Biochemistry and Biophysics Reports 18 (2019) 100616

Contents lists available at ScienceDirect

Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

PMab-219: A monoclonal antibody for the immunohistochemical analysis ofhorse podoplanin

TYoshikazu Furusawaa,b,c, Shinji Yamadaa, Shunsuke Itaia,d, Takuro Nakamuraa, Miyuki Yanakaa,
Masato Sanoa, Hiroyuki Haradad, Masato Fukuic, Mika K. Kanekoa, Yukinari Katoa,b,∗a

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapanNew Industry Creation Hatchery Center, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, JapancZENOAQ RESOURCE CO., LTD, 1-1 Tairanoue, Sasagawa, Asaka-machi, Koriyama, Fukushima, 963-0196, Japand
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku,
Tokyo, 113-8510, Japanb

A R T I C LE I N FO

A B S T R A C T

Keywords:
Horse podoplanin
PDPN
PMab-219

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), alymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse
PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present
study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/
horPDPN), and hybridomas producing mAbs against horPDPN were screened using ﬂow cytometry. One of themAbs, PMab-219 (IgG2a, kappa), speciﬁcally detected CHO/horPDPN cells via ﬂow cytometry and recognized
horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These ﬁndings suggest that PMab-219 is useful for investigating the function of horPDPN.

1. Introduction
Podoplanin (PDPN), a type I transmembrane glycoprotein, is expressed in normal tissues including renal podocytes, type I lung alveolar
cells, and lymphatic endothelial cells [1,2]. The interaction between
PDPN on lymphatic endothelial cells and C-type lectin-like receptor-2(CLEC-2) on platelets has been shown to facilitate embryonic blood/
lymphatic vessel separation [1,3–10].
The expression of human PDPN (hPDPN) has been reported inseveral malignant tumors, including oral squamous cell carcinomas[11], esophageal cancers [12], lung cancers [13], malignant mesotheliomas [14,15], osteosarcomas [16–18], chondrosarcomas [17], malignant brain tumors [19–22], and testicular tumors [23]. The expression of hPDPN is associated with malignant progression and cancer
metastasis [6,19,24].
Until now, we have developed monoclonal antibodies (mAbs)
against human [25], mouse [25], rat [26], rabbit [27], bovine [28], dog[29], and cat [30] PDPNs. Furthermore, an anti-cat PDPN mAb (PMab52) cross-reacted with tiger PDPN [31]. Although an anti-horse PDPN

(horPDPN) mAb, PMab-202 was recently established by immunizing
mice with synthetic peptides of horPDPN, it was not useful for immunohistochemical analysis [32]. Sensitive and speciﬁc mAbs against
horPDPN are necessary to investigate the expression and function of
horPDPN. In the present study, we immunized mice with CHO/
horPDPN cells and established hybridomas that could produce mAbs
against horPDPN.
2. Materials and methods
2.1. Cell lines
CHO-K1 and P3X63Ag8U.1 (P3U1) cells were obtained from theAmerican Type Culture Collection (ATCC, Manassas, VA, USA). The
horse kidney cell line, FHK-Tcl3.1, was established at Yamaguchi
University [33]. The horPDPN bearing an N-terminal PA16 tag (PA16horPDPN) was inserted into a pCAG-Ble vector (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan) [32]. The PA16 tag comprises 16
amino acids (GLEGGVAMPGAEDDVV) [34]. CHO-K1 cells wereAbbreviations: CBIS, Cell-Based Immunization and Screening; CHO, Chinese hamster ovary; CLEC-2, C-type lectin-like receptor-2; DAB, 3,3'-diaminobenzidine
tetrahydrochloride; ELISA, enzyme-linked immunosorbent assay; horPDPN, horse podoplanin; hPDPN, human podoplanin; mAb, monoclonal antibody; PBS,
phosphate-buﬀered saline; PDPN, podoplanin; PVDF, polyvinylidene diﬂuoride; SDS, sodium dodecyl sulfate∗
Corresponding author.New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2019.01.009
Received 2 November 2018; Received in revised form 8 December 2018; Accepted 27 January 20192405-5808/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/BY/4.0/).

