Biochemistry and Biophysics Reports 18 (2019) 100631

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Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

Establishment of a monoclonal antibody PMab-233 forimmunohistochemical analysis against Tasmanian devil podoplanin

TYoshikazu Furusawaa,b,c, Shinji Yamadaa, Shunsuke Itaia,d, Takuro Nakamuraa, Junko Takeid,
Masato Sanoa, Hiroyuki Haradad, Masato Fukuic, Mika K. Kanekoa, Yukinari Katoa,b,∗a

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapancZENOAQ RESOURCE CO., LTD., 1-1 Tairanoue, Sasagawa, Asaka-machi, Koriyama, Fukushima, 963-0196, Japand
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku,
Tokyo, 113-8510, Japanb

A R T I C LE I N FO

A B S T R A C T

Keywords:
Tasmanian devil podoplanin
PDPN
PMab-233

Monoclonal antibodies (mAbs) against not only human, mouse, and rat but also rabbit, dog, cat, bovine, pig, andhorse podoplanins (PDPNs) have been established in our previous studies. PDPN is used as a lymphatic endothelial cell marker in pathological diagnoses. However, mAbs against Tasmanian devil PDPN (tasPDPN),
which are useful for immunohistochemical analysis, remain to be developed. Herein, mice were immunized with
tasPDPN-overexpressing Chinese hamster ovary (CHO)-K1 (CHO/tasPDPN) cells, and hybridomas producing
mAbs against tasPDPN were screened using ﬂow cytometry. One of the mAbs, PMab-233 (IgG1, kappa), speciﬁcally detected CHO/tasPDPN cells by ﬂow cytometry and recognized tasPDPN protein by western blotting.
Furthermore, PMab-233 strongly detected CHO/tasPDPN cells by immunohistochemistry. These ﬁndings suggestthat PMab-233 may be useful as a lymphatic endothelial cell marker of the Tasmanian devil.

1. Introduction
Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in many cell types, including lymphatic endothelial cells [1].
Therefore, PDPN is extremely useful to distinguish lymphatic endothelial cells from vascular endothelial cells in pathological diagnoses[2]. We previously reported that C-type lectin-like receptor-2 (CLEC-2)
is an endogenous receptor of PDPN [3,4]. Importantly, the PDPN-CLEC2 interaction has been shown to facilitate the separation of embryonicblood and lymphatic vessels [5]. The expression of human PDPN
(hPDPN) has been reported in several malignant tumors, including
malignant brain tumors [6–9], oral squamous cell carcinomas [10],pulmonary cancers [11], esophageal cancers [12], malignant mesotheliomas [13,14], osteosarcomas [15–17], chondrosarcomas [16],and testicular tumors [18]. The expression of hPDPN is associated withcancer metastasis and malignant progression [4,6,19]. To date, we havedeveloped monoclonal antibodies (mAbs) against not only human [20]but also mouse [20], rat [21], rabbit [22], bovine [23], dog [24], cat[25], pig [26], and horse [27] PDPNs. Furthermore, an anti-cat PDPN
mAb (PMab-52) cross-reacted with a tiger PDPN [28], and an anti-

bovine PDPN mAb (PMab-44) cross-reacted with goat [29], sheep [30],and alpaca [31] PDPNs. However, anti-Tasmanian devil PDPN
(tasPDPN) mAb has not yet been reported. In this study, we immunized
mice with CHO/tasPDPN cells and established hybridomas that could
produce mAbs against tasPDPN.
2. Materials and methods
2.1. Cell lines and animals
CHO-K1 and P3X63Ag8U.1 (P3U1) cells were obtained from theAmerican Type Culture Collection (Manassas, VA, USA). The synthesized DNA of tasPDPN (accession No. XM_012545641.2) bearing an Nterminal PA16 tag (PA16-tasPDPN) was inserted into a pCAG-Ble vector(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) [32]. The
PA16 tag comprises 16 amino acids (GLEGGVAMPGAEDDVV) [33]. The
CHO-K1 cells were transfected with pCAG-Ble vector containing PA16tasPDPN using the Lipofectamine® LTX and Plus™ reagent (Thermo
Fisher Scientiﬁc Inc., Waltham, MA, USA). Stable transfectants wereselected by limiting dilution and cultivated in a medium containing∗
Corresponding author. Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi,
980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).

https://doi.org/10.1016/j.bbrep.2019.100631
Received 25 February 2019; Received in revised form 25 March 2019; Accepted 25 March 20192405-5808/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/BY/4.0/).

