Biochemistry and Biophysics Reports 14 (2018) 64–68

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

Detection of high CD44 expression in oral cancers using the novel
monoclonal antibody, C44Mab-5

T

Shinji Yamadaa, Shunsuke Itaia, Takuro Nakamuraa, Miyuki Yanakaa, Mika K. Kanekoa,⁎
Yukinari Katoa,b,abDepartment 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:
CD44
Monoclonal antibody
Immunohistochemistry
Oral cancer

CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration,
proliferation, diﬀerentiation, and survival. A large number of alternative splicing isoforms of CD44, containing
various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant
exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant
isoforms show tissue-speciﬁc expression patterns and have been extensively studied as both prognostic markers
and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines
overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C44Mab-5 (IgG1, kappa), recognized both CD44s and CD44v3-10. C44Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1,
SAS, HSC-2, HSC-3, and HSC-4 using ﬂow cytometry. Moreover, immunohistochemical analysis revealed that
C44Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C44Mab-5 antibody may be
useful for investigating the expression and function of CD44 in various cancers.

1. Introduction
CD44 is a transmembrane glycoprotein expressed on many cell
types, including endothelial cells, epithelial cells, ﬁbroblasts, keratinocytes, and leukocytes [1]. CD44 is believed to play important roles inphysiological processes such as cell proliferation, adhesion, migration,and lymphocyte activation [2]. The CD44 gene consists of 20 exons [3],with ten constitutively expressed to produce the smallest isoform,
which is the standard form of CD44 (CD44s). The other possible isoforms are categorized as CD44 variants (CD44v) and are generated byalternatively spliced transcripts [4]. The diversity of the CD44 protein isalso augmented by post-translational modiﬁcations such as N- and Oglycosylation and heparan sulfate [5,6].
Cancer cells often express a large variety of CD44 variants, particularly in the advanced stages. Indeed, the CD44v6 isoform was ﬁrst
identiﬁed as one of the metastatic determinants in cancer. CD44v6speciﬁc monoclonal antibodies (mAbs) have been shown to inhibit themetastasis of rat pancreatic tumor cells [7,8]. Furthermore, the transfection of CD44v4-7 cDNA has been shown to confer a metastatic

phenotype to non-metastatic cells [9]. In addition, CD44v6 isoforms
have been reported to act as co-receptors for receptor tyrosine kinases(RTKs), such as Met and VEGFR-2 [10–12]. Another variant isoform,
CD44v3, is able to bind several heparan sulfate-binding growth factors,
such as FGFs and HB-EGF [13,14]. These studies suggest that the coreceptor function of CD44v for RTKs is required for tumor progression.
Several reports have also indicated that CD44v likely functions as aprognostic marker in many tumors, including lung, colorectal, breast,
hepatocellular, and head and neck cancers [15–19]. Considering that
CD44 has multifunctional roles and a promising prognostic value invarious cancers, targeting CD44 for cancer therapy may prove to be a
promising approach. MAbs, which neutralize the binding of hyaluronic
acid to CD44, have been shown to inhibit anchorage-independent
growth of murine mammary carcinoma cells and human colon carcinoma cells, and to induce apoptosis in vitro [20]. In addition, mAbs
against CD44 or CD44v have been demonstrated to exert signiﬁcant
antitumor activity in in vivo animal models of human xenograft tumors[21,22]. Although a number of studies have been conducted on CD44,
CD44v isoforms have not been fully characterized. This is, in part, due

Abbreviations: mAb, monoclonal antibody; CBIS, cell-based immunization and screening; SCC, squamous cell carcinoma; ACC, adenoid cystic carcinoma; MEC, mucoepidermoid
carcinoma; 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⁎
Corresponding author at: New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
E-mail address: yukinari-k@bea.hi-ho.ne.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2018.03.007
Received 21 March 2018; Accepted 29 March 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 14 (2018) 64–68

S. Yamada et al.

Fig. 1. (A) Schematic illustration of CD44 standard (CD44s) and variants (CD44v3-10). (B) Procedure of Cell-Based Immunization and Screening (CBIS) method.

