Received: 14 October 2021

| Revised: 4 March 2022 | Accepted: 14 March 2022

DOI: 10.1111/cas.15340

ORIGINAL ARTICLE

Establishment of a monoclonal antibody against glycosylated
CD271 specific for cancer cells in immunohistochemistry
Keitaro Fujii1,2 | Shinkichi Morita1,2 | Mai Mochizuki1 | Rie Shibuya-­Takahashi1 |Haruna Fujimori1 | Kazunori Yamaguchi3 | Jiro Abe4 | Tomoko Yamazaki5 |Takayuki Imai2 | Kazuo Sugamura3 | Jun Yasuda3 | Kennichi Satoh6| Ikuro Sato7 |Ryoko Saito-­Koyama8| Fumiyoshi Fujishima9 | Hironobu Sasano8
|
10
2,11
2
Yukinari Kato
| Kazuto Matsuura| Yukinori Asada | Keiichi Tamai1
1

Division of Cancer Stem Cell, Miyagi Cancer Center Research Institute, Miyagi, Japan2

Department of Head and Neck Surgery, Miyagi Cancer Center, Miyagi, Japan3

Molecular and Cellular Oncology, Miyagi Cancer Center Research Institute, Miyagi, Japan

4

Department of Thoracic Surgery, Miyagi Cancer Center, Miyagi, Japan

5

Department of Head and Neck Medical Oncology, Miyagi Cancer Center, Miyagi, Japan

6

Division of Gastroenterology, Tohoku Medical and Pharmaceutical University, Miyagi, Japan

7

Department of Pathology, Miyagi Cancer Center, Miyagi, Japan

8

Department of Anatomic Pathology, Tohoku University Graduate School of Medicine, Miyagi, Japan

9

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Miyagi, Japan10

Department of Pathology, Tohoku University Hospital, Miyagi, Japan

11

Department of Head and Neck Surgery, National Cancer Center Hospital East, Kashiwanoha, Kashiwa, Chiba, Japan

Correspondence
Keiichi Tamai, Division of Cancer StemCell, Miyagi Cancer Center ResearchInstitute, 47-­1, Medeshima-­Shiote, Natori,
Miyagi, Japan.
Email: tamaikeiichi@med.tohoku.ac.jp
Funding information
JSPS KAKENHI, Grant/Award Number:
20K09701, 20H03756, 19K08430,
17K11166 and 21H02396; Takeda Medical
Foundation; Kobayashi Foundation forCancer Research; Japan Agency for
Medical Research and Development(AMED), Grant/Award Number:
JP21am0401013 and JP21am0101078

Abstract
Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor
receptor), a glycosylated protein, increases malignant behavior of cancer, particularly
stemlike phenotypes in squamous cell carcinoma (SCC). CD271 is expressed in SCC
and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer
cells; therefore, we attempted to establish a cancer-­specific anti-­glycosylated CD271
antibody. We purified recombinant glycosylated CD271 protein, immunized mice withthe protein, and screened hybridomas using an ELISA assay with cancer cell lines. We
established a clone G4B1 against CD271 which is glycosylated with O-­glycan andsialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, but not in normal esophageal basal cells. This specificity was confirmed in

Abbreviations: CIS, carcinoma in situ; CRD, cysteine-­rich domain; FBS, fetal bovine serum; FFPE, formalin-­f ixed paraffin-­embedded; IHC, immunohistochemistry; PBS, phosphate-­
buffered saline; RT, room temperature; SCC, squamous cell carcinoma.

This is an open access article under the terms of the Creative Commons Attribution-­NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.
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wileyonlinelibrary.com/journal/cas

Cancer Science. 2022;113:2878–2887.

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FUJII et al.

hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal
epithelium and Barrett's esophagus, which possess stem cell–­like characteristics. In
conclusion, G4B1 antibody could be useful for precise identification of dysplasia andcancer cells in SCC.
KEYWORDS

antibody, cancer stem cell, CD271, esophageal cancer, glycosylation

1|

I NTRO D U C TI O Nof glycosylation that occurs in CD271 in cancer cells and its biological and pathological significance.

