MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 5, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0033

Detection of Tiger Podoplanin Using the Anti-Cat
Podoplanin Monoclonal Antibody PMab-52

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Shinji Yamada,1 Shunsuke Itai,1,2 Yoshikazu Furusawa,1,3,4 Masato Sano,1 Takuro Nakamura,1Miyuki Yanaka,1 Saori Handa,1 Kayo Hisamatsu,1 Yoshimi Nakamura,1 Masato Fukui,4 Hiroyuki Harada,2Takuya Mizuno,5 Yusuke Sakai,6 Satoshi Ogasawara,7 Takeshi Murata,7 Hiroaki Uchida,8Hideaki Tahara,8 Mika K. Kaneko,1 and Yukinari Kato1,3

Podoplanin (PDPN) is expressed in type I alveolar cells of lung but not in type II alveolar cells. PDPN is also
known as a specific lymphatic endothelial cell marker because PDPN is not expressed in vascular endothelial
cells. PDPNs of several animals have been characterized using specific anti-PDPN monoclonal antibodies(mAbs): PMab-1, PMab-2, PMab-32, PMab-38, PMab-44, and PMab-52 for mouse, rat, rabbit, dog, bovine, andcat PDPNs, respectively. In this study, we investigated the possible crossreaction between these anti-PDPN
mAbs and tiger PDPN. Flow cytometry and western blot analyses revealed that the anti-cat PDPN mAb PMab52 (IgM, kappa) reacted with tiger PDPN, which is overexpressed in Chinese hamster ovary-K1 cells. Using
immunohistochemical analysis, type I alveolar cells of the tiger lung were strongly detected by PMab-52. These
results indicate that PMab-52 may be useful for the detection of tiger PDPN.
Keywords: tiger podoplanin, PDPN, PMab-52
IntroductionP

odoplanin (PDPN) is a specific marker for lymphatic
endothelial cells and type I alveolar cells of the lung.(1,2)
Therefore, anti-PDPN monoclonal antibodies (mAbs) areuseful in distinguishing lymphatic from vascular endothelial
cells or type I from type II alveolar cells of the lung. We
characterized the PDPNs of several animals using specificanti-PDPN mAbs, such as anti-mouse (PMab-1),(3) anti-rat(PMab-2),(4) anti-rabbit (PMab-32),(5) anti-dog [PMab-38(6)and PMab-48(7)], anti-bovine (PMab-44),(8) and anti-cat(PMab-52).(9) Furthermore, we developed many anti-human
PDPN mAbs, including anti-pan human PDPN mAbs, such as
NZ-1.2,(3) LpMab-7,(10,11) LpMab-10,(12) LpMab-13,(13) and
LpMab-17(14); anti-glycopeptide mAbs (GpMabs), such as
LpMab-3,(10) LpMab-9,(10) LpMab-12,(15) LpMab-19,(16)and LpMab-21(17,18); and cancer-specific mAbs (CasMabs),
such as LpMab-2(10,19) and LpMab-23.(20,21)
PDPN of all species is a type I transmembrane sialoglycoprotein that induces platelet aggregation through theC-type lectin-like receptor-2 (CLEC-2) of platelets.(22) It
comprises three platelet aggregation-stimulating (PLAG)
domains, termed PLAG1–3 (EDxxVTPG sequence).(2) Previously, we have shown that PLAG3 is the most important
domain for platelet aggregation by human PDPN.(2,23,24)
Recently, PLAG4 (EDxxT sequence) was reported to be theother critical sequence for the PDPN–CLEC-2 interaction.
However, PLAG4 may be categorized as ‘‘PLAG-like domain’’ because the original definition of PLAG domain is‘‘EDxxVTPG’’ sequence.(25) These functional assays havebeen performed using their original anti-PDPN mAbs, thebinding affinity of which may differ. Therefore, the mostcritical domain for the PDPN–CLEC-2 interaction remains
controversial. Cancer-specific anti-PDPN mAbs should be
used for PDPN-targeted therapy(10,19–21,26) because PDPN isexpressed in many healthy tissues.
Until now, anti-tiger PDPN mAbs have not been reported.
Although tiger tumors have been investigated in several
studies, the presence of tiger PDPN in cancers or type I alveolar cells of the lung has not been investigated.(27–33)

