to visually describe
molecular crowding was calculated. Ratiometric images ofthe mES cells showed a color variation within a colony (Fig.
1A). As mES cells differentiated by withdrawal of LIF, the
CFP/YFP1G ratio gradually decreased; however, the color
variation was still observed (Fig. 1B & C). The peak value ofthe histogram of the ratio gradually decreased during the
progression of differentiation, up to 10 days after removal of
LIF, indicating less molecular crowding in the differentiatedstate (Fig. 1D). Nonetheless, we detected no differences inthe diffusion coefficients between undifferentiated mES
cells and differentiated mES cells according to MP-FRAP ofGimRET (Fig. 1E), indicating that the mobility of proteinsin the mES cells did not change with differentiation despite121

the lower molecular crowding. These results indicated thatmolecular crowding changes during cell differentiation,
which cannot be detected by means of a diffusion coefficientof a fluorescent protein: a common method for estimation ofmolecular crowding in the cell.
Imaging of cell division with GimRET-fused H2B
To demonstrate the applicability of GimRET to bioimaging, we fused GimRET to histone 2B (H2B-GimRET), which
is one of the five main histone proteins controlling chromatin structure [19]. Figure 2A shows the intensity ratio of thewhole cell, where H2B-GimRET was localized to chromatin. The change in the H2B-GimRET intensity ratio wasquite different from that of GimRET: the intensity ratio transiently increased in the late G2 phase, which is when the
nucleospindle is formed (Fig. 2B & C). To confirm that
increase in the ratio is not due to the increase in the total
fluorescence intensity, we calculated the relationship between
the ratio and the total fluorescence intensity in different cells

Figure 1 Live cell imaging of mES cells expressing GimRET. (A–C) Images of undifferentiated mES cells (A, with LIF), mES cells after 3
days of differentiation (B, 3 days after LIF removal), and mES cells after 6 days of differentiation (C, 6 days after LIF removal). mES cells weretransfected with GimRET. Top panels, transmission images; middle panels, ratiometric images; bottom panels, Oct3/4 expression reporting mKate2
fluorescence images. The CFP/YFP1G ratio was calculated by dividing the data from the CFP channel (467–499 nm) by data from the YFP1G
channel (510–560 nm) in each pixel. (D) Histogram of the intensity ratio of the GimRET during differentiation. (E) The relation between the ratio
and the diffusion coefficient obtained by MP-FRAP of mES cells at 0 (circles) and 6 days (squares) after the removal of LIF, and that of COS7 cells(triangles). N= 66, 52, and 41 cells, respectively. Error bars denote standard deviation.

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Figure 2 Live cell imaging with GimRET-fused H2B. (A) Transmission (top), total fluorescence (middle), and ratiometric images (bottom) of
HeLa cells transfected with H2B-GimRET. (B) Time-lapse transmission (upper panel) and ratiometric images (lower panel) of HeLa cells transfected with H2B-GimRET. (C) Total fluorescence intensity (black), and the intensity ratio (red) during cell division. Error bars denote standard
deviation. (D) The relation between total fluorescence intensity and the ratio 2 h before metaphase (blue), at metaphase (red), and 2 h after metaphase (green).

at different stages of cell division, which showed that theratio and the total fluorescence intensity does not correlate(Fig. 2D). Because GimRET can sense protein and DNA
crowding [17], we hypothesized that the condensation of
DNA contributed to the increase in the H2B-GimRET intensity ratio. Rapid increase in the GimRET ratio during mitosis
contradicts the finding that the unfused-GimRET ratio only
show slow change during whole cell cycle [17], and indicates that when GimRET is fused to a functional protein, it
can reveal a change in crowding in the local nanoenvironment. On the other hand, unfused GimRET can report thecytoplasmic environment where proteins can freely diffuse,
which imply that observation of molecular crowding in
fused and unfused GimRET at the same cellular location isimportant in the in depth understanding of the molecularenvironment at the site of interest.

Molecular crowding at focal adhesion
To monitor this molecular crowding, we chose vinculin asa focal adhesion marker because the residence time of vinculin molecules at focal adhesion is much longer than that ofother focal adhesion proteins [20,21]. We examined HeLa
cells transfected with GimRET-vinculin by Total Internal
Reflection Fluorescence (TIRF) microscopy and calculated
the CFP/YFP1G ratio (Fig. 3A). Focal adhesion sites in thecell periphery showed a relatively higher CFP/YFP1G ratio
than did other cell regions including focal adhesion locatedinside the cell (Fig. 3A & B), indicating that focal adhesion
proteins are densely packed within focal adhesion in the cell
periphery. The higher CFP/YFP1G ratio at peripheral focal
adhesion sites was stable for 1 h (Fig. 3C). Because focal
adhesion at the base of the cell protrusion area tends to disassemble as new focal adhesion forms at the leading edge[22], low molecular crowding within the focal adhesion
inside the cell seems to indicate the process of disassembly-554-

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123

Figure 3 (A) Transmission (left), total fluorescence (center), and ratiometric images (right) of HeLa cells transfected with GimRET-vinculin.
Scale bar, 10 μm. (B) Line profiles of total fluorescence intensity (black) and the ratio (red) along the rectangular region indicated in panel A.
(C) Total fluorescence intensity (black) and the ratio (red) within focal adhesion in the cell periphery (bold curve) and inside the cell (solid curve)
are plotted against time. The inset shows a kymograph of the ratio profile in the rectangular region indicated in panel A.

of focal adhesion. Furthermore, considering that the majority of tyrosine-phosphorylated focal adhesion proteins andcatalytically activated Src kinases are located at peripheral
focal adhesion sites [23,24] and because protein phosphorylation is key to signal transduction, the above results suggestthat high molecular crowding within focal adhesion expectedly facilitates signal transduction at the interface between
the extracellular matrix and cell.
Molecular crowding in filopodia
To demonstrate that GimRET can visualize local crowing
conditions, we fused it to myosin-X, a vertebrate-specificunconventional myosin motor involved in filopodium production [25]. Filopodium is thin projection of cytoplasm andone may assume that biological characteristic should be
similar to the cytoplasm. However, protein composition infilopodium is expected to differ from cytoplasm in cell body,where actin filament bundle and actin binding proteins areabundant [26]. Thus, it is of interest whether molecular
crowding differ in filopodium and cytoplasm in cell body.
Myosin-X widely distributed in the cytoplasm, with a proportion assembled at the tips of filopodia, and the differences

in intensity ratio between the cell body and the filopodia tips
were apparent (Fig. 4A). Interestingly, the whole filopodium
showed a low degree of crowding (Fig. 4B). Since this difference was not observed in cells expressing GimRET only,
the observation of GimRET-myosin-X indicated that thecrowding condition around myosin-X was locally different
between the cell body and the filopodia. Thus, GimRETfused proteins enable monitoring of the temporal and spatial
distribution of local molecular crowding in cells. While
molecular crowding in the cell body was uniform [17], thedifferences between the filopodia and the cell body wereobserved by fusion of GimRET to myosin-X (Fig. 4). The
combination of GimRET and GimRET-fused proteins
allowed observation of differences in global and local intracellular crowding conditions.

Conclusion
Here, we showed the suitability of the GimRET molecular
crowding sensor for elucidation of molecular crowding inlive cells. Because GimRET is a genetically encoded probe, it
can be easily attached to functional proteins, thereby enabling

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Figure 4 (A) Transmission (left), total fluorescence (middle), and ratiometric images (right) of COS7 cells transfected with myosin-X-GimRET.
(B) One-dimensional cross-section of the total intensity (black) and the intensity ratio (red) for the purple dotted line shown in A.

detection of molecular crowding near this protein. To deeply
understand a biological function, it recently requires monitoring physiological and physical conditions at the sitewhere the function occur. Since molecular crowding affects
not only the mobility of molecules but also protein stability
and kinetics, quantification of molecular crowding using
GimRET fused to subcellular localization markers can be an
in situ comprehensive parameter of biological functions.
Thus, the genetically encoded molecular crowding sensor
GimRET should advance the understanding of local nanoenvironments.

Acknowledgments
The authors thank Keiko Yoshizawa for technical assistance.

Conflict of Interests
The authors declare that there is no conflict of interests
regarding the publication of this paper.

Author Contribution
H. M. performed imaging experiment, T. J. M. made theconstructs, K. O. made cells expressing constructs, T. M. W.
performed analysis and H.F. planned the project and wrote
the manuscript.

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Role of interleukin-25 in development of spontaneous arthritis ininterleukin-1 receptor antagonist-deﬁcient mice
Yasuharu Abea,c, Aya Nambud, Sachiko Yamaguchid, Ayako Takamorid, Hajime Sutoe,⁎⁎⁎
Sachiko Hirosef, Tadashi Yokosukaa, Susumu Nakaed,g, , Katsuko Sudob,aDepartment of Immunology, Tokyo Medical University, Tokyo 160-8402, Japan
Animal Research Center, Tokyo Medical University, Tokyo 160-8402, Japanc
Department of Pharmacy, Toyohashi Medical Center, National Hospital Organization, Aichi 440-8510, JapandLaboratory of Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639,
Japane
Atopy Research Center, Juntendo University, Tokyo 113-8412, JapanfToin Human Science and Technology Center, Department of Biomedical Engineering, Toin University of Yokohama, Yokohama 225-8502, Japang
Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama 332-0012, Japanb

A B S T R A C T
Interleukin (IL)-25, which is a member of the IL-17 family of cytokines, induces production of such Th2 cytokines as IL-4, IL-5, IL-9 and/or IL-13 by various types of cells, including Th2 cells, Th9 cells and group 2 innate
lymphoid cells (ILC2). On the other hand, IL-25 can suppress Th1- and Th17-associated immune responses byenhancing Th2-type immune responses. Supporting this, IL-25 is known to suppress development of experimental autoimmune encephalitis, which is an IL-17-mediated autoimmune disease in mice. However, the role ofIL-25 in development of IL-17-mediated arthritis is not fully understood. Therefore, we investigated this using IL1 receptor antagonist-deﬁcient (IL-1Ra-/-) mice, which spontaneously develop IL-17-dependent arthritis.
However, development of spontaneous arthritis (incidence rate, disease severity, proliferation of synovial cells,
inﬁltration of PMNs, and bone erosion in joints) and diﬀerentiation of Th17 cells in draining lymph nodes in IL25-/- IL-1Ra-/- mice were similar to in control IL-25+/+ IL-1Ra-/- mice. These observations indicate that IL-25
does not exert any inhibitory and/or pathogenic eﬀect on development of IL-17-mediated spontaneous arthritis
in IL-1Ra-/- mice.

1. Introduction
Interleukin (IL)-25 is a member of the IL-17 family of cytokines andbinds to IL-17 receptor (IL-17R) A and IL-17RB [1]. IL-25 is produced
by epithelial cells, Th2 cells, macrophages and mast cells [2,3]. It induces production of such Th2 cytokines as IL-4, IL-5 and/or IL-13 byvarious types of cells—including Th2 cells, Th9 cells, CD11c+ F4/80+
macrophages, natural killer T (NKT) cells and/or group 2 innate lymphoid cells (ILC2) [4,5]—that are involved in host defense against such
pathogens as Trichuris muris and Nippostrongylus brasiliensis [6–8]. On
the other hand, inappropriate/excessive activation of IL-25 leads todevelopment of certain Th2-type allergic diseases such as asthma. Expression of IL-25 mRNA/protein is elevated in specimens from asthmatics [4]. In addition, inhalation of recombinant IL-25 by mice

resulted in development of airway inﬂammation accompanied by accumulation of eosinophils in the lungs [9]. By contrast, IL-25-deﬁcient(IL-25-/-) mice and/or mice treated with anti-IL-25–neutralizing Ab
showed attenuation of allergic airway inﬂammation induced by ovalbumin [10,11]. On the other hand, IL-25 is thought to suppress Th1and Th17-associated immune responses by enhancing Th2-type immune responses [12,13]. For example, IL-25 suppressed development ofexperimental autoimmune encephalitis [12], colitis [14–16] and type I
diabetes [17] in mice.
Mice deﬁcient in IL-1 receptor antagonist (IL-1Ra), which binds to
IL-1R but does not induce cellular signal transduction, are known tospontaneously develop certain diseases such as arthritis [18], aortitis[19] and dermatitis [20]. In particular, the spontaneous arthritis seen in
IL-1Ra-/- mice is known to be caused by excessive Th17-associated⁎Corresponding author at: Laboratory of Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1
Shirokanedai, Minato, Tokyo 108-8639, Japan.
⁎⁎
Corresponding author.
E-mail addresses: snakae@ims.u-tokyo.ac.jp (S. Nakae), ksudo@tokyo-med.ac.jp (K. Sudo).

http://dx.doi.org/10.1016/j.bbrep.2017.08.006
Received 17 January 2017; Received in revised form 5 June 2017; Accepted 15 August 2017
Available online 25 August 20172405-5808/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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immune responses [21,22]. These observations suggested that IL-25
also may inhibit development of Th17-associated autoimmune arthritis.
Therefore, in the present study we investigated the role of IL-25 indevelopment of spontaneous autoimmune arthritis in IL-1Ra-/- mice.
2. Materials and methods
2.1. Mice
IL-25-/- IL-1Ra-/- mice were generated by crossing IL-25-/- mice [23]and IL-1Ra-/- mice [24] on the BALB/cA background (more than N8).
Gender- and age-matched littermates (IL-25+/+ IL-1Ra-/- mice) wereused as controls. All mice were housed in a speciﬁc pathogen-free environment at The Institute of Medical Science, The University of Tokyo.
The animal protocol for experiments was approved by the Institutional
Review Board of The Institute (A14-11) and met the ethical and safety
guidelines of The Institute.
2.2. Scoring of severity of arthritis
For each mouse paw, the severity of arthritis was scored on a scaleof 0–3 based on the macroscopic degree of redness and swelling, as
described previously [18]. Grade 0 = normal, grade 1 = light swellingof the joint and/or redness of the footpad, grade 2 = obvious swellingof the joint, and grade 3 = severe swelling and ﬁxation of the joint. The
total severity score was calculated for the four limbs of each mouse(maximum score of 12 per mouse). The scoring was performed by aninvestigator who was blinded to the mouse genotypes.
2.3. Histology
Joints of mice were ﬁxed in 10% neutral buﬀered formalin, decalciﬁed in 10% EDTA-4Na and embedded in paraﬃn. Sections wereprepared from the paraﬃn-embedded tissues and stained with hematoxylin and eosin.
2.4. Cell culture
Popliteal lymph nodes (LNs) were collected, and LN cells weresuspended in RPMI1640 (Sigma-Aldrich) supplemented with 10% heatinactivated FBS (Invitrogen), 50 μM 2-mercaptoethanol (Invitrogen),
50 μg/ml streptomycin and 50 U/ml penicillin (Invitrogen). The suspended LN cells (2 × 105 cells/well in 0.2 ml in 96-well ﬂat-bottom
plates (IWAKI)) were cultured in the presence and absence of 0.1 μg/ml
anti-mouse CD3 mAb (145-2C11; BioLegend) and anti-mouse CD28
mAb (37.51; BioLegend) at 37 °C for 48 h in a 5% CO2 incubator. Cell
proliferative responses were determined by pulsing with 0.25 μCi/ml[3H]-labeled thymidine for 6 h. The cells in each well were then harvested on a glass ﬁlter with a Micro 96 cell harvester (Skatron), and the[3H]-thymidine radioactivity on the ﬁlter was measured with a Micro
Beta counter (Pharmacia Biotech).

Fig. 1. IL-25 is not essential for development of spontaneous arthritis in IL-1Ra-/- mice.
(A) The incidence rate and (B) the disease severity score were compared between IL-25+/+
IL-1Ra-/- mice (n = 9–15) and IL-25-/- IL-1Ra-/- mice (n = 11–16). Data show themean ± SEM.

3. Results
As reported previously [18], IL-1Ra-/- mice (IL-25+/+ IL-1Ra-/mice) spontaneously developed autoimmune-like arthritis (Fig. 1).
Spontaneous development of arthritis in IL-1Ra-/- mice was shown to be
dependent on excessive activation of T cells and production of IL-17
and TNF [22,25,26]. IL-25 suppressed Th17 cell diﬀerentiation andthereby contributed to suppression of Th17-mediated autoimmune
diseases such as experimental autoimmune encephalitis in mice
[12,13]. Those ﬁndings suggested that IL-25 might suppress IL-17mediated spontaneous arthritis in IL-1Ra-/- mice. However, the incidence rate and disease severity of spontaneous arthritis were the samein IL-25-/- IL-1Ra-/- mice and IL-25+/+ IL-1Ra-/- mice (Fig. 1). In thehistological analysis, proliferation of synovial cells, inﬁltration of PMNs
and bone erosion were also similar in both groups (Fig. 2). It was reported that LN cells from IL-1Ra-/- mice excessively produced IL-17
compared with LN cells from wild-type mice [21]. The proliferative
responses and IL-17 production, as well as IFN-γ, IL-4 and IL-10 production, by LN cells from IL-25-/- IL-1Ra-/- mice were comparable tothose by LN cells from IL-25+/+ IL-1Ra-/- mice (Fig. 3), suggesting that
IL-25 does not inﬂuence diﬀerentiation of Th17 cells or Th1 and Th2

2.5. ELISA
Forty-eight hours after LN cell cultivation, the culture supernatants
were collected, and the levels of IFN-γ, IL-4, IL-10 and IL-17A in theculture supernatants were determined with ELISA kits according to the
manufacturer's instructions (BioLegend).
2.6. Statistics
The chi-square test and two-way ANOVA were used for statisticalevaluation of the incidence and score, respectively. For other results,
the unpaired Student's t-test, two-tailed, was used. The statistical analyses were performed using Prism (GraphPad Software, Inc.).
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Fig. 2. IL-25-deﬁciency did not aﬀect local inﬂammation in IL-1Ra-/- mice. Histology of the ankle
joints of IL-25+/+ IL-1Ra-/- mice and IL-25-/- IL1Ra-/- mice. Representative results for the mice in
Fig. 1 are shown. Scale bar = 100 µm.

Fig. 3. IL-25-deﬁciency did not aﬀect diﬀerentiation of helper Tcell subsets in draining LNs. Popliteal LN cells were cultured in
the presence and absence of anti-CD3 and anti-CD28 mAbs for
48 h. (A) Proliferative responses and (B) cytokine production of
LN cells from IL-25+/+ IL-1Ra-/- mice (n = 5) and IL-25-/- IL1Ra-/- mice (n = 5). Data show the mean ± SEM. Representative
results of three independent experiments.

