NOTE
Surgery

Podoplanin promotes cell proliferation,
survival, and migration of canine
non-tonsillar squamous cell carcinoma
Masahiro SHINADA1), Daiki KATO1)*, Masaya TSUBOI2), Namiko IKEDA1),
Susumu AOKI1), Takaaki IGUCHI1), Toshio LI1), Yuka KODERA1), Ryosuke OTA1),
Shoma KOSEKI1), Hayato SHIBAHARA1), Yosuke TAKAHASHI2), Yuko HASHIMOTO2),
James K CHAMBERS3), Kazuyuki UCHIDA3), Shunsuke NOGUCHI4),
Yukinari KATO5,6), Ryohei NISHIMURA1), Takayuki NAKAGAWA1)
1)Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo,

Tokyo, Japan

2)Veterinary Medical Center, The University of Tokyo, Tokyo, Japan

3)Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo,

Tokyo, Japan

4)Laboratory of Veterinary Radiology, Graduate School of Veterinary Science, Osaka Metropolitan University,

Osaka, Japan5)Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Miyagi, Japan6)Department of Molecular Pharmacology, Tohoku University Graduate School of Medicine, Miyagi, Japan

J. Vet. Med. Sci.
85(10): 1068–1073, 2023
doi: 10.1292/jvms.23-0062
Received: 7 February 2023
Accepted: 26 July 2023
Advanced Epub: 		
4 August 2023

ABSTRACT. Podoplanin (PDPN) is a prognostic factor and is involved in several mechanisms oftumor progression in human squamous cell carcinoma (SCC). Canine non-tonsillar SCC (NTSCC) is acommon oral tumor in dogs and has a highly invasive characteristic. In this study, we investigated
the function of PDPN in canine NTSCC. In canine NTSCC clinical samples, PDPN overexpression wasobserved in 80% of dogs with NTSCC, and PDPN expression was related to ki67 expression. In PDPN
knocked-out canine NTSCC cells, cell proliferation, cancer stemness, and migration were suppressed.
As the mechanism of PDPN-mediated cell proliferation, PDPN knocked-out induced apoptosis andG2/M cell cycle arrest in canine NTSCC cells. These findings suggest that PDPN promotes tumor
malignancies and may be a novel biomarker and therapeutic target for canine NTSCC.
KEYWORDS: canine squamous cell carcinoma, ki67, migration, podoplanin, proliferation

Podoplanin (PDPN), also known as PA2.26, gp38, T1a, and Aggrus is the type 1 transmembrane glycoprotein [22, 33] and isexpressed in various normal cell types such as renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells (LECs)[22, 33]. In the embryo, PDPN plays a crucial role in development of some organs [33]. In adults, PDPN promotes lymphangiogenesis,maintains high endothelial venule integrity, and regulates immune systems [12, 22, 25, 33]. PDPN has been reported to be overexpressedin several types of humans, canine and feline tumors including squamous cell carcinoma (SCC) [16, 17, 20, 21, 33]. In human SCC,
high PDPN expression was related to a frequency of tumor metastasis and poor prognosis of patients with SCC [6, 11, 20, 38].
Previous experimental results showed that PDPN was related to cell survival, migration, and invasion of human SCC cells in vitro andin vivo [23, 27, 28, 34]. In addition, it was reported that anti-PDPN antibody exhibited anti-tumor effects on human SCC cell growth
in mouse xenograft models [18]. Therefore, PDPN promotes tumor malignancy and can be a new therapeutic target for human SCC.
Canine oral SCC is the second most common epithelial malignancy of the canine oral cavity [29]. Canine oral SCC develops
predominantly in gingiva (non-tonsillar SCC; NTSCC) and tonsil, and more than a half of oral SCC were NTSCC [15, 29]. Surgicalresection is the first line of treatment for canine NTSCC, because lymph node and distant metastasis is not frequent [29]. However,canine NTSCC is locally aggressive with local bone invasion seen in 77% of cases, and complete tumor cell resection is challenging
when the tumor was advanced and aggressively invaded local bones [36]. Therefore, it is important to reveal the mechanism of thecanine NTSCC progression. Recently, we have revealed that canine NTSCC expresses PDPN in tumor cells [35]. This finding gives*Correspondence to: Kato D: adk@g.ecc.u-tokyo.ac.jp, Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The
University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan(Supplementary material: refer to PMC https://www.ncbi.nlm.nih.gov/pmc/journals/2350/)©2023 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd)
License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/)

