Biochemistry and Biophysics Reports 13 (2018) 123–128

Contents lists available at ScienceDirect

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

Detection of high PD-L1 expression in oral cancers by a novel monoclonal
antibody L1Mab-4
Shinji Yamada, Shunsuke Itai, Mika K. Kaneko, Yukinari KatoT⁎

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
Programmed cell death-ligand 1
Monoclonal antibody
Oral cancer

Programmed cell death-ligand 1 (PD-L1), which is a ligand of programmed cell death-1 (PD-1), is a type I
transmembrane glycoprotein that is expressed on antigen-presenting cells and several tumor cells, including
melanoma and lung cancer cells. There is a strong correlation between human PD-L1 (hPD-L1) expression on
tumor cells and negative prognosis in cancer patients. In this study, we produced a novel anti-hPD-L1 monoclonal antibody (mAb), L1Mab-4 (IgG2b, kappa), using cell-based immunization and screening (CBIS) method
and investigated hPD-L1 expression in oral cancers. L1Mab-4 reacted with oral cancer cell lines (Ca9-22, HO-1-u1, SAS, HSC-2, HSC-3, and HSC-4) in ﬂow cytometry and stained oral cancers in a membrane-staining pattern.
L1Mab-4 stained 106/150 (70.7%) of oral squamous cell carcinomas, indicating the very high sensitivity of
L1Mab-4. These results indicate that L1Mab-4 could be useful for investigating the function of hPD-L1 in oral
cancers.

1. Introduction
Oral cancer is the eleventh highest of all cancer types [1] andconstitutes approximately 2% of all cancer cases worldwide [2]. In oral
cancers, there are certain histological tumors, including squamous cell
carcinoma (SCC), adenosquamous cell carcinoma, adenoid cystic carcinoma, osteosarcoma, and mucoepidermoid carcinoma. SCC is themost common type, accounting for > 90% of oral cancers [3]. Becauseof improvements and progression in therapeutic techniques such as
surgery, chemotherapy, and radiotherapy, the 5-year survival rate hasreached > 80% [4,5]. In contrast, it is sometimes diﬃcult to provide
suﬃcient therapeutic eﬀects because of the risks of adverse events[6–8].
Recently, tumor immunotherapies, which is focused on several immune checkpoint molecules, such as programmed cell death-1 (PD-1),
programmed cell death-ligand 1 (PD-L1), and cytotoxic T-lymphocyteassociated antigen 4 (CTLA-4), have emerged. Clinically, nivolumab, acomplete human IgG4 against PD-1, was ﬁrstly approved for the treatment of recurrent and/or metastatic head and neck cancer, which waspreviously treated with platinum-based chemotherapy [9]. Avelumab, acomplete human IgG1 against PD-L1, was recently approved for thetreatment of metastatic Merkel cell carcinoma [10,11], which isdetected in oral cavity [12].
PD-L1, also known as B7-H1 and CD274, is a type I transmembrane
glycoprotein, which is expressed on antigen-presenting cells and sometumor cells, including melanoma, ovarian, and lung cancer cells[13–15]. PD-L1 is a ligand for PD-1 and is involved in inhibiting T-celleﬀector functions [16], leading to the escape of tumor cells from immune response. Recent studies have revealed a strong correlation between PD-L1/PD-L2 expression on tumor cells and negative prognosis
in cancer patients [17–19].
Lin et al. have revealed a correlation between high PD-L1 expression
and metastasis and poor prognosis in oral SCC [20]. Several reports
have shown that PD-L1 could be an eﬀective target for treatment[21–26]. However, PD-L1 expression in oral cancers has not been
completely investigated. In this study, we established a novel anti-PDL1 antibody and performed immunohistochemistry for oral cancers.
2. Materials and methods
2.1. Cell lines
Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, HSC-4, and HEK-293T cells
were obtained from the Japanese Collection of Research Bioresources

