MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 35, Number 2, 2016ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2015.0078

Original Articles

Critical Epitope of Anti-Rabbit Podoplanin Monoclonal
Antibodies for Immunohistochemical Analysis
Ryusuke Honma,1,2,* Yuki Fujii,1,* Satoshi Ogasawara,1,* Hiroharu Oki,1,2 Satoru Konnai,3
Yumiko Kagawa,4 Michiaki Takagi,2 Mika K. Kaneko,1 and Yukinari Kato1

Podoplanin (PDPN) is a type I transmembrane sialoglycoprotein, which is expressed in several normal cells,
including lymphatic endothelial cells throughout the body, podocytes of the kidney, and lung type I alveolar cellsof the lung. We have established many monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, and
rat PDPN. In addition, we recently produced an anti-rabbit PDPN mAb, PMab-32, which was established byimmunizing mice with recombinant proteins of rabbit PDPN. Herein, we compared the reactivity of PMab-32 withthat of newly established anti-rabbit PDPN mAbs, PMab-33 and PMab-21, against normal tissues in immunohistochemistry. PMab-32 reacted with podocytes, type I alveolar cells, and lymphatic endothelial cells, whereas
PMab-33 detected only podocytes and type I alveolar cells but not lymphatic endothelial cells. PMab-21 was notuseful in immunohistochemistry. We identified the epitope of PMab-32 and PMab-33 as Ser61-Ala68 of rabbit
PDPN using western blot and flow cytometric analyses. In contrast, the epitope of PMab-21 was identified as
Leu44-Glu48, which is corresponding to platelet aggregation-stimulating (PLAG) domain, indicating that Ser61Ala68 of rabbit PDPN is a more appropriate epitope for immunohistochemistry compared with PLAG domain.
PMab-32 could be useful for uncovering the function of rabbit PDPN.
reticular cells and lymph node microarchitecture.(22) The
physical elasticity of lymph nodes is maintained by PDPN ofstromal fibroblastic reticular cells.(23) PDPN is expressed inhuman fetal rib and chondrocytes of the proliferative andhypertrophic regions of the growth plate.(24) Binding of thecytoplasmic tail of PDPN to the ezrin, radixin, and moesin
proteins may change cytoskeletal organization, which alters
the phenotype of PDPN-expressing cells. This may contribute to morphological changes in the rudiment cartilages,
which lead to the establishment of the primary ossification
centers during the endochondral ossification.
The function of rabbit PDPN has not been clarified because
no anti-rabbit PDPN monoclonal antibody (mAb) has been
established. In this study, we investigated three anti-rabbit
mAbs and determined the critical epitope for immunohistochemistry.

IntroductionP

odoplanin (PDPN) is a type I transmembrane sialoglycoprotein, which is expressed in several normal cells, including lymphatic endothelial cells, renal podocytes, pulmonary
type I alveolar cells, osteocytes, and chondrocytes.(1) The expression of PDPN has been reported in many malignant tumors,such as oral squamous cell carcinomas,(2) malignant brain tumors,(3–6) lung cancers,(7) esophageal cancers,(8) malignant
mesotheliomas,(9,10) testicular tumors,(11) osteosarcomas,(1,12,13)
and chondrosarcomas.(1) PDPN expression is also associatedwith malignant progression and cancer metastasis.(3,14,15)
PDPN, also known as Aggrus,(16) activates platelet aggregation by binding to the C-type lectin-like receptor-2(CLEC-2) on platelet.(14,17,18) The interaction with CLEC-2
was mainly observed at Glu47 and Asp48 in the platelet
aggregation-stimulating (PLAG) domain and the a2,6-linked
sialic acid at Thr52 of human PDPN.(19) PDPN possesses
many other important functions. The interaction between
PDPN and CLEC-2 facilitates blood–lymphatic vessel separation.(20) The actin cytoskeleton is rearranged in CLEC-2expressing dendritic cells to promote efficient motility along
PDPN-expressing stromal surfaces.(21) The signal from
CLEC-2 to PDPN controls the contractility of fibroblastic

Materials and Methods
Cell lines, rabbit tissues, and animals

Chinese hamster ovary (CHO)-K1 and P3U1 were purchased from the American Type Culture Collection (ATCC,
Manassas, VA). CHO-K1 cells were transfected with the1

Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Orthopaedic Surgery, Yamagata University Faculty of Medicine, Yamagata, Japan.
3
Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
4
North Lab, Hokkaido, Japan.
*These authors contributed equally to this work.
2

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rabbit PDPN (rabPDPN)-PA tag plasmid using a Gene Pulser
Xcell Electroporation System (Bio-Rad Laboratories, Inc.,
Philadelphia, PA). CHO-K1, CHO/rabPDPN, and P3U1 were
cultured in the RPMI 1640 medium (Nacalai Tesque, Inc.,
Kyoto, Japan) supplemented with 10% heat-inactivated fetal
bovine serum (Thermo Fisher Scientific, Inc., Waltham,MA), 100 units/mL of penicillin, 100 mg/mL of streptomycin,
and 25 mg/mL of amphotericin B (Nacalai Tesque, Inc.) at
37C in a humidified atmosphere of 5% CO2 and 95% air.
Female BALB/c mice (4 weeks old) were purchased from
CLEA Japan (Tokyo, Japan). Animals for immunization
were housed under pathogen-free conditions. The Animal
Care and Use Committee of the Tohoku University approved
the animal experiments described herein.
Hybridoma production

BALB/c mice were immunized by intraperitoneal (i.p.) injection of 100 mg of recombinant rabbit PDPN(25) or synthesized rabbit PDPN peptides (39–52 amino acids) together with
Imject Alum (Thermo Fisher Scientific, Inc.). After several
additional immunizations of 50 mg, a booster injection of 50 mg
was given i.p. 2 days before spleen cells were harvested. The
spleen cells were fused with P3U1 cells using GenomONE-CF(Ishihara Sangyo Kaisha, Ltd., Osaka, Japan) against immu-

HONMA ET AL.

nization of recombinant rabbit PDPN or PEG1500 (Roche
Diagnostics, Indianapolis, IN) against immunization of synthesized rabbit PDPN peptides. The hybridomas were grown in
the RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.).
Enzyme-linked immunosorbent assay

Recombinant proteins of rabbit PDPN or synthesized
rabbit PDPN peptides were immobilized on Nunc Maxisorp
96-well immunoplates (Thermo Fisher Scientific, Inc.) at
1 mg/mL for 30 minutes. After blocking with 1% bovine serum albumin (BSA)/phosphate-buffered saline (PBS), theplates were incubated with culture supernatant, followed by
1:3000 diluted peroxidase-conjugated anti-mouse IgG(Dako; Agilent Technologies, Inc., Santa Clara, CA). The
enzymatic reaction was conducted with a 1-Step Ultra TMBELISA (Thermo Fisher Scientific, Inc.). The optical density
was measured at 655 nm using an iMark Microplate Reader(Bio-Rad Laboratories, Inc.).
Immunohistochemical analyses

Rabbit normal tissues were obtained from the Yamagata
University,(25) and one rabbit fibrosarcoma tissue was obtained

FIG. 1. Immunohistochemical analysis against normal tissues by PMab-32, PMab-33, and PMab-21. Sections of the rabbit
kidney, lung, and small intestine were incubated with 5 mg/mL of PMab-32, PMab-33, or PMab-21, followed by EnVision+
Kit, and color was developed using DAB and counterstained with hematoxylin. Arrows: glomerulus; arrowheads: lymphatic
vessels. Scale bar: 100 mm. DAB, 3,3¢-diaminobenzidine tetrahydrochloride.

ANTI-RABBIT PDPN MAB

from the North Lab. Four-micrometer-thick histological sections were deparaffinized in xylene and rehydrated. Then, they
were autoclaved in citrate buffer (pH 6.0, Dako; Agilent
Technologies, Inc.) for 20 minutes. Sections were incubatedwith PMab-32, PMab-33, and PMab-21 for 1 hour at room
temperature, followed by treatment with EnVision+ Kit for 30
minutes (Dako; Agilent Technologies, Inc.). Color was developed using 3,3¢-diaminobenzidine tetrahydrochloride(DAB, Dako; Agilent Technologies, Inc.) for 1 minute, andthen, the sections were counterstained with hematoxylin(Wako Pure Chemical Industries Ltd., Osaka, Japan).

