NOTE
Surgery

Expression of podoplanin in various types offeline tumor tissues
Satoshi KAMOTO1)#, Masahiro SHINADA1)#, Daiki KATO1)*, Masaya TSUBOI3),
Sho YOSHIMOTO1), Ryohei YOSHITAKE1), Shotaro ETO1), Namiko IKEDA1),
Yosuke TAKAHASHI3), Yuko HASHIMOTO3), James CHAMBERS2),
Kazuyuki UCHIDA2), Shinji YAMADA4), Mika K. KANEKO4),
Ryohei NISHIMURA1), Yukinari KATO4,5) and Takayuki NAKAGAWA1)
1)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

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

1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

3)Veterinary Medical Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan4)Department of Antibody Drug Development, Tohoku University Graduate School of Medicine,

2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan

5)New Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai,

Miyagi 980-8575, Japan

J. Vet. Med. Sci.
83(11): 1795–1799, 2021
doi: 10.1292/jvms.20-0608
Received: 14 October 2020
Accepted: 8 September 2021
Advanced Epub: 		
18 October 2021

ABSTRACT. Podoplanin is expressed in various human tumors where it promotes tumor
progression, epithelial-mesenchymal transition, and distant metastasis. Podoplanin is also
expressed in cancer-associated fibroblasts and induces tumor malignancy. The objective of
this study was to evaluate podoplanin expression in various types of feline tumor tissues.
Immunohistochemical analysis revealed that podoplanin was expressed in cells of 13/15 (87%)
squamous cell carcinomas and 5/19 (26%) fibrosarcomas. Moreover, cancer-associated fibroblasts
expressed podoplanin in most tumor types, including 18/21 (86%) mammary adenocarcinoma
tissues. Our findings demonstrate that various types of feline tumor tissues expressed podoplanin,
indicating the importance of the comparative aspects of podoplanin expression, which may be
used as a novel research model for podoplanin biology.
KEY WORDS: cat, feline tumor, immunohistochemistry, podoplanin

Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein expressed in renal podocytes, lymphatic endothelial cells, andpulmonary type I alveolar cells [5, 6, 37]. It has been widely used as a prominent marker of lymphatic endothelial cells, where
it is strongly expressed [24]. PDPN plays an essential role in the formation of a normal lymphatic system during embryonicdevelopment, as well as in platelet aggregation [2, 4, 12, 31].
In human medicine, PDPN was reported to be overexpressed in various types of tumors, including squamous cell carcinoma[18], malignant astrocytic tumors [19], malignant mesothelioma [25], hemangiosarcoma [6], osteosarcoma [1], and germinoma[17]. Many reports have demonstrated that PDPN expression is associated with tumor malignancy via promoting proliferation,epithelial-mesenchymal transition, and metastasis by inducing tumor cell migration and platelet aggregation; hence, PDPN has
been considered as a therapeutic target [32]. In addition, cancer-associated fibroblasts (CAFs), a major component of the tumor
stroma, have been reported to overexpress PDPN [11, 14, 28]. PDPN-positive CAFs have been reported to strongly contributeto tumor progression by promoting local invasion and lymph node metastasis [9, 21]. Importantly, a high ratio of PDPN-positive
CAFs have been reported to be associated with poor prognosis in human patients with lung adenocarcinoma [21].
In veterinary medicine, PDPN was reported to be overexpressed in some naturally occurring tumors in dogs [17]. Cats livingwith humans have also been reported to develop various types of tumors that resemble those found in humans and dogs. Naturally
occurring tumors in cats have increased in recent years [3], owing to increased life expectancy resulting from increased nutritional
value of food, availability of vaccines for common infectious diseases, and advancements in veterinary medicine. Recently,
analysis of the feline genome provided evidence of strong similarities with humans [22]. Because many features of tumors in*Correspondence to: Kato, D.: adk@g.ecc.u-tokyo.ac.jp#Thease authors contributed equally to this work.
(Supplementary material: refer to PMC https://www.ncbi.nlm.nih.gov/pmc/journals/2350/)©2021 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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cats are similar to those in humans, the study of feline tumors would contribute to our understanding of tumor pathogenesis,
progression, and therapy and could be considered as a novel research model [26].
Although the evaluation of PDPN expression in feline tumors has provided us very important findings, such as the comparative
aspect of PDPN expression among species and a potential novel research model, there is no research on PDPN expression in feline
tumor tissues because of the lack of feline PDPN-specific antibodies. To address this issue, we previously developed monoclonal
antibodies against feline PDPN (fPDPN) that can be used to specifically recognize fPDPN expression in normal tissues using
immunohistochemistry [7, 34]. Thus, the objective of this study was to evaluate the distribution of fPDPN expression in various
types of feline tumor tissues by performing immunohistochemical analysis.
A total of 145 paraffin-embedded tumor tissues were evaluated. These tumor tissues were extracted surgically from the catsat the Veterinary Medical Center of the University of Tokyo between 2011 and 2017. Permission for collection and use of theresected tissues was obtained from the owners. The tissue samples were diagnosed by two veterinary pathologists (J.C. and K.U.)
certified by the Japanese College of Veterinary Pathologists at the Department of Veterinary Pathology at The University of Tokyo.
The tumor types included mammary adenocarcinoma (n=21), oral and skin fibrosarcoma (n=19), head and neck squamous cell
carcinoma (n=15), mast cell tumor (n=11), ceruminous adenocarcinoma (n=10), colorectal adenocarcinoma (n=10), meningioma(n=10), nasal adenocarcinoma (n=10), osteosarcoma (n=10), peripheral nerve sheath tumor (n=10), thyroid adenoma (n=10),
thymoma (n=6), and pulmonary adenocarcinoma (n=3). Normal skin, intestine, mammary gland, thyroid, and cerebral tissue
from one healthy cat were used as normal controls. This study was approved by The University of Tokyo Animal Care and Use
Committee (approval number: P15-099).
Immunohistochemical staining was performed using primary antibodies specific for fPDPN (mouse monoclonal antibody,clone: PMab-52, 2 µg/ml) [34], anti-α-smooth muscle actin (α-SMA) (mouse monoclonal antibody, clone: 1A4, 1:400, Agilent
Technologies, Santa Clara, CA, USA), and anti-p63 (mouse monoclonal antibody, clone 4A4, 1:100, Biocare Medical, CA, USA).
All tumor tissues were fixed in 10% neutral buffered formalin, embedded in paraffin wax, and cut into 4 µm thick serial sections.
Each section was subsequently dewaxed and rehydrated in xylene and graded ethanol, followed by antigen retrieval using citratebuffer (pH 6.0) at 100°C for 30 min in boiling water. After washing with Tris-buffered saline and 0.1% Tween® 20 detergent(TBST), endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 10 min at 24–26°C. The specimens werethen washed with TBST and incubated in 8% skim milk for 1 hr at 24–26°C to reduce non-specific binding before incubation
with primary antibodies, including anti-fPDPN antibodies for 1 hr at 24–26°C or anti-α-SMA antibodies overnight at 4°C in ahumidified chamber. After washing with TBST, the sections were incubated with a horseradish peroxidase-conjugated anti-mouse
antibody (EnVision™+ System, HRP labeled polymer; K4001; Agilent Technologies) for 30 min at 24–26°C. Thereafter, thesections were washed with TBST, incubated with 3,3′-diaminobenzidine (Dojindo Laboratories, Kumamoto, Japan) solution for 3
min, and counterstained with Mayer’s hematoxylin. Sections of feline lung tissues were used as positive controls for fPDPN [10],while sections of feline small intestine were used as positive controls for α-SMA [16, 27]. Negative controls did not include theprimary antibodies. The specimens were considered to be positive for fPDPN if >10% of the tumor cells or stromal cells were
stained in five randomly selected independent high-magnification (400×) fields [11, 15]. Five independent fields were selected
randomly. Ratios of fPDPN-positive samples in each tumor type were investigated separately in tumor cells and stromal cells.
The fPDPN-positive sample ratios in the tumor cells varied among the tumor types. Feline PDPN expression was observed in thesamples of squamous cell carcinoma (13/15; 87%), fibrosarcoma (5/19; 26%), and colorectal adenocarcinoma (2/10; 20%) (Table 1);
in contrast, fPDPN-positive cells were not detected in peritumoral normal squamous epithelium (n=11) and normal intestinal glands(n=7), except in the lymph vessels (Supplementary Fig. 1). The ratio of positively stained tumor cells varied in all tumor samples.
More than 50% of the tumor cells derived from some samples of squamous cell carcinoma and fibrosarcoma stained positive for
fPDPN expression, but less than 10% of tumor cells tested positive for fPDPN expression in colorectal adenocarcinoma samples.
The expression of fPDPN in positively stained
samples was strong in the cell membranes of tumor
cells and moderate in the cytoplasm of tumor cells.
Table 1. Feline podoplanin expression in various types of feline tumors
This result was consistent with the staining observedin the positive control feline lung tissues (Fig. 1 andTumor types
Samples Tumor cells (%) Stromal cells (%)
Supplementary Fig. 1).
Mammary adenocarcinoma
21
0 (0)18 (86)In positively stained samples, the cell membranes
Fibrosarcoma
19
5 (26)*of tumor cells exhibited strong fPDPN expression,
Squamous cell carcinoma
15
13 (87)9 (60)
whereas their cytoplasm showed moderate fPDPN
Mast cell tumor
11
0 (0)
0 (0)
expression (Fig. 1). Staining patterns, including diffuse
Colorectal adenocarcinoma10
2 (20)
7 (70)
and focal staining patterns, were heterogeneous among
Nasal adenocarcinoma
10
0 (0)3 (30)
positively stained samples. In addition, we could not
Ceruminous adenocarcinoma
10
0 (0)2 (20)
identify any specific staining patterns based on the
Thyroid adenoma
10
0 (0)2 (20)
tumor type or clinical characteristics. The labeling
Meningioma
10
0 (0)1 (10)
Peripheral nerve sheath tumor
10
0 (0)1 (10)
intensity of the tumor cells was comparable in all
Osteosarcoma
100 (0)*
positively stained tumor tissues.
Thymoma60 (0)1 (10)In the tumor stroma, large spindle-shaped
Pulmonary adenocarcinoma
3
0 (0)
0 (0)
mesenchymal cells expressed fPDPN in almost all
tumor types. The fPDPN-positive sample ratios*We could not distinguish tumor cells from stromal cells.

