MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 4, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0019

PMab-247 Detects Bear Podoplaninin Immunohistochemical Analysis

Downloaded by Yukinari Kato from www.liebertpub.com at 08/13/19. For personal use only.

Junko Takei,1,2 Yoshikazu Furusawa,1,3 Shinji Yamada,1 Takuro Nakamura,1 Yusuke Sayama,1 Masato Sano,1Satoru Konnai,4,5 Atsushi Kobayashi,6 Hiroyuki Harada,2 Mika K. Kaneko,1 and Yukinari Kato1,3Podoplanin (PDPN) is utilized as a specific marker of type I alveolar cells of lung and lymphatic endothelial
cells of every tissue. Therefore, sensitive and specific monoclonal antibodies detecting PDPN are necessary for
immunohistochemical analyses, especially using formalin-fixed paraffin-embedded tissues. Recently, we developed an anti-bear PDPN (bPDPN) mAb, PMab-247, which is useful for Western blot, flow cytometry, andimmunohistochemical analyses. In this study, immunohistochemical analyses showed that PMab-247 strongly
detected bPDPN, which is expressed in type I alveolar cells and lymphatic endothelial cells of bear lung andpodocytes of bear kidney. These findings suggest that PMab-247 could be useful for pathophysiological analyses using immunohistochemistry.
Keywords: bear podoplanin, PDPN, type I alveolar cells, lymphatic endothelial cells, PMab-247

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 ofplatelet 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, andlymphatic endothelial cells.(1) PDPN is used to distinguish
lymphatic endothelial cells from vascular endothelial cells inpathophysiological studies.(8) The PDPN–CLEC-2 interaction was reported to facilitate the separation of embryonicblood 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 expressionof 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, wedeveloped an anti-bear PDPN (bPDPN) mAb, PMab-247.(28)In this study, we performed immunohistochemical analyses
using PMab-247 against bPDPN.
Materials and Methods
Immunohistochemical analyses

Brown bear (Ursus arctos) tissues were collected afterautopsy at Hokkaido University, fixed in 10% neutralbuffered formalin, and routinely processed to make paraffinembedded tissue sections. Histological sections (4-mm thick)were directly autoclaved in citrate buffer (pH 6.0; Nichirei
Biosciences, Inc., Tokyo, Japan) for 20 minutes. Then, sections were blocked using the SuperBlock T20 (phosphatebuffered saline) Blocking Buffer (Thermo Fisher Scientific,Inc., Waltham, MA), incubated with PMab-247 (5 mg/mL)
for 1 hour at room temperature, and treated with the EnVision+ Kit for mouse (Agilent Technologies, Inc., Santa Clara,CA) for 30 minutes. Color was developed using 3,3¢diaminobenzidine tetrahydrochloride (Agilent Technologies,Inc.) for 2 minutes, and counterstaining was performed using
hematoxylin (FUJIFILM Wako Pure Chemical Corporation,Osaka, Japan).

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University,Tokyo, Japan.
3
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
4
Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University,Sapporo, Japan.
5Department of Advanced Pharmaceutics, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
6
Laboratory of Comparative Pathology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
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Results and Discussion

Although we have established many anti-PDPN mAbs, suchas anti-human,(14) anti-mouse,(15) anti-rat,(16) anti-rabbit,(17)anti-bovine,(18) anti-dog,(19) anti-cat,(20) anti-pig,(21) antihorse,(22) anti-goat,(23) anti-tiger,(24) anti-alpaca,(25) antiwhale,(26) and anti-Tasmanian devil(27) PDPNs, sensitive andspecific 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 wereseeded into 96-well plates and cultivated for 9 days. Wells
positive for CHO/bPDPN and negative for CHO-K1 wereselected by flow cytometry. After several additional screenings, PMab-247 (IgG1, kappa) was finally selected. PMab247 specifically detected CHO/bPDPN cells by Western blot
and immunohistochemistry, suggesting that the potential
usefulness of PMab-247 for the functional analyses ofbPDPN.
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 bearkidney (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).

FIG. 2. Immunohistochemical analyses for bear kidney.
Bear kidney sections were incubated with 5 mg/mL ofPMab-247 (A, B) or with blocking buffer (C, D), followedby that with the EnVision+ Kit. (E, F) Hematoxylin andeosin staining. Scale bar = 100 mm.

FIG. 1. Immunohistochemical analyses for bear lung. Bear lung sections were incubated with 5 mg/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 mm.

PMAB-247 AGAINST BEAR PDPN

In conclusion, PMab-247 is useful for immunohistochemical analyses against the bPDPN. The epitope of PMab247 needs further investigation to clarify the sensitivity andspecificity of PMab-247 against bPDPN. PMab-247 could be
useful in elucidating the pathophysiological functions ofbPDPN in future studies.
Acknowledgments

This research was supported in part by AMED under Grant
Numbers JP19am0101078 (Y.K.), JP18am0301010 (Y.K.),
and JP19ae0101028 (Y.K.), and by JSPS KAKENHI Grant
Numbers 17K07299 (M.K.K.), 16K10748 (Y.K.), and19K07705 (Y.K.).
Author Disclosure Statement

Downloaded by Yukinari Kato from www.liebertpub.com at 08/13/19. For personal use only.

No competing financial interests exist.
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery CenterTohoku University
2-1, Seiryo-machi
Aoba-ku, Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: April 12, 2019
Accepted: June 18, 2019