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Production of rabbit PDPN mutants

The amplified rabbit PDPN cDNA was subcloned into a
pCAG vector (Wako Pure Chemical Industries Ltd.), and a
PA tag(26) was added at the N-terminus. Substitutions ofamino acids to alanine in rabbit PDPN were performed usinga HotStar HiFidelity polymerase chain reaction (PCR;
QIAGEN, Inc., Hilden, Germany) with oligonucleotides
containing the desired mutations. PCR fragments bearing thedesired mutations were inserted into a pCAG vector using InFusion PCR Cloning Kit (Clontech, Palo Alto, CA). CHO-K1

FIG. 2. Characterization of PMab-32 and PMab-33. (A) Epitope mapping of PMab-32 and PMab-33 using western blot
analysis. Cell lysate (10 mg) was electrophoresed on 5%–20% polyacrylamide gels and transferred onto a PVDF membrane.
After blocking, the membrane was incubated with 1 mg/mL of mAbs (PMab-32, PMab-33, anti-PA tag, and anti-b-actin) andthen with peroxidase-conjugated secondary antibodies; the membrane was developed with Pierce Western Blotting Substrate Plus or ImmunoStar LD Chemiluminescence Reagent and detected using a Sayaca-Imager. (B) Epitope mapping of
PMab-32 and PMab-33 using flow cytometry. Point mutants of rabbit PDPN were treated with PMab-32 (10 mg/mL), PMab33 (10 mg/mL), and anti-PA tag (1 mg/mL) for 30 minutes at 4C, followed by treatment with Oregon Green-conjugated antimouse IgG or anti-rat IgG. Fluorescence data were collected using a Cell Analyzer EC800. Red line: PMab-32, PMab-33, or
anti-PA tag. Black line: negative control. (C) Determination of binding affinities of PMab-32 and PMab-33 using flow
cytometry. mAbs, monoclonal antibodies; PDPN, podoplanin.

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cells were transfected with the plasmids using a Gene Pulser
Xcell Electroporation System (Bio-Rad Laboratories, Inc.).
Western blot analyses

Cell lysates (10 mg) were boiled in sodium dodecyl sulfate
sample buffer (Nacalai Tesque, Inc.). The proteins were
electrophoresed on 5%–20% polyacrylamide gels (Wako
Pure Chemical Industries Ltd.) and were transferred onto a
polyvinylidene difluoride (PVDF) membrane (EMD Millipore Corp., Billerica, MA). After blocking with 4% skim
milk (Nacalai Tesque, Inc.), the membrane was incubatedwith PMab-32, PMab-33, PMab-21, anti-PA tag (clone: NZ1), and anti-b-actin (clone AC-15; Sigma-Aldrich Corp., St.
Louis, MO) and then with peroxidase-conjugated anti-mouseor anti-rat antibodies (1:1000 diluted, Dako; Agilent Technologies, Inc.) and developed with the Pierce Western Blotting Substrate Plus (Thermo Fisher Scientific, Inc.) or the

HONMA ET AL.

ImmunoStar LD Chemiluminescence Reagent (Wako Pure
Chemical Industries Ltd.) using a Sayaca-Imager (DRC Co.
Ltd., Tokyo, Japan). NZ-1 was previously developed against
human PDPN(27) and has been used as anti-PA tag mAb.(26)
NZ-1 does not cross-react with the other PDPN proteins, suchas mouse PDPN, rat PDPN, and rabbit PDPN.
Flow cytometry

Cells were harvested by a brief exposure to 0.25% trypsin/
1 mM EDTA (Nacalai Tesque, Inc.). After washing with
0.1% BSA/PBS, the cells were treated with PMab-32(10 mg/mL), PMab-33 (10 mg/mL), PMab-21 (10 mg/mL), andanti-PA tag (clone: NZ-1, 1 mg/mL) for 30 minutes at 4C,
followed by treatment with Oregon Green-conjugated antimouse IgG or anti-rat IgG (1:1000 diluted; Thermo Fisher
Scientific, Inc.). Fluorescence data were collected using a
Cell Analyzer EC800 (Sony Corp., Tokyo, Japan).