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[15] S.S. McHenga, D. Wang, C. Li, F. Shan, C. Lu, Inhibitory eﬀect of recombinant IL-25
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665–669.
[21] S. Nakae, S. Saijo, R. Horai, K. Sudo, S. Mori, Y. Iwakura, IL-17 production from
activated T cells is required for the spontaneous development of destructive arthritis in mice deﬁcient in IL-1 receptor antagonist, Proc. Natl. Acad. Sci. USA 100(2003) 5986–5990.
[22] A. Akitsu, H. Ishigame, S. Kakuta, S.H. Chung, S. Ikeda, K. Shimizu, S. Kubo, Y. Liu,
M. Umemura, G. Matsuzaki, Y. Yoshikai, S. Saijo, Y. Iwakura, IL-1 receptor antagonist-deﬁcient mice develop autoimmune arthritis due to intrinsic activation of
IL-17-producing CCR2(+)Vgamma6(+)gammadelta T cells, Nat. Commun. 6(2015) 7464.
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K. Okumura, H. Saito, S. Nakae, Development of IL-17-mediated delayed-type hypersensitivity is not aﬀected by down-regulation of IL-25 expression, Allergol. Int.
59 (2010) 399–408.
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187 (1998) 1463–1475.
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Biol. 81 (2007) 1258–1268.
[26] R. Horai, A. Nakajima, K. Habiro, M. Kotani, S. Nakae, T. Matsuki, A. Nambu,
S. Saijo, H. Kotaki, K. Sudo, A. Okahara, H. Tanioka, T. Ikuse, N. Ishii,
P.L. Schwartzberg, R. Abe, Y. Iwakura, TNF-alpha is crucial for the development of
autoimmune arthritis in IL-1 receptor antagonist-deﬁcient mice, J. Clin. Investig.
114 (2004) 1603–1611.
[27] D. Liu, T. Cao, N. Wang, C. Liu, N. Ma, R. Tu, X. Min, IL-25 attenuates rheumatoid
arthritis through suppression of Th17 immune responses in an IL-13-dependent
manner, Sci. Rep. 6 (2016) 36002.

cells in this model. Taken together, our ﬁndings suggest that IL-25 exerts no inhibitory and/or pathogenic eﬀects on development of IL-17mediated spontaneous arthritis in IL-1Ra-/- mice.
4. Discussion
Recently, IL-25 levels were shown to be elevated in specimens frompatients with rheumatoid arthritis [27], suggesting that IL-25 somehow
contributes to development of rheumatoid arthritis. Since IL-25 caninhibit IL-17-producing Th17 cell diﬀerentiation that is dependent on
IL-13, thereby contributing to suppression of IL-17-mediated autoimmune diseases such as experimental autoimmune encephalitis inmice [13], it was thought that IL-25 might also suppress IL-17-mediatedarthritis. Indeed, it was recently reported that administration of recombinant IL-25 resulted in amelioration of development of collageninduced arthritis, which is one murine model of IL-17-mediated arthritis, by inhibiting Th17 cell diﬀerentiation in an IL-13–dependent
manner [27]. On the other hand, we demonstrated that IL-25-deﬁciency
did not exert any eﬀect on the incidence rate, disease severity or Th17
cell diﬀerentiation in another IL-17-mediated spontaneous arthritis
model in IL-1Ra-/- mice. In the setting, IL-25 mRNA could not be detected in the animals’ inﬂamed joints by quantitative PCR (data notshown). Therefore, in contrast to collagen-induced arthritis and experimental autoimmune encephalitis, our results suggest that IL-25
does not play any inhibitory role in development of IL-17-mediatedspontaneous arthritis in IL-1Ra-/- mice.
5. Conclusion
IL-25 does not play any inhibitory and/or pathogenic role in development of IL-17-mediated spontaneous arthritis in IL-1Ra-/- mice.
Acknowledgments
We thank Ayano Ishii for her skilled technical assistance and Dr.
Yoichiro Iwakura (Tokyo University of Biomedial Sciences, Chiba,
Japan) for providing IL-1Ra-/- mice. We are grateful to Lawrence W.
Stiver (Tokyo, Japan) for his critical reading of the manuscript. This
work was supported by PRESTO, JST (S.N.).
Appendix A. Transparency document
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.bbrep.2017.08.006.
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Immunol. Rev. 226 (2008) 172–190.
[4] J.M. Reynolds, P. Angkasekwinai, C. Dong, IL-17 family member cytokines: regulation and function in innate immunity, Cytokine Growth Factor Rev. 21 (2010)
413–423.
[5] C.S. Klose, D. Artis, Innate lymphoid cells as regulators of immunity, inﬂammationand tissue homeostasis, Nat. Immunol. 17 (2016) 765–774.
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D.J. Cua, M. Goldschmidt, C.A. Hunter, R.A. Kastelein, D. Artis, Interleukin 25
regulates type 2 cytokine-dependent immunity and limits chronic inﬂammation in
the gastrointestinal tract, J. Exp. Med. 203 (2006) 843–849.
[7] P.G. Fallon, S.J. Ballantyne, N.E. Mangan, J.L. Barlow, A. Dasvarma, D.R. Hewett,
A. McIlgorm, H.E. Jolin, A.N. McKenzie, Identiﬁcation of an interleukin (IL)-25-

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ReportBlockage of Core Fucosylation Reduces Cell-Surface
Expression of PD-1 and Promotes Anti-tumor
Immune Responses of T Cells
Graphical Abstract

Authors
Masahiro Okada, Shunsuke Chikuma,
Taisuke Kondo, ..., Tadashi Yokosuka,
Miyako Nakano, Akihiko Yoshimura

Correspondence
okada@z5.keio.jp (M.O.),
yoshimura@a6.keio.jp (A.Y.)

In Brief
Using a genome-wide loss-of-function
screening method, Okada et al. identify
genes involved in the core fucosylation
pathway as positive regulators of cellsurface PD-1 expression.

HighlightsdCore fucosylation pathway genes are involved in cell-surface
PD-1 expressiondTwo major sites of core fucosylated N-glycans for PD-1
expression are determinedd

Blocking core fucosylation enhances T cell activation under
antigen presentationdAnti-tumor immune responses are strengthened by inhibiting
fucosylation

Okada et al., 2017, Cell Reports 20, 1017–1028
August 1, 2017 ª 2017 The Authors.
http://dx.doi.org/10.1016/j.celrep.2017.07.027-562-

Cell Reports

ReportBlockage of Core Fucosylation Reduces
Cell-Surface Expression of PD-1 and Promotes
Anti-tumor Immune Responses of T Cells
Masahiro Okada,1,4,5,* Shunsuke Chikuma,1 Taisuke Kondo,1 Sana Hibino,1 Hiroaki Machiyama,2 Tadashi Yokosuka,2Miyako Nakano,3 and Akihiko Yoshimura1,*
1Department of Microbiology and Immunology, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan
2Department of Immunology, Tokyo Medical University, Shinjuku-ku, Tokyo 160-8402, Japan
3Graduate School of Advanced Sciences of Matter, Hiroshima University, Higashi-hiroshima, Hiroshima 739-8530, Japan
4Present address: Laboratory for Immunotherapy, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan
5Lead Contact*Correspondence: okada@z5.keio.jp (M.O.), yoshimura@a6.keio.jp (A.Y.)
http://dx.doi.org/10.1016/j.celrep.2017.07.027

SUMMARY

Programmed cell death 1 (PD-1) is highly expressedon exhausted T cells and inhibits T cell activation.
Antibodies that block the interaction between PD-1
and its ligand prevent this inhibitory signal andreverse T cell dysfunction, providing beneficial antitumor responses in a substantial number of patients.
Mechanisms for the induction and maintenance ofhigh PD-1 expression on exhausted T cells havenot been fully understood. Utilizing a genome-wideloss-of-function screening method based on the
CRISPR-Cas9 system, we identified genes involvedin the core fucosylation pathway as positive regulators of cell-surface PD-1 expression. Inhibition of
Fut8, a core fucosyltransferase, by genetic ablationor pharmacologic inhibition reduced cell-surface
expression of PD-1 and enhanced T cell activation,
leading to more efficient tumor eradication. Taken
together, our findings suggest that blocking corefucosylation of PD-1 can be a promising strategy
for improving anti-tumor immune responses.
INTRODUCTION
Understanding the molecular mechanisms by which the immune
system recognizes and attacks targets is of particular interest,
especially after the discovery that immune checkpoint blockadetargeting programmed cell death 1 (PD-1), cytotoxic T cell antigen-4 (CTLA-4), or both provides therapeutic benefits for tumor
patients (Brahmer et al., 2012; Hamid et al., 2013; Hodi et al.,2010; Sharma and Allison, 2015; Snyder et al., 2014; Topalian
et al., 2012; Wolchok et al., 2013). PD-1 and CTLA-4, expressedon activated lymphocytes, are major receptors of T cell inhibition
that avoid excessive immune responses and autoimmunity(Bour-Jordan et al., 2011; Chikuma, 2016). Upon ligation byphysiological ligands, these receptors transmit inhibitory signals
for T cell activation, resulting in T cell dysfunction (termed ‘‘T cell

exhaustion’’) in chronic immune responses against persistentinfection or cancer (Ahmadzadeh et al., 2009). Systemic administration of antibodies that block ligand engagement reduces theinhibitory signals, reactivates T cells, and, in some cases, reverses the immune-deficient state in cancer patients. This
concept has been approved for application in the treatment of
human tumors and has been shown to provide promising antitumor responses in a substantial number of patients (Chen andMellman, 2013; Pauken and Wherry, 2015; Sharma and Allison,2015).
T cell exhaustion is associated with high expression of PD-1
and other inhibitory receptors, including T cell immunoglobulin
and mucin domain-3 (Tim-3), lymphocyte activation gene 3(Lag-3), and T cell immunoreceptor with immunoglobulin and
ITIM domains (TIGIT) (Anderson et al., 2016). In spite of extensive
efforts to understand the mechanisms of T cell exhaustion(Crawford et al., 2014; Hoffmann et al., 2016; Im et al., 2016;
Pauken et al., 2016; Schietinger et al., 2016; Sen et al., 2016;
Singer et al., 2016), the basic mechanisms that lead to highexpression of these inhibitory receptors on chronically activatedT cells remain to be elucidated.
PD-1 expression is primarily induced by T cell receptor (TCR)induced activation (Agata et al., 1996). Nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) initiates TCR-induced
transcription of PD-1 by binding to its promoter (Austin et al.,2014; Martinez et al., 2015; Oestreich et al., 2008). Interferon-a(IFN-a), a type-I interferon, causes durable PD-1 expression by
activating interferon-stimulated responsive element (ISRE) at adifferent site within the PD-1 promoter (Terawaki et al., 2011).
After the initial activation, B-lymphocyte-induced maturation
protein 1 (Blimp-1) and T-bet are recruited to the Pdcd-1 promoter and suppress transcription (Kao et al., 2011; Lu et al.,2014). Inhibition of glycogen synthase kinase 3 (GSK-3) downregulates PD-1 expression through T-bet induction, resulting inenhanced cytotoxic T lymphocyte (CTL) activity (Taylor et al.,2016). Drugs inhibiting PD-1 expression are expected to reduce
negative signals on tumor-reactive T cells and enhance antiimmune responses in clinical settings. To date, the publishedstudies focus mostly on PD-1 regulation at the transcriptional
level, and there are few studies focusing on post-translational

Cell Reports 20, 1017–1028, August 1, 2017 ª 2017 The Authors. 1017
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Figure 1. Overview of Screening for Genes
that Positively Regulate PD-1 Expression(A) Schematic representation of the loss-of-function programmed cell death 1 (PD-1) expression
screen using the mouse genome-wide knockout
library. 68-41 T cell lines stably expressing Cas9P2A-Puro (68-41-Cas9) were transduced with alentiviral gRNA library (87,898 gRNAs/19,150
genes). PD-1low fractions were sorted andexpanded four times. The bulk gRNA sequences
extracted from the genome DNA of the PD-1low
cells were read by high-throughput sequencer.
(B) Representative flow cytometry analysis
showing gating strategy of first sort and purification of post-fourth sort.
(C) Representative sequence analysis of the number of read counts and gRNA sequence rank score.
(D) Average of PD-1 mean fluorescence intensity(MFI) in each gRNA-positive cell. 68-41-Cas9 was
individually transduced with gRNA against
candidate gene (nos. 1–3). gRNA-targeted genes
were listed in order of false discovery rate (FDR)
compared with cells transduced with control
gRNA (Luciferase and EGFP). The dashed red line
indicates the border of FDR < 0.15. The genes
involved in the core fucosylation pathway areemphasized in red. PD-1 and control gRNA areemphasized in orange and blue, respectively.
Data are shown as mean ± SD of gRNA nos. 1–3.
(E) Pathway of core fucosylation and GDP-L-fucose
biosynthesis. Gmds, Tsta3, Slc35c1, and Fut8
encode GMDS, FX, FUCT1, and FUT8, respectively.

modifications of PD-1 (i.e., glycosylation), which may regulate its
expression and function.
The CRISPR-Cas9 system induces a guide RNA (gRNA)
sequence-targeted double-strand DNA break (DSB) (Conget al., 2013). The DSB is then mainly repaired by non-homologous end joining, which causes stochastic gene disruption byframeshift or stop codon insertion. Thus, the CRISPR-Cas9 system yields complete knockout cells with high efficiency, and it
has recently been accepted as a useful tool for genome editing.
Designing pooled gRNA libraries that cover the entire proteincoding regions have been applied for comprehensive genomewide loss-of-function screening. Previous approaches haveidentified genes involved in biological events, such as cell
death, cancer drug resistance, growth, or protein expression(Ma et al., 2015; Parnas et al., 2015; Shalem et al., 2014; Tzelepis et al., 2016;
Wang et al., 2014).
We employed CRISPR-based screening to identify the genes involved in PD-1
expression. By targeting murine T cellline, we found a set of genes involved indirect core fucosylation on N-linked oligosaccharides that regulates cell-surface
expression of PD-1. Genetic and pharmacologic inhibition of core fucosylation validated the findings in primary mouse
T cells, as well as in cell line in vitro.
Furthermore, T cells treated with a cellular fucosylation inhibitor
possessed stronger anti-tumor reaction in vivo. We propose thatcore fucosylation is a post-translational modification required forPD-1 expression and that this pathway can become an attractive
target for enhancing anti-tumor immunity in future clinical settings.
RESULTSCRISPR-Cas9-Based Genome-wide Knockout
Screening for PD-1 Expression
Screening strategy and vectors are summarized in Figures 1Aand S1. We chose a 68-41 murine T cell line (Kubo et al.,1994), which we found to show high basal expression of PD-1
and efficient delivery of CRISPR components using viral vectors.

1018 Cell Reports 20, 1017–1028, August 1, 2017-564-

First, we transduced the 68-41 cells with a Streptococcus pyogenes (Sp) Cas9-P2A-Puro vector (Figure S1A) and establisheda stable cell line (named 68-41-Cas9) by puromycin selection.
Next, we transduced the 68-41-Cas9 cells with a gRNA library
containing 87,898 gRNA sequences that target 19,150 mousegenes (Figure S1A; Koike-Yusa et al., 2014). After 7–10 days,
the gRNA-transduced (blue fluorescence protein [+]) PD-1low
fractions were sorted and cultured for expansion (Figure 1B).
The PD-1low cells were enriched by repeating the sorting andexpansion processes for another 3 times, and genomic DNA
was extracted after the fourth sorting. The bulk gRNA sequencesfrom the genome DNA were amplified by PCR and then subjected to deep sequencing (Figure 1A). We repeated the sameenrichment procedures for separate 4 times, and deep
sequence analysis was independently performed twice (forpooled PCR amplicons from the first and second or third andfourth enrichment procedure). From the analysis of the retrieved
gRNA sequences, gRNAs targeting the PD-1 gene itself werereproducibly identified, indicating that the screening was successful (Figure 1C, red arrowheads).
To narrow down the candidate genes, we ranked them using
criteria as follows (summarized in Figure S1B). Among the genes
within the top 1,500 read, genes containing more than one gRNA
sequence were prioritized. We also enriched the genes expressed on T cells using BioGPS (http://biogps.org). In thisway, we ended up with 97 candidate genes. We validated eachgene using individual knockout experiments, with vectors targeting luciferase, GFP, and PD-1 as controls (Figure S1A). Three
independent gRNAs for each gene were newly designed (300constructs in total) and individually introduced into 68-41-Cas9
cells. Ten days after transduction, the degree of PD-1 expression
in gRNA-positive cells (using red fluorescent protein DsRed as areporter; Okada et al., 2017) was examined and ranked by false
discovery rate (FDR), compared with negative control gRNAs(Figure 1D). The genes ranked top 42 for which the FDR was<0.15 included genes (Paxip1, Atxn7l3, Usp7, Fut8, Ankmy2,
and Lrig1; Table S1) that are thought to be involved in many biological functions, such as chromatin modification. Among thesegenes, we were particularly interested in Fut8, Slc35c1 (encoding
FUCT1 protein), Gmds, and Tsta3 (encoding FX protein), whichwere ranked 5th, 16th, 22nd, and 42nd, respectively (Figure 1D).
The genes encode four critical proteins in the core fucosylation
pathway and GDP-L-fucose biosynthesis (Figure 1E; Kanda
et al., 2007; Noda et al., 2003). In addition, although the FDR
was higher than 0.15, primary candidate genes also contained
SPPL3, which is recently identified as a key regulator for glycosylation and degrades the number of glycosyltransferase (Vosset al., 2014), which implied the participation of global glycosylation for PD-1 expression. Because PD-1 is a type I transmembrane protein that is highly glycosylated, we focused on thegenes of the core-fucosylation pathway that might play a significant role in PD-1 expression and function.
Validation of Screening: The Genes that Regulate theCore Fucosylation Pathway Are Required for Core
Fucosylation and Expression of PD-1
We wished to confirm that the targeting experiment resulted in
genuine gene knockout of four genes (Tsta3, Fut8, Gmds, andSlc35c1). A report suggested that lectin from Lens culinaris(Lens culinaris agglutinin [LCA]), which recognizes the a-1,6-fucosylated trimannosyl-core structure of N-linked oligosaccharides, can be used to detect cells with defective corefucosylation (Yamane-Ohnuki et al., 2004). We utilized this reagent to confirm functional knockout of core fucosylation. LCA
staining of the DsRed(+) cells infected with gRNAs against thefour genes resulted in significant appearance of lectin-negative(LCA()) cells, suggesting that core fucosylation was defectivein these cells (Figures 2A and S2A). It should be noted that the
population numbers of LCA() cells differ among different
gRNAs designed to the same target gene, indicating the geneknockout efficiency depends on the individual gRNA sequence(Sander and Joung, 2014). Importantly, we found that PD-1
expression of the LCA() cells was significantly lower compared
to LCA(+) cells or the cells transduced with control vector(LCA(+); Figures 2A and S2A, left). We tested three different
monoclonal antibody (mAb) clones for PD-1 detection and got
the same results, suggesting the reduction of PD-1 was notdue to direct mutation of the epitope of the particular mAb (Figure S2A, right). Binding of PD-L1 immunoglobulin (Ig) (stained by
PD-L1 Ig, followed by anti-Ig Fc secondary antibody) was also
decreased in LCA() cells (Figures 2B and S2B). PD-1 mRNA
expression was comparable between sorted LCA(+) (control
gRNAs) and LCA() (gRNAs against cell core fucosylation), indicating that attenuation of PD-1 expression was not due to transcriptional inactivation (Figure S2C).
We then examined whether PD-1 is directly core fucosylated,because previous reports showed that core fucosylation isrequired for proper protein expression and/or ligand–receptor
interaction (Osumi et al., 2009; Wang et al., 2006). To identify
the detailed glycan structure on each glycan-binding aminoacid residue, we analyzed the digested peptides of purified murine PD-1 extracellular domain expressed in human cells by
liquid chromatography coupled to tandem mass spectrometry(LC-MS and MS/MS). Although glycopeptides containing N58
could not be analyzed deeply (Figure S3A), glycopeptides containing N49, N74, and N116 were clarified as carrying corefucose (Figures 3A, S3B, and S3C). Glycopeptides containing
N58 were also detected after other protease digestion (datanot shown). Taken together, all four N-glycosylation sites werehighly core fucosylated. We also sought to determine the
N-linked oligosaccharide-modified amino acid residues, which
control PD-1 expression or function. We engineered a murine
PD-1 protein fused with GFP at its C terminus (mPD-1-GFP)
and its point mutants lacking putative N-linked oligosaccharide-modified amino acid residues (N to Q) and introduced
them into 68-41 cells, and S60W and T76M were also investigated as human SNP homologs (Figure 3B). The immunoprecipitated wild-type (WT) PD-1-GFP showed bands with higher
molecular weight on immunoblot analysis than predicted by its
primary structure (Figure 3C, middle and lower blots). Theband shift was canceled by PNGase F treatment, confirming
the presence of N-linked glycosylation (Figure S3D). The higher
band was further detected by Aspergillus oryzae L-fucose-specific lectin (AOL), which was shown to have strong affinity tocore fucose (Figure 3C, upper blot; Matsumura et al., 2007).
Mutations in N49Q and N74Q decreased core fucosylation

