Biochemistry and Biophysics Reports 15 (2018) 93–96

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

Podocalyxin is crucial for the growth of oral squamous cell carcinoma cellline HSC-2

T

Shunsuke Itaia,b, Shinji Yamadaa, Mika K. Kanekoa, Masato Sanoa, Takuro Nakamuraa,
Miyuki Yanakaa, Saori Handaa, Kayo Hisamatsua, Yoshimi Nakamuraa, Yoshikazu Furusawaa,c,d,⁎Masato Fukuid, Tomokazu Ohishie, Manabu Kawadae, Hiroyuki Haradab, Yukinari Katoa,c,aDepartment of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
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, Japanc
New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapandZENOAQ RESOURCE CO., LTD., 1-1 Tairanoue, Sasagawa, Asaka-machi, Koriyama, Fukushima 963-0196, Japane
Institute of Microbial Chemistry (BIKAKEN), Numazu, Microbial Chemistry Research Foundation, 18-24 Miyamoto, Numazu, Shizuoka 410-0301, Japanb

A R T I C LE I N FO

A B S T R A C T

Keywords:
Oral squamous cell carcinoma
OSCC
HSC-2
Podocalyxin
PODXL
Monoclonal antibody

Oral cancers constitute approximately 2% of all cancers, with the most common histological type being oral
squamous cell carcinoma (OSCC), representing 90% of oral cancers. Although diagnostic technologies andtherapeutic techniques have progressed, the survival rate of patients with OSCC is still 60%, whereas the incidence rate has increased. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is
detected in normal tissues such as heart, breast, and pancreas as well as in many cancers, including lung, renal,
breast, colorectal, and oral cancers. This glycoprotein is associated with the progression, metastasis, and pooroutcomes of oral cancers. PODXL overexpression was strongly detected using our previously established antiPODXL monoclonal antibody (mAb), PcMab-47, and its mouse IgG2a-type, 47-mG2a. In previous studies, we also
generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC cell lines, in order to investigate the functionof PODXL in the proliferation of oral cancer cells. The growth of SAS/PODXL-KO cell lines was observed to belower than that of parental SAS cells. For this study, PODXL-KO OSCC cell lines were generated using HSC-2
cells, and the role of PODXL in the growth of OSCC cell lines in vitro was assessed. Decreased growth wasobserved for HSC-2/PODXL-KO cells compared with HSC-2 parental cells. The inﬂuence of PODXL on tumor
growth of OSCC was also investigated in vivo, and both the tumor volume and the tumor weight were observed tobe signiﬁcantly lower for HSC-2/PODXL-KO than that for HSC-2 parental cells. These results, taken together,indicate that PODXL plays an important role in tumor growth, both in vitro and in vivo.

1. Introduction
Globally, oral cancers constitute approximately 2% of all cancers[1]. They can be histologically classiﬁed into the following types:
squamous cell carcinoma, adenoid carcinoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, and osteosarcoma. Approximately
90% of oral cancer burden is borne by oral squamous cell carcinoma(OSCC) [2]. Although diagnostic technologies and therapeutic techniques have progressed by leaps and bounds in recent decades, the survival rate of patients with OSCC has not improved. Consequently, the 5year survival rate of patients with OSCC is still 60% [3]; however, theincidence rate of OSCC is increasing [4,5].
Podocalyxin (PODXL) is a CD34-related highly glycosylated type I
transmembrane protein [6–9]. PODXL can be detected in normal tissues
such as heart, breast, and pancreas [10] as well as in cancers including
lung, renal, breast, colorectal, and oral cancers [9,11–15]. PODXL
overexpression is associated with the progression, metastasis, and pooroutcomes of several cancer types [16–19].
In our previous study, we established speciﬁc and sensitive antiPODXL mAbs: PcMab-47 (mouse IgG1, kappa) [20], 47-mG2a (mouse
IgG2a-type of PcMab-47), and 47-mG2a-f (core fucose-deﬁcient type of
47-mG2a) [21]. We demonstrated that 47-mG2a-f signiﬁcantly reduced

Abbreviations: OSCC, oral squamous cell carcinoma; mAb, monoclonal antibody⁎
Corresponding author at: 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.2018.07.008
Received 20 July 2018; Accepted 27 July 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/).