Squamous cell carcinoma (SCC) is one of the major cancer types, andTherefore, in this study, we established an antibody against gly-

there are few driver mutations and oncogenes because of the highcosylated CD271 in cancer cells and analyzed the specificity of thiscomplexity of genome alterations compared with adenocarcinoma.

antibody in pathology specimens.

Cancer tissue is composed of different cancer cell types, and there
are certain types of therapy-­resistant cancer cells, known as cancer stem cells. We previously identified CD271 as a cancer stem cellmarker in hypopharyngeal and lung SCC.1,2 CD271, also known as
p75NTR or NGFR, is a common receptor for all neurotrophins. We

2|

M ATE R I A L S A N D M E TH O D S

2.1 | Ethics statements

demonstrated that CD271-­high cancer cells exhibit cisplatin resistance and high tumorigenicity, and loss of CD271 reduced cancer

This study was conducted in accordance with the Declaration ofmalignancy, including tumorigenicity, proliferation capacity, and mi-

Helsinki and was approved by the Ethics Committees of the Miyagi

gration activity. These data led us to hypothesize that CD271 could

Cancer Center (Natori, Japan). All procedures were approved by andbe a promising target for therapy in SCC. We previously establishedexecuted in accordance with the Miyagi Cancer Center (permit num-

a humanized anti-­CD271 antibody, which possesses antibody-­

ber: 2019–­0 81) and performed as per committee regulations.

dependent cellular cytotoxicity in vitro and in vivo. However, thisapproach is expected to affect both cancer cells and normal basal
cells because CD271 is also expressed in epithelial basal cells; there-

2.2 | Cell lines

fore, a more cancer-­specific method is warranted.
Squamous cell carcinoma is a common esophageal cancer type in

The human cell lines used were the hypopharyngeal patient-­

Japan, although adenocarcinoma in the setting of Barrett's esopha-

derived xenograft cell lines (HPCM1, 2,11 HPCM 2, 2,11 HPCM612

3

gus is higher in the United States. Intraepithelial neoplasia is used ascells), which were maintained in RPMI-­1640 medium (Wako) sup-an inclusive term for squamous dysplasia and carcinoma in situ (CIS),

plemented with 10% fetal bovine serum (FBS), 100 unit/mL peni-

and SCC is generally considered to develop through dysplastic lesion
progression or dysplasia-­carcinoma sequence.4
Aberrant glycosylation is generally known to occur in cancer
5

cillin, and 100 μg/mL streptomycin. SK-­MEL-­2-­Luc and MeWo-­Luc
cells (human malignant melanoma, maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin) were provided

cells. Numerous studies have demonstrated that the cancer-­specific

by JCRB. MCC148c cells12 (human lung squamous carcinoma)

glycosylation pathway could play an essential role in cancer devel-

were maintained in DMEM supplemented with 10% FBS, 0.4 mg/

opment,6 but evidence of proteins with cancer-­specific glycosylation

mL hydrocortisone, 2.5 mM Y-­27632 (Focus Biomolecules), andis markedly limited. The antibody against podoplanin LpMab-­2 canpenicillin/streptomycin. DMEM supplemented with 10% FBS andrecognize esophageal cancer, but not normal lymphatic endothe-

penicillin/streptomycin was used to maintain 293T cells (RIKEN

lial cells, both of which express normal or aberrantly glycosylated

BioResource Center). Chinese hamster ovary (CHO) cells andpodoplanin.7 The antibody against MUC21 heM21c, which binds

glycan-­deficient CHO cell lines (Lec1, Lec2, and Lec8) were ob-

to MUC21 with Tn, T, or sialyl-­T epitopes, but not the unmodified

tained from the American Type Culture Collection (ATCC) andcore polypeptide of MUC21, recognizes esophageal cancer but not

maintained in RPMI with 10% FBS.

normal epithelium.8 These data all suggested that esophageal cancer cells possessed several aberrantly glycosylated proteins not expressed in the normal epithelium.

