MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 3, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0014

Immunohistochemical Analysis of the Harbor Porpoise
Using Antipodoplanin Antibody PMab-237

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Shinji Yamada,1 Shunsuke Itai,1 Takuro Nakamura,1 Junko Takei,1 Masato Sano,1Satoru Konnai,2,3 Atsushi Kobayashi,4 Shotaro Nakagun,5Yoshiyasu Kobayashi,5 Mika K. Kaneko,1 and Yukinari Kato1,6Podoplanin (PDPN)/T1 alpha is known as a specific marker of lymphatic endothelial cells and type I alveolarcells. Sensitive and specific monoclonal antibodies (mAbs) for PDPN are needed for immunohistochemical
analyses. Recently, we developed an anticetacean PDPN mAb, PMab-237. Herein, immunohistochemical
analyses showed that PMab-237 strongly detected pulmonary type I alveolar cells, renal podocytes, andlymphatic endothelial cells of the harbor porpoise. These findings suggest that PMab-237 may be useful for
immunohistochemical analyses for cetacean tissues.
Keywords: cetacean podoplanin, PDPN, lymphatic endothelial cells, PMab-237

Introduction

Materials and Methods
Immunohistochemical analyses

Podoplanin (PDPN)/T1alpha is a type I transmembrane
glycoprotein.(1) PDPN is expressed in several normalcells, including renal epithelial cells, pulmonary type I alveolarcells, and lymphatic endothelial cells.(1) PDPN is used todistinguish lymphatic endothelial cells from vascularendothelial cells in pathophysiological studies.(2) C-type
lectin-like receptor-2 (CLEC-2) was previously reported as
an endogenous receptor of PDPN in our studies.(3,4) ThePDPN–CLEC-2 interaction facilitates the separation of embryonic blood and lymphatic vessels.(5) The expression ofhuman PDPN (hPDPN) has been reported in many malignant
cancers, such as oral cancers,(6) lung cancers,(7) and esophagealcancers.(8) The expression of hPDPN is associated with cancer
metastasis.(9,10) We previously developed monoclonal antibodies (mAbs) against human,(11) mouse,(12) rat,(13) rabbit,(14)bovine,(15) dog,(16) cat,(17) pig,(18) and horse(19) PDPNs. Furthermore, we developed an anticetacean PDPN mAb, PMab237 using Cell-Based Immunization and Screening (CBIS)
method.(20) In this study, we performed immunohistochemical
analyses using PMab-237 against the harbor porpoises (Phocoena phocoena).(21)Cetacean tissues were collected from two harbor porpoises,
and were fixed in 15% neutral buffered formalin at Obihiro
University of Agriculture and Veterinary Medicine (Hokkaido, Japan). Histological sections (4-mm thick) were directly autoclaved in EnVision FLEX Target Retrieval
Solution High pH (Agilent Technologies, Inc., Santa Clara,CA) for 20 minutes. Then, sections were blocked using theSuperBlock T20 (PBS) Blocking Buffer (Thermo Fisher
Scientific, Inc., Waltham, MA), incubated with PMab-237(5 mg/mL) for 1 hour at room temperature, and treated with theEnvision+Kit for mouse (Agilent Technologies, Inc.) for 30minutes. Color was developed using 3,3¢-diaminobenzidine
tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
Results and Discussion

Although we have established many anti-PDPN mAbs,
such as antihuman,(11) antimouse,(12) antirat,(13) antirabbit,(14)antibovine,(15) antidog,(16) anticat,(17) antipig,(18) and1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,Sapporo, Japan.
3
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
4
Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
5
Laboratory of Veterinary Pathology, Department of Basic Veterinary Medicine, Obihiro University of Agriculture and Veterinary
Medicine, Obihiro, Japan.
6
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2104

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FIG. 1. Immunohistochemical analyses for cetacean lung. Cetacean lung sections were incubated with 5 mg/mL of PMab237 (A, B) or with blocking buffer (C, D), followed by that with the Envision+Kit. (E, F) HE staining. Scale bar = 100 mm.
HE, hematoxylin and eosin.