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Figure 2. Validation of the Selected Genes Involved in the Core Fucosylation Process(A and B) Left: representative flow cytometry analysis of 68-41-Cas9 cells individually transduced with gRNA targeting the Luciferase or Fut8 genes showed
expression of core fucose (Lens culinaris agglutinin [LCA]) binding and PD-1 (A) or PD-L1 Ig binding (B). Right: PD-1 (A) or PD-L1 Ig (B) MFI in cells gated on LCA(+)
or LCA() were plotted. Note that Tsta3-targeted gRNA no. 3 yielded few LCA() cells, which could not be analyzed. Data are pooled from 2 independent
experiments and shown as mean ± SD.

(Figure 3C, left blots), and the double mutation diminished itcompletely, whereas glycosylation was detected to some extent
in these mutations (Figure 3C, right blots). Complete loss of corefucosylation of PD-1-GFP was observed by Fut8 deletion, supporting the notion that core fucosylation is truly affected by genetic loss of Fut8 (Figure S3E). Moreover, the cell-surface
expression of PD-1-GFP in 68-41 cells is decreased in 68-41
cells knocked out for endogenous PD-1 and Fut8 genes, andthis Fut8 knockout effect was cancelled by the N49Q/N74Q double mutation, but not by single mutations. N49Q/N74Q double
mutant or S60W mutant PD-1 showed dramatic decrease of
the cell surface expression, probably due to misfolding in endoplasmic reticulum (ER) (Figure 3D). In this setting, S60W showedfurther decrease upon induction of Fut8 deficiency, whereas the
N49Q/N74Q double mutant did not show any further decrease,suggesting core fucosylation of N49 and N74 together isinvolved for core fucosylation-dependent expression of PD-1
(Figures 3D and S3F). It is previously shown that the ligation by
PD-L1 induces participation of PD-1 into the TCR microcluster,formed at the interface of T cell and antigen-presenting cells,
which is important for T cell inhibition (Yokosuka et al., 2012).
We found that the formation of PD-1 microcluster was severely

impaired in N74Q mutant, indicating that N74 is the critical residue for ligand interaction (Figure S3G). Collectively, these results
suggest that PD-1 is highly glycosylated, and most of the N-glycans are core fucosylated. At least N49 and N74 glycosylation(speculating core fucosylation) are necessary for appropriate
cell-surface expression and functional localization of PD-1.
Genetic Deprivation or Pharmacologic Inhibition of Fut8
Attenuates PD-1 Expression and Strengthens T Cell
Activation
Genetic screening using a T cell line suggested post-translational modifications (core fucosylation) of PD-1 and its augmentative effect of cell-surface expression. We aimed to confirm thisfinding in primary mouse T cells. As shown in Figure S4A, overexpression of Fut8 in a T helper cell 1 (Th1)-skewed CD4 T cell
culture promoted PD-1 expression, but its loss-of-function
mutant (R365A; Ihara et al., 2007) did not, suggesting the
involvement of this enzyme in the pathway regulating PD-1
expression.
SpCas9 is a large protein (4.2 kb), which hampers efficient delivery into mouse primary T cells together with reporter genes.
Staphylococcus aureus (Sa) Cas9, a shorter genome editing

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Figure 3. N-Linked Glycosylation Analysis Determines Core Fucosylation Sites of PD-1 and Its Biological Function(A) Average MS spectrum of desialo-glycopeptides containing N74-glycans. Base peak chromatogram (BPC) used for obtaining the average MS spectrum wasshown in Figure S3A. MS/MS spectrum of monofucosylated glycans was shown in Figure S3B to show presence of core fucose. Average MS spectrum of
glycopeptides containing N49 glycans and N116 glycans and summary of relative glycan structures were shown in Figure S3C.
(B) Schematic diagram of PD-1-GFP and construction of its glycosylation site mutants. EC, TM, and IC refer to the extracellular, transmembrane, and intracellulardomain, respectively.
(C) Lectin and western blot analysis of immunoprecipitated PD-1-GFP and its glycosylation site mutants. Densitometry analysis of Aspergillus oryzae L-fucosespecific lectin (AOL)/GFP is plotted. Data are shown as mean ± SD; n = 3.
(D) Expression of rescued PD-1 wild-type (WT) or its glycosylation mutants in PD-1 knockout or PD-1 and Fut8 double-knockout 68-41-Cas9 cells were analyzed
by flow cytometry. Relative PD-1 MFI was plotted. NS means not statistically significant. Data are pooled from 3 independent experiments and shown asmean ± SD.

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tool (3.3 kb), has been suggested for basic research and therapeutic uses due to easy vector packaging (Ran et al., 2015).
We recombined SaCas9 with a P2A-Venus sequence into
pMXs vectors and constructed a retroviral vector for mouse primary T cells, allowing simultaneous expression of SaCas9,
Venus, and gRNA in the same cell (Figure S4B). We transferredsorted SaCas9-Venus(+)-activated CD4 T cells into rag2(/)mice and analyzed PD-1 expression. PD-1 mean fluorescenceintensity (MFI) was decreased in the LCA() fraction of cells after
transduction with gRNA against Fut8, especially in colonic lamina propria lymphocytes (LPLs), which are frequently activatedby antigen presentation of food antigen and intestinal bacteria(Figure S4C). Taken together, these data suggest that expression levels of Fut8 correlate with expression levels of PD-1.
Next, we investigated whether Fut8 knockout causes PD-1
downregulation and enhances antigen-specific T cell responses.
Activated OT-II T cells were transduced with SaCas9 and transferred into congenic mice. After active immunization with theovalbumin (OVA), we analyzed the transferred cells (Figure S4D).
Compared with LCA(+) cells or the cells transduced with control
vector, Fut8 knockout LCA() cells exhibited lower PD-1 expression (Figures 4A and S4E), higher interleukin-2 (IL-2) production(Figures 4B and S4F), and exhibited a more activated/memory
phenotype (CD44highCD62Llow; Figures 4C and S4G), similarly
to the phenotype of PD-1-deficient T cells (Charlton et al.,2013, 2015; Chikuma et al., 2009; Patsoukis et al., 2015). Therefore, inhibiting cell core fucosylation caused PD-1 deprivation
and augmented T cell activation.
2-fluoro-L-fucose (2F-Fuc) functions as a metabolic fucosylation inhibitor, which is taken up by cells and converted to GDP2F-Fuc through endogenous salvage pathways. 2F-Fuc inhibits
the natural GDP-Fuc production, resulting in a reduction of fucosylated structures (Rillahan et al., 2012). Th1 cells activated byanti-CD3/28 antibodies in the presence of 2F-Fuc showed
reduced LCA binding and PD-1 expression, but not reduced
other activation or inhibitory molecules (Figures 4D and 4E).
This was mainly due to reduction of cell-surface PD-1 expression, not at intracellular levels (Figure 4F). Moreover, after blocking transport from the ER to the Golgi apparatus using
brefeldin A, 2F-Fuc-treated cells showed a remarkable decayof cell-surface PD-1 expression (Figure 4G). Collectively, thesedata suggested that treatment with 2F-Fuc specifically prevents
stable cell-surface expression of PD-1. PD-L1 Ig binding was
also decreased with 2F-Fuc treatment (Figure 4H). WhenT cells were activated using allogeneic dendritic cells (DCs),
which express PD-L1, 2F-Fuc treatment enhanced T cell proliferation (as measured by carboxyfluorescein succinimidyl ester[CFSE] dilution) and cytokine production (as measured by intracellular IL-2 and interferon gamma [IFN-g]), as well as blocking
anti-PD-1 antibody treatment. This enhancement was partiallyreversed by co-treatment (Figure 4I), indicating that pharmacologic inhibition of core fucosylation enhances T cell activation
at least partially due to the prevention of PD-1-mediated inhibitory signals.
Application of Blocking Core Fucosylation Augments
Anti-tumor Immune Responses
T cell dysfunction in tumor microenvironments dampens efficienttumor attack, leading to immune evasion. Given that core fucosylation of PD-1 regulates its expression, we investigatedwhether blocking this modification impacts anti-tumor immunity.
To address this, we applied a well-established syngeneic B16
melanoma transplantation model. Tumor-infiltrating lymphocytes (TILs) showed the hallmark of dysfunction, the expressionof inhibitory receptors (PD-1 and Tim3; Figure 5A, left). The levelsof expression positively correlated with LCA staining, indicatingthat T cell exhaustion proceeded in concert with core fucosylation modifications (Figure 5A, right), and expression of the genes
involved in GDP-L-fucose biosynthesis were upregulated under
dysfunction process (Figure S5A). This correlation between T cell
exhaustion and increased core fucosylation levels was also
observed in 3LL transplantation model, another type of tumor(Figure S5B). CD8 T cells are the cytotoxic T lymphocytes(CTLs) that directly attack tumors. We confirmed that 2F-Fuc
also reduced PD-1 expression on activated CD8 T cells (Figure S5C). 2F-Fuc has been reported to inhibit tumor growth
directly (Okeley et al., 2013). To investigate whether 2F-Fuc
has additional effects on the immune function of cells such as
CTLs, we activated OT-I T cells in the presence of 2F-Fuc andtransferred them into mice bearing B16-OVA tumors (Figure 5B).
The OT-I T cells treated with 2F-Fuc did not show drasticchanges in T cell priming (as measured by cytokine and granzyme B expression) before the transfer (Figure S5D); however,
we observed that they inhibited the growth of B16-OVA to agreater extent than did the control OT-I T cells treated withvehicle alone (Figure 5C). We also observed that this treatment
partially prolonged survival in vivo (Figure S5E), although 2FFuc was administered only to in vitro cultures. Additionally, we
found that 2F-Fuc-treated OT-I T cells showed better expansion
within tumors (measured by greater numbers of TILs) as well as
in tumor-draining lymph nodes (TDLN) and stronger granzyme B
expression and inflammatory cytokine production (tumor necrosis factor [TNF]-a and IFN-g) than vehicle-treated cells, but not in
non-TDLNs (Figures 5D and S5F). Finally, we investigated the

Figure 4. Blocking Core Fucosylation Downregulates PD-1 Expression and Enhances T Cell Activation(A–C) MFI of PD-1 (A) or percentages of interferon-gamma (IFN-g)(+), interleukin-2 (IL-2)(+) (B), and memory fractions (C) were plotted. Data are representative of
2 independent experiments and shown as mean ± SD.
(D and E) Flow cytometry analysis of T helper cell 1 (Th1)-skewed CD4 cells treated with DMSO or 100 mM 2F-Fuc shows LCA and PD-1 (D) or indicates cellsurface markers (E). The numbers on the indicated panel show MFI.
(F) Flow cytometry analysis as in (E) shows surface and intracellular expression of PD-1. The numbers on the indicated panel show MFI.
(G) MFI of PD-1 and its relative percentage of initial MFI after brefeldin A chase experiments in Th1-skewed CD4 cells treated with DMSO or 100 mM 2F-Fuc. Data
are pooled from 5 independent experiments and shown as mean ± SD.
(H) MFI of PD-L1 Ig was plotted. Data are pooled from 5 independent experiments and shown as mean ± SD.
(I) Flow cytometry analysis of carboxyfluorescein diacetate succinimidyl diester (CFSE)-labeled CD4 T cells activated by allogeneic dendritic cells (DCs) underTh1-skewing condition shows CFSE dilution and IL-2 and IFN-g production.

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Figure 5. Blocking Core Fucosylation Strengthens Anti-tumor Immunity(A) Flow cytometry analysis of tumor-infiltrating lymphocytes (TILs) in B16 melanoma model 2 weeks after transplantation shows LCA binding gated on indicatedexhaustion markers. LCA MFI was plotted. Data are shown as mean ± SD; n = 6.
(B) Experimental scheme of B16 melanoma mice model. Eleven days after tumor inoculation, activated CD8 OT-I cells treated with DMSO or 100 mM 2F-Fuc
were intravenously transferred into B16-ovalbumin (OVA)-transplanted mice, and the mice were immunized with OVA emulsified with incomplete Freud’s
adjuvant (IFA/OVA).
(C) Tumor volume was measured every 2 days after transfer of the OT-I T cells. Data are pooled from 4 independent experiments and shown as mean ± SEM.
(D) Transferred OT-I T cells in TIL and lymph nodes were analyzed 6 days after transfer. Upper: flow cytometry analysis shows granzyme B, tumor necrosis
factor-a (TNF-a), and IFN-g. Lower: total cell numbers or percentages of indicated cell fractions were plotted. Data are pooled from 2 independent experiments
and shown as mean ± SEM; n = 9.

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anti-tumor combination effect of 2F-Fuc-treated OT-I T cells with
anti-PD-1 immunotherapy. In early phases within 8 days, additive effect was not obvious, suggesting that anti-tumor effects
of 2F-Fuc-treated OT-I T cells were partly dependent on PD-1
signaling (Figure S5G, left). However, durable therapeutic
response was observed by combination therapy during latephases (Figure S5G, right). This is probably because of the transient effect of 2F-Fuc due to in vitro treatment. From these data,
we concluded that blocking core fucosylation enhances anti-tumor immunity.
DISCUSSION
In this study, we applied CRISPR-Cas9-based screening to a
T cell line. Our results indicated that core fucosylation and its
related molecules are post-translational regulators of PD-1
expression and that this regulation mechanism can be utilized
to optimize anti-tumor immune responses.
We identified genes involved in post-translational modifications of proteins rather than transcriptional factors for two reasons. First, it took substantial time to repeatedly sort/expand
the T cell line to enrich the cells that turned PD-1low. Therefore,
genes that regulate PD-1 but do not primarily affect cell physiology and/or viability are selected after random knockout.
Indeed, almost no cell death or growth suppression wasobserved among the cells targeted for candidate genes. Second, like other T cell line (i.e., EL-4), 68-41 cells showed semiactivated phenotype with constitutive expression of PD-1
without de novo TCR-signal-driven transcription, mimicking activated T cells in vivo.
Among the genes identified, Fut8 was the only enzyme thatcatalyzes core fucosylation, and genetic Fut8 deficiency is
associated with various physiological defects and diseases (Fukuda et al., 2011; Gao et al., 2012; Li et al., 2015; Wang et al.,2005, 2015). Our biochemical analysis demonstrated direct
core fucosylation of PD-1 at two major sites that were shown
to be critical for stable cell-surface expression of PD-1, as
well as for binding of its ligands. We proposed that N49 andN74 are the primary candidate N-linked glycosylation sites
decorated with core fucose for PD-1 cell surface expression
dependent on Fut8. Furthermore, we showed that N74 is thecritical site for clustering to the immune synapse. Glycosylation
analysis demonstrated that both sites are partly decorated withcore-fucosylated N-glycosylation, and N74 contained corefucose- and Lewis-fucose-enriched glycan structure than othersites, implying distinctive feature of this site. However, aminoacid substitution may disrupt the proper structure of PD-1 tosome extent. Therefore, we have to carefully distinguish between the defects of glycosylation and defects of core fucosylation. Actually, protein crystal structure analysis showed thatnear N74 residue faces the interface between PD-1 and PDL1 (Lin et al., 2008). Although TCR-driven PD-1 expression during initial T cell activation is transcription dependent, the strong
PD-1 expression seen in the late phase of chronic infection isless dependent on transcription, suggesting a post-transcriptional regulation mechanism (Crawford et al., 2014; Im et al.,2016; Schietinger et al., 2016). We found that exhausted
T cells in tumors harbored highly core-fucosylated structures,

including PD-1. Because blocking core fucosylation of PD-1
enhanced T cell activation, we speculated that high levels of
PD-1 expression on highly activated, terminally exhausted
T cells were caused by core fucosylation. Considering thatrecent research demonstrated that PD-L1 expression andstability is also regulated by post-translational modification
mechanisms, such as glycosylation and ubiquitination in tumor
cells (Li et al., 2016; Lim et al., 2016), post-translational modification must be an essential factor for deciphering tumor
microenvironments.
Recent reports using Fut8 knockout mice suggested that thisenzyme was involved in pathways for T cell activation. T cells
lacking Fut8 showed impaired T cell activation and low cytokine
production, and the Fut8 knockout mice showed less severe
pathology in experimental colitis (Fujii et al., 2016). The results
suggest that core fucosylation plays an important role in various
receptors and ligands for T cell development and activation.
Core fucosylation has been shown to be necessary for various
protein functions. For example, one study found that core fucosylation of N-glycans is required for the binding of epidermal
growth factor (EGF) to its receptor, whereas no effect wasobserved for the expression levels of epidermal growth factor receptor (EGFR) on the cell surface (Wang et al., 2006). In alpha1,6-fucosyltransferase knockdown mouse pancreatic acinar
cell carcinoma cells, the expression of E-cadherin and E-cadherin-dependent cell-cell adhesion was decreased (Osumi
et al., 2009). E-cadherin was core fucosylated in highly metastatic lung cancer cells whereas absent in lowly metastatic lungcancer cells (Geng et al., 2004). These data suggest that core fucosylation is important for the function and stability of cell surface glycoproteins. Our data suggest that core fucosylation or
N-glycosylation of PD-1 is necessary for proper cell-surface
expression and/or stability on the cell surface as well as ligand
binding. The precise biochemical mechanisms of how core fucosylation affects PD-1 structure and function remains to be elucidated in future study.
2F-Fuc was shown to have anti-tumor activity in vivo by oral
administration (Okeley et al., 2013). Upregulation of Fut8 has
been observed in several malignancies (Geng et al., 2004; Itoet al., 2003; Muinelo-Romay et al., 2008; Takahashi et al.,2000), and inhibition of Fut8 in lung cancer cells reduces
their growth and metastasis in vivo (Chen et al., 2013). In addition to tumor-cell intrinsic mechanisms, our current study suggests that core fucosylation causes detrimental effects intumor-infiltrating immune cells. Thus, targeting core fucosylation in tumor treatment not only inhibits intrinsic tumor growth
but may also attenuate tumor immune evasion throughdown-modulation of PD-1/PD-L1 interaction. This provides
attractive therapeutic opportunities targeting the entire tumor
microenvironment.
In conclusion, combining CRISPR-mediated screening andimmunological assays, we found a valid pathway of PD-1
expression that can be targeted using a small-molecule inhibitor, leading to enhanced immune responses against cancer.
Our study provides an example of a successful exploration ofan unidentified gene regulatory pathway by genome-wide
screening that leads to the proposal of an attractive therapeuticstrategy.