2.3 | CD271 expression vector and its variants

CD271 has a single N-­glycosylation site in the first cysteine-­richdomain (CRD) and is rich in O-­glycans, particularly in the juxtamem-

The

brane region.9 An O-­glycosylated stalk domain was reported to be

hCD271WT12 and CD271 deletion mutants (Δ1, Δ1-­2, Δ1-­3, and Δ1-­

required for apical sorting.10 However, little is known about the type

human

CD271

expression4) have been described previously.12

vectorpcDNA4/myc-­HisA-­

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2.4 | Establishment of genetically modified
cell lines

2.7 | Small interfering RNAs
Two CD271 siRNAs (siCD271-­1 HSS107179 and siCD271-­2

Human CD271-­overexpressing cells (SP2/0-­hCD271, SK-­OV-­3-­

HSS107181) and nonsilencing control siRNA (siControl, 12935–­300)

hCD271, and HPCM2-­hCD271) were established using the retroviral

were purchased from Invitrogen. siRNA transfections were per-12vector pBABE-­puro-­CD271, as previously described.

Human CD271-­overexpressing CHO, Lec1, Lec2, and Lec8 cells
were established as follows: the cell lines were transfected with pcD-

formed using Lipofectamine RNAiMAX Reagent (Life Technologies)
in an antibiotic-­free medium for 48 hours. The siRNA knockdown
efficiency was confirmed by flow cytometry analysis.

NA3.1-­CD271 vector by electroporation, followed by adding G418
(0.5 mg/mL; Wako) and cell sorting (FACS AriaII, BD Biosciences).

2.5 | Purification of CD271 extracellular domain(ECD) recombinant protein

2.8 | Flow cytometry
Flow cytometry was performed as previously described.11
Fluorescence data were collected using a FACSCanto II or SA3800
cell analyzer (BD Biosciences or Sony Biotechnology, respectively).

Purification of recombinant proteins was performed as previously

Anti-­CD271 antibody (ME20.4) was purchased from BioLegend. The

described.13 Briefly, Escherichia coli DH10Bac (Invitrogen) was trans-

data were analyzed using FlowJo (BD Biosciences) or CytoExploreR

formed using the pFastBac-­CD271 ECD recombinant donor plasmid.

package on R software.14

To produce recombinant baculovirus, the precultured Sf21 cells weretransfected with recombinant bacmid DNA using the Cellfection II
reagent (Invitrogen) according to the manufacturer's instructions.

2.9 | Immunocytochemistry

Lysis buffer (20 mM Tris-­HCl, 10% glycerol, 1% NP-­4 0, 1 mM EGTA,
1 mM DTT, 150 mM NaCl, pH8.0) with protease inhibitor cocktail

The cells were seeded in a glass-­bottom dish (Matsunami). On the(Sigma-­Aldrich) was added to the collected cells, sonicated, andfollowing day, the cells were washed with phosphate-­buffered saline

centrifuged. The supernatant was then rotated with Glutathione(PBS) and fixed with 10% formalin for 15 minutes at room tempera-

Sepharose 4 B (Sigma-­Aldrich) for 60 minutes at 4 °C. PreScission

ture (RT). After washing with PBS, the cells were subsequently incu-

Protease (GE Healthcare) was added to the mixture with PreScissionbated with Image-­iT FX Signal Enhancer (Thermo Fisher Scientific)

Cleavage buffer (50 mM Tris-­HCl, 150 mM NaCl, 1 mM DTT, pH7.5).

for 30 minutes at RT, washed with PBS, and incubated with thefirst antibody (G4B1, 50 μg/mL) for 1 hour at RT. After washing

2.6 | Establishment of hybridoma cell lines

with PBS, the cells were further incubated with the secondary antibody (alexa488-­conjugated anti-­mouse IgG, 1:200, Thermo Fisher
Scientific) and DAPI (1 μg/mL; Dojindo). The cells were observed

The establishment of hybridoma cell lines was performed as de-using confocal microscopy (Nikon A1; Nikon).