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us a possible hypothesis that PDPN expressed in canine NTSCC promotes tumor malignancy and may be a therapeutic target as well
as a diagnostic and prognostic marker. The aim of this study was to investigate the role of PDPN in canine NTSCC.
The expression of PDPN in canine NTSCC was compared with that of normal canine squamous epithelial cells, and relationshipbetween PDPN expression and ki67 expression in canine NTSCC tissues was evaluated by immunohistochemistry. Further, to evaluate
the functions of PDPN in NTSCC cells, a PDPN knocked-out (PDPN-KO) canine NTSCC cell line, oSCC3 [32], was generated andchanges of cell migration, cancer stemness, cell proliferation, apoptosis, and cell cycle were analyzed. Detailed materials and methods
are described in a Supplementary file.
PDPN expression on epithelial cells of normal skin (n=4), esophagus (n=4), and tumor cells of NTSCC (n=10) was evaluated byimmunohistochemistry. Normal lung tissue was used as a positive control for PDPN staining. All NTSCC tissues were derived fromthe oral cavity and the primary tumor sites were as follows; mandible (n=6), buccal mucosa (n=2), maxilla (n=1), and pharynx andlarynx (n=1). Although no expression of PDPN was detected in normal skin and esophageal tissues, 80% (8/10) of NTSCC tissues
expressed PDPN in tumor cells (Fig. 1A). The expression of PDPN in positively stained samples was strong in the cell membranesof tumor cells and moderate in the cytoplasm of tumor cells, which was similar to the expression pattern observed in the positive
control canine lung tissues (Fig. 1A). The proportion of positively stained tumor cells was varied among tumor samples (mean: 48.5%,
range: 0–100%). Next, we investigated the relationship between PDPN expression and ki67 expression, which is the biomarker for