from the American Type Culture Collection (ATCC, Manassas, VA).
CD44v3-10 open reading frame (ORF) was provided by the RIKEN
BRC through the National Bio-Resource Project of the MEXT, Japan.
CD44s ORF was ampliﬁed from LN229 cDNA using HotStar HiFidelity
Polymerase Kit (Qiagen Inc., Hilden, Germany). CD44v3-10 ORF andCD44s ORF were subcloned into pCAG-cRAP-MAP vector possessing Cterminal RAP/MAP tag and pCAG-ssPA16 vector possessing signal sequence and N-terminal PA16 tag (GLEGGVAMPGAEDDVV), respectively. These plasmids were named as pCAG-CD44v3-10 and pCAGssPA16-CD44s, respectively, and were transfected into CHO-K1 cells
using a Neon transfection system (Thermo Fisher Scientiﬁc, Inc.,
Waltham, MA). The stable transfectant of CHO/CD44v3-10 was established by a cell sorter (SH800; Sony Corp., Tokyo, Japan).
Ca9-22, HSC-2, HSC-3, and HSC-4 cells were cultured in Dulbecco's
Modiﬁed Eagle's Medium (DMEM; Nacalai Tesque, Kyoto, Japan), andHO-1-u-1, SAS, CHO-K1, CHO/CD44v3-10, CHO/CD44s, and P3U1 celllines were cultured in RPMI 1640 medium (Nacalai Tesque, Inc.) at
37 °C in a humidiﬁed atmosphere containing 5% CO2 and 95% air, bothof which were supplemented with 10% heat-inactivated fetal bovine

to the lack of critical probes needed for the speciﬁc detection of CD44v
isoforms, as limited antibodies against CD44 variant exons are commercially available. Therefore, sensitive antibodies to CD44v-speciﬁc
isoforms are necessary.
Recently, we immunized mice with cat podoplanin-expressed CHOK1 cells (CHO/catPDPN) and performed screening using CHO/catPDPN
in ﬂow cytometry [23]. This method was named as the Cell-Based
Immunization and Screening (CBIS) method. Using this CBIS method,
we obtained highly sensitive mAbs against various membrane proteins.
In this study, we aimed to develop a novel anti-CD44 mAb using the
CBIS method.

2. Materials and methods
2.1. Cell lines and plasmids
Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank
(Osaka, Japan). CHO-K1, LN229, and P3U1 cell lines were obtained65

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Fig. 2. Flow cytometry of C44Mab-5 against oral cancer cell lines. Cells were treated with 10 μg/mL of C44Mab-5 (red line), followed by treatment with Alexa
Fluor 488-conjugated anti-mouse IgG; black line, negative control.

injections of CHO/CD44v3-10 (1 × 108 cells) together with Imject
Alum (Thermo Fisher Scientiﬁc Inc.). After three additional immunizations, a booster injection of CHO/CD44v3-10 was intraperitoneally administered 2 days before harvesting the spleen cells.
Spleen cells were then fused with 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, Inc.) were added to the medium. Plasmocin (5 μg/mL;
InvivoGen, San Diego, CA) was also used to prevent Mycoplasma contamination. Culture supernatants were screened by SA3800 Cell
Analyzers (Sony Corp.) using CHO-K1 and CHO/CD44v3-10. 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).

serum (FBS; Thermo Fisher Scientiﬁc, Inc.). One hundred units/mL
penicillin, 100 μg/mL streptomycin, and 25 μg/mL amphotericin B(Nacalai Tesque, Inc.) were added to the culture medium. G418
(0.5 mg/mL; FUJIFILM Wako Pure Chemical Industries Ltd., Osaka,
Japan) was added to the culture medium of CHO/CD44v3-10 and CHO/
CD44s.
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 (SPF) 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 39–85 from Cat. # OR601b or from Cybrdi, Inc.
(Frederick, MD): Cases 86–156 from Cat. # 27-10-001. The study examined 26 patients (Case-157-182) 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.

2.4. Flow cytometry

2.3. Hybridoma production

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-buﬀered

Two BALB/c mice were immunized using intraperitoneal (i.p.)
66

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S. Yamada et al.

Fig. 3. Immunohistochemical analysis of C44Mab-5 against oral SCCs. (A, B, E, F) After antigen retrieval, sections were incubated with 1 μg/mL of C44Mab-5
followed by treatment with the Envision+ kit. The color was developed using 3,3-diaminobenzidine tetrahydrochloride (DAB), and sections were counterstainedwith hematoxylin. (C, D, G, H) Hematoxylin & eosin staining (HE). (A-D, case 96; E-H, case 99). Scale bar = 100 µm.