Biochemistry and Biophysics Reports 18 (2019) 100616

Y. Furusawa et al.

PMab-219, 1 μg/mL of anti-PA16 tag (NZ-1), or 1 μg/mL of anti-β-actin(AC-15; Sigma-Aldrich Corp., St. Louis, MO, USA), and subsequently
with peroxidase-conjugated anti-mouse IgG (1:1000; Agilent
Technologies, Santa Clara, CA, USA) or anti-rat IgG (1:10000; SigmaAldrich Corp.). Bands were visualized with ImmunoStar LD (FUJIFILM
Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co.
Ltd., Tokyo, Japan).

transfected with pCAG-Ble/PA16-horPDPN using Lipofectamine LTX
with Plus Reagent (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA).
Stable transfectants were selected by limiting dilution and cultivated ina medium containing 0.5 mg/mL of zeocin (InvivoGen, San Diego, CA,USA).
P3U1, CHO-K1, and CHO/horPDPN cells were cultured in Roswell
Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc.,
Kyoto, Japan), and FHK-Tcl3.1 was cultured in Dulbecco's modiﬁed
Eagle's medium (DMEM; Nacalai Tesque, Inc.) [32]. All media were
supplemented with 10% heat-inactivated fetal bovine serum (Thermo
Fisher Scientiﬁc Inc.), 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B (Nacalai Tesque, Inc.). Cells
were grown at 37 °C in a humidiﬁed environment with an atmosphereof 5% CO2 and 95% ambient air.

2.7. Immunohistochemical analyses

Female BALB/c mice (6 weeks old) were purchased from CLEA
Japan (Tokyo, Japan). Animals were housed under speciﬁc pathogenfree conditions. The Animal Care and Use Committee of Tohoku
University approved all the animal experiments.

Cell blocks were produced using iPGell (Genostaﬀ Co., Ltd., Tokyo,
Japan) and processed to make 4-μm paraﬃn-embedded cell sections
that were directly autoclaved in citrate buﬀer (pH 6.0; Nichirei
Biosciences, Inc., Tokyo, Japan) for 20 min. These tissue sections were
blocked using SuperBlock T20 (PBS) Blocking Buﬀer (Thermo Fisher
Scientiﬁc Inc.), incubated with PMab-219 (1 μg/mL) for 1 h at room
temperature, and treated using an Envision + Kit (Agilent Technologies
Inc.) for 30 min. Color was developed using 3,3′-diaminobenzidine
tetrahydrochloride (Agilent Technologies Inc.) for 2 min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure
Chemical Corporation).

2.3. Hybridoma production

3. Results and discussion

Two BALB/c mice were immunized with CHO/horPDPN cells(1 × 108), which were intraperitoneally (i.p.) administered together
with Imject Alum (Thermo Fisher Scientiﬁc Inc.). The procedure included an additional three immunizations followed by a ﬁnal booster
injection administered i.p. 2 days prior to the harvest of spleen cells,
making a total of ﬁve immunizations. Subsequently, these spleen cells
were fused with P3U1 cells using PEG1500 (Roche Diagnostics,
Indianapolis, IN, USA), and the hybridomas were grown in RPMI
medium supplemented with hypoxanthine, aminopterin, and thymidine
for selection (Thermo Fisher Scientiﬁc Inc.). The culture supernatants
were screened using ﬂow cytometry.

