MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 5, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0030

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Immunohistochemical Analysis Using Antipodocalyxin
Monoclonal Antibody PcMab-47 Demonstrates
Podocalyxin Expression in Oral
Squamous Cell Carcinomas
Shunsuke Itai,1,2 Shinji Yamada,1 Mika K. Kaneko,1 Hiroyuki Harada,2 and Yukinari Kato1,3

Podocalyxin is a CD34-related type I transmembrane protein that is highly glycosylated with N-glycan, O-glycan,
and keratan sulfate. Podocalyxin was originally found in the podocytes of rat kidney and is reportedly expressed inmany types of tumors, including brain tumors, colorectal cancers, and breast cancers. Overexpression of podocalyxin is an independent predictor of progression, metastasis, and poor outcome. We recently immunized micewith recombinant human podocalyxin, which was produced using LN229 glioblastoma cells, and produced anovel antipodocalyxin monoclonal antibody (mAb), PcMab-47, which reacts with endogenous podocalyxinexpressing cancer cell lines and normal cell lines independent of glycosylation in Western blot, flow cytometry,
and immunohistochemical analyses. In this study, we performed immunohistochemical analysis against oral
cancers using PcMab-47. PcMab-47-stained oral squamous cell carcinoma cells in a cytoplasmic pattern anddetected 26/38 (68.4%) of oral squamous cell carcinoma cells on tissue microarrays. These results indicate that
PcMab-47 is useful in detecting podocalyxin of oral cancers for immunohistochemical analysis.
Keywords: podocalyxin, monoclonal antibody, immunohistochemistry, oral cancer

Introduction

P

odocalyxin is a type I transmembrane protein with amolecular weight of 150,000–200,000,(1–3) which was
first found in the podocytes of rat kidney,(4) and its homologues
have been reported in humans.(5,6) Podocalyxin is expressed innormal tissues, including podocytes and endothelial cells, andis similar to CD34, which is known as a hematopoietic stem
cell marker.(7) Podocalyxin is highly glycosylated with Oglycan and N-glycan.(8) Podocalyxin acts as an adhesive
molecule to bind to vascular endothelial cells or platelets(9,10);in contrast, podocalyxin functions as an antiadhesive molecule
through its negatively charged sialic acid and keratan sulfate,
for the formation and maintenance of the filtration slits between the podocyte processes in the kidney.(11)Several studies have developed antipodocalyxin monoclonal antibodies (mAbs).(12,13) The antigen of mAbs, TRA1-60 and TRA-1-81, is keratan sulfate, which is attached to
podocalyxin.(3) We recently immunized mice with recombinant human podocalyxin, which was produced using LN229
glioblastoma cells, and produced a novel antipodocalyxin

mAb, PcMab-47 (IgG1, kappa).(14) PcMab-47 reacts withendogenous podocalyxin-expressing cancer cell lines andnormal cells independently of glycosylation in Western blot,
flow cytometry, and immunohistochemical analyses.
In this study, we investigated whether the antipodocalyxin
mAb, PcMab-47, is useful for immunohistochemical detection of podocalyxin, which is expressed in oral cancers.
Materials and Methods
Production of hybridoma and purification of mAbs

The development of an antipodocalyxin mAb, PcMab-47,
was described previously.(14) In brief, 4-week-old female
BALB/c mice (CLEA, Tokyo, Japan) were immunized byintraperitoneal (i.p.) injection of the purified ectodomain ofhuman podocalyxin (100 mg) together with Imject Alum
(Thermo Fisher Scientific, Inc., Waltham, MA). After several
additional immunizations, a booster i.p. injection of LN229/
podocalyxin was given 2 days before the mice were euthanizedby cervical dislocation, and spleen cells were harvested. The

1

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Miyagi, 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, Miyagi, Japan.
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PCMAB-47 AGAINST OSCC

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spleen cells were fused with P3U1 (American Type Culture
Collection [ATCC], Manassas, VA) cells using PEG1500(Roche Diagnostics, Indianapolis, IN). Hybridomas weregrown in RPMI 1640 medium including L-glutamine withhypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.). Culture
supernatants were screened using enzyme-linked immunosorbent assay for binding to the purified ectodomain of
podocalyxin. MAbs were purified from supernatants ofhybridomas cultured in Hybridoma-SFM medium (Thermo
Fisher Scientific, Inc.) using Protein G Sepharose 4 Fast
Flow (GE Healthcare UK Ltd, Buckinghamshire, England).
Immunohistochemical analyses

Oral cancer tissues were purchased from US Biomax, Inc.
(Rockville, MD). Four-micrometer-thick histologic sections
were deparaffinized in xylene and rehydrated. After the antigen retrieval procedure (autoclave using citrate buffer, pH
6.0; Agilent Technologies, Inc., Santa Clara, CA), sections
were incubated with 5 mg/mL of PcMab-47 for 1 hour at room
temperature followed by treatment with Envision+ kit (Agilent Technologies, Inc.) for 30 minutes. Color was developed
using 3, 3-diaminobenzidine tetrahydrochloride (Agilent
Technologies, Inc.) for 2 minutes, and then the sections werecounterstained with hematoxylin (Wako Pure Chemical Industries Ltd., Osaka, Japan).