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Fig. 1. Evaluation of the expression pattern of feline podoplanin (fPDPN) in tumor cells. (A) A representative image of hematoxylin and eosin(HE) staining in feline squamous cell carcinoma. (B, C) Representative immunostaining images of podoplanin in squamous cell carcinoma.
Tumor cell membranes were broadly and strongly stained, while the cytoplasm of tumor cells were moderately stained in squamous cell
carcinoma. Black arrow heads indicate tumor cells which fPDPN strongly express on tumor cells membranes. (D) A representative imageof HE staining in feline fibrosarcoma. (E, F) Representative immunostaining images of fPDPN in fibrosarcoma are shown. In fibrosarcoma
samples, part of the cells was stained, but we could not distinguish tumor cells from stromal cells.

Fig. 2. Evaluation of the expression pattern of feline podoplanin (fPDPN) in stromal cells. A representative image of hematoxylin and eosin
staining (A) and immunostaining images of fPDPN (B) and α-smooth muscle actin (α-SMA) (C) in feline mammary adenocarcinoma areshown. Large spindle-shaped mesenchymal cells in the tumor stroma expressed α-SMA and part of the α-SMA positive cells expressed
fPDPN.

in the tumor stroam were 9/15 (60%) for squamous cell carcinoma, 7/10 (70%) for colorectal adenocarcinoma, 18/21 (86%) for
mammary adenocarcinoma, 3/10 (30%) for nasal adenocarcinoma, 2/10 (20%) for ceruminous adenocarcinoma and thyroid adenoma,
respectively, and 1/10 (10%) for meningioma, peripheral nerve sheath tumor, and thymoma, respectively (Table 1). In fibrosarcoma
and osteosarcoma samples, we did not evaluate the stromal cells separately, because we could not distinguish between tumor andstromal cells (Fig. 1). As controls to the tumor tissues, normal tissues of the skin, intestine, mammary gland, thyroid, and cerebral
tissue from one healthy cat were stained; fPDPN expression was not detected in any of these samples, except for endothelial cells in
the thyroid tissue (Supplementary Fig. 2).
The fPDPN-positive large spindle-shaped mesenchymal cells were suspected to be CAFs, which have been reported to express
PDPN in human tumor tissues. We evaluated α-SMA expression, which is generally utilized to detect CAFs [33], in fPDPNpositive spindle-shaped mesenchymal cells. Some parts of α-SMA positive cells in the stromal region co-expressed fPDPN (Fig. 2).
Feline PDPN was strongly expressed in both the membranes and cytoplasm of α-SMA-positive cells in the stromal region. The
staining intensity and the ratio of fPDPN positive cells in the stromal region were especially high in mammary adenocarcinoma
samples compared to other tumor types (Table 1 and Fig. 2). In mammary adenocarcinoma samples, p63 expression which is amarker of myoepithelial cells, was evaluated, and no positive staining was observed in the tumor tissues (Supplementary Fig. 3).