Abbreviations: PD-L1, Programmed cell death-ligand 1; PD-1, programmed cell death-1; CBIS, cell-based immunization and screening; SCC, squamous cell carcinoma; ACC, adenoid
cystic carcinoma; MEC, mucoepidermoid carcinoma; CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; HNC, head and neck cancer; APC, antigen-presenting cell; DMEM, Dulbecco's
Modiﬁed Eagle's Medium; EDTA, ethylenediaminetetraacetic acid; BSA, bovine serum albumin; PBS, phosphate-buﬀered saline; FBS, fetal bovine serum; DAB, 3,3-diaminobenzidine
tetrahydrochloride⁎
Correspondence to: New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
E-mail addresses: yukinari-k@bea.hi-ho.ne.jp, yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2018.01.009
Received 5 January 2018; Received in revised form 23 January 2018; Accepted 23 January 2018
2405-5808/ © 2018 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 13 (2018) 123–128

S. Yamada et al.

Fig. 1. Flow cytometry of L1Mab-4 against LN229/hPD-L1. (A) Procedure of cell-based immunization and screening (CBIS) methods. (B) LN229 and LN229/hPD-L1 were treated with
1 μg/mL of 29E.2A3 (positive control; blue line) and L1Mab-4 (red line), followed by treatment with Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control. (C) HEK293T and HEK-293T/hPD-L1 were treated with 1 μg/mL of 29E.2A3 (positive control; blue line) and L1Mab-4 (red line), followed by treatment with Alexa Fluor 488-conjugated antimouse IgG; black line, negative control. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web version of this article.)vs. clone: NZ-1), RAP tag system (DMVNPGLEDRIE (12 a.a.) vs. clone:
PMab-2), and MAP tag system GDGMVPPGIEDK (12 a.a.) vs. clone:
PMab-1) have been previously established in Tohoku University
Graduate School of Medicine and described in detail [27–29].
Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, HSC-4, LN229, LN229/hPDL1, HEK-293T, and HEK-293T/hPD-L1 cells were cultured in Dulbecco's
Modiﬁed Eagle's Medium (DMEM; Nacalai Tesque, Kyoto, Japan), andP3U1 cell line was cultured in RPMI 1640 medium (Nacalai Tesque) at
37 °C in a humidiﬁed atmosphere containing 5% CO2 and 95% air, bothof which were supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Thermo Fisher Scientiﬁc, Inc.). One hundred units/mL

Cell Bank (Osaka, Japan). LN229 and P3U1 cell lines were obtained
from the American Type Culture Collection (ATCC, Manassas, VA).
LN229/human PD-L1 (hPD-L1) was produced by transfecting pCAG/
PA-hPD-L1-RAP-MAP into LN229 cells using a Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Inc., Berkeley, CA). The
stable transfectant of LN229/hPD-L1 was established by limiting dilution. HEK-293T/hPD-L1 was produced by transfecting pCAG/PA-hPDL1-RAP-MAP into HEK-293T cells using Neon transfection system(Thermo Fisher Scientiﬁc, Inc., Waltham, MA). A few days after transfection, PA tag-positive cells were sorted using a cell sorter (SH800;
Sony Corp., Tokyo, Japan). PA tag system (GVAMPGAEDDVV (12 a.a.)124

Biochemistry and Biophysics Reports 13 (2018) 123–128

S. Yamada et al.

Fig. 2. Flow cytometry of L1Mab-4 against oral cancer cell lines. Cells were treated with 1 μg/mL of 29E.2A3 (positive control; blue line) and L1Mab-4 (red line), followed bytreatment with Alexa Fluor 488-conjugated anti-mouse IgG; black line, negative control. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the web
version of this article.)