FIG. 3. Characterization of PMab-21. (A) Epitope mapping of PMab-21 using western blot analysis. Cell lysate (10 mg)
was electrophoresed on 5%–20% polyacrylamide gels and transferred onto a PVDF membrane. After blocking, themembrane was incubated with 1 mg/mL of mAbs (PMab-21, anti-PA tag, and anti-b-actin) and then with peroxidaseconjugated secondary antibodies; the membrane was developed with Pierce Western Blotting Substrate Plus or ImmunoStar
LD Chemiluminescence Reagent and detected using a Sayaca-Imager. (B) Epitope mapping of PMab-21 using flow
cytometric analysis. Point mutants of rabbit PDPN were treated with PMab-21 (10 mg/mL) and anti-PA tag (1 mg/mL) for 30
minutes at 4C, followed by treatment with Oregon Green-conjugated anti-mouse IgG or anti-rat IgG. Fluorescence data
were collected using a Cell Analyzer EC800. Red line: PMab-21 or anti-PA tag. Black line: negative control. (C) Determination of binding affinity of PMab-21 using flow cytometry. PVDF, polyvinylidene difluoride.

ANTI-RABBIT PDPN MAB
Determination of the binding affinity using
flow cytometry

CHO/rabPDPN (2 · 105 cells) were resuspended in 100 mLof serially diluted antibodies (0.0061–100 mg/mL), followed
by secondary anti-mouse IgG (Thermo Fisher Scientific,Inc.). Fluorescence data were collected using a Cell Analyzer(EC800; Sony Corp.). The dissociation constants (KD) wereobtained by fitting the binding isotherms using the built-in
one-site binding models in GraphPad Prism 6 (GraphPad
Software, Inc., La Jolla, CA).
Results
Production of mAbs against rabbit PDPN

In this study, we used two different antigens for producing
mAbs against rabbit PDPN: one is recombinant rabbit PDPN
of full length; the other is the synthetic peptide (39–52 aminoacids) of PLAG domains of rabbit PDPN. After immunizing
mice with the recombinant protein, which was purified from
CHO/rabPDPN cells, the enzyme-linked immunosorbent

69

assay (ELISA) screening was performed. Twenty-four wells
reacted with CHO/rabPDPN in flow cytometry. Seventeen of
the 24 wells of IgG class were cloned using limiting dilution,and two clones, such as PMab-32 (mouse IgG1, kappa) and
PMab-33 (mouse IgG1, kappa), were established. After immunizing mice with the synthetic peptide of PLAG domainsof rabbit PDPN, we established 24 clones (PMab-8–PMab31), which react with CHO/rabPDPN cells in flow cytometry.
Immunohistochemical analysis against normal tissues

First, we investigated whether PMab-32 and PMab-33 detect rabbit PDPN of normal tissues, such as the kidney, lung,and small intestine. As shown in Figure 1, PMab-32 reactedwith podocytes of the kidney, type I alveolar cells of the lung,and lymphatic endothelial cells of the small intestine. In contrast, PMab-33 detected podocytes and type I alveolar cells but
not lymphatic endothelial cells. Second, we used 24 clones(PMab-8–PMab-31) for immunohistochemistry. However, nomAb stained PDPN-expressing cells. Typical results areshown in Figure 1. One of those clones, PMab-21 did not stain

FIG. 4. Immunohistochemical analysis against rabbit tumors using PMab-32. (A–C) Sections of rabbit fibrosarcoma wereincubated with 1 mg/mL of PMab-32, followed by EnVision+ Kit, and color was developed using DAB and counterstainedwith hematoxylin. (D–F) Hematoxylin and eosin staining. Arrows: blood vessels; arrowheads: lymphatic vessels. Scale bar:
100 mm.

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any PDPN-expressing cells, such as podocytes, type I alveolar
cells, and lymphatic endothelial cells.
Epitope mapping of anti-rabbit PDPN mAbs