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EXPERIMENTAL PROCEDURES
Genome-wide Loss-of-Function Screening
68-41-Cas9 cells were transduced with lentiviral gRNA pooled library (MOI =0.5). After 1 week of culturing, gRNA(+) (monitored by blue fluorescent protein[BFP]) PD-1low cells were sorted by cell sorter SH800 (SONY, Tokyo, Japan)
and expanded. PD-1low cells were further enriched by repeat sorting andexpansion 3 more times. Post-fourth sort PD-1low cells were lysed using
Wizard SV Lysis Buffer (Promega, Madison, WI, USA). Genomic DNAs wereisolated by phenol-chloroform extraction, isopropanol precipitation, and
70% ethanol purification. gRNA-containing sequences were amplified using
PCR (25 amplification cycles/16 PCR reactions/sample) with the following
primers: for, GGACTATCATATGCTTACCG; rev, TAACTTGCTATTTCTAGCTC.
Amplicons were purified by Agencourt AMPure XP (Beckman Coulter, Tokyo,
Japan), and amplicon libraries were constructed using Ion Plus Fragment
Library Kit (Thermo Fisher Scientific K.K., Kanagawa, Japan). gRNA sequences were deep sequenced using the Ion PGM System with Ion 318
Chip v2 (Thermo Fisher Scientific K.K.). gRNA sequence extraction and analysis were performed using samtools, fastx_trimmer, and Microsoft Excel.
BioGPS (http://biogps.org) was used for investigating the gene expression in
T cells and narrowing down of candidate genes.
Statistical Analysis
All values are presented as the means ± SDs or SEMs. Unpaired Student’s
t tests were used, and p < 0.05 was defined as statistically significant.
*p < 0.05; **p < 0.01.
Additional and more detailed information is provided under Supplemental
Experimental Procedures. Mice studies were approved by and performed inaccordance with the guidelines of the animal ethics committee of Keio University, Tokyo, Japan.
ACCESSION NUMBERS

Medical Innovation (AMED-CREST) (to A.Y.), the Takeda Science Foundation(to A.Y.), Uehara Memorial Foundation (to A.Y.), and Keio Gijuku Academic
Developmental Funds (to A.Y.).
Received: February 10, 2017
Revised: June 15, 2017
Accepted: July 12, 2017
Published: August 1, 2017
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The accession number for the screened gRNA sequences reported in thisstudy is DDBJ: DRA005931.

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SUPPLEMENTAL INFORMATIONCharlton, J.J., Tsoukatou, D., Mamalaki, C., and Chatzidakis, I. (2015). Programmed death 1 regulates memory phenotype CD4 T cell accumulation,
inhibits expansion of the effector memory phenotype subset and modulates
production of effector cytokines. PLoS ONE 10, e0119200.

Supplemental Information includes Supplemental Experimental Procedures,five figures, and three tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2017.07.027.
AUTHOR CONTRIBUTIONS
M.O. conceived and designed the study, performed most of experiments,
analyzed the data, and wrote the manuscript. S.C. offered advice on study
design, helped with method and material development, and wrote the manuscript. T.K. offered advice on study design and helped with method and material development. S.H. helped with method and material development. H.M.
and T.Y. performed microscopy analysis and advised study. M.N. performed
glycan analysis and advised glycomic study. A.Y. supervised the study andwrote and edited the manuscript. All authors reviewed and revised themanuscript.
ACKNOWLEDGMENTS
MO5 cells were a kind gift from Dr. Kenneth L. Rock. CRISPR vectors were akind gift from Dr. Feng Zhang. The CRISRP gRNA library was a kind gift fromDr. Kosuke Yusa. We thank N. Shiino, M. Asakawa, H. Yamane, C. Ohkura, and
Y. Hirata for their technical assistance. We acknowledge S. Usui and A. Nayafor their technical assistance for glycoproteomics and Y. Ushijima for manuscript preparation. This work was supported by special grants-in-aid from
the Ministry of Education, Culture, Sports, Science and Technology of Japan(no. 25221305 to A.Y.), Grant-in-Aid for Scientific Research on Innovative
Areas (no. 17H05801 to S.C.), Grant-in-Aid for Young Scientists (B) (no.
17K15451 to M.O.), Advanced Research and Development Programs for

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Chapter 4
Analyzing the Dynamics of Signaling Microclusters
Akiko Hashimoto-Tane, Tadashi Yokosuka, and Takashi Saito
Abstract
T cell antigen receptor (TCR) stimulation induces recruitment and accumulation of various types of signaling molecules and forms signaling microclusters. The dynamics of the microclusters are important for
regulating the quality and quantity of T cell activation. We describe here our protocols for analysis of signaling microclusters by using supported planar bilayers.
Key words TCR, Signal, Phosphorylation, Retroviral expression system, Supported planar bilayers,
Time-lapse imaging, TIRF microscopy, Confocal microscopy

1

Introduction
The in vitro analysis of T cell activation provides considerable
information about intracellular signaling events involved in thisprocess. Triggering of the T cell receptor (TCR) by peptide-MHC
induces formation of TCR microclusters and clustering of various
signaling molecules to mediate phosphorylation/activation. To
analyze the dynamics of such TCR and signaling microclusters inliving T cells, we have utilized the combination of TIRF fluorescence microscopy and a planar bilayer system for live cell imaging.
We choose retrovirus-mediated gene transfer to express fluorescently labeled signaling molecules in normal T cells. Because it isquite difficult to transfect genes into primary T cells by lipofection
or electroporation, virus transduction is a powerful tool for preparing cells expressing the desired fluorescently labeled molecules.
For the activation of T cells for real-time imaging by microscopy, we use a supported planar bilayer system. The planar bilayer
mimics the membrane surface of antigen presenting cells (APC) by
the expression of freely mobile GPI-anchored MHC proteins, integrins, and other co-stimulatory molecules. A supported planar
membrane containing only peptide-MHC and integrins is able toinduce T cell activation. Under these conditions, the immunologicalsynapse is generated on the surface of the T cells at the interface

Cosima T. Baldari and Michael L. Dustin (eds.), The Immune Synapse: Methods and Protocols, Methods in Molecular Biology,
vol. 1584, DOI 10.1007/978-1-4939-6881-7_4, © Springer Science+Business Media LLC 2017

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with the flat surface of the planar bilayer and this structure can beeasily analyzed, making this the most suitable system for microscopic analysis. To confirm the bilayer data under physiologicalconditions with authentic APC, we also perform T cell stimulation
using activated B cells as APC. We present here our protocols forthe preparation of T cell transductants and for live cell imaging byfluorescence microscopy.

2

Materials

2.1

T Cell Isolation

1. TCR transgenic mice for primary T cells: AND [1] or 5C. C7[2], both of which are cytochrome c-specific, I-Ek restricted T
cells, DO11.10 [3] or OT-1 [4]. Mice for APC: C3H, B10BR,
BALB/c, or C57BL/6.
2. Medium: RPMI 1640 supplemented with 8.5 μM of
2-Mercaptoethanol (2-ME), 100 mg/L of streptomycin, 57.6
mg/L of penicillin and 10% (v/v) fetal bovine serum (FBS).
3. Glass homogenizer (16 × 160 mm, 5 mL, e.g., Abe Kagaku
Inc.).
4. Ammonium-chloride-potassium (ACK) buffer: 150 mM
NH4Cl, 10 mM KHCO3, and 0.125 mM EDTA, adjusted to
pH 7.6 with HCl.
5. BioMag Goat Anti-Mouse IgG (Qiagen) or equivalent.
6. Biotinylated mAb cocktail: 2.5 μg/mL of biotin-labeled
CD11b, CD11c, CD45/B220, CD105, Gr.1, I-A/I-E (MHC
II), TER-119/Erythroid, TCR-γδ, ΝΚ1.1, CD19 andCD24 in RPMI 1640 with 2% FCS.
7. Mojo buffer: 0.5% (w/v) bovine serum albumin (BSA) and 2
mM EDTA in phosphate buffered saline (PBS).
8. Mojo streptavidin beads (BioLegend) or equivalent.
9. Antigen peptide: MCC peptide (88–103) for AND and 5C. C7
tg mice, MCC peptide mutant (K99A) for AND tg mice, OVA
peptide (323–339) for DO11.10 cells, and OVA peptide(257–264) for OT-I tg mice.
10. IL-2 containing medium: 1.5 or 3.1 ng/mL IL-2 (Peprotech)in RPMI 1640 with 10% FCS.
11. B220 MACS beads (Miltenyi Biotec) or equivalent.

2.2 Retrovirus
Preparation

1. Plasmids: we recommend pMX retroviral expression vector
series [5] (Cell Biolabs); pMX or pMX-IRES-human CD4/8(with deleted cytoplasmic region).
2. Packaging cells: we recommend Phoenix-ECO (ATCC),
Platinum-E (Plat-E) [6] (Cell Biolabs).

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3. Medium: Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 8.5 μM 2-ME, 100 mg/L streptomycin, 57.6
mg/L penicillin, and 10% (v/v) FBS.
4. Medium for lipofection: Opti-MEMs (ThermoFisher) supplemented with 50 μM 2-ME and 0.9 mM CaCl2 or equivalent.
5. Lipofection reagent: 2% (w/v) Polyethylenimine “Max” (MW
40,000) (PEImax) (Polyscience) in sterilized water, filteredand stored at 4 °C or equivalent.
6. Transduction reagent: 10 μg/mL polybrene in sterilized ultrapure water.
2.3 Liposome
Preparation

1. Plasmids: I-Adα-GPI, I-Adβ-GPI, H-2Kb-GPI, CD80-GPI,
CD86-GPI, or PD-L1-GPI was inserted in the pMX expression vector [7–9].
2. CHO or BHK cell lines.
3. Agarose beads: Cyanogen bromide activated agarose.
4. Antibodies for affinity purification of proteins: 14–4-4 for I-Ek,
YN1/1 for ICAM-1, MK-D6 for I-Ad, 20–8-4 for H-2Kb,
16-10A1 for CD80, PO3 for CD86, and MIH5 for PD-L1.
5. Coupling buffer: 0.1 M NaHCO3, 0.5 M NaCl pH 8.3–8.5.
6. Tris buffer: 25 mM Tris, 0.15 M NaCl adjusted to pH 8.0 withHCl.
7. Lysis buffer: 1% (w/v) TitonX-100, 25 mM Tris, 0.15 M
NaCl, protease inhibitor cocktail [0.2% (v/v) aprotinin, 50
μg/mL antipain, 50 μg/mL chymostatin, 20 μg/mL leupeptin, 10 μg/mL pepstatin A] pH 8.0.
8. OG wash buffer: 1% (w/v) n-octyl-Beta-D-glucopyranoside(OG), 25 mM Tris, 0.15 M NaCl, pH 8.0.
9. Elution buffer: 50 mM triethylamine, 0.15 M NaCl, 1% OG(pH 11.5) or 50 mM Glycine, 0.15 M NaCl, 1% OG (pH 3.0).
10. Neutralizing solution: 1 M Tris pH 7.0, 1% OG.
11. 1,2-dioleoyl-sn-glycero-3-phosphocholine
Avanti Polar Lipids).

(DOPC)(e.g.,

12. Dialysis tubing (14000 MWCO).
13. Argon gas tank and regulator.
2.4

Lipid Bilayer

1. Chromic sulfuric acid mixture.
2. Cover glass, 40 × 50 mm, thickness 0.12–0.17 mm.
3. Micro cover glass, diameter 12 mm, thickness 0.12–0.17 mm.
4. Silicone sheet with a thickness 0.2 mm, stiffness of 20 was cut
into strips ~4 mm × 25 mm.
5. Silica beads 4.86 μm diameter (e.g., Bangs Laboratory).

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Akiko Hashimoto-Tane et al.

6. Lab-marker felt tip pen.
7. Loading buffer: 1% (w/v) BSA, 50 μg/mL gentamycin in PBS
adjusted to pH 4.5 with 2.3 M citric acid.
8. Peptide solution: MCC peptide, 1% EDTA, 0.2% (v/v) aprotinin and 20 μg/mL leupeptin in loading buffer.
9. Acrylic tubing 35 mm outside and 25 mm inside diameters,
cut to 10 mm height.
10. High Vacuum grease (e.g., Dow Corning Toray).
11. HEPES buffered saline stock solution (10 × HBS): 200 mM
Hepes, 1.37 M NaCl, 50 mM KCl, 7 mM Na2HPO4, 60 mM
D-glucose, 10% (v/v) BSA, adjusted to pH 7.2 with hydrochloric acid (HCl).
12. 1× HBS: 10% (v/v) 10× HBS, 1 mM CaCl2, 1 mM MgCl2,
10% (v/v) FBS, prepared when needed.
13. 4% Paraformaldehyde (PFA) in PBS.
14. Mounting
medium:Permafluor,(ThermoScientific) or equivalent.

ProlongGold

15. Slide glass 76 × 26 mm, thickness 1.2–1.5 mm.
2.5

Cell Staining

1. H57(Fab)-Dy549: Purified Fab fragment from anti-mouse
TCRβ chain mAb (H57) labeled with Dy549.
2. Halo tag TMR ligand (Promega).
3. Permeabilizing buffer for fixed cells: 0.05% Digitonin (stock 1
mg/mL in DMSO) or 0.1% TritonX-100 in PBS containing
2% BSA, 0.02% NaN3.
4. Abs for intracellular staining: Abs were labeled with Alexa
series or DyLight series and diluted in permeabilizing buffer.

2.6 Microscopeand Software

The information below describes commercial microscope systems
and software we have used successfully. Many other options areavailable.
1. A TIRFM system with dual laser (488, 561 nm) (Olympus),
UAPON 100× OTIRF lens (NA1.49, Olympus) and CCD
camera (ORCA-Flash, Hamamatsu photonics), auto-focus
system ZDC (Olympus) were attached to an Olympus IX81
inverted microscope.
2. Confocal microscope: Leica TSC SP5 system with an oil
immersion objective (HCX PLAPO 100×, NA1.44, oil, Leica),
four lasers (405, 488, 561, 633 nm), and a HyD detector.
3. Software: Metamorph (Molecular Devices) for Olympus IX81
inverted microscope, Leica Application Suite (Leica) for Leica
TSC SP5 system, Fiji (ImageJ) (freeware), and Imaris (Bitplane)
for statistical analysis.

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3

Methods

3.1

Cell Preparation

55

1. T cell preparation: Take spleen and lymph nodes from a TCR
transgenic mouse and homogenize in 5 mL RPMI 1640. Treat
the cell suspensions with ACK solution (2 mL/mouse) to lyse
red cells. From the cell suspension, deplete B cells with 2 mL/
mouse of BioMag Goat Anti-Mouse IgG and other non-T cell
populations by staining with a biotinylated-Ab cocktail andMojo streptavidin magnetic beads (5 μL/107 cells) and thensort T cells on a magnetic stand. The purity of the isolated T
cells is between 85 and 95%.
2. APC preparation: Take spleen cells from a mouse for APC andlyse red cells with ACK solution. The remaining cell suspension is then irradiated with 15 Gy of gamma radiation and used
for APC. Alternatively, incubate the cells with 10 μg/mL
Mitomycin C for 2 h and wash well.
3. Cell culture: Culture 3.0 × 106 purified T cells with 3.0 × 106
of the APC in 500 μL RPMI 1640 containing antigen peptide
and 1.5 ng/mL IL-2 per well in a 48-well culture plate. We
always prepare two to four wells of T cells for the analysis ofeach signaling molecule.
4. B cell preparation: For preparation of B cells as APC, B220
positive cells sorted from spleen by using B220 MACS beads
were cultured at 3.0 × 106 in 600 μL RPMI 1640, adding 10
μg/mL of LPS to activate the cells and pulsing with 100 nM
to 10 μM MCC peptide for 12–24 h.