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Y. Furusawa, et al.

Fig. 1. Schematic illustration of the Cell-Based Immunization andScreening (CBIS) method. Stable transfectants expressing the protein of interest are used as an immunogen with no puriﬁcation procedure. The selectionof hybridomas secreting speciﬁc mAbs is performed by ﬂow cytometry using
parental and transfectant cells.

0.5 mg/mL of Zeocin (InvivoGen, San Diego, CA, USA).
The P3U1, CHO-K1, CHO/tasPDPN, CHO/hPDPN [34], CHO/mouse
PDPN (mPDPN) [34], CHO/rat PDPN (rPDPN) [21], CHO/rabbit PDPN
(rabPDPN) [22], CHO/dog PDPN (dPDPN) [24], CHO/bovine PDPN
(bovPDPN) [23], CHO/cat PDPN (cPDPN) [25], CHO/pig PDPN
(pPDPN) [26], CHO/horse PDPN (horPDPN) [32], CHO/tiger PDPN
(tigPDPN) [28], CHO/alpaca PDPN (aPDPN) [31], CHO/bear PDPN
(bPDPN) [26], CHO/goat PDPN (gPDPN) [29], CHO/sheep PDPN
(sPDPN) [30], and CHO/whale PDPN (wPDPN) [26] were cultured in a
Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque,Inc., Kyoto, Japan), which was supplemented with 10% of heat-inactivated fetal bovine serum (Thermo Fisher Scientiﬁc Inc.), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.). The cells were grown in an incubator at 37 °C with humidity and 5% CO2 and 95% air atmosphere.
Female BALB/c mice (6 weeks of age) were purchased from CLEA Japan(Tokyo, Japan). The animals were housed under speciﬁc pathogen-free
conditions. The Animal Care and Use Committee of Tohoku University
approved all animal experiments.

Fig. 2. Detection of tasPDPN by ﬂow cytometry using PMab-233. CHO/
tasPDPN and CHO-K1cells were treated with PMab-233 (red line) or anti-PA16
tag (NZ-1; blue line) at a concentration of 1 μg/mL or 0.1% BSA in PBS (gray)for 30 min, followed by incubation with secondary antibodies.

these spleen cells were fused with P3U1 cells using PEG1500 (Roche
Diagnostics, Indianapolis, IN, USA), and the hybridomas were grown inan RPMI medium supplemented with hypoxanthine, aminopterin, andthymidine for selection (Thermo Fisher Scientiﬁc Inc.). The culture
supernatants were screened by ﬂow cytometry.
2.3. Flow cytometry
The cells were harvested following a brief exposure to 0.25% trypsin
and 1 mM ethylendiaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.).
The cells were washed with 0.1% bovine serum albumin (BSA) in
phosphate-buﬀered saline (PBS) and treated with primary mAbs for
30 min at 4 °C. Thereafter, the cells were treated with Alexa Fluor 488conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc.,
Danvers, MA, USA) or Oregon Green anti-rat IgG (1:2000; Thermo
Fisher Scientiﬁc Inc.). Then, ﬂuorescence data were collected using theSA3800 Cell Analyzers (Sony Corp., Tokyo, Japan).

2.2. Hybridoma production
We employed a Cell-Based Immunization and Screening (CBIS)
method [25,33,35,36] to develop sensitive and speciﬁc mAbs against
tasPDPN. Brieﬂy, two BALB/c mice were immunized with CHO/
tasPDPN cells (1 × 108) intraperitoneally (i.p.) together with the Imject
Alum (Thermo Fisher Scientiﬁc Inc.). The procedure included three
additional immunizations, followed by a ﬁnal booster injection administered ip. 2 days prior to the harvest of spleen cells. Subsequently,

2.4. Determination of binding aﬃnity by ﬂow cytometry
CHO/tasPDPN was suspended in 100 μL of serially diluted PMab233. Then, Alexa Fluor 488-conjugated anti-mouse IgG (1:200; Cell
Signaling Technology, Inc.) was added. Fluorescence data were2

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Y. Furusawa, et al.