In the present study, we employed a Cell-Based Immunization andScreening (CBIS) method to develop sensitive and speciﬁc mAbs against
horPDPN to facilitate the immunohistochemical analysis of paraﬃnembedded tissue sections. Previously, we have successfully utilized a
CBIS method to establish mAbs against various membrane proteins suchas cat PDPN [30], CD44 [34], CD133 [35], and PD-L1 [36]. Two mice
were immunized with CHO/horPDPN cells using an immunization andscreening procedure (Fig. 1). Developed hybridomas were seeded into
96-well plates and cultivated for 10 days. Wells positive for CHO/
horPDPN and negative for CHO-K1 were selected using ﬂow cytometry.
Moreover, FHK-Tcl3.1 cells were used to identify antibodies that reacted with the endogenous horPDPN. Screening identiﬁed strong signals against CHO/horPDPN cells and weak or no signals against CHOK1 cells in 19 of 960 wells (2.0%). Of these 19 wells, nine hybridomas
were developed: one clone of IgG1, one clone of IgG2a, ﬁve clones of
IgG3, and two clones of IgM. One of these nine clones, PMab-219 (IgG2a,kappa), was ﬁnally selected using immunohistochemistry against horsetissues.
PMab-219 recognized CHO/horPDPN but showed no reaction with
CHO-K1, as assessed using ﬂow cytometry (Fig. 2). PMab-219 reactedwith FHK-Tcl3.1 cells, indicating that PMab-219 was able to recognize
endogenous horPDPN. PMab-219 did not react with human, mouse, rat,
rabbit, dog, cat, or bovine PDPNs (data not shown). Furthermore, it did
not react with pig, Tasmanian devil, tiger, alpaca, bear, goat, sheep, or
whale PDPNs (data not shown), indicating that PMab-219 is speciﬁc to
horPDPN.
Additionally, a kinetic analysis conducted using ﬂow cytometry
assessed the interaction of PMab-219 with CHO/horPDPN and FHKTcl3.1 cells. KD of PMab-219 for CHO/horPDPN and FHK-Tcl3.1 cells
were determined to be 8.6 × 10−8 and 6.1 × 10−7 M, respectively,
indicating moderate and low aﬃnity for CHO/horPDPN and FHKTcl3.1 cells, respectively.
Western blotting performed using PMab-219 (Fig. 3) demonstrated
that PMab-219 detects horPDPN as a 40 kDa band in CHO/horPDPN
cells. However, PMab-219 did not detect a 40 kDa band in FHKTcl3.1 cells; this might be attributed to low expression levels of
horPDPN in FHK-Tcl3.1 cells. NZ-1, an anti-PA16 tag mAb, detected 40
and 25 kDa bands in CHO/horPDPN cells. The 40 kDa band represents ahighly glycosylated form, whereas the 25 kDa band represents an unglycosylated one [3,22].
The immunohistochemical analyses revealed that PMab-219
strongly stained CHO/horPDPN cells (Fig. 4A) and did not react with

2.2. Animals

2.4. Flow cytometry
The cells were harvested following brief exposure to 0.25% trypsin/
1 mM EDTA (Nacalai Tesque, Inc.). The cells were washed with 0.1%
BSA/PBS and treated with primary mAbs for 30 min at 4 °C. Thereafter,
the cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG(1:2000; Cell Signaling Technology, Inc., Danvers, MA, USA) or Oregon
green anti-rat IgG (1:2000; Thermo Fisher Scientiﬁc Inc.). Fluorescencedata were collected using SA3800 Cell Analyzers (Sony Corp., Tokyo,
Japan).
2.5. Determination of binding aﬃnity using ﬂow cytometry
CHO/horPDPN or FHK-Tcl3.1 (2 × 105 cells) was suspended in
100 μL of serially diluted PMab-219, followed by addition of Alexa
Fluor 488-conjugated anti-mouse IgG (1:200; Cell Signaling
Technology, Inc.). Fluorescence data were collected using EC800 Cell
Analyzer (Sony Corp.). The dissociation constant (KD) was obtained byﬁtting the binding isotherms to built-in one-site binding models in
GraphPad PRISM 6 (GraphPad Software, Inc., La Jolla, CA, USA).
2.6. Western blotting
Cell lysates (10 μg) were boiled in sodium dodecyl sulfate (SDS)
sample buﬀer (Nacalai Tesque, Inc.). The proteins were subjected toelectrophoresis on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure
Chemical Corporation) and subsequently transferred onto a polyvinylidene diﬂuoride (PVDF) membrane (Merck KGaA, Darmstadt,
Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.),
each membrane was incubated with primary mAbs, such as 1 μg/mL of2