scribed previously.12 The culture supernatant of hybridoma cells
was screened by ELISA. To select the cancer-­specific antibody,
we performed the ELISA using hypopharyngeal cancer cell lines,
HPCM2. HPCM2-­hCD271 (human CD271-­overexpressing cell) and
HPCM2-­E V (empty vector) cells were seeded in plates and fixed in
4% paraformaldehyde. Fixed cells were incubated with supernatant,
HRP-­conjugated anti-­mouse IgG was applied, and the enzyme activity was detected using the substrate tetramethyl benzidine.
To determine the epitope, transiently transfected 293T cells(CD271 full length, Δ1, Δ1-­2, Δ1-­3, and Δ1-­4) were seeded inpolyethylenimine-­coated plates and incubated for 24 hours.
To determine the antibody concentration, plates were coatedwith anti-­mouse IgG2a IgG (ISO2-­1 Kit; Sigma-­Aldrich), washed, andincubated with the culture supernatant of hybridoma cells or anti-­
mouse IgG2a antibody as a standard (200 µg/mL, anti-­C A IX, H-­11,
Santa Cruz Biotechnology).
The isotype of the antibody in the hybridoma supernatant wasdetermined using mouse monoclonal antibody isotyping reagents(Sigma-­Aldrich) following the manufacturer's instructions.

F I G U R E 1 Binding affinity of the G4B1 antibody on SKOV3-­
CD271 cells. G4B1 and ME20.4 clones were used for ELISA assay.
Fluorescence data were collected using flow cytometry, and thedissociation constant (KD) values were calculated using GraphPad
Prism8

FUJII et al.

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FUJII et al.

F I G U R E 2 G4B1 antibody specifically detects overexpressing and endogenous CD271. A, Flow cytometry analysis of G4B1 antibody
using CD271-­overexpressing SP2/0 and SK-­OV-­3 cells. EV, empty vector. B, Flow cytometry analysis of G4B1 and ME20.4 antibodies in
the indicated cell lines. C, HPCM2 cells were knocked down by siRNA, and flow cytometry analysis was performed using G4B1 or ME20.4
antibody

2.10 | Immunohistochemistry (IHC)

Technology, 1:1000; G323A, polyclonal antibody, Promega, 1:8000). To
test nondenaturing CD271, cell lysates were boiled with sample buffer

An esophageal disease tissue array (ES2081) was purchased fromwithout 2-­mercaptoethanol (62.5 mM Tris-­HCl [pH 6.8], 2% SDS, 5%

Biomax. Barrett's esophagus and fetal esophagus (19-­21 weeks) 10%

sucrose) for 5 minutes. The samples were separated by SDS-­PAGE gel

formalin-­fixed paraffin-­embedded (FFPE) samples were obtained(Bio-­Rad) and transferred onto PVDF membranes (BioRad).

from Tohoku University Hospital. Hypopharyngeal and cervical disease FFPE samples were obtained from the Miyagi Cancer Center.
Immunostaining of human CD271 cells was performed as pre11

viously described.

2.12 | Statistical analysis

Anti-­CD271 antibody (C40-­1457) was pur-chased from BD Biosciences. First antibody was diluted: G4B1,

For the evaluation of IHC, statistical tests were performed using the

1:9000; C40-­1457, 1:2500. Anti-­SOX2 antibody was purchasedchi-­square test with R software (version 4.1.0), and statistical signifi-

from Millipore (6F1.2, 1:400). Cell blocks were prepared as follows:cance was set at p < 0.05.

a piece of paraffin was placed at the bottom of a 1.5-­mL tube, towhich cells were added. The tube was centrifuged, the supernatant
was removed, and agar was added to the tube. The cells were fixedwith 10% formaldehyde solution for 24 hours at RT. Next, the cell-­3|

R E S U LT S

containing blocks were processed in paraffin blocks.

3.1 | Production and characterization of novel anti-­
glycosylated CD271 monoclonal antibody clone G4B1

2.11 | Western blotting

To develop a novel anti-­CD271 mAb, we immunized mice with purified CD271ECD protein and prepared their splenic hybridoma

Western blotting was performed as previously described.11 The anti-

cell culture. The culture supernatants were screened by ELISA for

bodies used were as follows: anti-­CD271 antibody (D4B3, Cell Signalingbinding to hCD271-­overexpressed HPCM2 cells. After limiting theF I G U R E 3 Immunocytochemistry using
G4B1. A, Immunocytochemistry of empty
vector-­and human CD271-­overexpressing
SP2/0 cells using G4B1. Green, G4B1;
blue, 4’,6-­diamidino-­2-­phenylindole.
Bar: 10 μm. B, Immunohistochemistry offormalin-­fixed paraffin-­embedded SK-­
OV-­3 cells expressing CD271 using G4B1.
Bar: 100 μm

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FUJII et al.