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tumor growth in OSCC xenograft models. [21]. To investigate thefunction of PODXL in the growth of oral cancer cells, we previously
generated PODXL-knock out (PODXL-KO) cell lines using SAS OSCC celllines. The growth of SAS/PODXL-KO cell lines (BINDS-01) was observed to be lower than that of parental SAS cells. In this study, we
further established PODXL-KO OSCC cell lines using HSC-2 (oralsquamous cell carcinoma from oral cavity) cells and investigated therole of PODXL in the growth of OSCC cell lines both in vitro and in vivo.
2. Materials and methods
2.1. Cell lines
HSC-2 cells were obtained from Japanese Collection of Research
Bioresources Cell Bank (Osaka, Japan). HSC-2/PODXL-KO cells (BINDS02) were produced using CRISPR/Cas9 plasmids (Target ID:
HS0000056763), targeting human PODXL (Sigma-Aldrich Corp., St.
Louis, MO, USA). Three clones were selected for this study: HSC-2/
PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/PODXL-KO #3.
Parental HSC-2 and HSC-2/PODXL-KO cells were cultured in Dulbecco's
Modiﬁed Eagle's Medium (DMEM; Nacalai Tesque, Inc., Kyoto, Japan),which was supplemented with 10% heat-inactivated fetal bovine serum(Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA), 100 units/mL penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B(Nacalai Tesque, Inc.), at 37 °C in a humidiﬁed atmosphere containing
5% CO2 and 95% air. BINDS-01 and BINDS-02 can be obtained from
Kato's lab in Tohoku University Graduate School of Medicine (http://
www.med-tohoku-antibody.com/topics/001_paper_cell.htm).
2.2. Flow cytometry
HSC-2/PODXL-KO cell lines were harvested following a brief exposure to 0.25% trypsin in 1 mM ethylenediaminetetraacetic acid
(Nacalai Tesque, Inc.). After washing with 0.1% bovine serum albuminin phosphate-buﬀered saline (Nacalai Tesque, Inc.), cells were treatedwith primary mAbs for 30 min at 4 °C, followed by treatment with Alexa
Fluor 488-conjugated anti-mouse IgG (1:2000; Cell Signaling
Technology, Danvers, MA, USA). Fluorescence data were obtainedusing SA3800 Cell Analyzer (Sony Corp., Tokyo, Japan).
2.3. In vitro proliferation assay
In vitro cell proliferation was measured using Cell Cloning Kit-8(CCK-8; Dojindo, Kumamoto, Japan), which can assess cell viability.
Cells were plated (1500, 3000, and 6000 cells/100 μL/well) in quintuple wells in 96-well plates and were incubated for 48 h. After adding
10 μL of CCK-8 to each well, the plates were incubated for 4 h at 37 °C.
Subsequently, the absorbance was recorded at 450 nm using iMark
microplate reader (Bio-Rad Laboratories, Inc., Berkeley, CA). The mean
absorbance of the 5-well set was obtained at 48 h after cell seeding. Alldata were expressed as the mean ± SEM. Statistical signiﬁcance wasanalyzed using Tukey–Kramer's test. P-values < 0.05 were considered
statistically signiﬁcant.
2.4. In vivo proliferation assay
Five-week-old, female BALB/c nude mice were purchased from
CLEA Japan (Tokyo, Japan). Seven-week-old mice were used for the invivo proliferation assay. Cells (0.1 mL of 5 × 107 /mL in DMEM) weremixed with 0.1 mL of BD Matrigel Matrix Growth Factor Reduced (BDBiosciences, San Jose, CA, USA). A 200 μL suspension (containing 5 ×
106 cells) was subcutaneously injected into the left ﬂanks of nude mice.
In this experiment, the following four cell lines were used: HSC-2, HSC2/PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/PODXL-KO #3.
Each group included eight mice. The tumor diameter was measured atday 7, 14, and 21 after injecting cancer cell lines using calipers, and theFig. 1. Flow cytometry using HSC-2/PODXL-KO cell lines. HSC-2, HSC-2/
PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/PODXL-KO #3 were treatedwith PcMab-47 (10 μg/mL), followed by secondary antibodies. Gray peak, negative control Red peak, PcMab-47.