Biochemistry and Biophysics Reports 18 (2019) 100616

Y. Furusawa et al.

Fig. 2. Detection of horPDPN via ﬂow cytometry using PMab-219. CHO/
horPDPN, CHO-K1, and FHK-Tcl3.1 cells were treated with PMab-219 (red line)
or anti-PA16 tag (NZ-1; red line) at a concentration of 1 μg/mL or 0.1% BSA inPBS (gray) for 30 min, followed by incubation with secondary antibodies. (Forinterpretation of the references to colour in this ﬁgure legend, the reader isreferred to the Web version of this article.)

Fig. 1. Schematic illustration of Cell-Based Immunization and Screening(CBIS) method. Stable transfectants expressing the protein of interest are used
as an immunogen with no puriﬁcation procedure. The selection of hybridomas
secreting speciﬁc mAbs is performed by ﬂow cytometry using parental andtransfectant cells.

Fig. 3. Western blotting. Cell lysates of CHO-K1, CHO/horPDPN, and FHKTcl3.1 (10 μg) were electrophoresed and transferred onto PVDF membranes.
The membranes were incubated with l μg/mL of PMab-219, 1 μg/mL of antiPA16 tag (NZ-1), or 1 μg/mL of anti-β-actin and subsequently with peroxidaseconjugated anti-mouse or -rat IgG.

CHO-K1 cells (Fig. 4B). PMab-219 did not react with FHK-Tcl3.1 cells;
this might be also attributed to low expression levels of horPDPN in
FHK-Tcl3.1 cells. (Fig. 4C). No staining was observed without primary
antibodies (Fig. 4D, 4E, 4F). These results indicated that PMab-219 isuseful for the detection of horPDPN using immunohistochemistry.
In conclusion, we have established an mAb against horPDPN, PMab219, which is suitable for use in ﬂow cytometry, Western blotting, andimmunohistochemical analyses. The epitope of PMab-219 needs further
investigation to clarify the sensitivity and speciﬁcity of PMab-219
against horPDPN. PMab-219 should prove useful for elucidating thepathophysiological functions of horPDPN in future studies.
Funding
This research was supported in part by AMED under Grant
Numbers: JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), andJP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number
17K07299 (M.K.K.) and Grant Number 16K10748 (Y.K.).
3

Biochemistry and Biophysics Reports 18 (2019) 100616

Y. Furusawa et al.

Fig. 4. Immunohistochemical analyses. Cell sections of CHO/horPDPN (A, D), CHO-K1 (B, E), or FHK-Tcl3.1 (C, F) were incubated with 1 μg/mL of PMab-219(A–C) or with blocking buﬀer (D–F), followed by an Envision + kit. Scale bar = 100 μm.