3.2 Virus
Transduction

1. Prepare retrovirus vectors for expressing fluorescently labeledsignaling molecules. The position to insert the fluorescentmolecule should be optimized for each target molecule.
Practically, we always try both N- and C-termini with or without some linker, and choose the best one in terms of function
and/or brightness. For example, some transmembrane molecules like CD3ζ, CD28, and LAT were optimally labeled at the
C-terminus. Certain intracellular molecules such as Lck,
ZAP70, PKCθ, CTLA-4, dynein light chain, Pyk2, and paxillin
were labeled at the C-terminus, but some others such as SLP76
and α-tubulin were at the N-terminus. GFP (pEGFP-N1, e.g.,
Addgene) is our first choice for a fluorescent protein because it
provides sufficiently bright fluorescence and has less effect on
the function or localization of the target protein. Red fluorescent proteins are potential candidates as a second color; however, based on our experience with T cells, red fluorescentmolecules such as mRFP, mCherry, mStrawberry, and Kusabiraorange have a tendency to form artificial bright aggregates.
Although we know that these red proteins work well in other

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Akiko Hashimoto-Tane et al.

cell types, they are sometimes problematic, even in the monomeric form, in T cells for unknown reasons. Therefore, we areusing a combination of Halo-tag (pHTC HaloTag, Promega)
and TMR ligand or CFP (pECFP-N1, Addgene) as the counter color for GFP [7–13].
2. Packaging cells: 1 day before lipofection, seed Phoenix cells at
1 × 106/well in a 6-well culture plate. If using Plat-E, plating
3 × 106/well 3 hours before lipofection is also acceptable.
3. Lipofection: Mix 3–10 μg of pMX plasmid with 250 μL of
Opti-MEM. In another polystyrene tube, mix 10 μL PEImax
and 250 μL of Opti-MEM, gently mix by taping, and thenleave for 5 min. Next, add the plasmid solution to the PEImax
solution and wait for 20–30 min. Remove culture supernatant(sup) from the packaging cells until 500 μL/well remains andthen add the 500 μL plasmid/PEImax solution very gently
dropwise. Immediately, add 1.0 mL DMEM gently, incubate
for 2–4 h, and then add an additional 3.0 mL DMEM.
4. Harvesting Virus: On the next day (day 2), remove culture sup
and add fresh DMEM, 11 mL/well. Harvest the viruscontaining culture sup on day 3 and add fresh media to harvest
again on day 4. Combine the sups in a 50 mL tube and concentrate virus by overnight centrifugation, 8,000 × g at 4
°C. After discarding the supernatant by decantation, add 1.0
mL of fresh RPMI 1640 and use for transduction, or freeze at−80 °C.
5. Viral infection: 24–36 h after initiation of T cell cultures, collect the cells and replate in RPMI 1640 (50 to 100 μL/well)in the same plate. Add 250–500 μL/well virus sup supplemented with 10 μg/mL polybrene and 3.2 ng/mL IL-2 foreach well and centrifuge in a pre-warmed centrifugation at
1,400 × g, 25–32 °C for 2 h. Then add 400 μL/well RPMI
1640 with 3.2 ng/mL IL-2 and incubate for another 2 days.
If you intend to infect two differently labeled target molecules into the same cells, prepare virus separately and mix
them at the transduction step.
6. Cell sorting: Harvest cells and isolate the fluorescent positive
infected cells with a cell sorter (see Note 1). If you use pMXIRES-hCD4/8, you can use magnetic beads to enrich theinfected cells. Incubate the sorted cells for 1 day in RPMI
1640 without IL-2.
3.3 Liposome
Preparation

1. Prepare the expression construct in the pMX vector engineered
to express the GPI anchored form of the molecule of interest.
The extracellular domains of I-Adα, I-Adβ, CD80, CD86, or
PD-L1 were fused with a GPI-motif derived from human DAF
and the expression constructs were transfected into CHO or-580-

Signaling Microclusters

57

BHK cells. CHO cells expressing I-Ek-GPI and BHK cells
expressing ICAM1-GPI were kindly provided by Dr. Michael
Dustin [14].
2. Cell culture: Culture the CHO or BHK cells expressing the
GPI-constructs, harvest, wash the cells with PBS, and keep frozen at −80 °C. We usually repeat the cell harvest several times
until reaching a 3.0 g cell pellet to collect a large amount of themolecules of interest (see Note 2).
3. Affinity purification: Extract GPI-anchored molecules fromthe cell pellets by affinity purification. Prepare 1.0 mL agarose
beads coupled with 2.0 mg of mAb specific for the target protein. Lyse the cells by ultrasonic treatment in Lysis buffer.
Ultracentrifuge the cell lysates containing GPI-anchored molecules and then preclear using agarose beads coupled with
nonspecific IgG. Incubate the cell lysates overnight at 4 °C
with beads coupled with the specific mAb. After washing thebeads, the GPI-anchored molecules are eluted with elution
buffer, pH 11.5, or pH 3.0 (the optimal pH should be determined for each target protein). Collect eluates in
450-μL-fractions in tubes containing 50 μL neutralizing solution. Do not mix the fractions that have different concentrations of eluted GPI-anchored proteins. We use the fractions
whose OD280 is more than 0.08. If the OD280 is less than 0.08,
concentrate using pre-washed centrifugal filter tubes.
4. DOPC preparation: Prepare a 4.0 mM DOPC micelle stock
solution by making a thin film of 6.28 mg DOPC on a glass
bottle by evaporating chloroform, then add 2.0 mL of Tris
buffer with 1% OG, sonicate and filter. Mix the micelle solution with nine volumes of each purified GPI-anchored protein
fraction, which results in 0.4 mM DOPC, and dialyze against
Tris buffer. We always prepare protein-free 0.4 mM DOPC
liposomes for diluting GPI-anchored proteins in the next step.
5. Quantitation on glass beads: Check the concentration of GPIanchored proteins by FACS analysis after staining of reconstituted glass beads. Put 1.5 μL of glass beads on the bottom of a
PCR tube and let it stand at an angle for 3 min to allow thebeads to sink to the bottom side (Fig. 1a). Remove the fluid
from the beads (Fig. 1b) (do not allow them to dry) and add
3.0 μL of dialyzed liposome solution. Mix and invert the tube,
keeping the fluid as a hanging drop for 30 min (Fig. 1c) withgentle tapping every 10 min. Wash the liposome-coated beads
gently with HBS, then incubate with RPMI 1640–10% FCS for
blocking. Next, stain the beads with fluorescently labeled mAbs
specific for the GPI-anchored proteins (Fig. 1d). Based on the
fluorescent intensity and the fluorescent-protein (F/P) ratio of
the mAbs, evaluate the amount of GPI-anchored proteins in
the liposomes. Dilute each liposome to a predetermined ideal

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Akiko Hashimoto-Tane et al.

Fig. 1 Preparation of lipid bilayers reconstituted on glass beads for quantification of GPI-anchored proteins andT cell stimulation. (a) Put 1.5 μL of glass beads in the bottom of a PCR tube. (b) Lean the tube for 3 min to allow
the beads to sink to the bottom side and remove the fluid. (c) Add 3.0 μL of liposome solution, mix very gently,
and invert the tube. Incubate for 30 min at room temperature, tapping gently every 10 min. Wash the liposomecoated beads gently with HBS. (d) Add RPMI 1640-FBS medium to the beads for blocking, and stain with fluorescently labeled mAbs for FACS analysis. (e) Incubate with MCC antigen peptide in loading buffer for 12–24
h and use for T cell simulation

concentration (molecules/μm2) and store at 4 °C in screwcapped tubes with air replaced by Argon gas. In the case of
MHC-GPI and ICAM-1-GPI, we prepare 800 molecules/μm2
as stock.
3.4 Setting
Up the Planer Bilayer

1. Wash glass: Place glass (40 × 50 mm) and cover glass (diameter
12 mm) in chromic acid for at least 1 day (we routinely store
these items in a chromic acid chamber), rinse with water, 95%
ethanol and ultra-pure water, then air dry.
2. Reconstitution of the lipid bilayer: Mix the MHC (e.g., I-Ek)
and integrin ligand (e.g., ICAM-1) liposomes to make a final
concentration of 200 molecules/μm2 each [14]. Add other costimulatory molecules (e.g., CD80) as needed and incubate forseveral minutes at room temperature. Set three silicone sheetsin parallel about 6 mm apart on the 40 × 50 mm glass and put
1.2–1.5 μL of mixed liposome solutions in between them(Fig. 2a). Place a cover glass on each liposome and confirm
that the liposome drop touches both the slide glass and thecover glass. We always make two to four spots per glass. Markthe drop position with a felt tip lab marker and incubate atroom temperature for 30 min, taking special care to prevent-582-

Signaling Microclusters

A

cover glass
liposomes

silicon
sheet

D

B

Lab marker

lipid bilayerE
paper
napkinwet filter paperCloading buffer

glass

culture dish59

acrylic tubing
greaseFcellgrease
RPMI 1640

acrylic tubing
HBS

objective

Fig. 2 Preparation of planer bilayer for live cell imaging. (a) Set three silicone sheets in parallel approximately
6 mm apart on the glass and put 1.2–1.5 μL of mixed liposomes in between the two sheets. (b) Place coverglasses on each liposome, mark the position with a felt tip lab marker, and incubate for 30 min at room temperature. (c) Wash each drop by flowing pre-warmed loading buffer between the glass and the cover glass,and replace the peptide solution. (d) Incubate the glasses on wet filter paper in a culture dish at 37 °C for
24–48 h. (e) Wash the cover glasses with pre-warmed RPMI 1640 and remove silicone sheets from the coverglasses. Wipe excess liquid with paper towels, apply grease to the acrylic tubing, and pour in pre-warmed HBS.
(f) Set the chamber on a microscope, put cells on the lipid bilayer area, and collect images at the interface

drying (Fig. 2b). Warm loading buffer and peptide solution to
avoid emerging bubbles. After prepareing the spots as reconstituted lipid bilayers, flow more than 3.0 mL/spot of loading
buffer between glass and cover glass, and then peptide solution (Fig. 2c). Take special care to prevent bubbles (see Note
3). Put the glass on wet filter paper in a culture dish and incubate at 37 °C for 24–48 h (Fig. 2d).
3. Setting up chambers: Put grease uniformly on one side of the
cut acrylic tubing (Fig. 2e). Wash the position of the lipid
bilayers with pre-warmed RPMI1640 and remove the coverglasses from the silicone sheets (see Note 4). Submerge thecover glasses in pre-warmed HBS in a 12-well culture dish withthe lipid bilayer side up (Fig. 3a). Wipe the area surrounding
the spots with a paper napkin (e.g., Kimwipe) to approximate
the size of acrylic tubing (Fig. 2e). Push the greased side of theacrylic tubing onto the wiped area of the glass to make an assaychamber (Fig. 2e). Confirm the absence of air bubbles in thegrease from the bottom side and immediately add pre-warmed
HBS (Fig. 2f ). You can set up many chambers at once andkeep them at 37 °C. For fixed samples, keep the cover glasses
in a 12-well culture dish at 37 °C.

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Akiko Hashimoto-Tane et al.

A

B

cell

C

antibody solution

mounting
mediumHBS
cover glasswith lipid bilayerDobjective

parafilm

slide glass

slide glass
nail polish
mounting medium
cells
cover glass

Fig. 3 Analysis of fixed cells on a planar bilayer. (a) Immerse the lipid bilayerreconstituted cover glasses in 400 μL of HBS in a 12-well culture dish with thebilayer side up, then add the T cell suspension dropwise and incubate at 37°C. Add 800 μL of a PFA solution to fix the cells and then replace with PBS. (b)Place the cover glass with the cell side up on Parafilm, followed by treatment
with permeabilizing buffer. Stain the cells with 150 μL of the mixed Ab solution
and then wash. (c) Drop 10 μL of mounting medium on the slide glass and put
the cover glass on the medium with the cell side down. (d) Seal the edge withtransparent nail polish and examine on a microscope by setting the cover glassside to the objective lens
3.5 Imaging of Live T
Cells by Microscopy

1. Cell preparation: Collect the sorted GFP- (or other fluorescentprotein) positive T cells as described in step 6 of Subheading
3.2 and resuspend in 1.0–2.0 μL of 10 μg/mL H57(Fab)-Dy549. When Halo tag is used for the red color, incubate the cells with 1.0 μM TMR ligand for 15 min. Wash thecells and incubate them in fresh medium for at least 30 min,then collect and resuspend them in 1.0–2.0 μL HBS at a concentration between 2.5 × 104 and 2.5 × 105/μL.
2. Planar bilayer assay on the microscope: Warm the microscope
system. We use a custom-made acrylic box to warm the entire
microscope (see Note 5). Set the assay chamber on the microscope, drop a very small volume of cell suspension on the planar bilayer area, and adjust focus and laser direction (Fig. 2f ).
The TIRF-M instrument from Olympus has independent laser
illumination paths for 488 and 561 nm. Therefore, the best
angle for each laser can be adjusted. Choose the illumination
conditions and resolution from the viewpoint of image clarityand bleaching. Put 0.5 μL of the cells on the planar bilayer andstart collecting images. We take one image every 5 s for
10–15 min at a resolution of 99.6 nm/pixel and area of 1024
× 1024 pixels on the TIRF-M [11] (see Note 6).

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Signaling Microclusters61

3. APC stimulation on the microscope: For T cell stimulation by
B cells as APC, prepare a glass bottom dish precoated with 0.5
μg/mL B220 Ab. Incubate the LPS-activated and MCCloaded B cells on the glass for at least 20 min, change themedium to HBS buffer, and move onto the microscope. Afterconfirming that the B cells are immobilized on the B220
Ab-coated glass, fluorescent-labeled T cells are added. T cells
scan the surface of the B cells and eventually stop, forming afirm conjugate with the B cells and begin activation. Determine
a good time point and position to analyze molecular dynamics
at the interface [10, 12].
3.6 Imaging of Fixed
T Cells
on the Microscope

1. Planar bilayer: Change the buffer in the 12-well culture platedescribed in step 3 of Subheading 3.4 to 400 μL of fresh prewarmed HBS. Drop 1.0–2.0 μL of T cell suspension (~106
cells/μL) on the planar bilayer area and incubate for 1.5 min for
the analysis of the initial activation phase or for 5 min for the
analysis of cSMAC formation (Fig. 3a). To fix the cells, quickly
add 800 μL of pre-warmed 4% PFA solution and keep at 37 °C
for 10–20 min. We prepare many samples at once on the same
culture plate by sequential handling. Remove the PFA solution
by decantation, replace with PBS, and keep at 4 °C.
2. Stimulation with APC: Coat cover glasses with 0.5 μg/mL
B220 Ab and then immerse them in RPMI1640 in a 12-wellculture plate with the Ab-coated side up. Add a drop 2.0 × 106
LPS-activated and MCC-loaded B cells and incubate for atleast 20 min. Change the medium to pre-warmed HBS, andadd 1.0–2.0 μL of T cell suspension (~106 cells/μL) dropwise
onto the immobilized B cell area. The following procedure is
the same as in step 1 of Subheading 3.6.
3. Ab staining: Place the cover glasses with the fixed cells side up
on Parafilm, followed immediately by a drop of the permeabilization buffer and then incubation for 15 min (Fig. 3b). Stain
the cells by replacing the buffer with 150 μL of the mixed mAb
solution and incubate at room temperature for 20 min to several hours, depending on the target molecule. Choose themAb color combinations according to the microscope settings.
Wash the cover glass with PBS.
4. Mounting on slide glass: Drop 10 μL of mounting medium
onto a slide glass and put a cover glass on the medium with thecell side down (Fig. 3c). Wait for 30 min and seal the edge with
nail polish (Fig. 3d).
5. Confocal microscopy: Set the objective lens above the coverglass and detect fluorescent molecules at the interface between
the cell and the cover glass (Fig. 3d). We first adjust the focus
onto the TCR microclusters and then adapt the position toother signaling molecules.

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Akiko Hashimoto-Tane et al.

3.7

Data Analysis

Use Fiji (ImageJ) and Imaris for the analysis of fluorescent intensities, number of microclusters, cell adhesion area, and so on (seeNote 7).

3.8

Bioassay

To measure cellular responses under the same stimulation conditions by microscopy, we prepare glass beads as in step 4 of
Subheading 3.2 and incubate with antigen peptide in loading buffer for 12–24 h (Fig. 1e). The beads can be used in the same manner as APC for measurement of cytokine production or cell growth.

4

Notes
1. Infection efficiency is dependent on both virus titer and cell
viability. If the virus titer is low, several points should be
improved: reconstruct the plasmid, functionally select the
packaging cell lines by adding selection drug/antibiotics, or
simply increase virus titer per cell. If the viability of T cells
upon stimulation is low, check TCR expression and increase
the peptide concentration for stimulation. Sometimes, removalof cell debris derived from irradiated spleen by density gradientcentrifugation increases the frequency of infection.
2. To collect 3.0 g of cell pellet of CHO or BHK cells, we repeat
cell harvesting two to three times from cultures in 30 of
150 mm diameter dishes. To increase the quality of purified
GPI molecules, it is critical to avoid overgrowth, to wash
medium out before freezing and to tap well after thawing thecells.
The quality of the planar bilayer is critical for the dynamics
of all stages of IS formation. If the planer bilayer is badly prepared, the area of cell adhesion and movements of signaling
molecules are altered and one often fails to observe the typicalIS, particularly the cSMAC. Some reasons for failure are shorter
incubation time of liposome drop, stains on the surface of theglass, bilayer broken by air bubbles, and oxidization of liposomes, which prevents motility of the molecules. The motilityof molecules in liposomes can be monitored by fluorescencerecovery after photobleaching (FRAP). If the motility is not
sufficient, it is better to make a new preparation.
3. When setting up the chamber, pay caution to exposure to air.
If the planar bilayer is exposed to air for even a short period, it
will be broken. Although the acrylic tubing we use is nondisposable, the grease is ethanol-soluble and easily removed, thus
the acrylic tubing can be reused.
4. We use a TIRF microscope for live cell imaging and a confocal
microscope for fixed cell analysis. The reasons are as follows:
live cell imaging requires fast shutter speed, higher sensitivity,-586-

Signaling Microclusters

63

and relatively less harmful conditions for the target cells.
Confocal microscopy has advantages of many color channels
and application to 3-D reconstitution analysis; however, it isless sensitive and harmful by laser illumination than the TIRF
system.
5. Temperature strongly affects focus. The problem is serious,
especially for time-lapse imaging by TIRF microscopy. Because
this is due to thermal expansion of metals, we warm the whole
microscope system to keep the temperature constant. In addition, it is recommended to use an auto-focus system ZDC.
6. If T cells on the planer bilayer keep moving even after stimulation with a higher concentration of antigen peptide, they may
still be in an activated state. We incubate T cells in media without extra IL-2 for 1 day, sometimes longer, before assay to letthem return to the resting state. It is necessary to check the cellstatus by cell size and the surface expression of activation
markers.
7. Recently, sophisticated statistical analysis of imaging studies isoften requested by reviewers for publication. In the case of theanalysis of TCR microclusters, the definition of a microcluster
is not fixed. We use criteria that a TCR microcluster is a dot,
which can be fitted by Gaussian fitting and is more than 10%
brighter than a nonclustered area. Although this is not a universal criterion, the resulting count of our analysis is similar toother studies.