F I G U R E 4 G4B1 recognizes conformational CRD4. A, A schema for the CD271 deletion mutant. B, Determination of the CD271 domain
recognized by G4B1. The indicated human CD271 deletion mutants were transfected into 293T cells, and the affinity between the deletionmutants and the supernatant of G4B1 hybridoma cells was analyzed using ELISA. Values were normalized to the absorbance of hCD271 WT-­
expressing 293T cells. The boxes are shaded to indicate the values from minimum (white) to maximum (dark gray). C, Western blot analysis of
SK-­OV-­3 control and CD271-­overexpressing cells. EV, empty vector. 2ME, 2-­mercaptoethanol in sample buffer. D, Western blot analysis of
HPCM2 cells. WT, wild type. 2-­ME, 2-­mercaptoethanol in sample buffer [Correction added on 15 June 2022, after first online publication: infigure 4C, upper panel, the image was inverted vertically and molecular marker was corrected]

dilution of the hybridomas, clone G4B1 (IgG2a) was established. The

CD271-­k nockdown cells, which is similar to the affinity of the

dissociation curve of G4B1 was analyzed, and we found that G4B1

ME20.4 antibody (Figure 2C). Next, we tested whether the G4B1

possessed higher affinity than clone ME20.4 (commercially availa-

antibody could be used for immunofluorescent staining. The

ble; Figure S1). We analyzed the kinetics of G4B1 in SK-­OV3-­CD271

G4B1 antibody recognized overexpressed CD271 in 293T cells

cells using flow cytometry. The dissociation constant (KD) of G4B1

(Figure 3A). We also found that the G4B1 antibody recognized

was estimated to be 2.322 nM (Figure 1).

FFPE SK-­OV-­3 cells overexpressing CD271 (Figure 3B), indicating

Next, we examined the affinity of G4B1 for human CD271

that G4B1 antibody was useful for IHC in FFPE samples.

using cell lines by flow cytometry. When we used humanCD271-­overexpressing SP2/0 and SK-­OV-­3 cells, G4B1 clearlybound SP2/0-­C D271 and SK-­OV3-­C D271 cells (Figure 2A).

3.2 | Determination of the epitope

We checked the affinity of G4B1 for endogenous CD271-­
expressing human cancer cell lines. We found that the G4B1

To determine the epitope for the G4B1 antibody, deletion mutants ofand ME20.4 antibody bound endogenous CD271 (Figure 2B).

CD271 were constructed (Figure 4A) and the mutants were transfected

To confirm the specificity of the G4B1 antibody against CD271,

into 293T cells. We checked the affinity and found that the G4B1 an-

we examined its affinity to CD271-­k nockdown cells. The affin-

tibody did not react with the deletion of CRD1 to the CRD4 mutant

ity of G4B1 to CD271-­k nockdown cells was clearly reduced in(Δ1-­4; Figure 4B), suggesting that G4B1 binds to the CRD4 domain.

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FUJII et al.

F I G U R E 5 A, Western blot analysis
of Chinese hamster ovary (CHO) and its
derivative cell lines. G323A antibody
was purchased from Promega. B,
Immunohistochemistry (IHC) of CHO and
its derivative cell lines. Cell blocks wereprepared using indicated cell lines, and
IHC was performed using G4B1 or BD
antibodies (C40-­1457). Bar: 100 μm

Next, we checked whether G4B1 could be used for Western
blotting. We found that G4B1 antibody bound protein samples

3.4 | G4B1 binds fetal esophageal epithelium andBarrett's esophagus

without 2-­ME treatment, but not 2-­ME–­treated protein samples in
CD271-­overexpressing SK-­OV-­3 cells and CD271 endogenously ex-

We previously reported that CD271 played a pivotal role in cancer

pressing HPCM2 cells (Figure 4C and D), suggesting that G4B1 anti-

stem cells. Therefore, in this study, we hypothesized that the dif-body could bind only to the conformational CD271 protein.

ferent glycosylation patterns between CD271 in normal epithelium

We generated this antibody using glycosylated CD271 antigen.