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,
Tokyo, Japan.
3
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
4
ZENOAQ RESOURCE CO., LTD., Koriyama, Japan.
5
Laboratory of Molecular Diagnostics and Therapeutics, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
6
Laboratory of Veterinary Pathology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan.
7
Department of Chemistry, Graduate School of Science, Chiba University, Inage, Japan.
8
Project Division of Cancer Biomolecular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
2224

TIGER PDPN DETECTED USING PMAB-52

In the present study, we investigated the potential crossreaction between our anti-PDPN mAbs for many species andtiger PDPN using flow cytometry, western blot, and immunohistochemical analyses.
Materials and Methods
Cell line

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Chinese hamster ovary (CHO)-K1 cell line was purchasedfrom the American Type Culture Collection (ATCC; Manassas, VA). CHO-K1 and transfectants were cultured in
RPMI medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientific, Inc., Waltham, MA), 100 U/mL of
penicillin, 100 mg/mL of streptomycin, and 25 mg/mL ofamphotericin B (Nacalai Tesque, Inc.) at 37C in a humidified atmosphere of 5% carbon dioxide and 95% air.

225

autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences,
Inc., Tokyo, Japan) for 20 minutes. After blocking with SuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher Scientific, Inc.), sections were incubated with PMab-52 (0.5 mg/mL)
for 1 hour at room temperature and treated using Envision+ Kit(Agilent Technologies, Inc.) for 30 minutes. Next, color wasdeveloped using 3,3-diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed with Hematoxylin (FUJIFILM
Wako Pure Chemical Corporation).
Results and Discussion

We previously developed a mouse anti-cat PDPN mAb(PMab-52: IgM, kappa) using the Cell-Based Immunization

Overexpression of tiger PDPN in CHO-K1 cells

The synthesized DNA of tiger PDPN (accession no.:XM_007083790.2) plus the N-terminal LP tag (NSVTGIRIEDLPTSES), recognized by an anti-LP tag mAb [LpMab17(14)], was subcloned into a pCAG-Neo vector (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan). Plasmids
were transfected using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific, Inc.). Stable transfectants
were selected by limiting dilution and cultivated in a medium
containing 0.5 mg/mL of G418 (Nacalai Tesque, Inc.).
Flow cytometry

Cells were harvested after brief exposure to 0.25% trypsin/
1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai
Tesque, Inc.). After washing with phosphate-buffered saline(PBS) containing 0.1% bovine serum albumin, the cells weretreated with PMab-52 (1 or 10 mg/mL) for 30 minutes at 4C,
followed by treatment with Alexa Fluor 488-conjugated antimouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data were acquired using Cell
Analyzer SA3800 (Sony Corp., Tokyo, Japan).
Western blot analysis

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). The proteins were electrophoresed using 5%–20% polyacrylamide gels (FUJIFILM
Wako Pure Chemical Corporation) and transferred onto a
polyvinylidene difluoride membrane (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai
Tesque, Inc.), the membrane was initially incubated with 1 mg/
mL of PMab-52 or anti-b-actin (clone AC-15; Sigma-Aldrich,
Corp., St. Louis, MO) and subsequently with peroxidaseconjugated anti-mouse IgG (Agilent Technologies, Inc., Santa
Clara, CA; 1:1000 diluted) and developed using ImmunoStar
LD (FUJIFILM Wako Pure Chemical Corporation) and a
Sayaca-Imager (DRC, Co., Ltd., Tokyo, Japan).
Immunohistochemical analyses

Tiger lungs were collected at autopsy in Yamaguchi University, fixed in 10% neutral-buffered formalin, and processed routinely to make paraffin-embedded tissue sections.
Histological sections (4-mm thick) of tiger lungs were directly

