PMab-247 Detects Bear Podoplanin in Immunohistochemical Analysis

Introduction

Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein.⁽¹⁾ PDPN is also known as gp44 ⁽²⁾/Aggrus,⁽³⁾ which induces platelet aggregation through C-type lectin-like receptor-2 (CLEC-2).^(4,5) The sialic acid of platelet aggregation-stimulating domain of PDPN is known to be important for PDPN-induced platelet aggregation.^(6,7)

PDPN is expressed in several normal cells, including pulmonary type I alveolar cells, renal epithelial cells, and lymphatic endothelial cells.⁽¹⁾ PDPN is used to distinguish lymphatic endothelial cells from vascular endothelial cells in pathophysiological studies.⁽⁸⁾ The PDPN–CLEC-2 interaction was reported to facilitate the separation of embryonic blood and lymphatic vessels.⁽⁹⁾

The expression of human PDPN (hPDPN) has been reported in many malignant cancers, such as lung cancers,⁽¹⁰⁾ oral cancers,⁽¹¹⁾ and esophageal cancers.⁽¹²⁾ The expression of hPDPN is associated with cancer metastasis.^(3,13) We previously developed monoclonal antibodies (mAbs) against human,⁽¹⁴⁾ mouse,⁽¹⁵⁾ rat,⁽¹⁶⁾ rabbit,⁽¹⁷⁾ bovine,⁽¹⁸⁾ dog,⁽¹⁹⁾ cat,⁽²⁰⁾ pig,⁽²¹⁾ horse,⁽²²⁾ goat,⁽²³⁾ tiger,⁽²⁴⁾ alpaca,⁽²⁵⁾ whale,⁽²⁶⁾ and Tasmanian devil⁽²⁷⁾ PDPNs. Furthermore, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247.⁽²⁸⁾ In this study, we performed immunohistochemical analyses using PMab-247 against bPDPN.

Materials and Methods

Results and Discussion

Although we have established many anti-PDPN mAbs, such as anti-human,⁽¹⁴⁾ anti-mouse,⁽¹⁵⁾ anti-rat,⁽¹⁶⁾ anti-rabbit,⁽¹⁷⁾ anti-bovine,⁽¹⁸⁾ anti-dog,⁽¹⁹⁾ anti-cat,⁽²⁰⁾ anti-pig,⁽²¹⁾ anti-horse,⁽²²⁾ anti-goat,⁽²³⁾ anti-tiger,⁽²⁴⁾ anti-alpaca,⁽²⁵⁾ anti-whale,⁽²⁶⁾ and anti-Tasmanian devil⁽²⁷⁾ PDPNs, sensitive and specific anti-bPDPN mAbs, which are useful for immunohistochemical analyses, had not been developed. Recently, two mice were immunized with Chinese hamster ovary (CHO)/bPDPN cells.⁽²⁸⁾ The developed hybridomas were seeded into 96-well plates and cultivated for 9 days. Wells positive for CHO/bPDPN and negative for CHO-K1 were selected by flow cytometry. After several additional screenings, PMab-247 (IgG₁, kappa) was finally selected. PMab-247 specifically detected CHO/bPDPN cells by Western blot and immunohistochemistry, suggesting that the potential usefulness of PMab-247 for the functional analyses of bPDPN.

In this study, we performed immunohistochemical analyses against the brown bear. PDPN has been reported to be expressed in several tissues, including lung and kidney⁽¹⁾; we selected bear lung (Fig. 1) and bear kidney (Fig. 2) for immunohistochemical analyses. Expectedly, PMab-247 stained type I alveolar cells of bear lung (Fig. 1B), lymphatic endothelial cells of bear lung (Fig. 1C), and podocytes of bear kidney (Fig. 2B). These staining patterns are consistent with our previous immunohistochemical studies using several anti-PDPN mAbs.⁽²¹⁾ No staining was observed without primary antibodies against type I alveolar cells of bear lung (Fig. 1E), lymphatic endothelial cells of bear lung (Fig. 1F), and podocytes of bear kidney (Fig. 2D).

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FIG. 1.

Immunohistochemical analyses for bear lung. Bear lung sections were incubated with 5 μg/mL of PMab-247 (A–C) or with blocking buffer (D–F), followed by that with the EnVision+ Kit. (G–I) Hematoxylin and eosin staining. Red arrows, lymphatic endothelial cells. Scale bar = 100 μm.

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FIG. 2.

Immunohistochemical analyses for bear kidney. Bear kidney sections were incubated with 5 μg/mL of PMab-247 (A, B) or with blocking buffer (C, D), followed by that with the EnVision+ Kit. (E, F) Hematoxylin and eosin staining. Scale bar = 100 μm.

In conclusion, PMab-247 is useful for immunohistochemical analyses against the bPDPN. The epitope of PMab-247 needs further investigation to clarify the sensitivity and specificity of PMab-247 against bPDPN. PMab-247 could be useful in elucidating the pathophysiological functions of bPDPN in future studies.