Oral Oncology 45 (2009) 633–639

Contents lists available at ScienceDirect

Oral Oncology
journal homepage: www.elsevier.com/locate/oraloncology

Expansion and characterization of cancer stem-like cells in squamous cell

carcinoma of the head and neck
Atsushi Okamoto a, Kazuaki Chikamatsu a,*, Koichi Sakakura b, Kyosuke Hatsushika a, Goro Takahashi a,
Keisuke Masuyama a
a
Department of Otolaryngology-Head and Neck Surgery, University of Yamanashi, Faculty of Medicine, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan
b
University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA 15213, USA

a r t i c l e i n f o s u m m a r y

Article history: Evidence has accumulated indicating that only a minority of cancer cells with stem cell properties, cancer
Received 31 July 2008 stem cells (CSCs), are responsible for maintenance and growth of the tumor. CD44 is currently used to
Received in revised form 3 October 2008 identify CSCs as one of the cell surface markers for solid tumors. Here we report the identification, expan-
Accepted 3 October 2008
sion, and characterization of CD44+ cancer stem-like cells from a permanent squamous cell carcinoma of
Available online 21 November 2008
the head and neck (SCCHN) cell line. Under serum-free medium culture conditions, a small population
(less than 3%) of CD44+ cells in a permanent cancer cell line was dramatically increased up to around
Keywords:
40%. The CD44+ cell population also showed higher expression of CD133 and ABCG2 as compared with
Cancer stem cells
CD44
the CD44 cell population. Moreover, CD44+ cells possess not only a marked capacity for forming tumor
Squamous cell carcinoma of the head and spheres, proliferation, migration, and invasion in vitro, but also resistance to chemotherapeutic agents.
neck Four genes related to chemoresistance, ABCB1, ABCG2, CYP2C8, and TERT, were up-regulated in a
Chemoresistance CD44+ cell population. Our findings indicate that a subpopulation of CSCs is maintained in the SCCHN cell
line, and the presence of such CSCs has an important clinical implication for head and neck cancer treat-
ment. Further characterization of CSCs may provide new insights for novel therapeutic targets and prog-
nostic markers.
Ó 2008 Elsevier Ltd. All rights reserved.

Introduction in a hierarchy together with a spectrum of cells at different stages

Evidence has accumulated indicating that only a minority of
cancer cells with stem cell properties, cancer stem cells (CSCs),
are responsible for maintenance and growth of the tumor.1–3 Re-
cent advances in stem cell biology enable the identification of CSCs
in solid tumors as well as putative stem cells in normal organs.4–7
CD44 is currently used to identify CSCs as one of the cell surface
markers for solid tumors.4–6 With respect to squamous cell carci-
noma of the head and neck (SCCHN), Prince et al. demonstrated
that a small population of CD44+ cancer cells obtained from fresh
tumor tissues, but not CD44 cancer cells, gave rise to new tumors
in immunodeficient mice.8 Interestingly, CSCs have been also iden-
tified in cultured SCCHN cell lines. Pries et al. demonstrated that
permanent SCCHN cell lines constitutively expressed CD44.9 Alter-
natively, Zhou et al. have shown that CD133+ cells in a laryngeal
carcinoma cell line possess the capacity for self-renewal, extensive
proliferation, and multilineal differentiation potency in vitro.10
These findings suggest that a subpopulation of CSCs can persist
even with long-term culture in vitro; however, CSCs are not only
a small or rare subpopulation in tumors, but are also maintained
* Corresponding author. Tel.: +81 55 273 6769; fax: +81 55 273 9670.
E-mail address: chikamatsu@yamanashi.ac.jp (K. Chikamatsu).
of differentiation; therefore, it is too difficult to maintain an en-
riched status of CSCs in long-term culture. Recent reports have
demonstrated that CSCs from epithelial organs can be expanded
as sphere-like cellular aggregates in serum-free medium (SFM)
containing epidermal growth factor (EGF) and basic fibroblast
growth factor (bFGF).11–13 In the current study, we first sought to
detect CD44+ cells in the established SCCHN cell line, the Gun-1
cell line and, as expected, CD44+ cells represented a minority of
the tumor cell population. Surprisingly, CD44+ cells were able to
be propagated in vitro under SFM containing EGF and bFGF culture
conditions. We then focused on whether such expanded CD44+
cells have stem cell properties by comparing with CD44 cells.
Materials and methods
Cell line and culture conditions
A SCCHN cell line, Gun-1, was established from a squamous cell
carcinoma of the hypopharynx.14 Gun-1 was cultured in either
RPMI-1640 (Sigma-Aldrich, St. Louis, MO) supplemented with
10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM L-gluta-
mine, 100 units/mL penicillin, and 100 lg/mL streptomycin (all re-
agents were from Invitrogen, Grand Island, NY), or Serum Free