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Altogether, this study demonstrated the immunohistochemical expression of fPDPN in tumor cells and stromal cells invarious types of feline tumor tissues. The tumor cell membranes showed strong fPDPN expression in squamous cell carcinoma,
fibrosarcoma, and colorectal adenocarcinoma samples, similar to findings reported for canine and human tumors [13, 23]. However,feline meningioma and osteosarcoma, which were reported to cause the expression of PDPN in human homogenous tumor tissues,
did not demonstrate strong staining (Table 1). This discrepancy in PDPN expression in tumor tissues of different species indicatesdifferences in the pathophysiology of PDPN expression. Additionally, although the feline normal lung tissues were positively
stained, the feline lung tumor tissues were negatively stained. PDPN is expressed in type I alveolar epithelial cells in normal humanlung tissues [37]. Therefore, differences in origin between the PDPN expressing cells in feline normal lung tissue and the PDPN
negatively stained tumor cells in feline lung tumor tissues might cause a difference in PDPN expression between normal and tumor
tissues. Some feline tumor cells and α-SMA-positive cells in the stromal region expressed PDPN, which was reported to be related
to malignancy of various tumors. Because of the limited sample volume and clinical information, we could not determine theclinical significance of fPDPN expression in feline tumors. Further studies are required to clarify the clinical significance of fPDPN.
Squamous cell carcinoma is the most common type of oral tumor in cats, accounting for approximately 70% of tumors in thefeline oral cavity [29]. Feline oral squamous cell carcinoma is highly locally aggressive, often extending into the bone tissues, andcan be associated with pain and inability to eat. In this study, most tumor cells in feline squamous cell carcinoma expressed fPDPN.
High expression of PDPN in tumor cells in human oral squamous cell carcinoma tissues is strongly correlated with poor prognosis[8, 36]. PDPN expression in human squamous cell carcinoma is associated with various malignant mechanisms, including tumor
cell invasion, metastasis, proliferation, and cancer stemness [20, 30]. These findings indicate that fPDPN expression in tumor
cells may contribute to the aggressive behavior of feline squamous cell carcinoma, and fPDPN expressed on feline squamous cell
carcinoma might be a candidate therapeutic target. Moreover, fPDPN-expressing feline squamous cell carcinoma might be a novel
research model for PDPN-positive human oral squamous cell carcinoma.
We found that α-SMA positive cells in the stromal region expressed fPDPN in most types of tumors, as reported in humans anddogs [11, 13, 14, 28]. In mammary adenocarcinoma, colorectal adenocarcinoma, and squamous cell carcinoma samples, the ratiosof positively stained α-SMA cells in the stromal region of tumor tissues were higher than those in other tumor types. Although it
is difficult to completely distinguish between CAFs and myoepithelial cells from α-SMA expression in mammary adenocarcinoma[35], α-SMA positive cells in the stromal region of mammary adenocarcinoma were considered as CAFs because of their cellmorphology, histological structures, and negative expression of p63 (a myoepithelial cell marker). In human medicine, PDPNexpressing CAFs have been reported to promote local invasion and lymph node metastasis of tumor cells [14, 21]. Furthermore,
it has been demonstrated that CAFs exist in feline tumors, and the presence of CAFs in feline oral squamous cell carcinoma wassignificantly associated with poor prognosis [15]. Therefore, fPDPN expressing α-SMA positive cells in the stromal region may be
associated with malignancy of these tumors as well as human tumors.
This study showed the distribution of fPDPN expression in feline normal and tumor tissues. However, our data is limited. Firstly,
the co-expression of fPDPN and α-SMA in stromal cells could not be confirmed by double staining. Secondly, number of normal
control samples was small; for example, we obtained only one normal tissue sample from mammary gland, thyroid, and cerebrum
and no tissue from normal nasal cavity, ear canal, and thymus.
In conclusion, fPDPN expression was observed in various types of feline tumor tissues for the first time. The cell membranesof squamous cell carcinoma tumor cells strongly express fPDPN. Remarkably, α-SMA positive cells in the stromal region ofseveral feline tumors extensively expressed fPDPN. These novel findings suggest the importance of comparative aspects of PDPN
expression in naturally occurring tumors among humans, cats, and dogs, and the possibility of feline tumors as a new researchmodel for PDPN biology.
CONFLICTS OF INTEREST. The authors declare no conflicts of interest. The funders had no role in the study design; in thecollection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
ACKNOWLEDGMENTS. This study was supported by the Platform Project for Supporting Drug Discovery and Life Science
Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED (grant number
JP19am0101078), and JSPS KAKENHI (grant number 19K22361).
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