125

Biochemistry and Biophysics Reports 13 (2018) 123–128

S. Yamada et al.

2.3. Hybridoma production
One BALB/c mouse was immunized using intraperitoneal (i.p.) injections of LN229/hPD-L1 (1 × 108 cells) together with Imject Alum
(Thermo Fisher Scientiﬁc Inc.). After three additional immunizations, abooster injection of LN229/hPD-L1 was intraperitoneally administered
2 days before harvesting the spleen cells. Spleen cells were then fusedwith P3U1 cells using PEG1500 (Roche Diagnostics, Indianapolis, IN).
The resulting hybridomas were grown in RPMI medium supplemented
with 10% FBS and hypoxanthine, aminopterin, thymidine selection
medium supplement (Thermo Fisher Scientiﬁc Inc.), and 5% BriClone
Hybridoma Cloning Medium (QED Bioscience Inc., San Diego, CA). One
hundred units/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL
amphotericin B (Nacalai Tesque) were added to the medium. Plasmocin(5 μg/mL; InvivoGen) was also used to prevent Mycoplasma contamination. Culture supernatants were screened by SA3800 Cell
Analyzers (Sony Corp.) using LN229 and LN229/hPD-L1. MAbs were
puriﬁed from the supernatants of hybridomas and cultured inHybridoma-SFM medium (Thermo Fisher Scientiﬁc Inc.) using Protein
G Sepharose 4 Fast Flow (GE Healthcare UK Ltd, Buckinghamshire,
England).
2.4. Flow cytometry
Cells were harvested by brieﬂy exposing to 0.25% trypsin/1-mM
ethylenediaminetetraacetic acid (EDTA; Nacalai Tesque, Inc.). Afterwashing with 0.1% bovine serum albumin (BSA)/phosphate-buﬀeredsaline, the cells were treated with 1 μg/mL of anti-PD-L1 mAbs, such as
clones 29E.2A3 [30] (BioLegend, Inc., San Diego, CA) and L1Mab-4(produced in this study), for 30 min at 4 °C and subsequently with Alexa
Fluor 488-conjugated anti-mouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA). Fluorescence data were collected using
EC800 Cell Analyzers (Sony Corp.).
2.5. Immunohistochemical analyses
Fig. 3. Immunohistochemical analysis of L1Mab-4 against oral SCCs. After antigen
retrieval, sections were incubated with 10 μg/mL of L1Mab-4 followed by treatment withEnvision+ kitn. Color was developed using 3,3-diaminobenzidine tetrahydrochloride(DAB), and sections were counterstained with hematoxylin. (A, B) Antigen retrieval using
citrate buﬀer, pH 6; (C, D) Antigen retrieval using EnVision FLEX Target Retrieval
Solution High pH; (E, F) Control; (G, H) Hematoxylin & eosin (HE) staining. Scale bar =100 µm.

Histological sections of 4-μm thickness were deparaﬃnized in xylene and subsequently rehydrated and autoclaved in citrate buﬀer (pH
6.0; Nichirei Biosciences, Inc., Tokyo, Japan) or EnVision FLEX Target
Retrieval Solution, High pH (Agilent Technologies Inc., Santa Clara,CA) for 20 min. Sections were then incubated with 10 μg/mL of L1Mab4 for 1 h at room temperature, treated using an Envision+ kit (Agilent
Technologies Inc.) for 30 min. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies Inc.) for
2 min, and counterstained with hematoxylin (Wako Pure Chemical
Industries Ltd., Osaka, Japan).

penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B(Nacalai Tesque) were added to the culture medium. Zeocin (0.5 mg/
mL; InvivoGen, San Diego, CA) was added to the culture medium of
LN229/hPD-L1 and HEK-293T/hPD-L1.

3. Results and discussion
3.1. Production of novel anti-hPD-L1 mAbs using cell-based immunizationand screening (CBIS) method

2.2. Animals and human tissues
Female 4-week-old BALB/c mice were purchased from CLEA Japan(Tokyo, Japan) and kept under speciﬁc pathogen-free conditions. The
Animal Care and Use Committee of Tohoku University approved all
animal experiments described in this study. Oral cancer tissue arrays
were purchased from US Biomax, Inc. (Rockville, MD): Cases 1–38 from
Cat. # OR480, Cases 39–88 from Cat. # OR601b or from Cybrdi, Inc.
(Frederick, MD), and Cases 89–159 from Cat. # 27-10-001. The study
examined one patient (Case-160) with oral cancer who underwent
surgery at the Tokyo Medical and Dental University. The Tokyo Medical
and Dental University Institutional Review Board reviewed and approved the use of human cancer tissues, and written informed consent
was obtained for using the human cancer tissue samples.

