MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 6, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0044

Anti-Horse Podoplanin Monoclonal Antibody PMab-219
is Useful for Detecting Lymphatic Endothelial Cells
by Immunohistochemical Analysis

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Yukinari Kato,1,2 Shinji Yamada,1 Shunsuke Itai,1 Atsushi Kobayashi,3 Satoru Konnai,4,5 and Mika K. Kaneko1

Podoplanin (PDPN) is expressed in lymphatic endothelial cells, where it induces platelet aggregation through C-type
lectin-like receptor-2 (CLEC-2). This protein has been characterized for a number of animal species using specificanti-PDPN monoclonal antibodies (mAbs). We recently established the mAb against horse PDPN (horPDPN) named
PMab-219. Therefore, in this study, we investigated whether PMab-219 can detect lymphatic endothelial cells inhorse tissues. Immunohistochemical analysis demonstrated that PMab-219 strongly stained lymphatic endothelial
cells in horse colon tissues, indicating that it will be useful for investigating the function of horPDPN in these cells.
Keywords: horse podoplanin, PDPN, PMab-219

IntroductionHuman podoplanin expression has been reported inseveral malignant tumors, including oral squamouscell carcinomas,(1) esophageal cancers,(2) lung cancers,(3) andmalignant mesotheliomas,(4,5) and is associated with malignant progression and cancer metastasis.(6–8) However, PDPN
is also expressed in normal tissues, including lymphatic endothelial cells,(9,10) where it helps to facilitate embryonicblood/lymphatic vessel separation by interacting with C-type
lectin-like receptor-2 (CLEC-2) on the platelets.(6,9,11–17)
Monoclonal antibodies (mAbs) against human,(18)mouse,(18) rat,(19) rabbit,(20) bovine,(21) dog,(22) cat,(23) andhorse(24,25) PDPNs have been developed, and an anti-cat
PDPN mAb (PMab-52) cross-reacted with tiger PDPN.(26) The
anti-horse PDPN (anti-horPDPN) mAb PMab-219 has beenshown to be useful in flow cytometry, Western blot, and immunohistochemical analyses.(25) Therefore, in this study, weinvestigated whether PMab-219 can detect lymphatic endothelial cells in horse tissues.
Materials and Methods
Immunohistochemical analyses

Normal horse tissues were collected after autopsy at Hokkaido
University (Hokkaido, Japan) fixed in 10% neutral-bufferedformalin, and processed routinely to prepare paraffin-embedded
tissue sections. These sections were directly autoclaved incitrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan)
for 20 minutes, blocked using SuperBlock T20 (PBS) Blocking
Buffer (Thermo Fisher Scientific, Inc., Waltham, MA) for 15
minutes, incubated with PMab-219 (10 mg/mL) for 1 hour atroom temperature, and then treated using the Envision+ Kit(Agilent Technologies, Inc., Santa Clara, CA) for 30 minutes.
Color was developed with 3,3¢-diaminobenzidine tetrahydrochloride (Agilent Technologies, Inc.) for 2 minutes, andcounterstaining was performed with hematoxylin (FUJIFILM
Wako Pure Chemical Corporation, Osaka, Japan).
Results and Discussion

We recently developed a Cell-Based Immunization andScreening (CBIS) method to produce sensitive and specificmAbs against membrane proteins, such as CD44,(27) CD133,(28)and PD-L1,(29) which we used to design anti-horPDPN mAbs.(25)
Two mice were immunized with Chinese hamster ovary (CHO)/
horPDPN cells using an immunization and screening procedure, and the developed hybridomas were seeded into
96-well plates and cultivated for 10 days. Wells that werepositive for CHO/horPDPN and negative for CHO-K1 werethen selected using flow cytometry. Nine hybridomas were developed: one clone of IgG1, one clone of IgG2a, five clonesof IgG3, and two clones of IgM. Among these, PMab-219

1Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan.
3
Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
4
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,Sapporo, Japan.
5
Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
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PMAB-219 AGAINST HORSE PDPN273(IgG2a, kappa) successfully recognized the horse kidney cellline FHK-Tcl3.1 in flow cytometry and detected horPDPN asa 40-kDa band in CHO/horPDPN cells using Western blot
analysis.(25) Furthermore, immunohistochemical analysis
revealed that PMab-219 strongly stained CHO/horPDPNcells.(25) However, it remained unclear whether PMab-219
was useful for detecting lymphatic endothelial cells in horsetissues.
In this study, we found that PMab-219 strongly stained
lymphatic endothelial cells in equine colon tissues, whereasleaving vascular endothelial cells unstained (Fig. 1). Furthermore, it weakly stained podocytes in equine kidney tissues(Fig. 2). Although it has been reported that type I alveolar cells
in the lung also express PDPN,(9) PMab-219 did not stain thesecells in equine lung tissues (data not shown), possibly due to thevarying expression levels of horPDPN in different tissues.
These results indicate that PMab-219 will be useful for elucidating the pathophysiological functions of horPDPN in equine
lymphatic endothelial cells in future studies.
Acknowledgments

FIG. 1. Immunohistochemical analysis of equine colon tissues. Equine colon tissue sections were incubated with
10 mg/mL of PMab-219 (A, D) or blocking buffer (B, E), followed by the Envision+ Kit, or were stained with hematoxylin
and eosin (C, F). Scale bar = 100 mm. Arrows, lymphatic endothelial cells; arrowheads, vascular endothelial cells.

This research was supported in part by AMED under Grant
Numbers JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), andJP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number17K07299 (M.K.K.) and Grant Number 16K10748 (Y.K.).
Author Disclosure Statement

No competing financial interests exist.
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FIG. 2. Immunohistochemical analysis of equine kidney
tissues. Equine kidney tissue sections were incubated with
10 mg/mL of PMab-219 (A, D) or blocking buffer (B, E),
followed by the Envision+ Kit, or were stained with hematoxylin and eosin (C, F). Scale bar = 100 mm.

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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
MiyagiJapan
E-mail: yukinari-k@bea.hi-ho.ne.jp
Received: November 12, 2018
Accepted: November 29, 2018