and cancer could depend on the stemness of the cells examined. In

Thus, we tested whether the G4B1 antibody is specific for certain

order to further test this interesting hypothesis, we immunolocal-

glycosylation types. We established human CD271-­expressing

ized the fetal esophageal epithelium. The C40-­1457 antibody could

CHO cells that lack a specific glycosylation transferase (Lec1 lacks

not detect any fetal esophageal cells, but the G4B1 antibody clearly

GlcNAc glycosyl transferase, resulting in N-­linked carbohydratebound to these fetal esophageal epithelial cells (Figure 6B). These

deficiency; Lec2 lacks the CMP-­sialic acid transporter, resulting

cells were also immunohistochemically positive for SOX2, confirm-in sialic acid deficiency; and Lec8 lacks the UDP-­galactose trans-

ing that the fetal esophagus possesses stemlike characteristics.

porter, resulting in O-­glycan deficiency). In Western blotting, G4B1

We then tested the immunolocalization of CD271 in the esopha-

antibody detected CD271 overexpression in wild-­t ype CHO andgeal adenocarcinoma arising in Barrett's esophagitis. We previously

Lec1 cells, but not in Lec2 and Lec8 cells (Figure 5A). We confirmed

reported that CD271 was expressed specifically in SCC but not inthis specificity using IHC. CD271 protein was clearly observed inadenocarcinoma.1 In this study, G4B1 antibody yielded immuno-­

CHO and Lec1 cells, but not in Lec2 and Lec8 cells (Figure 5B), in-positivity in carcinoma cells of Barrett's adenocarcinoma. However,dicating that G4B1 can only detect CD271 with sialic acid and O-­

the C40-­1457 antibody marginally yielded positivity in adenocarci-

glycan modifications.

noma (Figure 6C). G4B1 also yielded positivity in Barrett's epithelial
cells undergoing intestinal metaplasia (Figure 6D). Both adenocar-

3.3 | Immunohistochemistry ofesophageal diseases
We then studied the specificity of the G4B1 antibody using histopathological specimens. In normal esophageal tissue, basal cells werecinoma and metaplasia were immunohistochemically positive for
SOX2, suggesting that these cells also possess a stemlike capacity.

3.5 | Immunolocalization in hypopharyngeal andcervical cancers

immunohistochemically positive for the C40-­1457 antibody, while
no positive cells were detected using the G4B1 antibody (Figure 6A).

We further examined the specificity of the G4B1 antibody in hy-

There was no immunoreactivity in the intraepithelial esophageal ne-popharyngeal and cervical cancers. In hypopharyngeal cancer

oplasms and CIS with G4B1 antibody, but positivity was detected(Figure 7A), C40-­1457 antibody yielded positivity in the basal cells

when using C40-­1457. In SCC, both G4B1 and C40-­1457 antibod-

of normal and dysplastic lesion and cancerous lesion. However, theies could detect positive carcinoma cells. We studied 26 cases of

G4B1 antibody yielded no positivity in normal epithelium, dysplasia,

normal esophagus, and no immunoreactivity was detected in normal

and CIS, while it yielded marked positivity in carcinoma cells, whichepithelium using G4B1 antibody, while 20 cases were positive using

is consistent with the results of IHC in esophageal mucosa in our

the C40-­1457 antibody (Table 1).

present study.

FUJII et al.

| 2885F I G U R E 6 A, Immunohistochemistry of normal esophagus and esophageal cancer. Immunostaining was performed with anti-­CD271(G4B1 and C40-­1457). SCC, squamous cell carcinoma. Bar, 100 μm. B-­D, Immunohistochemistry of fetal esophagus (B), Barrett's esophagus(C), and adenocarcinoma (D). Immunostaining was performed with anti-­CD271 (G4B1 and C40-­1457) and anti-­SOX2. Bar: 100 μm

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FUJII et al.

TA B L E 1 Number of positive cases using each anti-­CD271
antibody in immunohistochemistry

The G4B1 antibody recognized overexpressing CD271 protein in

G4B1

C40-­1457

P value

not human cancer. Previous studies demonstrated that an antibody

Normal (n = 26)020<0.001

IEN (n = 18)015<0.001

Tis (n = 16)

2

12

0.001

pressing CD271 in CHO cells could be glycosylated similar to the

Advanced SCC (n = 54)

1680.0699cancer cells.