FIG. 1. Immunohistochemical analysis of oral cancers by
PcMab-47. Sections of oral cancers (Case No. 34) were incubated with 5 mg/mL of PcMab-47, followed by the Envision+ kit. Color was developed using 3, 3-diaminobenzidine
tetrahydrochloride (DAB) and counterstained with hematoxylin. PcMab-47 (A, B) and control (C, D). Serial sections
were also stained using hematoxylin and eosin (E, F). Arrows: endothelial cells. Scale bar: 100 mm.

221Results

Until now, podocalyxin expression has not been fully investigated in oral cancers because specific and sensitive mAbs
against podocalyxin have not been established. Recently, wedeveloped a sensitive and specific antipodocalyxin mAb,
PcMab-47 (mouse IgG1, kappa), which is useful for immunohistochemical analysis.(14) In this study, we performed
immunohistochemical analysis against oral cancers using
PcMab-47. PcMab-47 stained oral squamous cell carcinoma
cells in a cytoplasmic staining pattern (Fig. 1, Supplementary
Fig. S1, and Supplementary Fig. S2). In addition, podocalyxin
expression was also observed in endothelial cells (ECs) in
those cases (Fig. 1). Only ECs were stained by PcMab-47
when no podocalyxin expression was observed in oral cancer
cells (Supplementary Fig. S3). Podocalyxin expression was
also detected by PcMab-47 in ECs from normal tissues.(14)Using oral cancer tissue arrays (Supplementary Table S1),
26/38 (68.4%) of oral squamous cell carcinoma cells were
stained by PcMab-47 (Supplementary Table S2). Malignant
pleomorphic adenoma and adenolymphoma also showed thepositive staining by PcMab-47. In contrast, PcMab-47 did notstain pleomorphic adenoma, adenocystic carcinoma, andmucoepidermoid carcinoma.
Discussion

Podocalyxin is known as a diagnostic marker and prognostic
indicator in several cancers, including breast cancers,(15–17)
colorectal cancers,(18–21) gastric cancers,(22) prostate cancers,(23)testicular tumors,(2) renal cancers,(24) thyroid cancers,(25) bladder cancers,(26) ovarian cancers,(27) pancreatic cancers,(28,29)
and oral cancers.(30) Previously, we have reported that podocalyxin is associated with the malignant progression in brain
tumors.(8,31) The glycans on podocalyxin bind to E-/P-/Lselectin expressed on endothelium, platelets, and leukocytes, respectively.(32–34) These interactions enhance theformation of leukocyte–tumor–platelet aggregates and tumor cell arrest in the microvasculature.(35) Therefore, podocalyxin overexpression in cancer cells is a potential target for
antibody therapy.
We previously developed our original technology for production of cancer-specific monoclonal antibodies (CasMabs)
against membrane proteins.(36) Antipodoplanin CasMabs,
including LpMab-2 and LpMab-23, specifically recognizes
cancer-type PDPN in tumor tissues.(36,37) In addition, theCasMab technology is useful for generating antiglycopeptide mAbs (GpMabs), such as LpMab-3, LpMab-9, LpMab12, LpMab-19, and LpMab-21, against podoplanin.(38–40)
Moreover, it can generate mAbs, which bind to various
novel epitopes of podoplanin, such as LpMab-7, LpMab-10,
and LpMab-17.(36,40–42) We further produced a sensitiveand specific antipodocalyxin mAb, PcMab-47 (mouse IgG1,
kappa).(15) PcMab-47 reacted with not only cancer celllines, such as Caco-2 (colon adenocarcinoma cell line) and
MDA-MB-468 (breast cancer cell line) but also normal celllines, including HEK-293T (renal epithelial cell line) andvascular endothelial cells. Immunohistochemical studies
showed that PcMab-47 not only stained normal cells such as
podocytes or ECs of the kidney but also reacted with colon
adenocarcinoma, in which a membrane/cytoplasmic-staining
pattern was observed.(15) Strong expression of podocalyxin
was also observed in ECs in colon adenocarcinoma,(15)222

indicating that PcMab-47 is very useful for immunohistochemical detection of podocalyxin.
In this study, we demonstrated that PcMab-47 is useful for
detecting podocalyxin in oral squamous cell carcinoma cells
by immunohistochemical analysis. In addition, we recently
produced a mouse-human chimeric PcMab-47 (chPcMab-47)
and investigated its antitumor activity against podocalyxinexpressing tumors.(43) chPcMab-47 exerted antitumor activity
against mouse xenograft models using Chinese hamster ovary(CHO)/podocalyxin and HCT-15 (a colon cancer cell line),(43)suggesting that chPcMab-47 is useful for antibody therapy
against oral squamous cell carcinoma.

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Acknowledgments

We thank Takuro Nakamura, Miyuki Yanaka, Noriko
Saidoh, Saori Handa, and Yoshimi Nakamura for excellent
technical assistance. This work was supported, in part, by the
Basic Science and Platform Technology Program for Innovative Biological Medicine from Japan Agency for Medical Research and development, AMED (Y.K.). This work
was also supported, in part, 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 (Y.K.), by projectfor utilizing glycans in the development of innovative drug
discovery technologies from AMED (Y.K.), by the Platformfor Drug Discovery, Informatics, and Structural Life Science(PDIS) from AMED (Y.K.), and by JSPS KAKENHI Grant
Number 17K07299 (M.K.K.) and 16K10748 (Y.K.).
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd.
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi
Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: May 27, 2017
Accepted: August 3, 2017