Expression of Cat Podoplanin in Feline Squamous Cell Carcinomas

Introduction

Oral squamous cell carcinoma is an aggressive tumor in cats,⁽¹⁾ for which no molecular targeted therapies have been developed. In particular, sensitive and specific monoclonal antibodies (mAbs) should be established for developing antibody-based therapies. Although many mAbs against human membrane proteins have been developed, almost all mAbs do not cross-react with the feline ones. Therefore, we need to develop sensitive and specific mAbs that can be directed against feline membrane proteins, which are overexpressed in feline cancers.

Podoplanin (PDPN/T1α/Aggrus), a type I transmembrane glycoprotein,^(2–6) activates platelet aggregation by binding to the C-type lectin-like receptor-2 (CLEC-2) on the platelets.^(7–10) Podoplanin is expressed in normal cells, including renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells.⁽³⁾ The interaction between podoplanin and CLEC-2 facilitates lymphatic/blood vessel separation.⁽¹¹⁾ The expression of human podoplanin has been reported in many malignant tumors, including brain tumors,^(12–15) oral cancers,⁽¹⁶⁾ esophageal cancers,⁽¹⁷⁾ lung cancers,⁽¹⁸⁾ mesotheliomas,^(19,20) testicular tumors,⁽²¹⁾ osteosarcomas,^(22–24) and chondrosarcomas.⁽²³⁾ Podoplanin expression is associated with cancer metastasis and malignant progression.^(8,12,25)

We have established several mAbs against human,⁽²⁶⁾ mouse,⁽²⁷⁾ rat,⁽²⁸⁾ rabbit,⁽²⁹⁾ dog,⁽³⁰⁾ bovine,⁽³¹⁾ and cat podoplanin.⁽³²⁾ Using these antipodoplanin mAbs, we have investigated podoplanin expression in cancer tissues. PMab-38, an anti-dog podoplanin mAb, reacted with 83% of squamous cell carcinomas (15/18 cases)⁽³³⁾ and 90% of melanomas (9/10 cases).⁽³⁴⁾ Recently, we have established an anti-cat podoplanin mAb (clone: PMab-52), which is very useful in flow cytometry, Western blot, and immunohistochemical analyses.⁽³²⁾ In this study, we investigated podoplanin expression in feline squamous cell carcinomas by immunohistochemical analysis using PMab-52.

Materials and Methods

Results

In our recent studies, we immunized mice with CHO/cat podoplanin cells and performed flow cytometry as the first screening. Using this cell-based immunization and screening (CBIS) method, we could obtain highly sensitive mAbs, which are useful not only in flow cytometry but also in Western blot and immunohistochemical analyses. Indeed, one of the established clones, PMab-52 (IgM, kappa), was sensitive and specific against cat podoplanin in flow cytometry, Western blot, and immunohistochemical analyses. PMab-52 reacted with only cat podoplanin, whereas it did not react with human, mouse, rat, rabbit, dog, and bovine podoplanin in flow cytometry and Western blot analyses.⁽³²⁾ Although we have investigated podoplanin expression in human, rabbit, and canine cancers, its expression in feline cancer remains to be clarified.

First, we investigated podoplanin expression in normal feline tissues, including the kidney, lung, and rectum, by immunohistochemical analysis. PMab-52 was bound to the Bowman's capsule and glomerular podocytes in feline kidneys (Fig. 1A, C). Also, it was bound to the type I alveolar cells of feline lungs (Fig. 2A, C) and lymphatic endothelial cells (Fig. 3A, C), indicating that PMab-52 can detect cat podoplanin in normal tissues because a similar expression pattern has been reported in other species, including human and mouse.⁽³⁵⁾

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FIG. 1.

Immunohistochemical analysis of podoplanin using PMab-52 in feline kidney. (A, C) Sections of feline kidneys were autoclaved in citrate buffer (pH 6.0). After blocking, they were incubated with 1 μg/mL of PMab-52, followed by treatment with EnVision+ kit. Color was developed using DAB, and the slides were counterstained with hematoxylin. (B, D) H&E staining was performed against serial sections. Scale bar: 100 μm. DAB, 3, 3-diaminobenzidine tetrahydrochloride; H&E, hematoxylin and eosin.

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FIG. 2.

Immunohistochemical analysis of podoplanin using PMab-52 in feline lungs. (A, C) Sections of feline lungs were autoclaved in citrate buffer (pH 6.0). After blocking, they were incubated with 1 μg/mL of PMab-52, followed by treatment with EnVision+ kit. Color was developed using DAB, and the slides were counterstained with hematoxylin. (B, D) H&E staining was performed against serial sections. Scale bar: 100 μm.