Acknowledgment
This work was supported by KAKENHI (Grant-in-Aid for Scientific
Research (S) 24229004 for T.S. and (C) 10415226 for A.H.-T.),and partly by Takeda Science Foundation, Novartis Foundation(Japan), and Hayashi Memorial Foundation for Female Natural
Scientists.
References
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Gascoigne NR, Hedrick SM (1989) Selective
development of CD4+ T cells in transgenic
mice expressing a class II MHC-restricted antigen receptor. Nature 341:746–749
2. Seder RA, Paul WE, Davis MM, de St. Groth
BF (1992) The presence of interleukin 4 during in vitro priming determines the lymphokineproducing potential of CD4+ T cells from T
cell receptor transgenic mice. J Exp Med
176:1091–1098-587-

3. Murphy KM, Heimberger AB, Loh DY (1990)
Induction by antigen of intrathymic apoptosisof CD4+CD8+TCRlo thymocytes in vivo.
Science 250:1720–1723
4. Hogquist KA, Jameson SC, Heath WR,
Howard JL, Bevan MJ, Carbone FR (1994) T
cell receptor antagonist peptides induce positive selection. Cell 76:17–27
5. Kitamura T (1998) New experimental
approaches in retrovirus-mediated expression
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6. Morita S, Kojima T, Kitamura T (2000) Plat-E:
an efficient and stable system for transient packaging
ofretroviruses.
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Takamatsu M, Hashimoto-Tane A, Dustin ML,
Tokunaga M, Saito T (2008) Spatiotemporal
regulation of T cell costimulation by
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theta translocation. Immunity 29:589–601
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Sakata-Sogawa K, Zeng H, Hashimoto-Tane
A, Yagita H, Tokunaga M, Saito T (2010)
Spatiotemporal basis of CTLA-4 costimulatory
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cell activation. Immunity 33:326–339
9. Yokosuka T, Takamatsu M, Kobayashi-Imanishi
W, Hashimoto-Tane A, Azuma M, Saito T(2012) Programmed cell death 1 forms negative
costimulatory microclusters that directly inhibit
T cell receptor signaling by recruiting phosphatase SHP2. J Exp Med 209:1201–1217
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Yokosuka T, Kimura Y, Ohara O, Saito T(2016) Micro adhesion rings surrounding TCR

microclusters are essential for T cell activation.
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M, Saito T (2011) Dynein-driven transport ofT cell receptor microclusters regulates immune
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Sakuma M, Kobayashi W, Saito T (2010) T-cell
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Hiroshima M, Hashimoto-Tane A, Tokunaga
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SCIENCE SIGNALING | RESEARCH ARTICLETCR SIGNALING

Inhibition of T cell activation and function by theadaptor protein CIN85
Mei Suen Kong1,2*†, Akiko Hashimoto-Tane1*, Yusuke Kawashima3,4, Machie Sakuma1,
Tadashi Yokosuka1‡, Kohei Kometani5§, Reiko Onishi1||, Nick Carpino6, Osamu Ohara3,4,
Tomohiro Kurosaki5,7, Kia Kien Phua2, Takashi Saito1,8¶Copyright © 2019
The Authors, somerights reserved;
exclusive licensee
American Association
for the Advancementof Science. No claim
to original U.S.
Government WorksINTRODUCTION

T cells play a central role in adaptive immune responses, which can
include the priming of naïve T cells, effector functions from activatedeffector T cells, and the recall responses by memory T cells (1). Antigen (Ag)–induced T cell stimulation initiates not only positive
signal cascades but also negative signal pathways that attenuate thestrength and duration of positive signals to prevent excessive T cell
activation. Fine-tuning/balance between such positive and negative
signals determines appropriate T cell responses. Adaptor molecules
mediate branches in signal cascades and are essential in propagating both positive and negative signals.
CIN85 (Cbl-interacting protein of 85 kDa) is a ubiquitously expressed adaptor protein that has been identified by four groups
from different tissues and named differently: CIN85 from human(2), regulator of ubiquitous kinase (Ruk) from rat (3), Src homology
3 (SH3) domain–containing gene expressed in tumorigenic astrocytes (SETA) from rat (4), and SH3 domain kinase binding protein
1 (SH3KBP1) from mouse (5). CIN85 shares ~54% amino acid se1

Laboratory for Cell Signaling, RIKEN Center for Integrative Medical Sciences,
Yokohama, Kanagawa 230-0045, Japan. 2Institute for Research in Molecular Medicine,
Main Campus, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia. 3Laboratory
for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama,
Kanagawa 230-0045, Japan. 4Department of Technology Development, Kazusa
DNA Research Institute, Kisarazu, Chiba 292-0818, Japan. 5Laboratory for Lymphocyte
Differentiation, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa
230-0045, Japan. 6Department of Molecular Genetics and Microbiology, Stony
Brook University, Stony Brook, NY 11794-8434, USA. 7Lymphocyte Differentiation,
Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan.
8
Cell Signaling, Immunology Frontier Research Center, Osaka University, Suita,
Osaka 565-0871, Japan.
*These authors contributed equally to this work.
†Present address: Microbiota Host Interactions, Nutrigenomics & Metabolism Laboratory, Lee Kong Chian School of Medicine, Nanyang Technological University,
Singapore 308232, Singapore.
‡Present address: Department of Immunology, Tokyo Medical University, Shinjuku,
Tokyo 160-8402, Japan.
§Present address: Department of Life Science Frontiers, Center for iPS Cell Researchand Application, Kyoto University, Kyoto 606-8507, Japan.
||Present address: Research and Development, Oncology Project Development,
Sanofi K.K., Shinjuku, Tokyo 163-1488, Japan.
¶Corresponding author. Email: takashi.saito@riken.jp
Kong et al., Sci. Signal. 12, eaav4373 (2019)quence similarity with CD2-associated protein (CD2AP) (6). Thus,
CIN85 and CD2AP constitute the CD2AP/CIN85 family of adaptor
proteins.
The CIN85 protein is composed of three SH3 domains at the N
terminus followed by a proline-rich (PR) domain, a serine-rich (SR)domain, and a coiled-coil (CC) domain at the C terminus. The CC
domain mediates homotypic oligomerization of CIN85 and heterotypic interactions with CD2AP (4, 7, 8). CIN85 has at least 10 isoforms
whose expression is regulated in either a tissue- or developmental
stage–specific manner (8, 9). T cells exclusively express one CIN85
isoform lacking SH3A (CIN85A) (10).
Functions of CIN85 are well known downstream of receptor tyrosine kinase (RTK) signaling (2). In fibroblasts, it promotes internalization of epidermal growth factor receptor (EGFR), hepatocyte
growth factor receptor (HGFR), and platelet-derived growth factor
receptor (PDGFR) through a clathrin-dependent mechanism, which
is necessary for RTK degradation and signaling termination (11–13).
CIN85 as an adaptor protein recruits endocytic regulatory proteins,
such as E3 ubiquitin ligase Cbl, endophilin, and dynamin, to regulate receptor trafficking and degradation (11, 12, 14–16). Furthermore, the other family member, CD2AP, inhibits T cell receptor (TCR)
signaling by promoting the endocytosis and degradation of the TCR(17, 18). These data provide a hint that CIN85 may also negatively
regulate TCR signaling and activation.
In contrast, CIN85 promotes B cell receptor (BCR) signaling,
particularly for nuclear factor B (NF-B) activation (10). Because
B and T cells have similar Ag receptor signaling properties and both
express the same CIN85 isoform, it seems reasonable to speculatethat CIN85 might have a positive regulatory function in T cells. The
physiological function of CIN85 in T cells is still unknown, andthere are controversial findings for its function depending on thecell types studied; therefore, we investigated the precise function of
CIN85 in T cells.
Some studies suggest that CIN85 and CD2AP can compensate
for each other in terms of their expression and functions. For example, CIN85 compensates for the loss of CD2AP in CD2AP-deficient[knockout (KO)] podocytes (19, 20), and the function of CIN85 in
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T cell activation is initiated by signaling molecules downstream of the T cell receptor (TCR) that are organized byadaptor proteins. CIN85 (Cbl-interacting protein of 85 kDa) is one such adaptor protein. Here, we showed that
CIN85 limited T cell responses to TCR stimulation. Compared to activated wild-type (WT) T cells, those that lacked
CIN85 produced more IL-2 and exhibited greater proliferation. After stimulation of WT T cells with their cognate
antigen, CIN85 was recruited to the TCR signaling complex. Early TCR signaling events, such as phosphorylationof -chain–associated protein kinase 70 (Zap70), Src homology 2 (SH2) domain–containing leukocyte protein of
76 kDa (SLP76), and extracellular signal–regulated kinase (Erk), were enhanced in CIN85-deficient T cells. The inhibitory function of CIN85 required the SH3 and PR regions of the adaptor, which associated with the phosphatase suppressor of TCR signaling–2 (Sts-2) after TCR stimulation. Together, our data suggest that CIN85 is recruited
to the TCR signaling complex and mediates inhibition of T cell activation through its association with Sts-2.

SCIENCE SIGNALING | RESEARCH ARTICLE
B cell activation is partially replaced by CD2AP (21). Moreover,
CD2AP regulates posttranslational modification of CIN85 (22). Therefore, it was also important to determine the functional relationshipbetween CIN85 and CD2AP and whether these adaptors had distinct or redundant roles in T cells.
In this study, we generated a T cell–specific CIN85-KO mouse to
analyze the function of CIN85 in T cells and its relationship with
CD2AP. We found that CIN85-KO, but not CD2AP-KO, T cells
exhibited hyperresponses to TCR stimulation. Further, we demonstrated that CIN85 was recruited to the TCR signaling clusters after
stimulation and inhibited T cell activation by associating with thephosphatase suppressor of TCR signaling–2 (Sts-2). Our results suggest that CIN85 facilitates the recruitment and association of Sts-2
clusters to TCR clusters during initial TCR activation, which dampens
T cell activation.
RESULTS

Kong et al., Sci. Signal. 12, eaav4373 (2019)

Increased effector/memory-type T cells in CIN85-cKO mice
In CIN85-cKO mice, although the frequency and cellularity of T and
B cells in peripheral lymphoid organs were normal, we found thatthe proportion of total CD4+ and CD8+ T cells and effector/memory
T cells was different from WT controls. We observed an increased
frequency of CD8 T cells and a decreased frequency of CD4 T cells
in the spleen of CIN85-cKO mice as compared to WT mice (fig.
S1D). In addition, CIN85-cKO mice had an increased frequency of
CD62Llo CD44hi effector/memory-type T cells in spleen than WT
mice (Fig. 1, E and F). These data suggest that CIN85 plays a role inmaintaining the homeostasis of CD4/CD8 T cells and effector/memory
T cells.
Studies on DT40 B cells suggest that CIN85 promotes BCR signaling and its loss could be partially replaced by the CD2AP adaptor
protein (21). However, this was not the case for T cells. We foundthat CIN85 expression was not altered in CD2AP-KO T cells (fig.
S1B), which indicated that loss of CD2AP did not stimulate compensatory CIN85 expression in T cells. When we compared T cell
development in CIN85-cKO, CD2AP-cKO, and CIN85/CD2AP–
double cKO (dcKO) mice, we observed distinct patterns of thymocyte development. Although we observed moderate increases in thefrequency of SP thymocytes, the proportion of CD8+ T cells, andthe proportion of effector/memory-type T cells in the periphery in
CIN85-cKO and dcKO mice, we found no effect on thymocyte development or peripheral CD4+ T cell function in CD2AP-cKO mice
(fig. S1, C to E). These data suggest that, in T cells, CIN85 andCD2AP are not functionally redundant, because only CIN85 augments T cell development.
Hyperresponsiveness of CIN85-KO T cells to TCR stimulation
CIN85-cKO mice had increased effector/memory T cells, suggesting that CIN85-KO T cells may have enhanced T cell signaling andfunction. We examined T cell activation after TCR stimulation of
WT and CIN85-KO T cells and found that CIN85-KO T cells produced more interleukin-2 (IL-2) and proliferated more than WT
T cells (Fig. 2, A and B, and fig. S2, A and B). These data indicate that
CIN85-KO T cells are hyperresponsive to TCR stimulation. CD8
T cells were especially affected, and KO CD8+ T cells produced about
fourfold more IL-2 than WT CD8+ T cells. We further analyzed Agspecific responses using AND-Tg CIN85-cKO mice, which express
a TCR specific for a moth cytochrome c (MCC) peptide. Similar to
Ab cross-linking, peptide + Ag-presenting cell (APC) stimulation
stimulated stronger IL-2 production and enhanced cell growth in
CIN85-KO T cells compared to WT T cells (Fig. 2, C and D). In
contrast to CIN85-KO T cells, CD2AP-KO and WT T cells produced
comparable amounts of IL-2 after TCR stimulation by either Ab
cross-linking or Ag stimulation (Fig. 2, A to D, and fig. S2, A and B).
The enhanced IL-2 production by CIN85-KO T cells was also detected after secondary stimulation (fig. S2C), indicating that CIN85
would also function in the effector phase of immune responses. To
confirm that these hyperresponses were not caused by activationinduced cell death, we analyzed cell viability. Naïve T cells were stimulated with CD3 and CD28 monoclonal Abs (mAbs) for 1 day andstained with propidium iodide (PI) and CD25 mAb (fig. S2D). The
results showed that most of the cells were activated and PI-positive
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T cell–specific loss of CIN85 enhanced thymocyte maturation
To understand T cell–specific functions of CIN85, we generated
T cell–specific CIN85–conditional KO (cKO) mice. CIN85fl/y mice(10) were crossed with CD4-cre transgenic (Tg) mice, and crossbreeding of CIN85 heterozygotes produced offspring according to
the expected Mendelian ratio, which indicated that this deletion did
not result in obvious developmental defects. Immunoblot analysis
using CIN85 antibody (Ab) confirmed the loss of CIN85 protein in
both CD4 and CD8 CIN85-KO T cells (Fig. 1A).
When we examined T cell development in CIN85-cKO mice, we
found that there was no alteration in the total cellularity of thymus,
spleen, or lymph nodes (LNs) compared to control littermates (Fig. 1B).
Examination of thymic development by flow cytometry revealed amoderate increment in the frequency of both CD4 and CD8 singlepositive (SP) cells and a slight decrease in CD4 CD8 double-positive(DP) cells in thymocytes from CIN85-cKO when compared to wildtype (WT) controls. (Fig. 1, C and D). Note that CD4-cre–mediatedCIN85 depletion took place in the DP stage and did not occur in the
double-negative (DN) stage. CD24 expression in SP cells decreases
along with cell maturation. Surface staining of CD24 and TCR on
SP cells of CIN85-cKO thymocytes showed that the maturationof both CD4 and CD8SP cells was notably enhanced. We foundthat the ratio of mature (CD24lo) to immature (CD24hi) SP cells was
substantially increased in CIN85-cKO mice as compared to WT mice
(Fig. 1, C and D). These data imply that CIN85 augments with thymocyte maturation. In addition, when we examined thymocyte development using CD3 and CD69 markers, we found that the numberof CD3lo CD69lo cells (DN and preselection DP cells) and CD3int
CD69hi cells (thymocytes undergoing selection) was unchanged in
CIN85-cKO mice, whereas the CD3hi CD69hi (post-positive selection) and CD3hi CD69lo thymocytes (mature SP) were increased
compared to WT (Fig. 1, C and D). CD5 expression at the DP stage
is proportional to the TCR signal strength during positive selection,and it plays a role in thymocyte selection (23); however, CD5 expression on CIN85-cKO SP and DP cells was normal, which suggeststhat there was no difference in positive selection of CIN85-cKO andWT mice (fig. S1A). Overall, no obvious effect of CIN85 deficiency
in thymic selection was observed. Furthermore, the expression of
Bcl-2 and Bcl-xL in CIN85-cKO thymocytes was not altered compared to WT (fig. S1A); therefore, the increase of CIN85-cKO SP
cells is not due to the enhanced expression of these prosurvival mol-ecules. Collectively, these observations suggest that CIN85 plays arole in repressing thymocyte maturation but has no substantial rolein selection or survival of thymocytes.

SCIENCE SIGNALING | RESEARCH ARTICLE
A

B

E

C

F

Fig. 1. Increase of SP thymocytes and CD44hieffector/memory T cells in CIN85-cKO mice. (A) Western blot for CIN85 protein in lysates of WT and CIN85-KO T cells.
Blots are representative of three independent experiments. (B) Total cellularity of thymus, spleen, and LNs in WT and CIN85-cKO littermates. Data are means ± SD ofseven experiments. (C and D) Flow cytometry analysis of thymic T cell subsets in CIN85-cKO mice stained for the indicated markers. Dot plots including the percentage ofcells within the given gate (C) are representative of seven experiments. Quantification of % CD4 and CD8 SP thymocytes, the ratio of CD24lo (mature) to CD24hi (immature)
SP thymocytes, and % of CD69hi cells (positively selected) among CD3+ cells (D) are means ± SD from all experiments. (E and F) Flow cytometry analysis of splenic memory
T cell subsets in WT and CIN85-cKO littermates. Dot plots including the percentage of cells within the given gate (E) are representative of five experiments. Quantification of % CD4 and CD8 memory-type T cells (CD62Llo CD44hi) (F) is the mean ± SD from all experiments. *P < 0.05 and **P < 0.01, Student’s t test.

cells were very rare, although observed at similar frequency in WT,
CD2AP-KO, and CIN85-KO T cell cultures.
The hyperresponsiveness of CIN85-KO T cells could affect thedifferentiation of effector T cells. To examine this possibility, CD4naïve T cells were stimulated and differentiated into T helper 1(TH1), TH2, or TH17 cells under appropriate skewing conditions
and then restimulated and analyzed for cytokine production. As expected, few T cells produced cytokines in the control TH0 cells. In
T cells under TH1-skewed conditions, we found that a higher proportion of CIN85-KO T cells produced interferon- (IFN-) than WT
T cells. In contrast, we found that WT and CIN85-KO T cells generated a comparable frequency of IL-4+ and IL-17+ T cells when
cultured under TH2- and TH17-skewed conditions, respectively(Fig. 2E and fig. S2E). Thus, in addition to enhanced IL-2 producKong et al., Sci. Signal. 12, eaav4373 (2019)tion and cell growth, TH1 differentiation was also enhanced in theabsence of CIN85.
CIN85 inhibits TCR signaling
Because CIN85-KO T cells are hyperresponsive to TCR stimulation, we analyzed TCR signaling intermediates. We found that thephosphorylation of -chain–associated protein kinase 70 (Zap70),
which is one of the earliest events after TCR stimulation, was more
pronounced in CIN85-KO T cells than in WT T cells (Fig. 3, A to D,
and fig. S3A). In addition, phosphorylation of other TCR signaling
molecules, SLP76 (SH2 domain–containing leukocyte protein of
76 kDa), PLC (phospholipase C), and Erk (extracellular signal–
regulated kinase), was also significantly enhanced upon TCR stimulation when compared to WT T cells (Fig. 3, A to D, and fig. S3A).
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D

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A

C

B

D

Fig. 2. CIN85-KO T cells showed hyperresponsiveness to TCR stimulation. (A and B) Naïve (CD44lo CD62Lhi) CD8
T cells isolated from WT, CD2AP-cKO, and CIN85-cKO mice were stimulated as indicated with Abs against CD3 andCD28 or with phorbol 12-myristate 13-acetate (PMA) plus ionomycin. Supernatants were analyzed by enzyme-linked
immunosorbent assay (ELISA) for IL-2 production after 24 hours (A), and proliferation was assessed at 46 hours (B).
Data are means ± SD pooled from four independent experiments performed in triplicate. OD450, optical density at
450 nm. (C and D) Naïve CD4 AND-Tg T cells were stimulated with MCC peptide–pulsed irradiated splenocytes from
B10.BR mice, and IL-2 production (C) and proliferation (D) were measured as in (A) and (B). Data are means ± SD
pooled from three independent experiments performed in triplicate. (E) Flow cytometry analysis of CD4 WT/CIN85KO T cells cultured for 6 days under skewing conditions for TH0 (neutral), TH1, and TH2 cells and then restimulated for6 hours with Ab against CD3. Dot plots including the % of cytokine-positive cells are representative of four independent experiments. *P < 0.05 and **P < 0.01, Student’s t test.