Fig. 3. Cross-reaction of PMab-233 to PDPNs of the other species by ﬂow cytometry. CHO-K1 cells transfected with PDPNs of the other species were treated with
PMab-233 (red line) or each positive control (blue line) at a concentration of 1 μg/mL or 0.1% BSA in PBS (gray) for 30 min, followed by incubation with secondary
antibodies.

2.5. Western blottingcollected using the EC800 Cell Analyzer (Sony Corp.). The dissociation
constant (KD) was calculated by ﬁtting the binding isotherms to built-in
one-site binding models in the GraphPad PRISM 6 (GraphPad Software,Inc., La Jolla, CA, USA).

Cell lysates (10 μg) were boiled in a sodium dodecyl sulfate (SDS)
sample buﬀer (Nacalai Tesque, Inc.). The proteins were electrophoresed
on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical
Corporation) and subsequently transferred onto a polyvinylidene
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Y. Furusawa, et al.

Japan) and processed to make 4-μm thick paraﬃn-embedded cell sections that were directly autoclaved in a citrate buﬀer (pH 6.0; Nichirei
Biosciences, Inc., Tokyo, Japan) for 20 min. These tissue sections were
blocked using the SuperBlock T20 (PBS) Blocking Buﬀer (Thermo
Fisher Scientiﬁc Inc.), incubated with PMab-233 (1 μg/mL) for 1 h at
the room temperature, and then treated with the Envision + Kit(Agilent Technologies Inc.) for 30 min. Color was developed using 3,3′diaminobenzidine tetrahydrochloride (Agilent Technologies Inc.) for
2 min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
3. Results and discussion
Most cancers are somatic in origin, and only a few transmissible
cancers have been documented [37]. Transmissible cancers have beenreported only in natural cases, such as canine transmissible venereal
tumor in dogs [38] or devil facial tumor disease in Tasmanian devils[39]. Tasmanian devils (Sarcophilus harrisii) are endangered owing to
the emergence of two clonally transmissible cancers: devil facial tumor
disease 1 (DFT1) and devil facial tumor disease 2 (DFT2). DFT1 andDFT2 are infectious diseases that spread via biting [40]. DFT1 was ﬁrst
discovered in northeastern Tasmania in 1996 and has since then spreadto more than 80% of the area across the island, causing a signiﬁcantdecrease in the population [41]. DFT2 was discovered in 2014 and iscurrently restricted to a small region of southeastern Tasmania [42].
Although we had previously developed mAbs against human [20],mouse [20], rat [21], rabbit [22], bovine [23], dog [24], cat [25], pig[43], and horse [27] PDPNs, mAbs against tasPDPN has not yet beendeveloped. The development of anti-tasPDPN mAbs will enable us toperform pathophysiological studies about the lymphatic metastasis or
lymphangiogenesis.
In the present study, we employed the CBIS method to develop
sensitive and speciﬁc mAbs against tasPDPN to facilitate the immunohistochemical analysis of paraﬃn-embedded tissue sections. Twomice were immunized with CHO/tasPDPN cells using an immunizationand screening procedure (Fig. 1). The developed hybridomas wereseeded into 96-well plates and cultivated for 9 days. Wells positive for
CHO/tasPDPN and negative for CHO-K1 were selected by ﬂow cytometry. The screening approach identiﬁed strong signals from CHO/
tasPDPN cells and weak or no signals from CHO-K1 cells in 19 of the
960 wells (2.0%). After limiting dilution of 19 wells, we developed nineclones. One of these nine clones, PMab-233 (IgG1, kappa), was ﬁnallyselected via immunohistochemistry against the paraﬃn-embedded
sections of CHO/tasPDPN cell.
PMab-233 recognized CHO/tasPDPN cells, but showed no reaction
with CHO-K1 cells, as assessed by ﬂow cytometry (Fig. 2). PMab-233
did not react with human, mouse, rat, rabbit, dog, bovine, cat, pig,
horse, tiger, alpaca, bear, goat, sheep, or whale PDPNs (Fig. 3), whichindicates that PMab-233 is speciﬁc to tasPDPN. The identity of PDPN
amino acid sequence between tasPDPN and PDPNs of the other species
is shown as below: 45% (vs. hPDPN), 41% (vs. mPDPN), 38% (vs.
rPDPN), 36% (vs. rabPDPN), 45% (vs. dPDPN), 35% (vs. bovPDPN),
43% (vs. cPDPN), 39% (vs. pPDPN), 47% (vs. horPDPN), 44% (vs.
tigPDPN), 49% (vs. aPDPN), 44% (vs. bPDPN), 39% (vs. gPDPN), 35%(vs. sPDPN), and 43% (vs. wPDPN).
In addition, kinetic analysis conducted by ﬂow cytometry was employed to assess the interaction of PMab-233 with CHO/tasPDPN cells.
KD of PMab-233 for CHO/tasPDPN cells was determined to be
1.1 × 10−6, indicating a low aﬃnity of PMab-233 for CHO/tasPDPN
cells.
Western blotting performed using PMab-233 (Fig. 4) demonstrated
that PMab-233 detects tasPDPN as a 40-kDa band in CHO/tasPDPN
cells. NZ-1, an anti-PA16 tag mAb also detected a 40 kDa band. The
immunohistochemical analyses revealed that PMab-233 strongly
stained CHO/tasPDPN cells (Fig. 5A) and did not react with CHO-K1
cells (Fig. 5B). No staining was observed without primary antibodies