FIG. 2. Immunohistochemical analyses for cetacean kidney. Cetacean kidney sections were incubated with 5 mg/mL of PMab237 (A, B) or with blocking buffer (C, D), followed by that with the Envision+Kit. (E, F) HE staining. Scale bar = 100 mm.
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YAMADA ET AL.

FIG. 3. Immunohistochemical analyses for cetacean colon. Cetacean colon sections were incubated with 5 mg/mL ofPMab-237 (A, B) or with blocking buffer (C, D), followed by that with the Envision+Kit. (E, F) HE staining. Scalebar = 100 mm.
antihorse(19) PDPNs, cetacean PDPN was not recognizedby those anti-PDPN mAbs (data not shown). Then, twomice were recently immunized with cetacean PDPNexpressing CHO-K1 (CHO/wPDPN) cells using CBIS method.(20) Wells positive for CHO/wPDPN and negative for CHOK1 were selected by flow cytometry. PMab-237 (IgG1, kappa)
was finally selected. Western blot analysis performed using
PMab-237 demonstrated that PMab-237 detects wPDPN as a40-kDa band in CHO/wPDPN cells. Furthermore, the immunohistochemical analyses revealed that PMab-237 strongly
stained CHO/wPDPN cells, whereas it did not react with CHOK1 cells, suggesting that PMab-237 might be applicable for
immunohistochemical analyses against cetacean tissues.
In this study, we performed immunohistochemical analyses against the harbor porpoise. PDPN has been reported
to be expressed in several tissues, including lung, kidney,and colon(1); we selected those tissues for immunohistochemical analyses. Expectedly, PMab-237 stained type I
alveolar cells of lung (Fig. 1A, B), podocytes of kidney(Fig. 2A, B), and lymphatic endothelial cells of colon(Fig. 3A, B). These staining patterns are consistent with
our previous immunohistochemical studies using several
anti-PDPN mAbs.(18,22) No staining was observed without
primary antibodies against type I alveolar cells of lung(Fig. 1C, D), podocytes of kidney (Fig. 2C, D), and lymphatic endothelial cells of colon (Fig. 3C, D).
Our previous studies showed that PMab-237 cross-reactedwith human, bovine, goat, sheep, alpaca, and tiger PDPNs,

which were overexpressed in CHO-K1 cells.(20) PMab-237
also reacted with hPDPN, which was overexpressed in a
glioblastoma cell line, LN229 (data not shown). Then, we investigated the reactivity of PMab-237 with hPDPN-expressing
cancer cell lines, such as a glioblastoma cell (LN319), a lungsquamous cell carcinoma cell line (PC-10), and a malignant
mesothelioma cell line (NCI-H226) or hPDPN-expressing
normal cells, such as lymphatic endothelial cells, a kidney
epithelial cell (HEK-293T), and a mesothelial cell line (Met5A) by flow cytometry. However, PMab-237 did not react any
endogenous PDPN by flow cytometry (data not shown), indicating that only overexpressed hPDPN might be detected byPMab-237. Those results demonstrated that PMab-237 could
be specific for cetacean PDPN.
In conclusion, PMab-237 is useful for immunohistochemical analyses against the harbor porpoise. The epitopeof PMab-237 needs further investigation to clarify thesensitivity and specificity of PMab-237 against cetacean
PDPN. PMab-237 could be useful in elucidating the pathophysiological functions of cetacean PDPN in futurestudies.
Acknowledgments

This research was supported, in part, by AMED underGrant Nos. JP18am0101078 (Y.K.), JP18am0301010 (Y.K.),
and JP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant
No. 17K07299 (M.K.K.) and Grant No. 16K10748 (Y.K.).

PMAB-237 AGAINST CETACEAN PDPN
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 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: March 3, 2019
Accepted: April 30, 2019