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FIG. 3.

Immunohistochemical analysis of podoplanin using PMab-52 in feline rectum. (A, C) Sections of feline rectum were autoclaved in citrate buffer (pH 6.0). After blocking, they were incubated with 1 μg/mL of PMab-52, followed by treating with EnVision+ kit. Color was developed using DAB, and the slides were counterstained with hematoxylin. (B, D) H&E staining was performed against serial sections. Arrows: lymphatic endothelial cells. Scale bar: 100 μm.

Next, we investigated cat podoplanin expression in various squamous cell carcinomas because its expression has been reported in many previous studies with other species.^(18,33) All specimens of squamous cell carcinomas used in this study are listed in Table 1. Among the 40 cases analyzed, 14 were from mouth floor, 13 from skin, 9 from ear, and 4 from tongue (Table 2). PMab-52 was observed in squamous cell carcinoma cells in a membrane-staining pattern (Fig. 4). All staining patterns are depicted in Supplementary Figure S1 and S2 (Supplementary Data are available online at www.liebertpub.com/mab). In total, 95% (38/40) of squamous cell carcinomas were detected using PMab-52 (Table 2).

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FIG. 4.

Immunohistochemical analysis of cat podoplanin using PMab-52 in squamous cell carcinomas. (A, C, E, G) Sections of feline squamous cell carcinomas were autoclaved in citrate buffer (pH 6.0). After blocking, they were incubated with 1 μg/ml of PMab-52, followed by treatment with EnVision+ kit. Color was developed using DAB, and the slides were counterstained with hematoxylin. (B, D, F, H) H&E staining was performed against serial sections. (A–D) case 9; (E–H) case 11. Arrows: lymphatic endothelial cells; arrowheads: vascular endothelial cells. Scale bar: 100 μm.

PMab-52 also detected lymphatic endothelial cells around the squamous cell carcinoma cells, but it could not detect vascular endothelial cells (Fig. 4C). PMab-52 very weakly detected normal lymphatic endothelial cells (Fig. 3), indicating that the expression level of podoplanin is different between normal and cancerous tissues.

Discussion

In this study, we first investigated podoplanin expression in feline normal tissues. PMab-52 strongly bound to feline kidneys (Fig. 1) and lungs (Fig. 2). These results are consistent with our previous study regarding podoplanin expression in rabbit normal tissues.⁽²⁹⁾ In contrast, anti-dog podoplanin mAb PMab-38 reacted with canine kidney, but not with canine lungs.⁽³⁰⁾ PMab-52 was weakly bound to normal lymphatic endothelial cells (Fig. 3), although podoplanin is known to be a specific marker for lymphatic endothelial cells. Likewise, PMab-38 did not detect normal lymphatic endothelial cells of canine normal tissues.⁽³⁰⁾ NZ-1, an anti-human podoplanin mAb, detects lymphatic endothelial cells but not those of the lung and kidney.⁽²⁶⁾ Moreover, cancer-specific mAbs LpMab-2⁽¹⁵⁾ and LpMab-23⁽³⁶⁾ do not react with human podoplanin in all normal tissues. Taken together, two main reasons can explain the different reaction patterns of anti-podoplanin mAbs: (1) the expression level of podoplanin is different among tissues and (2) the epitopes recognized by antipodoplanin mAbs vary due to many reasons, including tissue-specific posttranslational modification or cancer-specific glycosylation.

We investigated cat podoplanin expression in 40 squamous cell carcinoma tissues. PMab-52 strongly detected lymphatic endothelial cells around squamous cell carcinoma cells (Fig. 4C), whereas it weakly detected normal lymphatic endothelial cells (Fig. 3C), indicating that the expression level of podoplanin differs between normal and cancer tissues. We have previously reported that podoplanin expression can be induced by transforming growth factor-β,⁽³⁷⁾ which might explain the different expression levels of podoplanin in cancer tissues. Cancer-associated fibroblasts were also detected in some cases (case 14; Supplementary Fig. S1), which have been observed in many previous studies using antipodoplanin mAbs in human cancer tissues^(38–40) and using PMab-38 in canine squamous cell carcinomas.⁽³³⁾ Targeted therapy against cancer-associated fibroblasts is also important because cancer-associated fibroblasts have been associated with malignancy.^(38–40)

In conclusion, PMab-52 can be useful for clarifying the pathophysiological function of podoplanin in feline squamous cell carcinomas. Furthermore, it is a potential candidate for developing antibody-based therapies against feline squamous cell carcinomas.