The total protein abundance of these molecules was not altered. In
contrast, the phosphorylation of CD3 and Lck (lymphocyte-specificprotein tyrosine kinase), the most proximal phosphorylation eventsin TCR signaling, was not affected by the loss of CIN85 (Fig. 3, C
and D, and fig. S3A). Collectively, these data demonstrated that
TCR signaling molecules downstream of Zap70 were enhanced in
CIN85-KO T cells after TCR activation, which implies that CIN85
inhibits proximal TCR signaling.
No detectable contribution of CIN85 in TCR internalization
CIN85 participates in Cbl-mediated internalization/degradationof RTKs (11–16). CIN85 forms a ternary complex with Cbl andendophilin, which mediates ligand-dependent internalization of RTKs
Kong et al., Sci. Signal. 12, eaav4373 (2019)

SH3 and/or PR regions are
responsible for the inhibitory
function of CIN85
To identify the molecular basis of CIN85mediated inhibition of T cell signaling,
we assessed CIN85 structure-function
relationships using truncation mutants
lacking different domains: SH3B, SH3C,
PR, SR, and CC, respectively (Fig. 4A).
These internal ribosomal entry site
(IRES)–green fluorescent protein (GFP) fusion constructs, which
also had a FLAG-tag at the N terminus, were retrovirally transfected
into CIN85-KO T cells, and IL-2 production was then measured
after TCR stimulation (Fig. 4B). We confirmed that CIN85-KO
T cells (empty vector control) had an enhanced IL-2 response to TCR
stimulation, and this enhancement was rescued to the level of WT
T cells by expression of either full-length CIN85 or the CIN85SR mutant. However, other truncated mutants lacking either SH3 (SH3B
and SH3BC) or PR (PR) regions failed to correct this overproduction of IL-2 by CIN85-KO T cells (Fig. 4B). These results strongly suggested that CIN85 inhibited TCR signaling through both SH3
and PR regions and that these regions are required for its inhibitory
function.
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(11, 12). Because our data showed that
CIN85 inhibits T cell signaling and function, it was possible that CIN85 promoted
TCR internalization/degradation, similar to its function in receptor internalization in nonimmune cells. Thus, weinvestigated whether TCR internalization
after TCR stimulation was affected by
CIN85 deficiency. We confirmed thatthe surface TCR abundance before stimulation was equivalent in WT and CIN85KO T cells and found that about 10 to
50% of the surface TCR in both WT and
CIN85-KO CD8 T cells was internalized
during 5 to 30 min after stimulation byanti-TCR cross-linking (fig. S3B). When
AND-Tg T cells were stimulated with
Ag peptide and APC, the surface TCR
abundance was similarly decreased in
WT and CIN85-KO T cells. We observed
maximum internalization of 25% of surface TCRs at 90 min (fig. S3C). When
we analyzed the efficiency of receptor
degradation in cells treated with cycloheximide for 1 hour and then stimulatedwith Abs against CD3/CD28, we foundthat the amount of CD3 detected byWestern blot was similar in WT and
CIN85-KO T cells (fig. S3D). These results suggest that CIN85 is dispensablefor TCR internalization and degradation and that the inhibitory function of
CIN85 in T cell activation is not due toits effect on TCR internalization anddegradation.

SCIENCE SIGNALING | RESEARCH ARTICLE
peptide abundance found in cells that
expressed CIN85 to cells that expressed
CIN85PR, we could identify proteins
with stronger binding to CIN85 than
CIN85PR (ratio > 1) and vice versa(ratio < 1). The analysis identified known
CIN85-binding proteins, including Cbl,
CapZ, and CD2AP in both unstimulated
and stimulated T cells (fig. S4, A and B).
In addition, we observed CIN85 interacBtion with a novel potential candidate Sts-2
in stimulated T cells (fig. S4B). Sts-2 isa cytoplasmic phosphatase that binds to
Cbl and inhibits Zap70 activation and
TCR signaling (24). MS analysis revealed
that Sts-2 associates with CIN85 but notwith CIN85PR and that this association
occurred only after T cell stimulation.
The TCR stimulation–induced assoC
ciation of CIN85 with Sts-2 was further
confirmed by biochemical analysis. 2D12
T hybridoma cells were transfected with
FLAG-tagged CIN85 and stimulated with
Abs against CD3 and CD28, and FLAGor His (control) immunoprecipitates wereanalyzed by Western blot (Fig. 4C). We
found that cCbl and Sts-2 associated with
CIN85 in a TCR activation–dependent
manner. Because Sts-1 has high aminoDacid sequence homology (75%) to Sts-2
and much stronger phosphatase activity than Sts-2 (24–27), we also tested theassociation of Sts-1 with CIN85. However,
we could not detect any specific association between CIN85 and Sts-1 in thepull-down analysis (Fig. 4C). These results
Fig. 3. CIN85 suppressed phosphorylation of early TCR downstream signal molecules. (A and B) Western blot
confirmed the results of MS analysis—for phosphorylated TCR signaling molecules in lysates of WT and CIN85 KO T cells after stimulation with Abs againstthat CIN85 associates with Sts-2 but not
CD3 and CD28 for the indicated times. Blots (A) are representative of three to seven independent experiments.
Sts-1.
Quantified data (B) are means ± SEM of all experiments. a.u., arbitrary units. (C and D) Flow cytometry analysis ofphosphorylated TCR signaling molecules in WT and CIN85-KO CD8 T cells after stimulation with Abs against CD3We determined which domains ofand CD28 for the indicated times. Histograms of staining in WT (black) and CIN85-KO (red) T cells at each time point
CIN85 were necessary for its association(C) are representative of four to eight independent experiments. Quantified values are means ± SEM from all experiwith Sts-2 in 2D12 cells transfected withments. *P < 0.05 and **P < 0.01, Student’s t test.
the four CIN85-truncated mutants, i.e.,SH3B, SH3BC, PR, and SR. AlCIN85 inhibits TCR signaling by recruiting thethough Sts-2 coimmunoprecipitated with CIN85 or CIN85SR afphosphatase Sts-2
ter TCR stimulation, we found no association with the other CIN85
We identified CIN85-interacting partners to further clarify the mutants, SH3B, SH3BC, or PR (Fig. 4D). Control immunopremechanism of CIN85-mediated inhibition of T cell activation. Be- cipitation confirmed the presence of each truncated or WT CIN85.
cause of our functional reconstitution results (Fig. 4B), we investi- Furthermore, immunoprecipitation of the lysates from normal T cells
gated the proteins that bind to WT and CIN85SR but not to the transduced with these same constructs confirmed that CIN85, but
SH3B, SH3BC, and PR mutants by immunoprecipitation and not CIN85PR, associated with endogenous Sts-2 in T cells after
liquid chromatography–tandem mass spectrometry (LC-MS/MS) anal- TCR stimulation (Fig. 4E). These results indicated that CIN85 assoysis. CIN85PR was used for comparison because it did not rescue ciates with Sts-2 through the SH3 and PR domains.
the hyperresponsiveness of CIN85-KO T cells, and nontransfected
CIN85-KO T cells were used as the background control. Among the CIN85 colocalizes in TCR microclusters and enhances
list of associated proteins detected, we selected the CIN85PR- and recruitment of Sts-2 to dampen TCR activation
CIN85-interacting proteins with quantity values at least twofold higher After Ag recognition, TCR microclusters (MCs) form at the interthan those of the background control, and each value was normalized face between T cell and APC, which contain TCR, kinases, and adapto that of CIN85 in each sample. When we compared the ratio of the tors to initiate T cell activation signals (28–31). CIN85 may functionA

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Fig. 4. Critical domains of CIN85 for its inhibitory function and its association of Sts-2 upon TCR stimulation. (A) Schematic of WT CIN85, which contains two SH3(SH3B and SH3C) domains, a PR domain, a SR domain, and a CC domain, and the indicated deletion mutant constructs, which include an N-terminal FLAG-tag. (B) ELISA
analysis of IL-2 production by WT or CIN85-KO T cells retrovirally transduced with CIN85 mutants or GFP vector control and restimulated with Abs against CD3 and CD28,
as indicated. Data are means ± SD from four experiments performed in triplicate. (C) Coimmunoprecipitation analysis of proteins associated with CIN85 in lysates of 2D12
T cell hybridoma cells transduced with FLAG-CIN85 and stimulated with Abs against CD3 and CD28. Blots (left) are representative of three independent experiments.
Quantified band intensity values (right) are means ± SD from all experiments. (D and E) Coimmunoprecipitation analysis of CIN85 association with Sts-2 in lysates of 2D12
T cell hybridoma (D) and mouse CD8 T cells (E) transduced with the indicated FLAG-tagged CIN85 mutant and stimulated with Abs against CD3 and CD28. Blots are
representative of at least two independent experiments. *P < 0.05 and **P < 0.01, Student’s t test.

as an adaptor protein to create nodes for the recruitment of signaling proteins into the TCR-MCs to direct the signals toward inactivation. The spatial and temporal dynamics of CIN85 were analyzed
in CIN85-GFP–transduced AND-Tg T cells on a supported planar
bilayer containing glycosylphosphatidylinositol (GPI)–anchored I-Ek
pulsed with MCC peptide and intercellular adhesion molecule–1
Kong et al., Sci. Signal. 12, eaav4373 (2019)(ICAM-1). The images of CIN85-GFP and TCR stained with a
DyLight 549–conjugated Fab fragment specific for TCR (H57) werecollected by total internal reflection fluorescence (TIRF) microscopy. TCR-MCs were generated at the contact region during the earlystage of activation and then translocated to the center to form
the cSMAC (28, 32). CIN85-GFP formed clusters colocalizing with
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TCR-MCs at the early stage of TCR stimulation, but later, they did
not accumulate into the cSMAC and most of them disappeared(Fig. 5A), as do other signaling molecules such as Zap70 and SLP76(28). As early as 2 min after stimulation, almost all of the CIN85
clusters colocalized with TCR-MCs (Fig. 5A, right bars). Similarly,
dynamic movement of Sts-2–GFP was analyzed. Sts-2 formed clus-

ters after TCR-MCs were generated, which were partly (about 30%
of TCR-MCs and 60% of Sts-2 clusters) colocalized with TCR-MCs,
and then translocated into the cSMAC (Fig. 5B). Simultaneous
analysis of CIN85-GFP and Sts-2–Halo revealed that around 85%
of Sts-2–Halo clusters were colocalized with CIN85-GFP clusters in
the early phase of activation (Fig. 5C, left). Meanwhile, CIN85-GFP
B

A

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C

D

E

Fig. 5. CIN85 formed clusters at TCR MCs and enhanced recruitment of Sts-2 clusters to dampen TCR activation signals. (A and B) TIRF microscopy analysis of ANDTg T cells transduced with CIN85-GFP (A) or Sts-2–GFP (B) and stimulated on a supported lipid bilayer containing I-Ek with MCC peptide. Images (left) of GFP-tagged
protein and TCR at early (top, 1 to 2 min) and later (bottom, 8 to 12 min) times after membrane interaction are representative of 24 cells from four independent experiments (A) and 21 cells from three independent experiments (B). Quantification of GFP and TCR colocalization clusters per cell at 2 min (right) are pooled from all experiments. (C) TIRF microscopy analysis of AND-Tg T cells cotransfected with CIN85-GFP and Sts-2–Halo tag and stimulated on an MCC–I-Ek–containing supported lipid bilayer
at the indicated times. Images (top) and fluorescent intensity profiles between white arrows in the merge images (bottom) are representative of 17 cells from three independent experiments. Quantification of GFP and Halo colocalized clusters per cell at 2 min (middle) are pooled from all experiments. (D) TIRF microscopy analysis of
CIN85-cKO/AND-Tg T cells transduced with Sts-2–GFP stimulated on an MCC–I-Ek–containing supported lipid bilayer early after membrane interaction. Images (top) arerepresentative of three independent experiments. Quantification of Sts-2–GFP and TCR colocalized clusters and the ratio of colocalized clusters among Sts-2 clusters(bottom) are means ± SEM of 18 to 24 cells from all experiments. (E) ELISA analysis of IL-2 production by WT, Sts-2–cKO, CIN85-cKO, and CIN85/Sts-2–dKO T cells at
24 hours after stimulation as indicated with Abs against CD3 and CD28. Data are means ± SD pooled from four independent experiments. Scale bars, 2.5 m. *P < 0.05,
Student’s t test.
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SCIENCE SIGNALING | RESEARCH ARTICLEDISCUSSION

The present study aimed to elucidate the role of CIN85 in T cell
activation and functions. Despite extensive studies on CIN85 over a
decade (2–5), its function remains nebulous because of its various
properties in different cell types. CIN85 hinders receptor kinase endocytosis in nonimmune cells, whereas it also promoted NF-B activation in B cells (10, 33). Our analysis using T cell–specific CIN85-KO
mice unexpectedly revealed that CIN85 restricts TCR signaling andT cell function. CIN85 deficiency induced hyperresponsiveness of
peripheral T cells in terms of cell growth and IL-2 production, as
well as enhanced TH1 differentiation and increased numbers ofeffector/memory T cells. CIN85-KO T cells showed hyperresponses
in early TCR signaling, i.e., enhanced phosphorylation of Zap70,
SLP76, and other downstream molecules such as PLC and Erk;
however, CD3 and Lck were unaffected, which indicates that CIN85
inhibits events downstream of Zap70 in TCR signaling.
CIN85 and CD2AP have strong amino acid sequence homology
and similar structural features. However, it was unclear whether
CIN85 has any physiological relationship with CD2AP in T cellfunction. Because both CIN85 and CD2AP bind to Cbl and theCIN85/CD2AP-Cbl complex restricts EGFR signaling through receptor endocytosis, it was proposed that CIN85 and CD2AP might
have similar functions in T cells. CD2AP deficiency causes hyperreKong et al., Sci. Signal. 12, eaav4373 (2019)

sponsiveness to Ag stimulation and augmented cell growth by preventing cSMAC formation necessary for TCR degradation (18).
Accordingly, we postulated that CIN85-KO and CD2AP-KO T cells
would have a similar phenotype. In this regard, it was interesting toknow whether CIN85 and CD2AP are compensatory in their T cellfunctions. However, in our analysis, CD2AP-KO T cells were not
hyperresponsive to TCR stimulation, in contrast to CIN85-KO T cells.
Furthermore, simultaneous deletion of CIN85 and CD2AP in T cells
did not cause additional difference in T cell function or development compared to those seen in CIN85-cKO T cells. Most T cell
development and function is normal in CD2AP-KO mice, which isconsistent with our results, and the development of a small subset offollicular helper T cells after infection is increased in CD2AP-KOmice (34). However, our analysis of TCR signaling clusters after
stimulation on a planar bilayer revealed that there was no difference
between WT and CD2AP-KO T cells in the generation of TCR-MCs
and the cSMAC. Therefore, unlike previous reports (18, 35), CD2AP
plays no obvious role in T cell function or activation, whereas CIN85
serves as a negative regulator of T cell function.
We postulated that the augmented response of CIN85-KO T cells
might be attributed to defective TCR endocytosis and degradation,a high survival rate, or impaired inhibition of TCR signaling. Although CIN85 limits receptor internalization in nonimmune cells(11–13), we found no defect in either TCR internalization or degradation, nor did we find enhanced activation-induced cell death in
CIN85-KO T cells after TCR stimulation. The enhanced phosphorylation of TCR-proximal signaling molecules such as Zap70, SLP76,
PLC, and Erk in CIN85-KO T cells showed that CIN85 has an inhibitory effect on early TCR signaling. Because CIN85 is an adaptor
protein, we postulated that the enhanced phosphorylation could be
mediated by recruiting negative effector molecule(s).
CIN85 has both SH3 domains and a PR region that can specifically interact with proteins containing PR regions and SH3 domains,respectively. Analysis of a series of CIN85 mutants revealed that theSH3 and PR regions were critical for the inhibitory function of
CIN85 in T cells. The PR region contains multiple PxxP motifs that
could possibly bind to internal SH3 domains (36). The fact that the
CIN85 mutants lacking both SH3 and PR regions could not rescue
the hyperresponsiveness of CIN85-KO T cells might be due to thestructural conformation of CIN85 with its inter- or intramolecular
contacts between SH3 and PR regions (36).
We identified several molecules that bind to CIN85 in T cells by
MS analysis. Among these candidates, the phosphatase Sts-2 was particularly interesting because of its specific binding to CIN85 upon
TCR stimulation and its inhibitory function. Sts-2 associated morewith WT CIN85 than the CIN85 mutant lacking the PR region
upon TCR stimulation, and the association was activation dependent. Although many proteins have been found to bind with CIN85
in various cell types, Sts-2 had not been reported. This may be because Sts-2 is predominantly expressed in T cells but not in Helacells (37) or B cells (38).
Sts-2 limits TCR signaling (24), and Sts-2 contains singlenucleotide polymorphisms that are highly associated with several
autoimmune diseases such as celiac disease, rheumatoid arthritis,and diabetes (39–45). There are many functional similarities between
CIN85-KO and Sts-2–KO T cells, such as hyperphosphorylation of
Zap70, increased TH1 differentiation and numbers of effector T cells(24), and elevated cytokine responses. In addition, both CIN85 andSts-2 can bind Cbl (46). These similarities, together with the specific
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and Sts-2–Halo were not colocalized in the later phase (Fig. 5C,
right). These data suggested that CIN85 transiently recruits and colocalizes with Sts-2 during early activation.
To further understand the dynamic relationship between CIN85
and Sts-2, Sts-2–GFP was expressed in CIN85-KO T cells and the
dynamics were similarly analyzed. Sts-2 clusters were generated
similarly in the absence of CIN85 (Fig. 5D, top). Statistical analysis
revealed that the number of TCR-MCs and Sts-2 clusters was similar in WT and CIN85-KO T cells, whereas the Sts-2 clusters thatcolocalized with TCR-MCs were significantly reduced in CIN85KO T cells (Fig. 5D, bottom). These data suggested that CIN85 facilitates the recruitment of Sts-2 clusters to TCR-MCs.
Because Cbl is a known binding partner of CIN85, we also analyzed
the dynamics of cCbl. cCbl-GFP generated MCs upon TCR stimulation that colocalized with TCR-MCs and Sts-2–GFP clusters (fig.
S5A). We confirmed the molecular assembly of CIN85-cCbl complexes by immunoprecipitation for cCbl-GFP in the lysate of 2D12
cells. Immunoprecipitation also showed that cCbl-GFP associatedwith endogenous CIN85 and Sts-2 in a TCR stimulation–dependent
manner. These data are consistent with the results of the FLAG-CIN85
immunoprecipitation MS analysis (fig. S4B) and suggest that Cbl isincluded in the transient CIN85–Sts-2 complex in TCR-MCs.
Last, we performed functional analysis of CIN85-cKO, Sts-2–KO,
and CIN85/Sts-2–dKO (double knockout) T cells. Cytokine production by naïve CD4 and CD8 T cells from each mouse was measured (Fig. 5E). Especially in naïve CD8 T cells, the amount of IL-2
produced by Sts-2–KO T cells was greater than that produced by
CIN85-KO T cells. However, dKO T cells produced the most IL-2.
However, during secondary activation of effector T cells, the loss of
CIN85 promoted greater IL-2 production than the loss of Sts-2 in
CD4 T cells (fig. S5C), whereas dKO effector T cells still produced
the most IL-2, similar to naïve T cells (Fig. 5E). Collectively, thesedata indicated that CIN85 and Sts-2 cooperatively reduced IL-2
production after T cell activation.