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tumor volume was calculated using the following formula: volume= W2 × L/2, where W is the short diameter, and L is the long diameter.
All mice were euthanized 21 days after cell implantation, and the tumor
weight was measured. All data were expressed as mean ± SEM.
Statistical analysis was performed using Tukey–Kramer's test. P-values < 0.05 were considered statistically signiﬁcant.
2.5. Hematoxylin and eosin staining
After euthanizing mice at day 21, histopathological specimen sections (thickness, 4 µm) were deparaﬃnized in xylene, rehydrated, andstained with hematoxylin and eosin.
3. Results and discussion
For this study, CRISPR/Cas9 plasmids targeting human PODXL wereused to generate three PODXL-KO OSCC cell lines: HSC-2/PODXL-KO#1, #2, and #3. Before initiating the study, PODXL expression was
conﬁrmed in these cell lines. As shown in Fig. 1, anti-human PODXL
mAb, PcMab-47 reacted with the HSC-2 parental cell line, but did notreact with any of the three HSC-2/PODXL-KO cell lines.
Next, we investigated the potential involvement of PODXL in thestimulation of in vitro OSCC cell growth. At every plated cell density(1500, 3000, and 6000 cells/well), the growth of HSC-2/PODXL-KO #1
and #2 cells was signiﬁcantly lower than that of HSC-2 cells (Fig. 2). In
contrast, HSC-2/PODXL-KO #3 showed signiﬁcantly less growth thanHSC-2 cells only when at a plated cell density of 1500 cells/well. These
results indicate that while PODXL is an important factor for in vitro
HSC-2 cell growth, many other factors may be involved in the induction
or inhibition of HSC-2 cell growth.
The role of PODXL in OSCC tumor growth in vivo was further examined by comparing the growth of four cell lines, parental HSC-2,
HSC-2/PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/PODXL-KO#3, which were transplanted subcutaneously into nude mice. On days
7, 14, and 21 after inoculation, tumor volumes of them were measured(Fig. 3). On day 7, no diﬀerence in tumor volumes were observed between parental HSC-2 and HSC-2/PODXL-KO cell lines. The tumor
volume of HSC-2/PODXL-KO #1 cell lines was signiﬁcantly lower thanthat of parental HSC-2 cell lines on day 14, although the tumor volumesfor HSC-2/PODXL-KO #2 and #3 cell lines were similar to those arisingfrom the parental cell line, HSC-2 (Fig. 3A). On day 21, tumor volumes
arising from the transplantation of HSC-2/PODXL-KO #1, #2, and #3
cell lines were signiﬁcantly lower than those arising from the parental

Fig. 3. In vivo functional analysis of PODXL using PODXL-KO OSCC lines. HSC2, HSC-2/PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/PODXL-KO #3 celllines were injected subcutaneously into female BALB/c mice. (A) The tumor
volume was measured at day 7, 14, and 21 after inoculation. The values arepresented as mean ± SEM. Asterisks indicate statistical signiﬁcance between
HSC-2 and HSC-2/PODXL-KO cell lines (*P < 0.05, **P < 0.01,
Tukey–Kramer's test). (B) Mice were euthanized at day 21 and their photos
were taken at the same day.

HSC-2 cell lines (Fig. 3A). Subcutaneous tumors arising from parental
HSC-2, HSC-2/PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/
PODXL-KO #3 on day 21 are shown in Fig. 3B. In vivo analysis revealed
that HSC-2/PODXL-KO #1 cell lines resulted in the smallest tumor
growth among the three HSC-2/PODXL-KO cell lines (Fig. 3A), which isconsistent with the in vitro result (Fig. 2).
On day 21 after inoculation, all tumors were measured, following
the resection of mice (Fig. 4A). As depicted in Fig. 4B, the tumor weightfrom HSC-2/PODXL-KO #1 and #2 cell lines were signiﬁcantly lower
than those from parental HSC-2 cell lines. The tumor weight from HSC2/PODXL-KO #3 was not signiﬁcantly diﬀerent from that of parental
HSC-2 cell line (Fig. 4B), whereas the tumor volumes from HSC-2/
PODXL-KO #3 cell lines were signiﬁcantly lower than those from parental HSC-2 cell lines (Fig. 3A). Hematoxylin and eosin staining of resected tumors of parental HSC-2, HSC-2/PODXL-KO #1, HSC-2/
PODXL-KO #2, and HSC-2/PODXL-KO #3 on day 21 are shown in
Supplementary Fig. 1.
Taken together, our results indicate that PODXL plays an importantrole in the growth of tumor in oral cancers. The HSC-2/PODXL-KO celllines, which were generated in this study, are promising tools that canbe used in future studies to elucidate the function of PODXL in the
proliferation of oral cancer.