We immunized one mouse with LN229/hPD-L1 cells and performed
ﬂow cytometric screening. We previously named this cell-based
strategy as the CBIS method, as shown in Fig. 1A [31,32]. Culture supernatants of 672 wells were mixed with LN229/hPD-L1 and LN229
cells, and 27 wells (27/672; 4.0%), showing a stronger reaction against
LN229/hPD-L1 cells than LN229 cells, were selected. After limiting
dilution, 10 clones were established, of which seven were classiﬁed as
IgG1 subclass and three as IgG2b subclass. Only one clone, L1Mab-4(IgG2b, kappa), reacted with lung SCC tissue, which was previously
diagnosed as PD-L1-positive in an immunohistochemical analysis (datanot shown). Although both L1Mab-4 and 29E.2A3 (mouse IgG2b, kappa;
positive control) [30] reacted with LN229 and LN229/hPD-L1, the reactivity of L1Mab-4 was higher for LN229/hPD-L1 than LN229(Fig. 1B). Both L1Mab-4 and 29E.2A3 also reacted with HEK-293T/PD126

Biochemistry and Biophysics Reports 13 (2018) 123–128

S. Yamada et al.

Fig. 4. Immunohistochemical analysis of L1Mab-4 against oral cancers (tissue microarray). After antigen retrieval, sections were incubated with 10 μg/mL of L1Mab-4 followed bytreatment with Envision+ kit. Color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB), and sections were counterstained with hematoxylin. Scale bar = 100 µm.

3.3. Immunohistochemical analysis against oral cancer tissues

L1, but not with HEK-293T (Fig. 1C), indicating the speciﬁcity of
L1Mab-4 against hPD-L1.

We further investigated the immunohistochemical utility of L1Mab4 in human oral cancers. First, we performed immunohistochemical
analysis using two diﬀerent conditions of antigen retrieval. Fig. 3 showsthat antigen retrieval using citrate buﬀer (pH 6) revealed a better
staining pattern (Fig. 3A and B) than did EnVision FLEX Target Retrieval Solution, High pH (Fig. 3C and D). Therefore, we employed citrate buﬀer (pH 6) for antigen retrieval in this study. L1Mab-4 stainedoral cancer cells in a membrane-staining pattern, indicating that L1Mab4 is very useful for immunohistochemical analysis against oral cancers.

3.2. Flow cytometric analysis against oral cancer cell lines
L1Mab-4 recognized endogenous hPD-L1 in oral cancer cell lines,
such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 (Fig. 2). Although 29E.2A3 also reacted with these cell lines, the reaction of
L1Mab-4 was higher than that of 29E.2A3, indicating the usefulness of
L1Mab-4 for the detection of hPD-L1 in ﬂow cytometry.
127

Biochemistry and Biophysics Reports 13 (2018) 123–128

S. Yamada et al.

We then stained oral cancer tissue arrays, and the typical results(staining level, 3+) are shown in Fig. 4. L1Mab-4 strongly stained thecell membranes of oral cancers. As shown in Supplementary Tables 1
and 2, 106/150 (70.7%) of SCCs were stained by L1Mab-4 and 26/106(24.5%) were diagnosed as 3+. Although the samples were limited,adenoid cystic carcinomas (ACCs) and mucoepidermoid carcinomas(MECs) also showed positive staining by L1Mab-4.
In conclusion, we successfully produced L1Mab-4, a novel anti-hPDL1 mAb, using the CBIS method. This method is very advantageous
because it does not require the membrane protein to be puriﬁed in all
steps of mAb production. L1Mab-4 is very useful in the detection ofhPD-L1 using ﬂow cytometry and immunohistochemical analysis. In thenear future, studies investigating the reactivity of L1Mab-4 for otherhuman cancers need to be conducted. Furthermore, we should consider
the possibility that L1Mab-4 might cross-react with other membraneproteins or react with post-translational modiﬁcation, including glycosylation, on PD-L1 because the reaction of L1Mab-4 is much higher
than those of 29E.2A3 against LN229 and HO-1-u-1 (Figs. 1 and 2).