Because PMab-32 and PMab-33 are useful for immunohistochemical analysis, we performed epitope mapping using
western blot analysis. Figure 2A showed that several point
mutants, such as S61A, G65A, T67A, and A68G, completely
lost the reaction by PMab-32. In contrast, PMab-33 did notrecognize the point mutants from Ser61 to Ala68. Flow cytometric analysis revealed that PMab-32 did not react with
G65A (Fig. 2B). PMab-33 did not react with S61A andweakly reacted with G65A, T67A, and A68G. Taken together, the epitope of PMab-32 is Ser61-Ala68, and Gly65 is
the most important amino acid. In contrast, the epitope ofPMab-33 is Ser61-Ala68, and Ser61, Gly65, Thr67, andAla68 are the most important amino acids. Flow cytometricanalysis showed that the KD of PMab-32 and PMab-33 were
determined to be 3.1 · 10-8 M and 9.8 · 10-8 M, respectively,
demonstrating that the apparent binding affinity of PMab-32
is higher than that of PMab-33 (Fig. 2C).
To determine the epitope of PMab-21, we performed
western blot analysis. PMab-21 reaction was lost in point
mutations of 44–48 amino acids (Fig. 3A). Flow cytometricanalysis revealed that PMab-21 did not react with L44A,
G46A, and V47A and weakly reacted with T45A and E48G
(Fig. 3B). Taken together, the epitope of PMab-21 is Leu44Glu48, and Leu44, Gly46, and Val47 are the most importantamino acids. Flow cytometric analysis showed that the KD of
PMab-21 was determined to be 7.8 · 10-9 M (Fig. 3C).
Immunohistochemical analysis against tumors

We finally investigated whether PMab-32 is applicable for
immunohistochemical staining against rabbit tumors. PMab32 stained lymphatic endothelial cells of fibrosarcoma tissues, whereas it did not recognize blood vessels (Fig. 4A). Of
interest, PMab-32 also stained tumor cells of rabbit fibrosarcoma in a membranous staining pattern (Fig. 4B, C).
Discussion

We have established mAbs against human PDPN,(6,27–31)
mouse PDPN,(32) and rat PDPN.(33) In addition, we previously reported a novel anti-rabbit PDPN mAb, PMab-32,
which was established by immunizing mice with recombinant proteins of rabbit PDPN.(25) Furthermore, anti-rabbit
PDPN mAbs were established in this study and were wellcharacterized. Many anti-human PDPN mAbs, such as
LpMab-9, LpMab-10, NZ-1, and D2-40, react with PLAG
domain of human PDPN.(30,31,34) In contrast, anti-human
PDPN mAbs against non-PLAG domains, such as LpMab-2,
LpMab-3, LpMab-7, and LpMab-17, are also useful forwestern blot, flow cytometry, and immunohistochemistry.(6,28,29,35) Especially, LpMab-7 possesses a much higher
sensitivity in immunohistochemistry compared with NZ-1
and D2-40.(12)Because anti-rabbit PDPN mAbs have not been establishedand characterized, we herein investigated three anti-rabbit
PDPN mAbs and determined the critical epitope for immunohistochemistry. PMab-32 and PMab-33 are useful to detectrabbit PDPN of normal tissues in immunohistochemical

HONMA ET AL.

analysis, although PMab-33 did not detect lymphatic endothelial cells. In contrast, 24 clones (PMab-8–PMab-31), whichwere established by immunizing PLAG domain, did not stain
any PDPN-expressing cells (data not shown). The PLAG domains of PDPN have been known to be suitable for producing
mAbs, which are useful for immunohistochemical analysis:
NZ-1 mAb against human PLAG domain,(27) PMab-1 mAb
against mouse PLAG domain,(32) and PMab-2 mAb against rat
PLAG domain.(33) Therefore, PLAG domain of rabbit PDPN
might be different from those of other species.
Epitope mapping of anti-rabbit PDPN mAbs demonstrated that the critical epitope of anti-rabbit PDPN mAb for
immunohistochemistry is Ser61-Ala68, and Gly65 is the mostimportant amino acid. The binding affinity of PMab-21 ismuch higher than those of PMab-32 and PMab-33, althoughPMab-21 is not applicable to immunohistochemistry. These
results demonstrate that the high binding affinity is not sufficient for the usefulness in immunohistochemistry against
rabbit PDPN. PMab-32 also stained lymphatic endothelial
cells and tumor cells of fibrosarcoma,(36) demonstrating that
PMab-32 could be useful for diagnostic tool for rabbit tumors.
In conclusion, PMab-32 is very useful for not only western
blot analysis and flow cytometry but also immunohistochemical analysis. The critical epitope of anti-rabbit PDPN
mAb for immunohistochemistry is Ser61-Ala68, and Gly65
is the most important amino acid. PMab-32 could be usefulfor uncovering the function of rabbit PDPN.
Acknowledgments