1368-8375/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.oraloncology.2008.10.003
634 A. Okamoto et al. / Oral Oncology 45 (2009) 633–639
Expansion Medium (StemCell Technologies, Inc., Vancouver, Can-
ada) supplemented with EGF (Calbiochem, Darmstadt, Germany)
and bFGF (Calbiochem) (20 ng/ml each). For tumor sphere culture,
cells were seeded at a density of 1  103 cells/ml in uncoated plas-
tic dishes and cultured for 7 days.
Flow cytometry
The trypsinized cells were resuspended, incubated with mono-
clonal antibodies for 30 min at 4 °C, washed twice with phosphate-
buffered saline (PBS) containing 0.1% FBS and 0.1% NaN3, and fixed
with 1% paraformaldehyde in PBS. The antibodies used were anti-
CD44 fluorescein isothiocyanate (FITC), -phycoerythrin (PE), or -
allophycocyanin (APC), anti-CD24-PE, anti-HLA class I-FITC, anti-
CD133-PE, anti-ABCG2 (all purchased from BD Pharmingen).
FITC-conjugated goat anti-mouse monoclonal antibody was used
as a secondary antibody in some experiments. Respective immuno-
globulin G (IgG) isotype-matched controls (BD Pharmingen) were
used as negative controls.
Immunocytochemistry
Dishes containing tumor spheres were washed with PBS (Invit-
rogen), and incubated with antibodies against CD44-FITC (BD
Pharmingen, San Diego, CA), CD133-PE (Miltenyi Biotec, Gladbach,
Germany), and ABCG2 (BD Pharmingen) for 30 min. FITC-conju-
gated goat anti-mouse monoclonal antibody was used as the sec-
ondary antibody for ABCG2 staining. After two additional washes,
fluorescence microscopy was performed.
Magnetic cell sorting
The cells were incubated with anti-CD44-biotin (BD Pharmin-
gen) for 5 min at 4 °C. After washing once, 20 ll of anti-biotin
microbeads (Miltenyi Biotec)/1  107 cells was added for
15 min at 4 °C. Subsequently, cells were washed once, resus-
pended, and applied into MACS separation columns (Miltenyi
Biotec). Positive (CD44+) and negative (CD44 ) fractions were
resuspended in SFM with EGF and bFGF for further experiments,
respectively.
Cell proliferation assays
Cell proliferation was determined using the CellTiter 96 Aque-
ous One Solution Cell Proliferation Assay (Promega). Cells were
plated at a density of 500 cells/well in 96 flat-bottomed plates,
and cell proliferation assays were performed on days 1, 3, 5, and
7 using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS. Twenty
microliters of MTS solution was added to each well and the plate
was incubated for 2 h at 37 °C, and viable cells were quantified
by measuring absorbance at 490 nm.
Migration and Invasion assays
The sorted cells were plated at a density of 2.5  104 cells/well
onto 8-lm Transwell filters in a 24-well plate. Medium containing
10% FBS was added to the bottom wells as a chemoattractant.
Twenty-four hours later, the filters were removed and, then
stained with a Diff-Quik kit (Sysmex Corp., Kobe, Japan) according
to the manufacturer’s instructions. The migratory cells were
counted in four random fields per insert under a microscope at
20 magnification. The invasion assay was performed in a similar
fashion using BD BioCaot Matrigel Invasion Chamber (BD Biosci-
ence), and the results are expressed as the total number of cells
that had invaded each filter.
Drug sensitivity assay
The number of viable cells following drug treatment was as-
sessed using a MTS proliferation assay as described above. Cells
were plated at a density of 5  103 cells/well in 96 flat-bottomed
plates, allowed to attach overnight, and finally chemotherapeutic
agents at various concentrations were added. The cultures were
incubated at 37 °C for 48 h, 20 ll of MTS solution was added for
the last 2 h, and absorbance at 490 nm was measured. The chemo-
therapeutic agents tested were carboplatin (kindly supplied by
Bristol-Myers Squibb), paclitaxel, docetaxel (both kindly supplied
by Sanofi-Aventis), 5-fluorouracil (kindly supplied by Kyowa Hak-
ko), and cisplatin (kindly supplied by NIPPON KAYAKU).
Real-time quantitative RT-PCR
To assess the expression of genes related to stem cell and cancer
drug resistance in CD44+ cells, real-time quantitative RT-PCR was
performed. We used the commercially available RT2 Profiler PCR TM
Array (Super Array Bioscience Corp). A list of genes on this PCR
array is available from http://www.superarray.com/rt_pcr_prod-
uct/HTML/PAHS-405A.html and http://www.superarray.com/
rt_pcr_product/HTML/PAHS-004A.html. The relative expression le-
vel of the target gene in CD44+ cells to that in CD44-cells was
determined by 2 deltadelta Ct method.
Statistical analysis
Two-tailed Student’s t test was used for statistical analysis of
data. p-values <0.05 were considered significant.
Results
Identification and expansion of CD44+ cell in Gun-1 cell line
We first examined CD44 expression in a SCCHN cell line, Gun-1,
using flow cytometry, and it was detected in 2.1%, as shown in
Figure 1A. Gun-1 was then cultured in SFM containing EGF and
bFGF. After 5 weeks of culture, the CD44+ population was in-
creased up to around 40%, as shown in Figure 1B.
Expression of CSC-related markers on CD44+ and CD44 cells
To examine the difference between CD44+ and CD44 subpopula-
tions, the expression of CSC-related markers was analyzed by flow
cytometry (Fig. 2). The expression of CD133 and ABCG2 on CD44+
cells was higher than on CD44 cells. In contrast, the expression of
CD24 was lower in CD44+ cells. There was no difference in the expres-
sion of HLA class I molecules between these two subpopulations.
Tumor sphere and expression of CSC-related markers
After plating at low densities in uncoated dishes, tumor cells
readily proliferated and formed spheres in within 5 days (Fig. 3A
and B), whereas the original parental cells cultured with RPMI-
1640 containing 10% FBS neither proliferated nor formed spheres
(data not shown). Moreover, as expected, tumor spheres showed
immunoreactivity for CD44, CD133, and ABCG-2 (Fig. 3C–E).
CD44+ cell population exhibits higher potential for proliferation,
migration, and invasion
To investigate the biological significance of the CD44+ popula-
tion, we sorted CD44+ and CD44 cell populations using magnetic
bead cell sorting. After sorting, CD44+ cells were only 0.8% in
CD44 populations (data not shown). CD44+ and CD44 popula-
 A. Okamoto et al. / Oral Oncology 45 (2009) 633–639 635