W.J. Geese, J. Kopit, J.W. Shaw, M.L. Gillison, Nivolumab for recurrent squamouscell carcinoma of the head and neck, N. Engl. J. Med. 375 (2016) 1856–1867.
[10] B. Boyerinas, C. Jochems, M. Fantini, C.R. Heery, J.L. Gulley, K.Y. Tsang, J. Schlom,
Antibody-dependent cellular cytotoxicity activity of a novel anti-PD-L1 antibody
Avelumab (MSB0010718C) on human tumor cells, Cancer Immunol. Res. 3 (2015)
1148–1157.
[11] H.L. Kaufman, J. Russell, O. Hamid, S. Bhatia, P. Terheyden, S.P. D'Angelo,
K.C. Shih, C. Lebbe, G.P. Linette, M. Milella, I. Brownell, K.D. Lewis, J.H. Lorch,
K. Chin, L. Mahnke, A. von Heydebreck, J.M. Cuillerot, P. Nghiem, Avelumab inpatients with chemotherapy-refractory metastatic Merkel cell carcinoma: a multicentre, single-group, open-label, phase 2 trial, Lancet Oncol. 17 (2016) 1374–1385.
[12] M. Li, N. Saghaﬁ, E. Freymiller, J.R. Basile, Y.L. Lin, Metastatic Merkel cell carcinoma of the oral cavity in a human immunodeﬁciency virus-positive patient and thedetection of Merkel cell polyomavirus, Oral Surg. Oral Med. Oral Pathol. Oral
Radiol. 115 (2013) e66–e71.
[13] J.A. Brown, D.M. Dorfman, F.R. Ma, E.L. Sullivan, O. Munoz, C.R. Wood,
E.A. Greenﬁeld, G.J. Freeman, Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production, J. Immunol. 170(2003) 1257–1266.
[14] H. Dong, S.E. Strome, D.R. Salomao, H. Tamura, F. Hirano, D.B. Flies, P.C. Roche,
J. Lu, G. Zhu, K. Tamada, V.A. Lennon, E. Celis, L. Chen, Tumor-associated B7-H1
promotes T-cell apoptosis: a potential mechanism of immune evasion, Nat. Med. 8(2002) 793–800.
[15] J. Konishi, K. Yamazaki, M. Azuma, I. Kinoshita, H. Dosaka-Akita, M. Nishimura,
B7-H1 expression on non-small cell lung cancer cells and its relationship withtumor-inﬁltrating lymphocytes and their PD-1 expression, Clin. Cancer Res. 10(2004) 5094–5100.
[16] B.T. Fife, K.E. Pauken, The role of the PD-1 pathway in autoimmunity and peripheral tolerance, Ann. N. Y. Acad. Sci. 1217 (2011) 45–59.
[17] J. Hamanishi, M. Mandai, M. Iwasaki, T. Okazaki, Y. Tanaka, K. Yamaguchi,
T. Higuchi, H. Yagi, K. Takakura, N. Minato, T. Honjo, S. Fujii, Programmed cell
death 1 ligand 1 and tumor-inﬁltrating CD8+ T lymphocytes are prognostic factorsof human ovarian cancer, Proc. Natl. Acad. Sci. USA 104 (2007) 3360–3365.
[18] T. Okazaki, T. Honjo, PD-1 and PD-1 ligands: from discovery to clinical application,
Int. Immunol. 19 (2007) 813–824.
[19] R.H. Thompson, M.D. Gillett, J.C. Cheville, C.M. Lohse, H. Dong, W.S. Webster,
K.G. Krejci, J.R. Lobo, S. Sengupta, L. Chen, H. Zincke, M.L. Blute, S.E. Strome,
B.C. Leibovich, E.D. Kwon, Costimulatory B7-H1 in renal cell carcinoma patients:
indicator of tumor aggressiveness and potential therapeutic target, Proc. Natl. Acad.
Sci. USA 101 (2004) 17174–17179.
[20] Y.M. Lin, W.W. Sung, M.J. Hsieh, S.C. Tsai, H.W. Lai, S.M. Yang, K.H. Shen,
