Biochemistry and Biophysics Reports 18 (2019) 100644

Contents lists available at ScienceDirect

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

Development of an anti-bear podoplanin monoclonal antibody PMab-247 forimmunohistochemical analysis

TYoshikazu Furusawaa,b, Junko Takeia, Yusuke Sayamaa, Shinji Yamadaa, Mika K. Kanekoa,
Yukinari Katoa,b,∗ab

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, JapanA R T I C LE I N FO

A B S T R A C T

Keywords:
Bear podoplanin
PDPN
ImmunohistochemistryPMab-247

Sensitive and speciﬁc monoclonal antibodies (mAbs) targeting podoplanin (PDPN) are needed for immunohistochemical analyses using formalin-ﬁxed paraﬃn-embedded tissues because PDPN is known as alymphatic endothelial cell maker in pathology. Recently, we established anti-PDPN mAbs against many species,
such as human, mouse, rat, rabbit, dog, cat, bovine, pig, horse, goat, tiger, alpaca, and Tasmanian devil.
However, anti-bear PDPN (bPDPN) has not been established yet. In this study, we immunized mice with bPDPNoverexpressing Chinese hamster ovary (CHO)-K1 (CHO/bPDPN) cells, and screened mAbs against bPDPN using
ﬂow cytometry. One of the mAbs, PMab-247 (IgG1, kappa), speciﬁcally detected CHO/bPDPN cells by ﬂow
cytometry and immunohistochemistry. Our ﬁndings suggest the potential usefulness of PMab-247 for thefunctional analyses of bPDPN.

1. Introduction

2. Materials and methods

A type I transmembrane sialo-glycoprotein, podoplanin (PDPN), isexpressed in many cell types, such as renal podocytes, pulmonary type I
alveolar cells, and lymphatic endothelial cells of every organ [1]. PDPN
has been reported to distinguish lymphatic endothelial cells from vascular endothelial cells in pathophysiological studies [2]. C-type lectinlike receptor-2 (CLEC-2) was previously reported as an endogenous
receptor of PDPN in our studies [3,4]. The PDPN-CLEC-2 interaction
facilitates the separation of embryonic blood and lymphatic vessels [5].
Human PDPN (hPDPN) is expressed in many malignant tumors, including brain tumors [6] and mesotheliomas [7], and is associated withmalignant progression and cancer metastasis [8].
We have developed monoclonal antibodies (mAbs) against human[6], mouse [9], rat [10], rabbit [11], bovine [12], dog [13], cat [14],pig [15], horse [16], goat [17], tiger [18], alpaca [19], whale [20], andTasmanian devil [21] PDPNs. In this study, we immunized mice with
CHO/bear PDPN (bPDPN) cells and established mAbs against bPDPN.

2.1. Cell lines
CHO-K1 and P3X63Ag8U.1 (P3U1) cells were obtained from theAmerican Type Culture Collection (ATCC; Manassas, VA, USA).
Synthesized DNA (Euroﬁns Genomics KK, Tokyo, Japan) encoding
bPDPN (accession No.: XM_008694703.1) plus an N-terminal BAP tag,which are recognized by an anti-BAP tag mAb (PMab-44), was subcloned into a pCAG-Neo vector (FUJIFILM Wako Pure Chemical
Corporation, Osaka, Japan). Plasmids were transfected using
Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientiﬁc Inc.,
Waltham, MA, USA). Stable transfectants (CHO/bPDPN) were selectedby limiting dilution and cultivation in a medium containing 0.5 mg/mL
of G418 (Nacalai Tesque, Inc., Kyoto, Japan).
The P3U1, CHO-K1, CHO/bPDPN, CHO/hPDPN [22], CHO/mouse
PDPN (mPDPN) [22], CHO/rat PDPN (rPDPN) [10], CHO/rabbit PDPN
(rabPDPN) [11], CHO/dog PDPN (dPDPN) [13], CHO/bovine PDPN
(bovPDPN) [12], CHO/cat PDPN (cPDPN) [14], CHO/pig PDPN
(pPDPN) [15], CHO/horse PDPN (horPDPN) [16], CHO/tiger PDPN
(tigPDPN) [18], CHO/alpaca PDPN (aPDPN) [19], CHO/TasmanianAbbreviations: BAP, bovine aggrus/podoplanin; DAB, 3,3′-diaminobenzidine tetrahydrochloride; ELISA, enzyme-linked immunosorbent assay; PBS, phosphatebuﬀered saline; PDPN, podoplanin; mAb, monoclonal antibody∗
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.2019.100644
Received 6 April 2019; Received in revised form 20 April 2019; Accepted 22 April 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) 100644