Abbreviations: IEN, intraepithelial neoplasia; SCC, squamous cell
carcinoma.

CHO and 293T (human embryonic kidney) cells, whose origins areagainst cancer-­specific glycosylated podoplanin recognized overexpressing human podoplanin in CHO cells.7,15 Therefore, overex-

In this study, we found that the G4B1 antibody could bind to
Barrett's epithelium and adenocarcinoma. We previously reported
that CD271 was not expressed in adenocarcinomas. A previous report demonstrated that the expression of SOX2 and OCT3, both ofIn the cervical normal epithelium (Figure 7B), positive cells werewhich are essential for maintaining pluripotency, was elevated in

detected in the suprabasal area, but not in basal cells, using G4B1

Barrett's esophagus and esophageal adenocarcinoma as compared

antibody. Using C40-­1457, positive cells were detected in the basal

with normal epithelium.16 In a mouse model, Lgr5+ gastric stem cells

layer. In dysplasia or CIS, the staining area increased from the su-

were reported to be present in Barrett's esophagus developed byprabasal to the middle area in stratified squamous epithelium andbile acid exposure.17 These data all suggested that Barrett's esopha-

approximately all regions in CIS. In SCC, cancerous regions were

gus and adenocarcinoma possessed stemlike characteristics. We also

positive in using both G4B1 and C40-­1457 antibodies, which is also

demonstrated that the G4B1 antibody recognized the fetal esopha-

compatible with esophageal specimens.

gus. These data indicated that the G4B1 antibody could recognize
esophageal epithelial cells harboring stemlike features by IHC.

4|DISCUSSION

In cervical cancer, G4B1 antibody did not react with the normal
basal layer, but the staining was positive in the suprabasal layer ofdysplasia/CIS, which is different from esophageal and hypopharyn-

We demonstrated that the recognition site of CD271 by the G4B1

geal cancer. The staining intensity in the suprabasal layer graduallyantibody is the CRD4 domain. We also revealed that the G4B1 an-increased from normal to CIS, suggesting that the G4B1 antibody

tibody was specific to the conformational epitope of CRD4 and gly-

could recognize not only cancer cells but also early atypical cells.

cosylated CD271. The G4B1 antibody recognizes cancerous regions,

Further studies are required to elucidate the role of aberrant glyco-

but not in the normal epithelium, both of which express the CD271

sylated CD271 in premalignant cervical lesions.

protein. These data suggested that specific glycosylation, including

In this study, we demonstrated that the G4B1 antibody recog-

CD271, was specifically increased through esophageal oncogenesis.

nized esophageal carcinoma cells but not non-­neoplastic epithelial

F I G U R E 7 Immunohistochemistry ofhypopharyngeal (A) and cervical cancer(B). Immunostaining was performed with
anti-­CD271 (G4B1 and C40-­1457). CIS,
carcinoma in situ; SCC, squamous cell
carcinoma. Bar: 100 μm

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FUJII et al.

cells. This antibody can be useful as a prominent marker that clearly
distinguishes between normal and malignant epithelium by IHC.
Future analysis of aberrant glycosylated CD271 will shed light on
the mechanisms of oncogenesis in the esophagus.
AC K N OW L E D G M E N T S
This research was supported in part by JSPS KAKENHI under
grant numbers JP 20K09701, 20H03756, 19K08430, 17K11166,
21H02396; Takeda Medical Foundation, Kobayashi Foundation
for Cancer Research; and Japan Agency for Medical Research andDevelopment (AMED) under grant numbers JP21am0401013 andJP21am0101078. We thank Ms. Shima-­Nakamura for her excellent
technical assistance.
D I S C LO S U R E
The authors have no conflict of interest to disclose.
ORCID
https://orcid.org/0000-0003-1324-6387