M.K. Chen, H. Lee, K.T. Yeh, C.J. Chen, High PD-L1 expression correlates withmetastasis and poor prognosis in oral squamous cell carcinoma, PLoS One 10 (2015)
e0142656.
[21] J. Chen, Y. Feng, L. Lu, H. Wang, L. Dai, Y. Li, P. Zhang, Interferon-gamma-induced
PD-L1 surface expression on human oral squamous carcinoma via PKD2 signal
pathway, Immunobiology 217 (2012) 385–393.
[22] Y.A. Cho, H.J. Yoon, J.I. Lee, S.P. Hong, S.D. Hong, Relationship between the expressions of PD-L1 and tumor-inﬁltrating lymphocytes in oral squamous cell carcinoma, Oral Oncol. 47 (2011) 1148–1153.
[23] M. Hirai, H. Kitahara, Y. Kobayashi, K. Kato, G. Bou-Gharios, H. Nakamura,S. Kawashiri, Regulation of PD-L1 expression in a high-grade invasive human oral
squamous cell carcinoma microenvironment, Int. J. Oncol. 50 (2017) 41–48.
[24] C. Jiang, F. Yuan, J. Wang, L. Wu, Oral squamous cell carcinoma suppressed antitumor immunity through induction of PD-L1 expression on tumor-associated macrophages, Immunobiology 222 (2017) 651–657.
[25] S.I. Pai, D.P. Zandberg, S.E. Strome, The role of antagonists of the PD-1:PD-L1/PDL2 axis in head and neck cancer treatment, Oral Oncol. 61 (2016) 152–158.
[26] M. Troeltzsch, T. Woodlock, A. Pianka, S. Otto, M. Troeltzsch, M. Ehrenfeld,
T. Knosel, Is there evidence for the presence and relevance of the PD-1/PD-L1
pathway in oral squamous cell carcinoma? Hints from an immunohistochemical
study, J. Oral Maxillofac. Surg. 75 (2017) 969–977.
[27] Y. Fujii, M. Kaneko, M. Neyazaki, T. Nogi, Y. Kato, J. Takagi, PA tag: a versatile
protein tagging system using a super high aﬃnity antibody against a dodecapeptide
derived from human podoplanin, Protein Expr. Purif. 95 (2014) 240–247.
[28] Y. Fujii, M.K. Kaneko, S. Ogasawara, S. Yamada, M. Yanaka, T. Nakamura,N. Saidoh, K. Yoshida, R. Honma, Y. Kato, Development of RAP tag, a novel tagging
system for protein detection and puriﬁcation, Monoclon. Antib. Immunodiagn.
Immunother. 36 (2017) 68–71.
[29] Y. Fujii, M.K. Kaneko, Y. Kato, MAP tag: a novel tagging system for protein puriﬁcation and detection, Monoclon. Antib. Immunodiagn. Immunother. 35 (2016)
293–299.
[30] Y. Latchman, C.R. Wood, T. Chernova, D. Chaudhary, M. Borde, I. Chernova,
Y. Iwai, A.J. Long, J.A. Brown, R. Nunes, E.A. Greenﬁeld, K. Bourque,
V.A. Boussiotis, L.L. Carter, B.M. Carreno, N. Malenkovich, H. Nishimura,T. Okazaki, T. Honjo, A.H. Sharpe, G.J. Freeman, PD-L2 is a second ligand for PD-1
and inhibits T cell activation, Nat. Immunol. 2 (2001) 261–268.
[31] 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. Antib. Immunodiagn. Immunother. 36 (2017)
224–230.
[32] S. Itai, S. Yamada, M.K. Kaneko, H. Harada, Y. Kagawa, S. Konnai, Y. Kato,
Expression of cat podoplanin in feline squamous cell carcinomas, Monoclon. Antib.
Immunodiagn. Immunother. 36 (2017) 243–250.