We thank Takuro Nakamura, Noriko Saidoh, Hazuki
Kanno, Kanae Yoshida, and Eiko Saito for their excellent
technical assistance. This work was supported in part by the
Platform for Drug Discovery, Informatics, and Structural
Life Science (PDIS) from the Japan Agency for Medical
Research and Development, AMED (Y.K.); by the Basic
Science and Platform Technology Program for Innovative
Biological Medicine from AMED (Y.K.); by the Regional
Innovation Strategy Support Program from the Ministry of
Education, Culture, Sports, Science and Technology(MEXT) of Japan (Y.K.); by JSPS KAKENHI grant numbers
26440019 (M.K.K.) and 25462242 (Y.K.); and by the Takeda
Science Foundation (S.O.).
Author Disclosure Statement

No competing financial interests exist.
References

1. Ariizumi T, Ogose A, Kawashima H, Hotta T, Li G, Xu Y,
Umezu H, Sugai M, and Endo N: Expression of podoplaninin human bone and bone tumors: New marker of osteogenic
and chondrogenic bone tumors. Pathol Int 2010;60:193–202.
2. Ochoa-Alvarez JA, Krishnan H, Pastorino JG, Nevel E,
Kephart D, Lee JJ, Retzbach EP, Shen Y, Fatahzadeh M,
Baredes S, Kalyoussef E, Honma M, Adelson ME, Kaneko
MK, Kato Y, Young MA, Deluca-Rapone L, Shienbaum
AJ, Yin K, Jensen LD, and Goldberg GS: Antibody andlectin target podoplanin to inhibit oral squamous carcinoma
cell migration and viability by distinct mechanisms. Oncotarget 2015;6:9045–9060.
3. Mishima K, Kato Y, Kaneko MK, Nishikawa R, Hirose T,
and Matsutani M: Increased expression of podoplanin in

ANTI-RABBIT PDPN MAB

malignant astrocytic tumors as a novel molecular marker ofmalignant progression. Acta Neuropathol 2006;111:483–
488.
4. Mishima K, Kato Y, Kaneko MK, Nakazawa Y, Kunita A,
Fujita N, Tsuruo T, Nishikawa R, Hirose T, and Matsutani
M: Podoplanin expression in primary central nervous systemgerm cell tumors: A useful histological marker for the diagnosis of germinoma. Acta Neuropathol 2006;111:563–568.
5. Kato Y, Vaidyanathan G, Kaneko MK, Mishima K, Srivastava N, Chandramohan V, Pegram C, Keir ST, Kuan
CT, Bigner DD, and Zalutsky MR: Evaluation of antipodoplanin rat monoclonal antibody NZ-1 for targeting
malignant gliomas. Nucl Med Biol 2010;37:785–794.
6. Kato Y, and Kaneko MK: A cancer-specific monoclonal
antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep 2014;4:5924.
7. Kato Y, Kaneko M, Sata M, Fujita N, Tsuruo T, and Osawa
M: Enhanced expression of Aggrus (T1alpha/podoplanin),a platelet-aggregation-inducing factor in lung squamouscell carcinoma. Tumor Biol 2005;26:195–200.
8. Schoppmann SF, Jesch B, Riegler MF, Maroske F,
Schwameis K, Jomrich G, and Birner P: Podoplanin expressing cancer associated fibroblasts are associated withunfavourable prognosis in adenocarcinoma of the esophagus. Clin Exp Metastasis 2013;30:441–446.
9. Kimura N, and Kimura I: Podoplanin as a marker for mesothelioma. Pathol Int 2005;55:83–86.
10. Abe S, Morita Y, Kaneko MK, Hanibuchi M, Tsujimoto Y,
Goto H, Kakiuchi S, Aono Y, Huang J, Sato S, Kishuku M,
Taniguchi Y, Azuma M, Kawazoe K, Sekido Y, Yano S,
Akiyama S, Sone S, Minakuchi K, Kato Y, and Nishioka Y:
A novel targeting therapy of malignant mesothelioma using
anti-podoplanin antibody. J Immunol 2013;190:6239–6249.
11. Kato Y, Sasagawa I, Kaneko M, Osawa M, Fujita N, andTsuruo T: Aggrus: A diagnostic marker that distinguishes
seminoma from embryonal carcinoma in testicular germ
cell tumors. Oncogene 2004;23:8552–8556.
12. Kaneko MK, Oki H, Ogasawara S, Takagi M, and Kato Y:
Anti-podoplanin monoclonal antibody LpMab-7 detects
metastatic legions of osteosarcoma. Monoclon Antib Immunodiagn Immunother 2015;34:154–161.
13. Kunita A, Kashima TG, Ohazama A, Grigoriadis AE, andFukayama M: Podoplanin is regulated by AP-1 and promotes platelet aggregation and cell migration in osteosarcoma. Am J Pathol 2011;179:1041–1049.
14. Kato Y, Kaneko MK, Kunita A, Ito H, Kameyama A,
Ogasawara S, Matsuura N, Hasegawa Y, Suzuki-Inoue K,
Inoue O, Ozaki Y, and Narimatsu H: Molecular analysis of
the pathophysiological binding of the platelet aggregationinducing factor podoplanin to the C-type lectin-like receptor CLEC-2. Cancer Sci 2008;99:54–61.
15. Kunita A, Kashima TG, Morishita Y, Fukayama M, Kato
Y, Tsuruo T, and Fujita N: The platelet aggregationinducing factor aggrus/podoplanin promotes pulmonary
metastasis. Am J Pathol 2007;170:1337–1347.
16. Kato Y, Fujita N, Kunita A, Sato S, Kaneko M, Osawa M,
and Tsuruo T: Molecular identification of Aggrus/T1alphaas a platelet aggregation-inducing factor expressed in colorectal tumors. J Biol Chem 2003;278:51599–51605.
17. Kaneko MK, Kunita A, Abe S, Tsujimoto Y, Fukayama M,
Goto K, Sawa Y, Nishioka Y, and Kato Y: Chimeric antipodoplanin antibody suppresses tumor metastasis throughneutralization and antibody-dependent cellular cytotoxicity.
Cancer Sci 2012;103:1913–1919.