Fig. 4. Western blotting. Cell lysates of CHO-K1 and CHO/tasPDPN (10 μg)
were electrophoresed and transferred onto PVDF membranes. The membranes
were incubated with l μg/mL of PMab-233, anti-PA16 tag (NZ-1), or anti-β-actin
and subsequently, with peroxidase-conjugated anti-mouse or -rat IgG.

Fig. 5. Immunohistochemical analyses. Cell sections of CHO/tasPDPN (A, C)
and CHO-K1 (B, D) were incubated with 1 μg/mL of PMab-233 (A, B) or withblocking buﬀer (C, D), followed by that with the Envision + Kit. Scalebar = 100 μm.

diﬂuoride (PVDF) membrane (Merck KGaA, Darmstadt, Germany).
After blocking with 4% skim milk (Nacalai Tesque, Inc.), each membrane was incubated with primary mAbs, including 1 μg/mL of PMab233, 1 μg/mL of anti-PA16 tag (NZ-1), or 1 μg/mL of anti-β-actin (AC15; Sigma-Aldrich Corp., St. Louis, MO, USA), and subsequently withperoxidase-conjugated anti-mouse IgG (1:1000; Agilent Technologies,
Santa Clara, CA, USA) or anti-rat IgG (1:10000; Sigma-Aldrich Corp.).
The developed bands were visualized with the ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC
Co. Ltd., Tokyo, Japan).
2.6. Immunohistochemical analyses
Cell blocks were produced using iPGell (Genostaﬀ Co., Ltd., Tokyo,
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Y. Furusawa, et al.

(Fig. 5C). These results cumulatively indicate that PMab-233 is usefulfor the detection of tasPDPN by immunohistochemistry.
In conclusion, we established an mAb, PMab-233, against tasPDPN,
which is suitable for use in ﬂow cytometry, Western blotting, and immunohistochemical analyses. The epitope of PMab-233 needs further
investigation to clarify the sensitivity and speciﬁcity of PMab-233
against tasPDPN. We believe that PMab-233 should prove to be usefulin elucidating the pathophysiological functions of tasPDPN in futurestudies.

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Conﬂicts of interest
Y.K. received research funding from ZENOAQ RESOURCE CO., LTD.
The other authors have no conﬂict of interest.
Acknowledgments
We thank Miyuki Yanaka, Kayo Hisamatsu, Saori Handa, andYoshimi Nakamura. for their excellent technical assistance. This research was supported in part by AMED under Grant Numbers:
JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), and JP18ae0101028(Y.K.), and by JSPS KAKENHI Grant Number 17K07299 (M.K.K.) and
Grant Number 16K10748 (Y.K.).
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