MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 5, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2016.0036

PMab-38 Recognizes Canine Podoplaninof Squamous Cell Carcinomas
Mika K. Kaneko,1,* Ryusuke Honma,1,2,* Satoshi Ogasawara,1 Yuki Fujii,1 Takuro Nakamura,1Noriko Saidoh,1 Michiaki Takagi,2 Yumiko Kagawa,3 Satoru Konnai,4 and Yukinari Kato1Podoplanin, a type I transmembrane protein, is expressed in lymphatic endothelial cells. Although we previously developed an anticanine podoplanin monoclonal antibody (mAb), PMab-38, immunohistochemistry(IHC) showed that it did not react with canine lymphatic endothelial cells. Here, we determined whether PMab38 recognizes canine podoplanin of squamous cell carcinomas (SCCs) and clarified its epitope. In IHC, PMab38 reacted with 83% of SCCs (15/18 cases). Flow cytometry showed that the epitope of PMab-38 was different
from that of the platelet aggregation-stimulating domain of the N-terminus, which was detected by almost all
antipodoplanin mAbs such as D2–40 or NZ-1. PMab-38 is expected to be useful for investigating the functionof podoplanin in canine tumors.
Keywords: canine podoplanin, monoclonal antibody, immunohistochemistry

Podoplanin is a type I transmembrane sialoglycoprotein, also known as Aggrus/T1a.(1–4) Canine podoplanin
was first reported as gp40.(5) It is expressed in normal cells
including renal podocytes, lymphatic endothelial cells, andpulmonary type I alveolar cells.(3) Podoplanin activates platelet aggregation by binding to C-type lectin-like receptor-2(CLEC-2) on platelets,(6,7) and the interaction between podoplanin and CLEC-2 facilitates blood/lymphatic vessel
separation.(8) The expression of human podoplanin has beenreported in many malignant tumors, such as oral squamouscell carcinomas (SCCs),(9) malignant brain tumors,(10–12) lungcancers,(13) esophageal cancers,(14) malignant mesotheliomas,(15)testicular tumors,(16) and osteosarcomas.(17) Podoplanin expression is also associated with malignant progression andcancer metastasis.(6,10) However, canine podoplanin in tumors has not been investigated because of the absence of aspecific and sensitive monoclonal antibody (mAb). We recently reported anticanine podoplanin mAb, PMab-38,(18)which is useful for immunohistochemistry (IHC). Furthermore, PMab-38 specifically reacts with canine podoplanin inflow cytometry and Western blotting.(18)Here, we determined whether PMab-38 can recognize
podoplanin of canine SCCs because human podoplanin ishighly expressed in these cells and is involved in tumor
malignancy.(9) We first checked the PMab-38 reactivity

against SCCs (8 oral SCCs and 10 skin SCCs) in IHC. (Fig. 1and Supplementary Fig. S1). Among them, only case 16 (1/18; 5.6%) was detected by PMab-38 in >50% of tumor cells.
In total, tumor cells in 15 out of 18 cases (83%) were stainedwith PMab-38 in a membrane-staining pattern. Similarly,
cancer-associated fibroblasts in 14 out of 18 cases (78%)
were detected by PMab-38. Human podoplanin expression inseveral cancers contributes to poor prognosis(19); therefore,
PMab-38 might be useful for investigating the pathological
function of canine podoplanin in the tumor microenvironment.
Next, we investigated the epitope of PMab-38 using flow
cytometry. To this end, we produced several deletion mutantsof canine podoplanin, which are expressed in CHO-K1 cells,including dN23 (corresponding to 23–169 amino acids [aa]),dN40 (corresponding to 40–169 aa), dN60 (corresponding to60–169 aa), dN80 (corresponding to 80–169 aa), and dN100(corresponding to 100–169 aa). Antipodoplanin mAbs usually detect the platelet aggregation-inducing (PLAG) domain, particularly PLAG1–PLAG3 (corresponding to 36–61aa) near the N-terminus(20); in contrast, PMab-38 reactedwith dN23, dN40, dN60, and dN80. When antipodoplaninmAbs reacted with the PLAG domain, they could not reactwith dN60, dN80, and dN100, indicating that the PMab-38
epitope is far from PLAG1–PLAG3 (Fig. 2). Previously, we

1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Orthopaedic Surgery, Faculty of Medicine, Yamagata University, Yamagata, Japan.
3
North Lab, Sapporo, Japan.
4
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
*Contributed equally to this work.
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KANEKO ET AL.