Conﬂict of Interest

Canc. 113 (2005) 899–910.
[6] Y. Kato, M.K. Kaneko, A. Kunita, H. Ito, A. Kameyama, S. Ogasawara, N. Matsuura,
Y. Hasegawa, K. Suzuki-Inoue, O. Inoue, Y. Ozaki, H. Narimatsu, Molecular analysisof the pathophysiological binding of the platelet aggregation-inducing factor podoplanin to the C-type lectin-like receptor CLEC-2, Cancer Sci. 99 (2008) 54–61.
[7] M.K. Kaneko, A. Kunita, S. Abe, Y. Tsujimoto, M. Fukayama, K. Goto, Y. Sawa,
Y. Nishioka, Y. Kato, Chimeric anti-podoplanin antibody suppresses tumor metastasis through neutralization and antibody-dependent cellular cytotoxicity, Cancer
Sci. 103 (2012) 1913–1919.
[8] K. Suzuki-Inoue, Y. Kato, O. Inoue, M.K. Kaneko, K. Mishima, Y. Yatomi,
Y. Yamazaki, H. Narimatsu, Y. Ozaki, Involvement of the snake toxin receptor
CLEC-2, in podoplanin-mediated platelet activation, by cancer cells, J. Biol. Chem.
282 (2007) 25993–26001.
[9] M. Nagae, K. Morita-Matsumoto, M. Kato, M.K. Kaneko, Y. Kato, Y. Yamaguchi, A
Platform of C-Type lectin-like receptor CLEC-2 for binding O-glycosylated podoplanin and nonglycosylated rhodocytin, Structure 22 (2014) 1711–1721.
[10] C.C. Bertozzi, A.A. Schmaier, P. Mericko, P.R. Hess, Z. Zou, M. Chen, C.Y. Chen,
B. Xu, M.M. Lu, D. Zhou, E. Sebzda, M.T. Santore, D.J. Merianos, M. Stadtfeld,
A.W. Flake, T. Graf, R. Skoda, J.S. Maltzman, G.A. Koretzky, M.L. Kahn, Platelets
regulate lymphatic vascular development through CLEC-2-SLP-76 signaling, Blood
116 (2010) 661–670.
[11] J.A. Ochoa-Alvarez, H. Krishnan, J.G. Pastorino, E. Nevel, D. Kephart, J.J. Lee,
E.P. Retzbach, Y. Shen, M. Fatahzadeh, S. Baredes, E. Kalyoussef, M. Honma,
M.E. Adelson, M.K. Kaneko, Y. Kato, M.A. Young, L. Deluca-Rapone,
A.J. Shienbaum, K. Yin, L.D. Jensen, G.S. Goldberg, Antibody and lectin target
podoplanin to inhibit oral squamous carcinoma cell migration and viability by
distinct mechanisms, Oncotarget 6 (2015) 9045–9060.
[12] S.F. Schoppmann, B. Jesch, M.F. Riegler, F. Maroske, K. Schwameis, G. Jomrich,
P. Birner, Podoplanin expressing cancer associated ﬁbroblasts are associated withunfavourable prognosis in adenocarcinoma of the esophagus, Clin. Exp. Metastasis
30 (2013) 441–446.
[13] Y. Kato, M. Kaneko, M. Sata, N. Fujita, T. Tsuruo, M. Osawa, Enhanced expressionof Aggrus (T1alpha/podoplanin), a platelet-aggregation-inducing factor in lung

The authors declare no conﬂicts of interest involving this article.
Acknowledgments
We thank Kayo Hisamatsu, Saori Handa, and Yoshimi Nakamura. fortheir excellent technical assistance.
References[1] S. Breiteneder-Geleﬀ, K. Matsui, A. Soleiman, P. Meraner, H. Poczewski, R. Kalt,
G. Schaﬀner, D. Kerjaschki, Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis, Am. J. Pathol.
151 (1997) 1141–1152.
[2] S. Breiteneder-Geleﬀ, A. Soleiman, H. Kowalski, R. Horvat, G. Amann, E. Kriehuber,
K. Diem, W. Weninger, E. Tschachler, K. Alitalo, D. Kerjaschki, Angiosarcomas
express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a speciﬁc marker for lymphatic endothelium, Am. J. Pathol. 154 (1999)
385–394.
[3] Y. Kato, N. Fujita, A. Kunita, S. Sato, M. Kaneko, M. Osawa, T. Tsuruo, Molecular
identiﬁcation of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors, J. Biol. Chem. 278 (2003) 51599–51605.
[4] M.K. Kaneko, Y. Kato, T. Kitano, M. Osawa, Conservation of a platelet activating
domain of Aggrus/podoplanin as a platelet aggregation-inducing factor, Gene 378(2006) 52–57.
[5] E. Martin-Villar, F.G. Scholl, C. Gamallo, M.M. Yurrita, M. Munoz-Guerra, J. Cruces,
M. Quintanilla, Characterization of human PA2.26 antigen (T1alpha-2, podoplanin), a small membrane mucin induced in oral squamous cell carcinomas, Int. J.