Fig. 1. Podoplanin (PDPN) expression in canine normal and non-tonsillar squamous cell carcinoma (NTSCC) tissues and a relationship between
PDPN and ki67 expression in canine NTSCC tissues. (A) Representative images of PDPN staining for canine normal lung (a positive control ofstaining for PDPN), skin, esophagus, and NTSCC tissues. An enlarged image is shown. Black arrowheads indicate PDPN expression in cellular
membranes. (B) Left panel: representative images of PDPN and Ki67 expression in NTSCC tissues. Right panel: The percentage of Ki67positive tumor cells in canine NTSCC tissues was compared between the PDPN high-expression group and the PDPN low-expression group.
Box plots where box limits show 25th and 75th percentiles, the horizontal line shows the median, and dot plots in boxplots indicate a percentage
of each tissue. Welch’s t test, *: P<0.05.
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lymph node metastasis characteristics of canine oral SCC [26]. Ten NTSCC tissues were divided in PDPN high expression or low
expression according to the median percentage (42%) of PDPN positive tumor cells. The percentage of ki67 positive tumor cells in
the tissues with high PDPN expression was significantly higher than that in the tissues with low PDPN expression (Fig. 1B). This
result suggested that PDPN was related to proliferation of canine NTSCC cells.
To investigate the functions of PDPN in canine NTSCC cells, PDPN-KO canine NTSCC cells were generated using CRISPR/Cas9
technology from the primary canine NTSCC cell line: oSCC3. The single guide RNA targeting the canine PDPN gene depleted the
PDPN protein expression on the cell surface (Fig. 2A) with confirmation of genetic mutations in PDPN genes in the canine NTSCC
cell line (Fig. 2B). Cell proliferation of PDPN-KO canine NTSCC cells was strongly suppressed, and the number of tumor cells was
reduced to one-fourth compared to normal oSCC3 cells (control cells) (Fig. 2C). In addition, PDPN-KO canine NTSCC cells also
exhibited reduced sphere-forming ability (Supplementary Fig. 1A) and a lower migratory ability in wound-healing assay compared
to control cells (Supplementary Fig. 1B). These results indicated that PDPN regulated cell proliferation, cancer stemness, and cell
migration in canine NTSCC cells.
To investigate the mechanism of PDPN-KO related suppression of cell proliferation in canine NTSCC cells, induction of apoptosis
and changes of cell cycle were evaluated in PDPN-KO canine NTSCC cells. As a result, both early and late apoptotic cells weresignificantly increased in PDPN-KO canine NTSCC cells compared to those in control cells (Fig. 3A and 3B). In addition, expressionof cleaved caspase 3, an indicator for initiation of apoptosis, was also upregulated in PDPN-KO canine NTSCC cells. Moreover,expression of Bax, a pro-apoptotic protein, was upregulated and expression of Bcl-2 and Bcl-xL, anti-apoptotic proteins, were
downregulated in PDPN-KO canine NTSCC cells, compared to control cells by western blot analysis (Fig. 3C). In cell cycle analysis,
the proportion of G2/M-phase cells was significantly increased and the proportion of G0/G1-phase cells was significantly decreased in
PDPN-KO canine NTSCC cells compared to control cells, indicating the induction of G2/M cell cycle arrest (Fig. 3D). The expressionof G2/M cell cycle related proteins was evaluated by western blot analysis and the expression of phospho-chk2 (Thr68), phospho-cdc2(Tyr15), p53, and p27 was upregulated, and cyclin B1 expression was downregulated in PDPN-KO canine NTSCC cells compared tocontrol cells (Fig. 3E). Overall, these findings suggested that the suppression of cell proliferation observed in PDPN depleted canine
NTSCC cells would be caused by the induction of apoptosis and G2/M cell cycle arrest through the alteration of G2/M related proteins.
In this study, we first demonstrated the association between the PDPN expression and the ki67 expression in the NTSCC clinicaltissue samples. Moreover, the abrogation of PDPN in canine NTSCC cells suppressed tumor cell proliferation. In addition, PDPN-KO
canine NTSCC cells showed induction of both apoptosis and G2/M cell cycle arrest. These results suggested that PDPN is related totumor malignancies of canine NTSCC through the several mechanisms.
We revealed that PDPN expression was related to ki67 expression in canine NTSCC tissues. Ki67 is one of the representative
proliferation markers, and ki67 is used as a prognostic marker in various tumors such as canine melanoma and human breast cancer[4, 8]. Therefore, PDPN would be related to canine NTSCC progression by promoting tumor cell proliferation. In addition, ki67 isFig. 2. Investigating the association between podoplanin(PDPN) and tumor malignancy in canine non-tonsillar
squamous cell carcinoma (NTSCC) cells. (A) PDPN
expression of control and podoplanin knocked-out(PDPN-KO) oSCC3 cells. PDPN expression was detected by flowcytometry. (B) Base sequences of control
and PDPN-KO oSCC3 cells. The region targeted bysingle guide RNA is shown. (C) The number of live
cells of control and PDPN-KO oSCC3 cells after 24,
48, and 72 hr of cell seeding (n=3). Representative images are shown under the graph. All experiments were
performed in triplicate. Bar graphs are indicated as themean ± SD. Welch’s t test, **: P<0.01.
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Fig. 3. Effects of podoplanin knocked-out (PDPN-KO) on apoptosis and cell cycle of canine non-tonsillar squamous cell carcinoma (NTSCC)
cells. (A) Annexin V-FITC/propidium iodide analysis of apoptosis in control and PDPN-KO oSCC3 cells. The lower-right panel presents early
apoptotic cells, and the upper-right panel shows late apoptotic cells. Non-stained cells were used as a control (n=3). (B) The percentage of early
apoptotic cells (left panel) and late apoptotic cells (right panel) in control and PDPN-KO oSCC3 cells (n=3). (C) Apoptotic related protein
expression of control and PDPN-KO oSCC3 cells detected by western blot. (D) Proportions of each cell cycle phase of control and PDPN-KO
oSCC3 cells (n=3). (E) G2/M cell cycle-related protein expression of control and PDPN-KO oSCC3 cells detected by western blot. All experiments were performed in triplicate. Bar graphs are indicated as the mean ± SD. Welch’s t test, **: P<0.01.

reported to be the predictive marker of lymph node metastasis in canine NTSCC [26]. Therefore, PDPN expression might be a poor
prognostic factor of canine NTSCC, similar to findings in medicine that PDPN expression was associated with the frequency of lymph
node metastasis and poor prognosis in human SCC [6, 11, 20, 38]. We could not find the relationship between PDPN expression andclinicopathological features of dogs with NTSCC due to a small sample size and further large cohort investigation would give us
significant clinical value of PDPN expression in canine NTSCC.
In PDPN-KO canine NTSCC cells, cell proliferation was suppressed through the induction of apoptosis and G2/M cell cycle
arrest. Previous reports indicated that PDPN promoted cell proliferation in human SCC cells and canine melanoma cells [14, 35],
but the regulating mechanism of PDPN in tumor cell proliferation had not been elucidated. In this study, p27 expression was also