FIG. 1. Detection of tiger PDPN using PMab-52. (A) Flowcytometry. CHO/cPDPN and CHO/tigPDPN cells weretreated with PMab-52 at a concentration of 1 mg/mL (blue) or
10 mg/mL (red) for 30 minutes at 4C, followed by the addition of secondary antibodies. (B) Western blot with PMab52. Cell lysates (10 mg) were electrophoresed and transferred
onto a PVDF membrane. After blocking, the membrane wasinitially incubated with 1 mg/ml of PMab-52 or anti-b-actin(AC-15) and subsequently with peroxidase-conjugated antimouse IgG. CHO, Chinese hamster ovary; cPDPN, cat
PDPN; IgG, immunoglobulin G; PDPN, podoplanin; PVDF,
polyvinylidene difluoride; tigPDPN, tiger PDPN.

226(PMab-1),(3) anti-rat (PMab-2),(4) anti-rabbit (PMab-32),(5)anti-dog [PMab-38(6) and PMab-48(7)], and anti-bovine(PMab-44),(8) did not react with CHO/tigPDPN (data notshown). Western blot analysis revealed that PMab-52 also
detected specific bands of CHO/cPDPN and CHO/tigPDPN(Fig. 1B). These results indicate that PMab-52 is useful forthe detection of tiger PDPN. Subsequently, we investigatedthe expression of tiger PDPN in the tiger lung. Previously, it
was shown that PMab-52 reacted with type I alveolar cells of
the feline lung.(9) Similarly, PMab-52 strongly stained type I
alveolar cells of the tiger lung in a membrane-staining pattern(Fig. 2A, B).
In conclusion, PMab-52 is useful for the detection of tiger
PDPN using flow cytometry, Western blot, and immunohistochemical analyses. Further studies are necessary to showthat PMab-52 is able to detect tiger PDPN in other healthytiger tissues or tiger cancers, such as mesotheliomas.(33)

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and Screening method.(9) PMab-52 specifically detects cat
PDPN using flow cytometry(9) and successfully recognizes cat
PDPN in feline squamous cell carcinomas.(34) Furthermore, aseries of deletion or point mutants of cat PDPN were utilized toinvestigate the binding of PMab-52 epitopes using flow cytometry and western blotting.(35) The PLAG4 of cat PDPN was
identified as a critical epitope of PMab-52. Comparison ofamino acid sequences revealed that 99% homology is observed
between the tiger and cat PDPNs (Supplementary Fig. S1).
Therefore, in this study, we investigated the potential reaction
between PMab-52 and tiger PDPN.
We initially produced a tiger PDPN-stable transfectant
using CHO-K1 cells plus an N-terminal LP tag, recognizedby an anti-LP tag mAb (LpMab-17). Flow cytometry revealed that PMab-52 reacted with CHO/cat PDPN (cPDPN)
and CHO/tiger PDPN (tigPDPN) in a dose-dependent manner(Fig. 1A). The other anti-PDPN mAbs, namely, anti-mouse

YAMADA ET AL.

FIG. 2. Immunohistochemical analyses using tiger lung tissues. Histological sections of the tiger lung were directly
autoclaved in citrate buffer for 20 minutes. After blocking, sections were incubated with 0.5 mg/mL of PMab-52 (A, B) or
control phosphate-buffered saline (C, D), followed by detection using Envision+ Kit. (E, F) Hematoxylin and Eosin
staining. Scale bar = 100 mm.

TIGER PDPN DETECTED USING PMAB-52
Acknowledgments

This research was supported in part by AMED under Grant
Nos.: JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), andJP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant Nos.
17K07299 (M.K.K.) and 16K10748 (Y.K.). This work waspartially performed by the Grant for Joint Research Project of
the Institute of Medical Science, the University of Tokyo.
Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: August 30, 2018
Accepted: September 29, 2018