4

Biochemistry and Biophysics Reports 18 (2019) 100616

Y. Furusawa et al.

Development of sensitive monoclonal antibody PMab-2 against rat podoplanin,
Monoclon. Antibodies Immunodiagn. Immunother. 34 (2015) 396–403.
[27] R. Honma, Y. Fujii, S. Ogasawara, H. Oki, X. Liu, T. Nakamura, M.K. Kaneko,
M. Takagi, Y. Kato, Establishment of a novel monoclonal antibody PMab-32 against
rabbit podoplanin, Monoclon. Antibodies Immunodiagn. Immunother. 35 (2016)
41–47.
[28] R. Honma, S. Ogasawara, M. Kaneko, Y. Fujii, H. Oki, T. Nakamura, M. Takagi,
S. Konnai, Y. Kato, PMab-44 detects bovine podoplanin in immunohistochemistry,
Monoclon. Antibodies Immunodiagn. Immunother. 35 (2016) 186–190.
[29] R. Honma, M.K. Kaneko, S. Ogasawara, Y. Fujii, S. Konnai, M. Takagi, Y. Kato,
Speciﬁc detection of dog podoplanin expressed in renal glomerulus by a novel
monoclonal antibody PMab-38 in immunohistochemistry, Monoclon. Antibodies
Immunodiagn. Immunother. 35 (2016) 212–216.
[30] S. Yamada, S. Itai, T. Nakamura, M. Yanaka, N. Saidoh, Y.W. Chang, S. Handa,
H. Harada, Y. Kagawa, O. Ichii, S. Konnai, M.K. Kaneko, Y. Kato, PMab-52: speciﬁcand sensitive monoclonal antibody against cat podoplanin for immunohistochemistry, Monoclon. Antibodies Immunodiagn. Immunother. 36 (2017)
224–230.
[31] S. Yamada, S. Itai, Y. Furusawa, M. Sano, T. Nakamura, M. Yanaka, S. Handa,
K. Hisamatsu, Y. Nakamura, M. Fukui, H. Harada, T. Mizuno, Y. Sakai,
S. Ogasawara, T. Murata, H. Uchida, H. Tahara, M.K. Kaneko, Y. Kato, Detection oftiger podoplanin using the anti-cat podoplanin monoclonal antibody PMab-52,
Monoclon. Antibodies Immunodiagn. Immunother. 37 (2018) 224–228.
[32] Y. Furusawa, S. Yamada, S. Itai, M. Sano, T. Nakamura, M. Yanaka, S. Handa,
T. Mizuno, K. Maeda, M. Fukui, H. Harada, M.K. Kaneko, Y. Kato, Establishment of
monoclonal antibody PMab-202 against horse podoplanin, Monoclon. Antibodies
Immunodiagn. Immunother. 37 (2018) 233–237.
[33] K. Andoh, K. Kai, T. Matsumura, K. Maeda, Further development of an equine cellline that can be propagated over 100 times, J. Equine Sci. 20 (2009) 11–14.
[34] S. Yamada, S. Itai, T. Nakamura, M. Yanaka, M.K. Kaneko, Y. Kato, Detection ofhigh CD44 expression in oral cancers using the novel monoclonal antibody,
C44Mab-5, Biochem Biophys Rep 14 (2018) 64–68.
[35] S. Itai, Y. Fujii, T. Nakamura, Y.W. Chang, M. Yanaka, N. Saidoh, S. Handa,
H. Suzuki, H. Harada, S. Yamada, M.K. Kaneko, Y. Kato, Establishment of CMab-43,
a sensitive and speciﬁc anti-CD133 monoclonal antibody, for immunohistochemistry, Monoclon. Antibodies Immunodiagn. Immunother. 36 (2017)
231–235.
[36] S. Yamada, S. Itai, T. Nakamura, M. Yanaka, Y.W. Chang, H. Suzuki, M.K. Kaneko,
Y. Kato, Monoclonal antibody L1Mab-13 detected human PD-L1 in lung cancers,
Monoclon. Antibodies Immunodiagn. Immunother. 37 (2018) 110–115.

