MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 6, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0040

Podoplanin Expression in Canine Melanoma
Satoshi Ogasawara,1,* Ryusuke Honma,1,* Mika K. Kaneko,1 Yuki Fujii,1Yumiko Kagawa,2 Satoru Konnai,3 and Yukinari Kato1

A type I transmembrane protein, podoplanin (PDPN), is expressed in several normal cells such as lymphatic
endothelial cells or pulmonary type I alveolar cells. We recently demonstrated that anticanine PDPN monoclonal antibody (mAb), PMab-38, recognizes canine PDPN of squamous cell carcinomas, but does not reactwith lymphatic endothelial cells. Herein, we investigated whether PMab-38 reacts with canine melanoma.
PMab-38 reacted with 90% of melanoma cells (9/10 cases) using immunohistochemistry. Of interest, PMab-38
stained the lymphatic endothelial cells and cancer-associated fibroblasts in melanoma tissues, although it did notstain any lymphatic endothelial cells in normal tissues. PMab-38 could be useful for uncovering the function ofPDPN in canine melanomas.
Keywords: canine podoplanin, melanoma, monoclonal antibody, immunohistochemistry

Atype I transmembrane sialoglycoprotein, podoplanin (PDPN), is also known as gp40/T1a/Aggrus.(1–5)
PDPN is expressed in normal cells including renal podocytes,
pulmonary type I alveolar cells, and lymphatic endothelialcells.(4) PDPN activates platelet aggregation by binding to Ctype lectin-like receptor-2 (CLEC-2) on platelets,(6,7) and thePDPN–CLEC-2 interaction facilitates blood/lymphatic vessel separation.(8) The expression of human PDPN was reported in many tumors, including oral cancers,(9) malignant
brain tumors,(10–12) lung cancers,(13) esophageal cancers,(14)malignant mesotheliomas,(15) testicular tumors,(16) and osteosarcomas.(17) PDPN expression is also associated withcancer metastasis and malignant progression.(6,10)
We previously developed an anticanine PDPN monoclonal antibody (mAb), PMab-38,(18) which is useful for
immunohistochemistry (IHC), flow cytometry, and Western blotting. Recently, we demonstrated that PMab-38 canrecognize PDPN of canine squamous cell carcinomas using IHC.(19) Tumor cells in 15 out of 18 canine squamouscell carcinomas (83%) were stained by PMab-38 in IHC.
Cancer-associated fibroblasts in 14 out of 18 cases (78%)
were detected by PMab-38. In this study, we investigatedwhether canine melanoma was stained by PMab-38
because mouse PDPN expression and human PDPN
expression were observed in melanomas.(20,21) Watanabe
et al. reported that high expression of mouse PDPN is
associated with the metastatic ability in metastatic variantsof B16 melanomas.(20)We stained 10 canine oral melanomas, which are themost frequent oral cancers in dog.(22) As depicted in Figures 1 and 2A, melanoma cells were stained by PMab-38 in
9 of 10 cases. Although melanoma cells were not stained by
PMab-38 in case 9 (Fig. 1), cancer-associated fibroblasts
were recognized by PMab-38 (Fig. 2B). Both melanoma
cells and cancer-associated fibroblasts were stained in case
5 (Fig. 1). Kan et al. reported that PDPN expression incancer-associated fibroblasts correlates with aggressive
behavior in human melanoma,(21) indicating that PDPN incancer-associated fibroblasts of melanoma tissues mightserve as a useful prognostic factor not only in humanmelanoma but also in canine melanoma. As shown in
Figure 2C, PDPN of lymphatic endothelial cells was detected by PMab-38 in melanoma tissues, although lymphatic endothelial cells in normal tissues(18) or squamouscell carcinomas(19) were not stained by PMab-38, indicating that PDPN expression might be upregulated inmelanoma, or post-translational modification of canine
PDPN might be different between squamous cell carcinomas and melanomas.
We recently reported that the PMab-38 epitope is far fromthe platelet aggregation-stimulating domain of PDPN.(19)However, we have not clarified whether the epitope of PMab38 includes the post-translational modification. In the nearfuture, we should determine the PMab-38 epitope using alanine scanning or glycan-deficient cells and uncover thedifference of PDPN expression in several tissues.

1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Miyagi, Japan.
North Lab, Hokkaido, Japan.
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
*These authors contributed equally to this work.

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FIG. 2. PMab-38 reacted with PDPN of melanoma cells,cancer-associated fibroblasts, and lymphatic endothelial cells.
Sections of melanomas (A, case 7; B, case 9; C, case 2) wereincubated with 10 mg/mL of PMab-38 overnight at 4C followed by treatment with Envision+ kit for 30 minutes (Dako).
As a control, blocking buffer was used. Color was developed
using 3,3-diaminobenzidine tetrahydrochloride (Dako) for 2minutes, after which the sections were counterstained withhematoxylin (Wako). H&E staining was also performed.
Scale bar: 100 mm. (A) staining of melanoma cells by PMab38, (B) staining of cancer-associated fibroblasts by PMab-38,and (C) staining of lymphatic endothelial cells by PMab-38.
Acknowledgments

FIG. 1. Immunohistochemical analysis against canine
melanoma using PMab-38. Canine melanomas (10 cases)
were obtained from North Lab (Hokkaido, Japan). Four
micrometers thick histologic sections were deparaffinized inxylene, rehydrated, and autoclaved in citrate buffer (pH 6.0;
Dako, Glostrup, Denmark) for 20 minutes. Sections wereincubated with 10 mg/mL of PMab-38 overnight at 4Cfollowed by treatment with Envision+ kit for 30 minutes(Dako). As a control, blocking buffer was used in this study.
Color was developed using 3,3-diaminobenzidine tetrahydrochloride (Dako) for 2 minutes, after which the sections
were counterstained with hematoxylin (Wako Pure Chemical Industries Ltd., Osaka, Japan). H&E staining was alsoperformed. Scale bar: 100 mm. mAb, monoclonal antibody;
PDPN, podoplanin.

We thank Takuro Nakamura, Noriko Saidoh, and KanaeYoshida for their excellent technical assistance. This work
was supported, in part, by the Regional Innovation Strategy
Support Program from the Ministry of Education, Culture,Sports, Science and Technology (MEXT) of Japan (Y.K.), byJSPS KAKENHI Grant Numbers 26440019 (M.K.K.) and
16K10748 (Y.K.), by the Platform for Drug Discovery, Informatics, and Structural Life Science (PDIS) from Japan
Agency for Medical Research and Development, AMED(Y.K.), by Project for utilizing glycans in the development ofinnovative drug discovery technologies from AMED (Y.K.),
and by the Basic Science and Platform Technology Programfor Innovative Biological Medicine from AMED (Y.K.). This
work was performed, in part, under the Cooperative Research
Program of Institute for Protein Research, Osaka University,

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CR-16-05 and by the Grant for Joint Research Project of theInstitute of Medical Science, the University of Tokyo.
Author Disclosure Statement

No competing financial interests exist.
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Address correspondence to:
Dr. Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai, Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: September 9, 2016
Accepted: October 21, 2016