Acknowledgments
We thank Takuro Nakamura, Miyuki Yanaka, Yao-Wen Chang,
Noriko Saidoh, Saori Handa, and Yoshimi Nakamura for excellent
technical assistance.
Funding
This research was supported in part by AMED under Grant numbers:
JP17am0301010 (Y.K.), JP17am0101078 (Y.K.), and JP17ae0101028(Y.K.), and by JSPS KAKENHI Grant number 17K07299 (M.K.K.) and
Grant number 16K10748 (Y.K.).
Appendix A. Transparency document
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.bbrep.2018.01.009.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at http://dx.doi.org/10.1016/j.bbrep.2018.01.009.
References[1] S. Warnakulasuriya, Global epidemiology of oral and oropharyngeal cancer, Oral
Oncol. 45 (2009) 309–316.
[2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016, CA Cancer J. Clin. 66(2016) 7–30.
[3] C. Rivera, Essentials of oral cancer, Int. J. Clin. Exp. Pathol. 8 (2015) 11884–11894.
[4] M. Gorsky, J.B. Epstein, C. Oakley, N.D. Le, J. Hay, P. Stevenson-Moore, Carcinomaof the tongue: a case series analysis of clinical presentation, risk factors, staging,and outcome, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 98 (2004)
546–552.
[5] M. Sasaki, T. Aoki, K. Karakida, M. Otsuru, M. Takahashi, T. Akamatsu,
H. Sakamoto, Y. Ota, Postoperative follow-up strategy in patients with oral squamous cell carcinoma, J. Oral Maxillofac. Surg. 69 (2011) e105–e111.
[6] J. Bernier, C. Domenge, M. Ozsahin, K. Matuszewska, J.L. Lefebvre, R.H. Greiner,
J. Giralt, P. Maingon, F. Rolland, M. Bolla, F. Cognetti, J. Bourhis, A. Kirkpatrick,M. van Glabbeke, R. European Organization for, T. Treatment of Cancer,
Postoperative irradiation with or without concomitant chemotherapy for locallyadvanced head and neck cancer, N. Engl. J. Med. 350 (2004) 1945–1952.
[7] J.H. Lee, J.H. Song, S.N. Lee, J.H. Kang, M.S. Kim, D.I. Sun, Y.S. Kim, Adjuvant
postoperative radiotherapy with or without chemotherapy for locally advanced
squamous cell carcinoma of the head and neck: the importance of patient selection
for the postoperative chemoradiotherapy, Cancer Res. Treat. 45 (2013) 31–39.
[8] J.P. Pignon, J. Bourhis, C. Domenge, L. Designe, Chemotherapy added to locoregional treatment for head and neck squamous-cell carcinoma: three meta-analyses
of updated individual data. MACH-NC Collaborative Group. Meta-Analysis of
Chemotherapy on Head and Neck Cancer, Lancet 355 (2000) 949–955.
[9] R.L. Ferris, G. Blumenschein Jr., J. Fayette, J. Guigay, A.D. Colevas, L. Licitra,
K. Harrington, S. Kasper, E.E. Vokes, C. Even, F. Worden, N.F. Saba, L.C. Iglesias
Docampo, R. Haddad, T. Rordorf, N. Kiyota, M. Tahara, M. Monga, M. Lynch,128