Kennichi Satoh

Ryoko Saito-­Koyama
Hironobu Sasano

https://orcid.org/0000-0003-3627-1788
https://orcid.org/0000-0002-6600-8641

Yukinari Kato

https://orcid.org/0000-0001-5385-8201

Keiichi Tamai

https://orcid.org/0000-0003-0813-5885

REFERENCES
1. Mochizuki M, Nakamura M, Sibuya R, et al. CD271 is a negative
prognostic factor and essential for cell proliferation in lung squamous cell carcinoma. Lab Invest. 2019;99(9):1349-­1362.
2. Imai T, Tamai K, Oizumi S, et al. CD271 defines a stem cell-­like population in hypopharyngeal cancer. PLoS One. 2013;8(4):e62002.
3. Shimizu M, Nagata K, Yamaguchi H, Kita H. Squamous intraepithelial neoplasia of the esophagus: past, present, and future. J
Gastroenterol. 2009;44(2):103-­112.
4. Taylor PR, Abnet CC, Dawsey SM. Squamous dysplasia–­the precursor lesion for esophageal squamous cell carcinoma. Cancer
Epidemiol Biomarkers Prev. 2013;22(4):540-­552.
5. Stowell SR, Ju T, Cummings RD. Protein glycosylation in cancer.
Annu Rev Pathol. 2015;10:473-­510.
6. Pinho SS, Reis CA. Glycosylation in cancer: mechanisms and clinicalimplications. Nat Rev Cancer. 2015;15(9):540-­555.
7. Kato Y, Kaneko MK. A cancer-­specific monoclonal antibody recognizes the aberrantly glycosylated podoplanin. Sci Rep. 2014;4:5924.

8. Tian Y, Denda-­Nagai K, Kamata-­Sakurai M, et al. Mucin 21
in esophageal squamous epithelia and carcinomas: analysis
with glycoform-­specific monoclonal antibodies. Glycobiology.
2012;22(9):1218-­1226.
9. Johnson D, Lanahan A, Buck CR, et al. Expression and structure of
the human NGF receptor. Cell. 1986;47(4):545-­554.
10. Yeaman C, Le Gall AH, Baldwin AN, Monlauzeur L, Le Bivic A,
Rodriguez-­Boulan E. The O-­glycosylated stalk domain is required
for apical sorting of neurotrophin receptors in polarized MDCK
cells. J Cell Biol. 1997;139(4):929-­940.
11. Mochizuki M, Tamai K, Imai T, et al. CD271 regulates the proliferation and motility of hypopharyngeal cancer cells. Sci Rep.
2016;6:30707.
12. Morita S, Mochizuki M, Wada K, et al. Humanized anti-­CD271
monoclonal antibody exerts an anti-­tumor effect by depleting cancer stem cells. Cancer Lett. 2019;461:144-­152.
13. Morita S, Mochizuki M, Shibuya-­Takahashi R, et al. Establishmentof a monoclonal antibody that recognizes cysteine-­rich domain
1 of human CD271. Monoclon Antib Immunodiagn Immunother.
2020;39(1):6-­11.
14. Hammill D. CytoExploreR: Interactive Analysis of Cytometry Data
[Internet]. 2020. Available from: https://github.com/Dillo​nHamm​
ill/CytoE​xploreR
15. Kaneko MK, Nakamura T, Kunita A, et al. ChLpMab-­23: Cancer-­
specific human-­mouse chimeric anti-­podoplanin antibody exhibits
antitumor activity via antibody-­dependent cellular cytotoxicity.
Monoclon Antib Immunodiagn Immunother. 2017;36(3):104-­112.
16. Wang X, Yang S, Zhao X, et al. OCT3 and SOX2 promote the transformation of Barrett’s esophagus to adenocarcinoma by regulating
the formation of tumor stem cells. Oncol Rep. 2014;31(4):1745-­1753.
17. Quante M, Bhagat G, Abrams JA, et al. Bile acid and inflammation
activate gastric cardia stem cells in a mouse model of Barrett-­like
metaplasia. Cancer Cell. 2012;21(1):36-­51.

S U P P O R T I N G I N FO R M AT I O N
Additional supporting information may be found in the online
version of the article at the publisher’s website.

How to cite this article: Fujii K, Morita S, Mochizuki M, et al.
Establishment of a monoclonal antibody against glycosylated
CD271 specific for cancer cells in immunohistochemistry.
Cancer Sci. 2022;113:2878–­2887. doi:10.1111/cas.15340