Y. Furusawa, et al.

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

devil PDPN (tasPDPN) [21], CHO/goat PDPN (gPDPN) [17], CHO/
sheep PDPN (sPDPN) [23], and CHO/whale PDPN (wPDPN) [20] were
cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium(Nacalai Tesque, Inc.), supplemented with 10% of heat-inactivated fetal
bovine serum (FBS; Thermo Fisher Scientiﬁc Inc.), 100 units/mL of
penicillin, 100 μg/mL of streptomycin, and 25 μg/mL of amphotericin B(Nacalai Tesque, Inc.). The cells were grown in an incubator at 37 °C
with humidity and 5% CO2 and 95% air atmosphere.

Fig. 2. Detection of bPDPN by ﬂow cytometry using PMab-247. CHO/
bPDPN and CHO-K1 were treated with PMab-247 (red line) or anti-BAP tag(PMab-44; blue line) at a concentration of 1 μg/mL or 0.1% BSA in PBS (gray)for 30 min, followed by incubation with secondary antibodies.

mouse was immunized with CHO/bPDPN cells (1 × 108) intraperitoneally (i.p.) together with the Imject Alum (Thermo Fisher
Scientiﬁc Inc.). The procedure included three additional immunizationsof CHO/bPDPN cells (1 × 108) together with the Imject Alum, followed
by a ﬁnal booster injection of CHO/bPDPN cells (1 × 108) together
with the Imject Alum 2 days prior to the harvest of spleen cells. Furthermore, one BALB/c mouse was immunized with CHO/bPDPN cells(1 × 108) intraperitoneally (i.p.) together with chitosan (Koyo Chemical Co., Ltd., Osaka, Japan). The procedure included three additional
immunizations of CHO/bPDPN cells (1 × 108) together with chitosan,
followed by a ﬁnal booster injection of CHO/bPDPN cells (1 × 108)
together with chitosan 2 days prior to the harvest of spleen cells.
Subsequently, these spleen cells were fused with P3U1 cells using
PEG1500 (Roche Diagnostics, Indianapolis, IN, USA), and the2.2. Animals
Female BALB/c mice (6 weeks of age) were purchased from CLEA
Japan (Tokyo, Japan). The animals were housed under speciﬁc pathogen-free conditions. The Animal Care and Use Committee of Tohoku
University approved all animal experiments.
2.3. Hybridoma production
We employed a Cell-Based Immunization and Screening (CBIS)
method [14,24–26] to develop mAbs against bPDPN. One BALB/c2

Biochemistry and Biophysics Reports 18 (2019) 100644

Y. Furusawa, et al.

Fig. 3. Cross-reaction of PMab-247 to PDPNs of the other species by ﬂow cytometry. CHO-K1 cells transfected with PDPNs of the other species were treated with
PMab-247 (red line) or each positive control (blue line) at a concentration of 1 μg/mL or 0.1% BSA in PBS (gray) for 30 min, followed by incubation with secondary
antibodies.
3