Funding
Fig. 2. In vitro functional analysis of PODXL using PODXL-KO OSCC lines. HSC2, HSC-2/PODXL-KO #1, HSC-2/PODXL-KO #2, and HSC-2/PODXL-KO #3 celllines were plated (1500, 3000, and 6000 cells/100 μL/well) in quintuple wells
in 96-well plates and were incubated for 48 h. After adding 10 μL of CCK-8 toeach well, plates were incubated for 4 h at 37 °C. The values are mean ± SEM.
The absorbance was recorded at 450 nm (*P < 0.05, **P < 0.01,
Tukey–Kramer's test).

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.).

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[3] S.B. Chinn, J.N. Myers, Oral cavity carcinoma: current management, controversies,
and future directions, J. Clin. Oncol. 33 (2015) 3269–3276.
[4] T. Hakulinen, L. Tryggvadottir, M. Gislum, H.H. Storm, F. Bray, A. Klint,
G. Engholm, Trends in the survival of patients diagnosed with cancers of the lip,oral cavity, and pharynx in the Nordic countries 1964–2003 followed up to the endof 2006, Acta Oncol. 49 (2010) 561–577.
[5] A.A. Hussein, M.N. Helder, J.G. de Visscher, C.R. Leemans, B.J. Braakhuis,
H.C.W. de Vet, T. Forouzanfar, Global incidence of oral and oropharynx cancer inpatients younger than 45 years versus older patients: a systematic review, Eur. J.
Cancer 82 (2017) 115–127.
[6] K.M. McNagny, I. Pettersson, F. Rossi, I. Flamme, A. Shevchenko, M. Mann, T. Graf,
Thrombomucin, a novel cell surface protein that deﬁnes thrombocytes and multipotent hematopoietic progenitors, J. Cell Biol. 138 (1997) 1395–1407.
[7] W.M. Schopperle, W.C. DeWolf, The TRA-1-60 and TRA-1-81 human pluripotent
stem cell markers are expressed on podocalyxin in embryonal carcinoma, Stem Cells
25 (2007) 723–730.
[8] W.M. Schopperle, D.B. Kershaw, W.C. DeWolf, Human embryonal carcinoma tumor
antigen, Gp200/GCTM-2, is podocalyxin, Biochem. Biophys. Res. Commun. 300(2003) 285–290.
[9] N. Hayatsu, M.K. Kaneko, K. Mishima, R. Nishikawa, M. Matsutani, J.E. Price,
Y. Kato, Podocalyxin expression in malignant astrocytic tumors, Biochem. Biophys.
Res. Commun. 374 (2008) 394–398.
[10] R. Doyonnas, D.B. Kershaw, C. Duhme, H. Merkens, S. Chelliah, T. Graf,
K.M. McNagny, Anuria, omphalocele, and perinatal lethality in mice lacking theCD34-related protein podocalyxin, J. Exp. Med. 194 (2001) 13–27.
[11] Y.H. Hsu, W.L. Lin, Y.T. Hou, Y.S. Pu, C.T. Shun, C.L. Chen, Y.Y. Wu, J.Y. Chen,
T.H. Chen, T.S. Jou, Podocalyxin EBP50 ezrin molecular complex enhances themetastatic potential of renal cell carcinoma through recruiting Rac1 guanine nucleotide exchange factor ARHGEF7, Am. J. Pathol. 176 (2010) 3050–3061.
[12] L.K. Koch, H. Zhou, J. Ellinger, K. Biermann, T. Holler, A. von Rucker, R. Buttner,
I. Gutgemann, Stem cell marker expression in small cell lung carcinoma and developing lung tissue, Hum. Pathol. 39 (2008) 1597–1605.
[13] C.W. Lin, M.S. Sun, H.C. Wu, Podocalyxin-like 1 is associated with tumor aggressiveness and metastatic gene expression in human oral squamous cell carcinoma,Int. J. Oncol. 45 (2014) 710–718.