Corp.).

Table 1
Summary of immunostaining using C44Mab-5.
Tumor type

SCC
ACC
MEC
Total

No. of
cases

172
7
3
182

C44Mab-5 immunostaining

No. of positive
cases

−

1+

2+

3+

14
2
0
16

20
3
1
24

61
1
0
62

77
1
2
80

2.5. Immunohistochemical analyses
Histological sections of 4-μm thickness were autoclaved in citrate
buﬀer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 min.
Sections were then incubated with 1 μg/mL of primary mAbs for 1 h atroom 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 (FUJIFILM Wako Pure
Chemical Industries Ltd.).

158/172 (91.9%)
5/7 (71.4%)
3/3 (100%)
166/182 (91.2%)

SCC; squamous cell carcinoma, ACC; adenocystic carcinoma MEC; mucoepidermoid carcinoma.
The intensity of staining was evaluated as −, 1+, 2+, 3+.

saline, the cells were treated with 10 μg/mL of primary antibodies, for
30 min at 4 °C and subsequently with Alexa Fluor 488-conjugated antimouse IgG (1:1000; Cell Signaling Technology, Inc., Danvers, MA).
Fluorescence data were collected using EC800 Cell Analyzers (Sony67

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S. Yamada et al.

3. Results and discussion

online version at http://dx.doi.org/10.1016/j.bbrep.2018.03.007.

3.1. Production of novel anti-CD44 mAbs using the Cell-Based
Immunization and Screening (CBIS) method

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We immunized two mice with CHO/CD44v3-10 cells (Fig. 1A), andperformed ﬂow cytometric screening. We previously named this cellbased strategy as the CBIS method, as shown in Fig. 1B. Culture supernatants of 1008 wells were mixed with CHO/CD44v3-10 and CHOK1 cells, and 15 wells (15/1008; 1.5%) showing a stronger reaction
against CHO/CD44v3-10 cells than CHO-K1 cells were ultimately selected. After limiting dilution, 7 clones were established, all of whichwere classiﬁed as IgG1 subclass. Clone C44Mab-5 (IgG1, kappa) reactedwith oral cancer tissues in further immunohistochemical analyses (datanot shown).
3.2. Flow cytometric analysis against oral cancer cell lines
C44Mab-5 reacted not only with CHO/CD44v3-10 but also with
CHO/CD44s (Fig. 2A), indicating that C44Mab-5 can detect all CD44
isoforms (Fig. 1A). C44Mab-5 also recognized endogenous CD44 in oral
cancer cell lines, such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, andHSC-4 (Fig. 2B).
3.3. Immunohistochemical analysis against oral cancer tissues
We further investigated the immunohistochemical utility of
C44Mab-5 in human oral cancers. C44Mab-5 stained oral cancer cells ina membrane-staining pattern (Fig. 3), indicating that C44Mab-5 may bevery useful for immunohistochemical analysis against oral cancers. We
then stained oral cancer tissue arrays; typical results (staining level,3+, 2+, 1+, −) are shown in Supplementary Fig. 1. As shown in
Table 1 and Supplementary Table 1, 158/172 (91.9%) of SCCs were
stained by C44Mab-5, and 80/182 (44.0%) were diagnosed as 3+. Although the samples were limited, adenoid cystic carcinomas (ACCs) andmucoepidermoid carcinomas (MECs) also showed positive staining by
C44Mab-5.
In conclusion, we successfully produced C44Mab-5, a novel antiCD44s mAb, using the CBIS method. This method is advantageous because it does not require the membrane protein to be puriﬁed in all
steps of mAb production. C44Mab-5 appears to be promising for thedetection of CD44s, as well as many CD44 variants using ﬂow cytometry and immunohistochemical analysis. Future studies investigating
the reactivity of C44Mab-5 towards other human cancers are warranted.
Acknowledgments
We thank Saori Handa and Yoshimi Nakamura for excellent technical assistance.
Funding
This research was supported in part by AMED under Grant nos.:
JP17am0301010 (Y.K.), JP17am0101078 (Y.K.), and JP17ae0101028(Y.K.), and by JSPS KAKENHI Grant no. 17K07299 (M.K.K.) and Grant
no. 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.2018.03.007.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the68