71

18. Suzuki-Inoue K, Kato Y, Inoue O, Kaneko MK, Mishima
K, Yatomi Y, Yamazaki Y, Narimatsu H, and Ozaki Y:
Involvement of the snake toxin receptor CLEC-2, in
podoplanin-mediated platelet activation, by cancer cells. J
Biol Chem 2007;282:25993–26001.
19. Nagae M, Morita-Matsumoto K, Kato M, Kaneko MK,
Kato Y, and Yamaguchi Y: A platform of C-type lectin-likereceptor CLEC-2 for binding O-glycosylated podoplanin
and nonglycosylated rhodocytin. Structure 2014;22:1711–
1721.
20. Bertozzi CC, Schmaier AA, Mericko P, Hess PR, Zou Z,
Chen M, Chen CY, Xu B, Lu MM, Zhou D, Sebzda E,
Santore MT, Merianos DJ, Stadtfeld M, Flake AW, Graf T,
Skoda R, Maltzman JS, Koretzky GA, and Kahn ML:
Platelets regulate lymphatic vascular development through
CLEC-2-SLP-76 signaling. Blood 2010;116:661–670.
21. Acton SE, Astarita JL, Malhotra D, Lukacs-Kornek V,
Franz B, Hess PR, Jakus Z, Kuligowski M, Fletcher AL,
Elpek KG, Bellemare-Pelletier A, Sceats L, Reynoso ED,
Gonzalez SF, Graham DB, Chang J, Peters A, Woodruff M,
Kim YA, Swat W, Morita T, Kuchroo V, Carroll MC, Kahn
ML, Wucherpfennig KW, and Turley SJ: Podoplanin-richstromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2. Immunity
2012;37:276–289.
22. Astarita JL, Cremasco V, Fu J, Darnell MC, Peck JR,
Nieves-Bonilla JM, Song K, Kondo Y, Woodruff MC,
Gogineni A, Onder L, Ludewig B, Weimer RM, Carroll
MC, Mooney DJ, Xia L, and Turley SJ: The CLEC-2podoplanin axis controls the contractility of fibroblastic
reticular cells and lymph node microarchitecture. Nat Immunol 2015;16:75–84.
23. Acton SE, Farrugia AJ, Astarita JL, Mourao-Sa D, Jenkins
RP, Nye E, Hooper S, van Blijswijk J, Rogers NC, Snelgrove KJ, Rosewell I, Moita LF, Stamp G, Turley SJ, Sahai
E, and Reis e Sousa C: Dendritic cells control fibroblastic
reticular network tension and lymph node expansion. Nature 2014;514:498–502.
24. Smith SM, and Melrose J: Podoplanin is expressed by a
sub-population of human foetal rib and knee joint rudimentchondrocytes. Tissue Cell 2011;43:39–44.
25. Honma R, Fujii Y, Ogasawara S, Oki H, Liu X, Nakamura
T, Kaneko MK, Takagi M, and Kato Y: Establishment of anovel monoclonal antibody PMab-32 against rabbit podoplanin. Monoclon Antib Immunodiagn Immunother 2016;35:41–47.
26. Fujii Y, Kaneko M, Neyazaki M, Nogi T, Kato Y, andTakagi J: PA tag: A versatile protein tagging system using a
super high affinity antibody against a dodecapeptide derived from human podoplanin. Protein Expr Purif 2014;
95:240–247.
27. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano K,
Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H, Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody
reacting with its platelet-aggregation-stimulating domain.
Biochem Biophys Res Commun 2006;349:1301–1307.
28. Oki H, Ogasawara S, Kaneko MK, Takagi M, Yamauchi M,
and Kato Y: Characterization of monoclonal antibody
LpMab-3 recognizing sialylated glycopeptide of podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:
44–50.
29. Oki H, Kaneko MK, Ogasawara S, Tsujimoto Y, Liu
X, Sugawara M, Takakubo Y, Takagi M, and Kato Y:

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Characterization of a monoclonal antibody LpMab-7 recognizing non-PLAG domain of podoplanin. Monoclon AntibImmunodiagn Immunother 2015;34:174–180.
30. Kaneko MK, Oki H, Hozumi Y, Liu X, Ogasawara S,
Takagi M, Goto K, and Kato Y: Monoclonal antibody
LpMab-9 recognizes O-glycosylated N-terminus of human
podoplanin. Monoclon Antib Immunodiagn Immunother2015;34:310–317.
31. Ogasawara S, Oki H, Kaneko MK, Hozumi Y, Liu X,
Honma R, Fujii Y, Nakamura T, Goto K, Takagi M, andKato Y: Development of monoclonal antibody LpMab-10
recognizing non-glycosylated PLAG1/2 domain including
Thr34 of human podoplanin. Monoclon Antib Immunodiagn Immunother 2015;34:318–326.
32. Kaji C, Tsujimoto Y, Kato Kaneko M, Kato Y, and Sawa
Y: Immunohistochemical examination of novel rat monoclonal antibodies against mouse and human podoplanin.
Acta Histochem Cytochem 2012;45:227–237.
33. Oki H, Honma R, Ogasawara S, Fujii Y, Liu X, Kaneko MK,
Takagi M, and Kato Y: Development of a sensitive monoclonal antibody PMab-2 against rat podoplanin. Monoclon
Antib Immunodiagn Immunother 2015;34:396–403.
34. Ogasawara S, Kaneko MK, Price JE, and Kato Y: Characterization of anti-podoplanin monoclonal antibodies:
Critical epitopes for neutralizing the interaction between
podoplanin and CLEC-2. Hybridoma 2008;27:259–267.

HONMA ET AL.

35. Kato Y, Ogasawara S, Oki H, Honma R, Takagi M, Fujii Y,
Nakamura T, Saidoh N, Kanno H, Umetsu M, Kamata S,
Kubo H, Yamada M, Sawa Y, Morita K, Harada H, Suzuki
H, and Kaneko MK: Novel monoclonal antibody LpMab17 developed by CasMab technology distinguishes human
podoplanin from monkey podoplanin. Monoclon AntibImmunodiagn Immunother 2016.
36. Suzuki H, Kato Y, Kaneko MK, Okita Y, Narimatsu H, andKato M: Induction of podoplanin by transforming growth
factor-beta in human fibrosarcoma. FEBS Lett 2008;582:341–
345.

Address correspondence to:
Dr. Yukinari Kato
Department of Regional Innovation
Tohoku University Graduate School of Medicine
2-1 Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp;
yukinarikato@med.tohoku.ac.jp
Received: December 17, 2015
Accepted: January 19, 2016