Biochemistry and Biophysics Reports 18 (2019) 100644

Y. Furusawa, et al.

Fig. 4. Western blot analysis. Cell lysates of CHO-K1 and CHO/bPDPN (10 μg)
were electrophoresed and transferred onto PVDF membranes. The membranes
were incubated with l μg/mL of PMab-247, 1 μg/mL of anti-BAP tag (PMab-44),or 1 μg/mL of anti- β-actin and subsequently with peroxidase-conjugated antimouse IgG.

hybridomas were grown in an RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine (HAT) for selection (Thermo
Fisher Scientiﬁc Inc.). The culture supernatants were screened by ﬂow
cytometry.
2.4. Flow cytometry
The cells were harvested following a brief exposure to 0.25% trypsin
and 1 mM ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque,Inc.). The cells were washed with 0.1% bovine serum albumin (BSA) in
phosphate-buﬀered saline (PBS) and treated with primary mAbs for
30 min at 4 °C. Thereafter, the cells were treated with Alexa Fluor 488conjugated anti-mouse IgG (1:2000; Cell Signaling Technology, Inc.,
Danvers, MA, USA) or Oregon Green anti-rat IgG (1:2000; Thermo
Fisher Scientiﬁc Inc.). Then, ﬂuorescence data were collected using theSA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).
2.5. Western blot analysis
Cell lysates (10 μg) were boiled in sodium dodecyl sulfate (SDS)
sample buﬀer (Nacalai Tesque, Inc.). Proteins were then electrophoresed on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure
Chemical Corporation) and transferred onto polyvinylidene diﬂuoride(PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking
with 4% skim milk (Nacalai Tesque, Inc.), membranes were incubatedwith 1 μg/mL of PMab-247, 1 μg/mL of PMab-44 (anti-BAP tag), and1 μg/mL of anti-β-actin (clone AC-15; Sigma-Aldrich Corp., St. Louis,MO), followed by incubation with peroxidase-conjugated anti-mouse
IgG (Agilent Technologies Inc., Santa Clara, CA; diluted 1:1000), andwere ﬁnally developed using ImmunoStar LD (FUJIFILM Wako Pure
Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo,
Japan).

Fig. 5. Immunohistochemical analyses for cell lines. CHO-K1 cells andCHO/bPDPN cells were incubated with 0.01–1 μg/mL of PMab-247 or blocking
buﬀer, followed by the Envision + Kit. HE, Hematoxylin and eosin staining.
Scale bar = 100 μm.

xylene, then rehydrated, and autoclaved in citrate buﬀer (pH 6.0;
Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min. Then, sections
were blocked using the SuperBlock T20 (PBS) Blocking Buﬀer (Thermo
Fisher Scientiﬁc Inc.), incubated with PMab-247 for 1 h at the room
temperature, and treated with the Envision + Kit for mouse (Agilent
Technologies Inc.) for 30 min. Color was developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for
2 min, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

2.6. Immunohistochemical analyses
Cell blocks were produced using iPGell (Genostaﬀ Co., Ltd., Tokyo,
Japan). Histologic sections of 4-μm thickness were deparaﬃnized in4Biochemistry and Biophysics Reports 18 (2019) 100644