squamous cell carcinoma, Tumor Biol. 26 (2005) 195–200.
[14] N. Kimura, I. Kimura, Podoplanin as a marker for mesothelioma, Pathol. Int. 55(2005) 83–86.
[15] S. Abe, Y. Morita, M.K. Kaneko, M. Hanibuchi, Y. Tsujimoto, H. Goto, S. Kakiuchi,
Y. Aono, J. Huang, S. Sato, M. Kishuku, Y. Taniguchi, M. Azuma, K. Kawazoe,
Y. Sekido, S. Yano, S. Akiyama, S. Sone, K. Minakuchi, Y. Kato, Y. Nishioka, A novel
targeting therapy of malignant mesothelioma using anti-podoplanin antibody, J.
Immunol. 190 (2013) 6239–6249.
[16] M.K. Kaneko, H. Oki, S. Ogasawara, M. Takagi, Y. Kato, Anti-podoplanin monoclonal antibody LpMab-7 detects metastatic legions of osteosarcoma, Monoclon.
Antibodies Immunodiagn. Immunother. 34 (2015) 154–161.
[17] T. Ariizumi, A. Ogose, H. Kawashima, T. Hotta, G. Li, Y. Xu, H. Umezu, M. Sugai,
N. Endo, Expression of podoplanin in human bone and bone tumors: new marker ofosteogenic and chondrogenic bone tumors, Pathol. Int. 60 (2010) 193–202.
[18] A. Kunita, T.G. Kashima, A. Ohazama, A.E. Grigoriadis, M. Fukayama, Podoplanin isregulated by AP-1 and promotes platelet aggregation and cell migration in osteosarcoma, Am. J. Pathol. 179 (2011) 1041–1049.
[19] K. Mishima, Y. Kato, M.K. Kaneko, R. Nishikawa, T. Hirose, M. Matsutani, Increased
expression of podoplanin in malignant astrocytic tumors as a novel molecularmarker of malignant progression, Acta Neuropathol. (Berl) 111 (2006) 483–488.
[20] K. Mishima, Y. Kato, M.K. Kaneko, Y. Nakazawa, A. Kunita, N. Fujita, T. Tsuruo,
R. Nishikawa, T. Hirose, M. Matsutani, Podoplanin expression in primary central
nervous system germ cell tumors: a useful histological marker for the diagnosis ofgerminoma, Acta Neuropathol. (Berl) 111 (2006) 563–568.
[21] Y. Kato, G. Vaidyanathan, M.K. Kaneko, K. Mishima, N. Srivastava,
V. Chandramohan, C. Pegram, S.T. Keir, C.T. Kuan, D.D. Bigner, M.R. Zalutsky,
Evaluation of anti-podoplanin rat monoclonal antibody NZ-1 for targeting malignant gliomas, Nucl. Med. Biol. 37 (2010) 785–794.
[22] Y. Kato, M.K. Kaneko, A cancer-speciﬁc monoclonal antibody recognizes theaberrantly glycosylated podoplanin, Sci. Rep. 4 (2014) 5924.
[23] Y. Kato, I. Sasagawa, M. Kaneko, M. Osawa, N. Fujita, T. Tsuruo, Aggrus, A diagnostic marker that distinguishes seminoma from embryonal carcinoma in testiculargerm cell tumors, Oncogene 23 (2004) 8552–8556.
[24] A. Kunita, T.G. Kashima, Y. Morishita, M. Fukayama, Y. Kato, T. Tsuruo, N. Fujita,
The platelet aggregation-inducing factor aggrus/podoplanin promotes pulmonary
metastasis, Am. J. Pathol. 170 (2007) 1337–1347.
[25] C. Kaji, Y. Tsujimoto, M. Kato Kaneko, Y. Kato, Y. Sawa, Immunohistochemical
examination of novel rat monoclonal antibodies against mouse and human podoplanin, Acta Histochem. Cytoc. 45 (2012) 227–237.
[26] H. Oki, R. Honma, S. Ogasawara, Y. Fujii, X. Liu, M. Takagi, M.K. Kaneko, Y. Kato,

5