FIG. 1. Immunohistochemical analysis against canine SCCs by PMab-38. Canine tissues (A, oral SCCs; B, skin SCCs)
were obtained from North Lab (Hokkaido, Japan). In total, 4 mm thick histologic sections were deparaffinized in xylene,rehydrated, and autoclaved in citrate buffer (pH 6.0; Dako, Glostrup, Denmark) for 20 minutes. Sections were incubatedwith 10 mg/mL of PMab-38 overnight at 4C followed by treatment with Envision+ kit for 30 minutes (Dako). Color wasdeveloped using 3, 3-diaminobenzidine tetrahydrochloride (DAB; Dako) for 2 minutes, after which the sections werecounterstained with hematoxylin (Wako Pure Chemical Industries Ltd., Osaka, Japan). Hematoxylin and eosin (HE) staining
was also performed. Scale bar: 100 mm.

PMAB-38 AGAINST CANINE PODOPLANIN IN SCCs265

produced an antihuman podoplanin mAb, LpMab-7, theepitope of which is Arg79-Leu83 of human podoplanin,(17)which corresponds to His88-Gly90 of canine podoplanin.
LpMab-7 demonstrated high sensitivity compared with otherantihuman podoplanin mAbs,(21) suggesting that mAbs against
these epitopes have high sensitivity and specificity against not
only human podoplanin but also canine podoplanin.
Taken together, these data show that PMab-38 could be
useful for uncovering the pathophysiological function ofpodoplanin in canine tumors. PMab-38 did not react with thelymphatic endothelium in our previous study(18); therefore,
the PMab-38 epitope might be involved in cancer specificityof canine podoplanin.
Acknowledgments

We thank Kanae Yoshida for their excellent technical assistance. This work was performed, in part, under the Cooperative Research Program of Institute for Protein Research,Osaka University, CR-16-05. This work was supported, inpart, by the Regional Innovation Strategy Support Programfrom the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan (Y.K.), by JSPS KAKENHI
Grant Number 26440019 (M.K.K.) and 16K10748 (Y.K.), bythe Platform for Drug Discovery, Informatics, and Structural
Life Science (PDIS) from Japan Agency for Medical Research and development, AMED (Y.K.), and by the Basic
Science and Platform Technology Program for Innovative
Biological Medicine from AMED (Y.K.).
Author Disclosure Statement

No competing financial interests exist.
References

FIG. 2. Epitope mapping of PMab-38. Amplified canine
podoplanin cDNA was subcloned into a pCAG vector (Wako)
with an MAP tag, which was added to the N-terminus. MAPtag is a peptide: GDGMVPPGIEDK, which is derived frommouse podoplanin. The signal sequence of IL-2 was inserted
into the N-terminus. Deletion mutants of canine podoplanin
were generated using sense primers and an antisense primer
for dN23, dN40, dN60, dN80, and dN100, as summarized in
Supplementary Table S1. CHO-K1 cells were transfectedwith the plasmids using electroporation. Each deletion mutantof CHO/canine podoplanin was cultured in RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with
10% heat-inactivated fetal bovine serum (Thermo Fisher
Scientific Inc., Waltham, MA) at 37C in a humidified atmosphere of 5% CO2 and 95% air. Each cell was treated with
PMab-38 or PMab-1 (anti-MAP tag) at a concentration of1 mg/mL for 30 minutes at 4C followed by treatment withOregon green-conjugated antimouse IgG or antirat IgG(1:1000 diluted; Thermo). Fluorescence data were collectedusing a Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

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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-kuSendaiMiyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: September 6, 2016
Accepted: September 15, 2016