Y. Furusawa, et al.

3. Results

M. Lee, Y. Jin Hong, Y. Kato, J. Kipnis, M.J. Davis, A.K. Wong, Y.K. Hong,
Development and characterization of A novel prox1-EGFP lymphatic and schlemm's
canal reporter rat, Sci. Rep. 7 (2017) 5577.
[3] 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.
[4] 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.
[5] 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.
[6] Y. Kato, M.K. Kaneko, A. Kuno, N. Uchiyama, K. Amano, Y. Chiba, Y. Hasegawa,J. Hirabayashi, H. Narimatsu, K. Mishima, M. Osawa, Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its
platelet-aggregation-stimulating domain, Biochem. Biophys. Res. Commun. 349(2006) 1301–1307.
[7] S. Abe, M.K. Kaneko, Y. Tsuchihashi, T. Izumi, S. Ogasawara, N. Okada, C. Sato,
M. Tobiume, K. Otsuka, L. Miyamoto, K. Tsuchiya, K. Kawazoe, Y. Kato,
Y. Nishioka, Antitumor eﬀect of novel anti-podoplanin antibody NZ-12 against
malignant pleural mesothelioma in an orthotopic xenograft model, Cancer Sci. 107(2016) 1198–1205.
[8] 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.
[9] 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.
[10] H. Oki, R. Honma, S. Ogasawara, Y. Fujii, X. Liu, M. Takagi, M.K. Kaneko, Y. Kato,Development of sensitive monoclonal antibody PMab-2 against rat podoplanin,
Monoclon. Antibodies Immunodiagn. Immunother. 34 (2015) 396–403.
[11] 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.
[12] 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.
[13] 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.
[14] 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.
[15] Y. Kato, S. Yamada, Y. Furusawa, S. Itai, T. Nakamura, M. Yanaka, M. Sano,
H. Harada, M. Fukui, M.K. Kaneko, PMab-213: a monoclonal antibody for immunohistochemical analysis against pig podoplanin Monoclon, Antib.
Immunodiagn. Immunother. 38 (2019) 18–24.
[16] Y. Furusawa, S. Yamada, S. Itai, T. Nakamura, M. Yanaka, M. Sano, H. Harada,
M. Fukui, M.K. Kaneko, Y. Kato, PMab-219: a monoclonal antibody for the immunohistochemical analysis of horse podoplanin, Biochem. Biophys. Rep. 18(2019), https://doi.org/10.1016/j.bbrep.2019.1001.1009.
[17] Y. Furusawa, S. Yamada, T. Nakamura, M. Sano, S. Itai, J. Takei, H. Harada, M.
Fukui, M.K. Kaneko, Y. Kato, PMab-235: a Monoclonal Antibody for
Immunohistochemical Analysis against Goat Podoplanin, (submit).
[18] Y. Furusawa, M.K. Kaneko, T. Nakamura, S. Itai, M. Fukui, H. Harada, S. Yamada,Y. Kato, Establishment of a monoclonal antibody PMab-231 for tiger podoplanin,
Monoclon. Antibodies Immunodiagn. Immunother. 38 (2019) 89–95.
[19] Y. Kato, Y. Furusawa, S. Yamada, S. Itai, J. Takei, M. Sano, M.K. Kaneko,
Establishment of a monoclonal antibody PMab-225 against alpaca podoplanin for
immunohistochemical analyses, Biochem. Biophys. Rep. 18 (2019) 100633.
[20] Y. Kato, Y. Furusawa, S. Itai, J. Takei, T. Nakamura, M. Sano, H. Harada, S. Yamada,
M.K. Kaneko, Establishment of an Anti-cetacean Podoplanin Monoclonal Antibody
PMab-237 for Immunohistochemical Analysis, (submit).
[21] Y. Furusawa, S. Yamada, S. Itai, T. Nakamura, J. Takei, M. Sano, H. Harada,
M. Fukui, M.K. Kaneko, Y. Kato, Establishment of a monoclonal antibody PMab-233
for immunohistochemical analysis against Tasmanian devil podoplanin, Biochem.
Biophys. Rep. 18 (2019), https://doi.org/10.1016/j.bbrep.2019.100631.
[22] 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.
[23] Y. Kato, S. Yamada, S. Itai, A. Kobayashi, S. Konnai, M.K. Kaneko,
Immunohistochemical detection of sheep podoplanin using an antibovine podoplanin monoclonal antibody PMab-44, Monoclon. Antibodies Immunodiagn.
Immunother. 37 (2018) 265–268.
[24] 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.
[25] S. Itai, Y. Fujii, T. Nakamura, Y.W. Chang, M. Yanaka, N. Saidoh, S. Handa,