[14] K.A. Snyder, M.R. Hughes, B. Hedberg, J. Brandon, D.C. Hernaez, P. Bergqvist,
F. Cruz, K. Po, M.L. Graves, M.E. Turvey, J.S. Nielsen, J.A. Wilkins, S.R. McColl,
J.S. Babcook, C.D. Roskelley, K.M. McNagny, Podocalyxin enhances breast tumor
growth and metastasis and is a target for monoclonal antibody therapy, Breast
Cancer Res. 17 (2015) 46.
[15] T. Tschernig, R. Pabst, M. Kasper, M. El-Hadi, B. Singh, Expression of caveolin-1 andpodocalyxin in rat lungs challenged with 2-kDa macrophage-activating lipopeptide
and Flt3L, Cell Tissue Res. 356 (2014) 207–216.
[16] T.N. Flores-Tellez, T.V. Lopez, V.R. Vasquez Garzon, S. Villa-Trevino, Co-expressionof ezrin-CLIC5-podocalyxin is associated with migration and invasiveness in hepatocellular carcinoma, PLoS One 10 (2015) e0131605.
[17] H. Kusumoto, Y. Shintani, R. Kanzaki, T. Kawamura, S. Funaki, M. Minami,
I. Nagatomo, E. Morii, M. Okumura, Podocalyxin inﬂuences malignant potential bycontrolling epithelial-mesenchymal transition in lung adenocarcinoma, Cancer Sci.
108 (2017) 528–535.
[18] K. Taniuchi, M. Tsuboi, M. Sakaguchi, T. Saibara, Measurement of serum PODXL
concentration for detection of pancreatic cancer, Onco Targets Ther. 11 (2018)
1433–1445.
[19] J. Zhang, Z. Zhu, H. Wu, Z. Yu, Z. Rong, Z. Luo, Y. Xu, K. Huang, Z. Qiu, C. Huang,
PODXL, negatively regulated by KLF4, promotes the EMT and metastasis and serves
as a novel prognostic indicator of gastric cancer, Gastric Cancer (2018).
[20] S. Ogasawara, M.K. Kaneko, S. Yamada, R. Honma, T. Nakamura, N. Saidoh,
M. Yanaka, K. Yoshida, Y. Fujii, Y. Kato, PcMab-47: novel antihuman podocalyxin
monoclonal antibody for immunohistochemistry, Monoclon. Antib. Immunodiagn.
Immunother. 36 (2017) 50–56.
[21] S. Itai, T. Ohishi, M.K. Kaneko, S. Yamada, S. Abe, T. Nakamura, M. Yanaka,
Y.W. Chang, S.I. Ohba, Y. Nishioka, M. Kawada, H. Harada, Y. Kato, Anti-podocalyxin antibody exerts antitumor eﬀects via antibody-dependent cellular cytotoxicity
in mouse xenograft models of oral squamous cell carcinoma, Oncotarget 9 (2018)
22480–22497.

Fig. 4. Comparison of the tumor size in the in vivo functional analysis of PODXL
using PODXL-KO OSCC lines. (A) After euthanizing mice at day 21, tumors were
resected. Scale bar = 1 cm. (B) Tumor weights were measured after tumor resection on day 21. The values are presented as mean ± SEM. Asterisks indicate
statistical signiﬁcance between HSC-2 and HSC-2/PODXL-KO cell lines(*P < 0.05, **P < 0.01, Tukey–Kramer's test).

Appendix A. Transparency document
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.bbrep.2018.07.008.
References[1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016, CA Cancer J. Clin. 66(2016) 7–30.
[2] C. Rivera, Essentials of oral cancer, Int. J. Clin. Exp. Pathol. 8 (2015) 11884–11894.

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