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upregulated by PDPN-KO. Previous studies showed that p27 expression was upregulated by p53 and an accumulation of p27 induced
G2/M cell cycle arrest [13, 30]. Moreover, phosphor-chk2, phosphor-cdc2, and p53 expression were upregulated by PDPN-KO. Oneof the major regulators of the chk/p53 cascade is ataxia telangiectasia (ATM), a DNA damage sensor [3, 19]. When DNA damage
occurs, the chk/p53 cascade is activated by ATM, and the activated chk/p53 cascade suppresses the activation of cyclin B1 and cdc2
complex, which induces G2/M cell cycle arrest [3, 19]. Therefore, it was possible that G2/M cell cycle arrest induced by PDPN-KO
was depended on the activation of the chk/p53 cascade. Other possible mechanisms of inhibition of tumor cell proliferation induced
by PDPN-KO are NF-kB pathways and Rho associated kinase (ROCK) signaling. In the LECs, PDPN promotes lymphatic growththrough mitogen-activated protein kinase and NF-kB pathways [25]. In lymph node stroma, PDPN regulates lymph node expansion
through Rho associated kinase (ROCK) signaling [1]. It was also reported that PDPN promoted human SCC cell invasion and thegrowth of human mesothelioma cells through ROCK signaling [24, 37]. Since these pathways were reported to regulate apoptosis and/or G2/M cell cycle arrest in human tumor cells [5, 7, 31, 40, 41]. These pathways might also regulate PDPN-related cell proliferationin canine NTSCC cells. Identification of the specific signaling pathway regulating PDPN-mediated cell proliferation is desired and it
would allow PDPN to be used as a novel therapeutic target in canine NTSCC.
PDPN-KO suppressed the migration of canine NTSCC cells in wound-healing assay. Canine NTSCC has locally aggressive clinicalcharacteristics, and approximately 80% of canine NTSCC invaded local bones [36]. In addition, up to 36% of NTSCC showed distantmetastasis [9]. PDPN has been reported to promote tumor cell migration and invasion through various mechanisms in human SCC.
One reported mechanism was that PDPN activated Rho-GTPase family proteins to remodel cellular cytoskeleton into a suitable formof cell migration [24]. Another report showed that PDPN regulated epithelial-mesenchymal transition to acquire highly migrative andinvasive capabilities [39]. The detailed mechanism of PDPN promoting migration in canine NTSCC cells needs to be investigated infurther study and PDPN promoting migration might be involved in aggressive local invasion and metastatic characteristics of canine
NTSCC.
PDPN-KO canine NTSCC cells also showed lower sphere forming capacity compared to control cells, indicating that PDPN was
related to cancer stemness of canine NTSCC cells. Previous reports suggested that PDPN is one of the cancer stem cell markers ofhuman SCC, and PDPN enhanced cancer stemness to support cell survival [27]. Cancer stem cells are subpopulations of tumor cells
which have highly resistant capacities to chemotherapy and radiotherapy [2]. Although canine NTSCC is responsive to radiotherapy,
16% and 53% of dogs showed local recurrence and tumor regrowth, respectively, even after adjuvant radiotherapy [9, 10]. Furthermore,canine NTSCC is often unresponsive to systemic chemotherapy [36]. Therefore, it is possible that PDPN is also related to resistanceto chemotherapy and radiotherapy by regulating cancer stemness in canine NTSCC cells.
In this study, we performed experiments with only one PDPN-KO cell line, thus; an off-target effect of CRISPR/Cas9 technology
could not denied completely. To show the function of the PDPN more clearly, further studies were required.
Overall, we found the association between PDPN expression and proliferative marker expression in canine NTSCC and the novel
malignant mechanisms of PDPN in canine NTSCC cells which can explain the aggressive clinical behaviors of canine NTSCC. Our
results suggested the potential of PDPN as a novel prognostic and diagnostic marker and therapeutic target.
CONFLICTS OF INTEREST. The authors have nothing to disclose.
ACKNOWLEDGMENTS. We thank all the clinicians who treated the dogs, pathologists who processed the samples, and ownersof the dogs. This research was supported in part by JSPS KAKENHI under Grant Number: 19K22361 and 21K20614, and by Japan
Agency for Medical Research and Development (AMED) under Grant Numbers: JP22ama121008 and JP22am0401013.
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