SCIENCE SIGNALING | RESEARCH ARTICLE
binding of Sts-2 with CIN85 after TCR stimulation, implied thatthese two molecules may function cooperatively in downstream TCR
signaling. CIN85/Sts-2–dKO T cells produced more IL-2 than CIN85KO and Sts-2–KO T cells after activation, which indicated thatboth synergistic cooperation of CIN85 and Sts-2 as well as CIN85independent Sts-2 functions inhibit T cell activation.
Sts-2 associated with CIN85 through the SH3 and PR regions of
CIN85, and its binding was essential for inhibitory functions.
CIN85 constitutively binds cCbl through its SH3 domain (11), andour results in T cells showed that the association of cCbl and CIN85
was increased by TCR activation and reduced by loss of the CIN85
PR domain, which similarly decreased association with Sts-2. We
think that all these possible binding partners may form a molecularcomplex; CIN85 may directly bind both cCbl and Sts-2, cCbl may
mediate Sts-2 binding to CIN85, or Sts-2 may mediate cCbl binding
to CIN85. Imaging analysis showed that CIN85, cCbl, and Sts-2 accumulated together within TCR-MCs during early T cell activation.
Therefore, these results suggest that a negative feedback mechanism through the association of CIN85, Sts-2, and Cbl may generate
an “inhibitory complex” that regulates the inhibitory function in T cell
activation.
The cellular function of CIN85 appears to be cell type–specific,
probably depending on the binding partners present in different cells.
For example, CIN85 binds to SLP65/BLNK expressed in B cells, but
not to SLP76 expressed in T cells (21). This difference may result in
the functional differences of CIN85 between B and T cells. Such differences have also been observed in humans. where CIN85-deficientpatients have defects in B cell activation and Ab production but not
in T cells (47). Cell type–specific function was also revealed in thymocyte development, where CIN85 associates with pre-TCR tomediate signals (48).
Specific blockade of the inhibitors of TCR signaling could induce
enhanced T cell immunity, as in the case of Csk (49). Our finding
suggests that CIN85 restricts T cell function only after TCR stimulation and not during the resting stage. Therefore, targeting CIN85
or its associated proteins like Sts-2 may be an untapped therapeuticstrategy to promote T cell immunity in diseases like cancer.

alyzed using FlowJo software (Tree Star). Fluorophore Abs against
CD4 (RM4-5), CD8a (53-6.7), CD24 (M1/69), TCR (H57-597), CD3
(145-2C11), CD69 (H1.2F3), CD5 (53-7.3), CD45R (RA3-6B2),
CD62L (MEL-14), CD44 (IM7), CD11b (M1/70), CD11c (N418),
NK1.1 (PK136), TCR (GL3), CD25 (PC61), Ly-6G (Gr-1), andCD49b (DX5) were purchased from BD Biosciences, BioLegend,
or eBioscience. Intracellular staining with Abs against Bcl-2 and
Bcl-xL was conducted following the manufacturer’s protocol (BDPharmingen).

MATERIALS AND METHODS

Western blot
T cells were left either unstimulated or stimulated by 2C11 and PV-1
Abs followed by cross-linking with goat anti-hamster (GAH) immunoglobulin G (IgG) Ab at 37°C for the indicated times and lysed with
1% NP-40– or Triton X-100–containing lysis buffer. The cell lysates
were immunoprecipitated or boiled at 95°C for 5 min in SDS sample buffer. The samples were separated by SDS–polyacrylamide gelelectrophoresis, and the transferred membrane was blotted by reacting with the indicated Abs and developed with enhanced chemiluminescence assay according to the manufacturer’s procedure (Pierce).
For Western blotting, anti-CIN85 Ab was developed by K.K. (RIKEN,
Japan). The anti-CD2AP (Santa Cruz Biotechnology, sc-9137), actin,
FLAG (M1) (Sigma-Aldrich, F3040), phospho-Zap70 (Cell Signaling
Technology, 2701s), phospho-PLC (Cell Signaling Technology,
2821s), phospho-Erk (Cell Signaling Technology, 9101s), SLP76 (Cell
Signaling Technology, 4958), phospho-SLP76 (GeneTex, 300076),
Zap70 (BD Biosciences, 610239), PLC (BD Biosciences, 610027),
Erk (Promega, V114A), Sts-2 (Proteintech, 15823-1-AP), Sts-1(Proteintech, 19563-1-AP), and cCbl (Cell Signaling Technology,8447) Abs were purchased.

Flow cytometry
Cell sorting and analysis were performed using FACSAria and
FACSCalibur (BD Biosciences), respectively, and the data were anKong et al., Sci. Signal. 12, eaav4373 (2019)

Helper T cell differentiation and intracellular
staining of cytokinesNaïve (CD4+ CD62Lhi CD44lo CD25− NK1.1−) T cells were collected from spleens and LNs and sorted using FACSAria. For both TH1
and TH2 cells, T cells were stimulated with plate-bound anti-CD3
(10 g/ml) (145-2C11) and anti-CD28 (10 g/ml) (PV-1) Abs,
whereas TH17 cells were stimulated with plate-coated 2C11 (10 g/ml)
and anti-CD28 (5 g/ml) (clone 37.51) Abs. The medium was supplemented with IL-12 (10 ng/ml; PeproTech) and anti–IL-4 Ab(10 g/ml) for TH1 cells; IL-4 (10 ng/ml; PeproTech) for TH2 cells;
and transforming growth factor– (TGF) (3 ng/ml; R&D Systems),
IL-6 (20 ng/ml; PeproTech), IL-1 (20 ng/ml; PeproTech), anti–
IL-4 (10 g/ml), and anti–IFN- Abs (10 g/ml) for TH17 cells.
On day 7, the cells were restimulated with plate-bound 2C11 andPV-1 Abs in the presence of 2 M monensin (Wako, Osaka, Japan)
for 6 hours. The restimulated cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100 permeabilizing
buffer, and then stained with fluorophore Abs specific to cytokines,
i.e., anti-mouse IL-4, IFN- (XMG1.2), and IL-17A (TC11-18H10).
Abs were purchased from BD Biosciences and eBioscience. Cells
were analyzed using FACSCalibur.

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Mice and cells
CIN85-floxed and CD2AP-KO mice on the C57BL/6 (B6) backgroundwere generated by T.K. (RIKEN, Japan). CIN85-floxed mice have beenpreviously described (10), and CD2AP-floxed mice were designedwith the LoxP sites flanking the same sites of cd2ap as shown in (35).
To generate T cell–specific CIN85/CD2AP-KO mice, CD4-cre-Tg
mice were crossed with the flox-CIN85/CD2AP mice. Sts-2–KO mice
were from N.C. and were crossed with CIN85-floxed mice. C57BL/6,
B10.A, and B10.BR mice were purchased from CLEA Japan Inc. All
mice were maintained under specific pathogen–free conditions, andall the animal experiments were conducted in accordance with RIKEN
Animal Research Committee–approved protocols. The 2D12 T cellhybridoma cell line expressing the AND-Tg TCR was established byfusing AND-Tg CD4+ T cells (specific for MCC 88-103 on major
histocompatibility complex class II I-Ek) (50–52).

ELISA and proliferation assay
T cells were stimulated with plate-bound Abs against CD3 (1452C11) and CD28 (PV-1) for 24 hours; then, the culture supernatant
was analyzed for IL-2 production by ELISA (BD Biosciences). Cell
proliferation was measured using a Cell Counting Kit-8 (CCK-8)
assay kit (Dojindo Laboratories) after 48 hours of T cell stimulation.

SCIENCE SIGNALING | RESEARCH ARTICLE
Intracellular staining of phospho-proteins
CD4+ or CD8+ naïve (CD44lo) T cells were stimulated by TCR crosslinking, fixed with 2% PFA, permeabilized with pre-cold methanol,
blocked with 1% bovine serum albumin in phosphate-buffered saline (PBS), and stained with fluorophore-conjugated Abs specificfor phospho-proteins. The Abs used in this study are as follows:anti–phospho-CD247 (BD Phosflow, 558489), phospho-Lck (BDPhosflow, 558577), phospho-Zap70 (BD Phosflow, 557817), phosphoSLP76 (BD Phosflow, 558438), phospho-Erk (BD Phosflow, 561991),
phospho–c-Jun N-terminal kinase (JNK) (BD Phosflow, 562481),
and goat anti-rabbit IgG (H+L) secondary Ab (Thermo Fisher
Scientific, 65-6120). Cells were analyzed using FACSCalibur.

Plasmid construction and transduction
CIN85 complementary DNA was obtained by polymerase chain reaction (PCR) and subcloned into the pMX-IRES-hCD8 retroviral
expression vector. GFP was added to the C terminus of CIN85 andsubcloned into the pMX retroviral vector (provided by T. Kitamura,Tokyo University, Japan). The constructs of mutant CIN85 (CIN85SH3B,
CIN85SH3BC, CIN85PR, and CIN85SR) were created by PCR.
The mutant CIN85 constructs were cloned into pMX-FLAG-IRESGFP to generate in-frame fusion with FLAG peptide (1 kDa) at the
N terminus.
For retrovirus infection, the expression vectors were transfected
into Platinum-E packaging cells (provided by T. Kitamura, Tokyo
University) with Lipofectamine 2000 (Invitrogen) and VSV-G(vesicular stomatitis virus glycoprotein) retroviral vector. Viral supernatants were collected and concentrated by centrifugation at 8000g(4°C) overnight. One day after T cells or AND-Tg T cells were stimulated with plate-bound anti-CD3 (10 g/ml) and anti-CD28(10 g/ml) Abs or irradiated B10.BR splenocytes with 3 M MCC
mutant (K99A) peptide, the cells were resuspended into the concentrated retroviral supernatants in the presence of polybrene(10 g/ml) (Sigma). T cell hybridomas were retrovirally transfected similarly without stimulation. Transfectants were sorted byfluorescence-activated cell sorting on the basis of expression of theselection markers.

MS analysis
Proteins in the eluate were digested by trypsin/Lys-C mix for 18 hours
at 37°C. Peptides were injected onto a PicoFrit emitter (New Objective, MA, USA) and then separated with an Eksigent ekspert
nanoLC 400 HPLC system. Peptides eluting from the column wereanalyzed on a TripleTOF 5600+ mass spectrometer for both shotgunMS and SWATH-MS analyses (53, 54). All shotgun-MS files weresearched against mouse UniProt Swiss-Prot database (September
2015 release) using ProteinPilot software v.4.5 with the Paragon algorithm for protein identification (55). The SWATH-MS data files
were then annotated using the expanded protein/peptide library generated above using PeakView v.2.2. Proteins in the protein/peptide
library were quantified from summed peak areas (56).
Supported planar bilayer system and imaging analysis
GPI-anchored proteins of mouse I-Ek and ICAM-1 were transfected
into and purified from Chinese hamster ovary or baby hamster kidney cells and then incorporated into dioleoylphosphatidylcholine
liposomes (DOPS; Avanti Polar Lipids) (57). Planar bilayer formation was carried out on a glass slip with 0.16-mm thickness. Planar
bilayers were then loaded with MCC peptide in citrate buffer (pH 4.5)
overnight at 37°C, followed by blocking with 5% nonfat dried milk
in PBS at 37°C. Imaging analysis on planar bilayers was performed on
an IX81 microscope (Olympus, Japan) with a TIRF objective lens
(UAPON 100×/NA1.49OTIRF, 1.49 numerical aperture) (Olympus,
Japan) with dual excitation lasers: Ar 488-nm laser and DPSS 561-nm
laser, connected to a charge-coupled device camera (Hamamatsu
Photonics), were used. Images were taken every 10 s.
SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/12/567/eaav4373/DC1
Fig. S1. Subpopulations of thymocytes and splenocytes in CIN85 and/or CD2AP-cKO mice.
Fig. S2. CIN85-KO T cells showed hyperresponsiveness to TCR stimulation.
Fig. S3. CIN85 is not involved in TCR internalization.
Fig. S4. Analysis of CIN85-binding molecules by MS.
Fig. S5. Sts-2 partially colocalized with TCR-MCs.

REFERENCES AND NOTES

Immunoprecipitation
The 2D12 T cell hybridomas expressing FLAG-CIN85 or its mutants
with and without TCR stimulation (cross-linking with anti-CD3,Kong et al., Sci. Signal. 12, eaav4373 (2019)

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TCR internalization assay
Whole splenocytes were stained with phycoerythrin (PE)–conjugatedanti-TCR (PE-H57) on ice and were stimulated with prewarmed
GAH Ab (5 g/ml) at 37°C for the indicated duration. For Ag stimulation, B cells were purified from B10.BR mice by using MicroBeads[Mouse CD45R (B220), Miltenyi Biotec] and activated with lipopolysaccharide (LPS) (1 g/ml) for 24 hours with MCC peptide on
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TCR internalization (%) = [(mean fluorescence intensity (MFI) of
acid-treated sample at each time − MFI of acid-treated sample attime 0)/(MFI of the non–acid-treated sample at each time)] × 100.

anti-CD28, and GAH Abs) were lysed and treated with anti–FLAGM2 Ab-conjugated magnetic beads (Sigma-Aldrich, M8823). Anti–
His-tag Ab-conjugated magnetic beads (Sigma-Aldrich, H9914)
were used as the negative control. For immunoblotting, the precipitated proteins were released from the beads by boiling in SDS sample
buffer. For MS analysis, the precipitated proteins were released fromthe magnetic beads using the elution buffer by rotating for 20 min
at room temperature. The Sts-2–GFP– or cCbl-GFP–expressing 2D12
T cell hybridomas were activated and lysed as mentioned above,and the immunoprecipitations were done using anti-GFP Abconjugated magnet beads (MBL, D153-11).

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Kong et al., Sci. Signal. 12, eaav4373 (2019)

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Acknowledgments: We are grateful to T.K. and N.C. for providing KO mice; T. Kitamura forvectors and cells; T. Imanishi and M. E. S. Badr for technical advice; M. Unno and
W. Kobayashi for technical support; and M. Yoshioka and H. Yamaguchi for secretarial
assistance. Funding: This work was supported by a Grant-in-Aid for Scientific Research fromJapan Society for the Promotion of Science KAKENHI (S 24229004 to T.S.). Author
contributions: T.S., M.S.K., and A.H.-T. designed experiments. M.S.K., A.H.-T., M.S., Y.K., and R.O.
performed experiments. T.Y., O.O., and K.K.P. provided help/advice. K.K., T.K., and N.C. provided
materials. T.S., M.S.K., and A.H.-T. wrote the manuscript. Competing interests: The authors
declare that they have no competing interests. Data and materials availability: The raw data
files of the LC-MS/MS analyses have been deposited in the ProteomeXchange Consortium
through the jPOST partner repository (http://jpostdb.org) with the dataset identifier
PXD012369. A material transfer agreement (MTA) was required to obtain the CIN85-KO andCD2AP-KO mice from T.K. and the Sts-2–KO mice from N.C. An MTA was also required to obtainthe 2D12 hybridoma T cells from T.S. All other data needed to evaluate the conclusions in thepaper are present in the paper or the Supplementary Materials.
Submitted 16 September 2018
Accepted 21 January 2019
Published 5 February 201910.1126/scisignal.aav4373
Citation: M. S. Kong, A. Hashimoto-Tane, Y. Kawashima, M. Sakuma, T. Yokosuka, K. Kometani,
R. Onishi, N. Carpino, O. Ohara, T. Kurosaki, K. K. Phua, T. Saito, Inhibition of T cell activation andfunction by the adaptor protein CIN85. Sci. Signal. 12, eaav4373 (2019).

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Inhibition of T cell activation and function by the adaptor protein CIN85
Mei Suen Kong, Akiko Hashimoto-Tane, Yusuke Kawashima, Machie Sakuma, Tadashi Yokosuka, Kohei Kometani, Reiko
Onishi, Nick Carpino, Osamu Ohara, Tomohiro Kurosaki, Kia Kien Phua and Takashi Saito

Sci. Signal. 12 (567), eaav4373.
DOI: 10.1126/scisignal.aav4373

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Thou shalt not CIN
The ubiquitously expressed adaptor protein CIN85 has distinct roles in different cell types. This adaptor inhibits theactivation of the receptor tyrosine kinases EGFR and PDGFR but promotes B cell receptor signaling. To determine its
function in T cells, Kong et al. compared the activation of mouse T cells lacking CIN85 or the highly related adaptor
CD2AP. Only loss of CIN85 augmented T cell growth and IL-2 production. CIN85 associated with the inhibitory
phosphatase Sts-2 and promoted its recruitment to T cell receptor microclusters after activation. These data define apreviously unknown inhibitory interaction, which could be targeted to augment T cell function in cancer and immunity.

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