Two mice were immunized with CHO/bPDPN cells (Fig. 1). The
procedure in this study included four additional immunizations of
CHO/bPDPN cells together with the Imject Alum (one mouse) or chitosan (one mouse) as adjuvants, followed by a ﬁnal booster injection of
CHO/bPDPN cells together with the Imject Alum (one mouse) or chitosan (one mouse). The developed hybridomas were seeded into 96-wellplates and cultivated for 9 days. Wells positive for CHO/bPDPN andnegative for CHO-K1 were selected by ﬂow cytometry. The ﬁrst
screening approach identiﬁed strong signals from CHO/bPDPN cells
and weak or no signals from CHO-K1 cells in 24 of the 480 wells (5.0%)
for Imject Alum (one mouse) or in 4 of the 480 wells (0.83%) for
chitosan (one mouse), indicating that Imject Alum was found to be abetter adjuvant for this immunization. After several additional screenings, PMab-247 (IgG1, kappa) was ﬁnally selected from a mouse using
chitosan.
PMab-247 recognized CHO/bPDPN cells, but showed no reaction
with CHO-K1 cells, as assessed by ﬂow cytometry (Fig. 2). PMab-247
did not cross-react with the other PDPNs (Fig. 3). The expression levelsof PDPNs were conﬁrmed by each positive control mAb.
Western blot analysis performed using PMab-247 demonstrated that
PMab-247 detects bPDPN as a 48-kDa band in CHO/bPDPN cells(Fig. 4). An anti-BAP tag mAb (PMab-44) also detected a 48-kDa band in
CHO/bPDPN cells. The immunohistochemical analyses revealed that
PMab-247 strongly stained CHO/bPDPN cells even in a concentration of
0.05 μg/mL (Fig. 5), whereas it did not react with CHO-K1 cells.
4. Discussion
In this study, we established PMab-247 against bPDPN, which issuitable for ﬂow cytometry, Western blot, and immunohistochemical
analyses using CBIS method. Previously, we tried to develop antibPDPN mAbs using CBIS method, and added Imject Alum as an adjuvant only in a ﬁrst injection in the same way with our previous studies[14,24–26]. However, we could not establish anti-bPDPN mAbs, which
are useful for immunohistochemical analyses (data not shown). Herein,
we used Imject Alum or chitosan as adjuvants for every immunization,and successfully obtained high positive rates in ﬂow cytometry, indicating that repeated use of adjuvants is also advantageous in CBIS
method. The epitope of PMab-247 needs further investigation to clarify
the sensitivity and speciﬁcity of PMab-247 against bPDPN.
We believe that PMab-247 should prove to be useful in elucidating
the pathophysiological functions of bPDPN in some bear tumors such as
osteosarcomas [27,28] or squamous cell carcinoma [29].
Conﬂicts of interest
Y.K. received research funding from ZENOAQ RESOURCE CO., LTD.
The other authors have no conﬂict of interest.
Acknowledgments
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.), Grant Number 16K10748 (Y.K.), and Grant
Number 19K07705 (Y.K.).
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] E. Jung, D. Gardner, D. Choi, E. Park, Y. Jin Seong, S. Yang, J. Castorena-Gonzalez,
A. Louveau, Z. Zhou, G.K. Lee, D.P. Perrault, S. Lee, M. Johnson, G. Daghlian,5

Biochemistry and Biophysics Reports 18 (2019) 100644

Y. Furusawa, et al.

[27] E. Momotani, H. Aoki, Y. Ishikawa, T. Yoshino, Osteosarcoma in the maxilla of abrown bear (Ursus arctos), Vet. Pathol. 25 (1988) 527–529.
[28] V.I. Ponomar'kov, A.A. Khutorianskii, [A case of osteosarcoma in a white polarbear], Arkh. Patol. 57 (1995) 81–83.
[29] N.D. Mylniczenko, A.L. Manharth, L.A. Clayton, R. Feinmehl, M. Robbins,
Successful treatment of mandibular squamous cell carcinoma in a Malayan sun bear
(Helarctos malayanus), J. Zoo Wildl. Med. 36 (2005) 346–348.

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.
[26] 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.

6