Figure 1 Detection of CD44+ cells in a permanent SCCHN cell line, Gun-1. The Gun-1 cell line was cultured in RPMI-1640 supplemented with 10% FBS (A) or SFM
supplemented with EGF and bFGF (B).

Figure 2 Flow cytometry analysis of CD44+ and CD44 populations in the Gun-1 cell line. On the histograms, the thin line represents expression on the CD44 population,
while the bold line represents expression on the CD44+ population.

tions were then collected and cultured separately under the same
conditions, as described above. As shown in Figure 4, CD44+ pop-
ulations demonstrated increased proliferative, migratory, and
invasive capacity as compared with CD44 populations.
CD44+ cell population exhibits resistance to chemotherapeutic
agents
Both CD44+ and CD44 cells were treated with chemothera-
strated more drug resistance to all five chemotherapeutic agents
tested, as compared with CD44 cells.
Expression of genes related to stem cell and cancer drug resistance in
CD44+ cells
To examine the difference in the expression of genes related to
stem cell and cancer drug resistance between CD44+ and CD44-
cells, we used a PCR array, the RT2 Profiler PCR Array. The up-reg-
TM

peutic agents for 48 h, and then viable cells were assessed using ulated (rate >2.0) genes in CD44+ cells compared with CD44 cells
MTS proliferation assay. As shown in Figure 5, CD44+ cells demon- are listed in Table 1.
636 A. Okamoto et al. / Oral Oncology 45 (2009) 633–639

Figure 3 Representative images of tumor spheres from the Gun-1 cell line cultured in SFM supplemented with EGF and bFGF. (A, original magnification, 20; B, original
magnification, 100). Tumor spheres expressed cancer stem cell markers, CD44 (C), CD133 (D), and ABCG2 (E), as assessed by immunofluorescence. (Original magnification,
40).

Discussion Ricci-Vitiani et al. succeeded in expanding CD133+ colon-cancer-

The objective of this study was the characterization of CSCs in
the SCCHN cell line. We succeeded in achieving this objective,
but three points are of particular importance: (1) The serum-free
culture condition was capable of expanding CD44+ cells from the
permanent SCCHN cell line; (2) These CD44+ cells also expressed
both CD133 and ABCG2, and possessed a marked capacity for form-
ing tumor spheres, proliferation, migration, and invasion in vitro;
(3) CD44+ cells were more resistant to chemotherapeutic agents
than CD44 cells.
The first point reveals that SFM selection may be useful for CSC
expansion. Since serum causes irreversible differentiation of stem
cells, SFM allows for the maintenance of an undifferentiated stem
cell status. Moreover, the addition of EGF and bFGF has been re-
ported to induce the proliferation of multipotent and self-renewing
stem cells.15,16 Using dissociated tumor cells from cancer tissue,
initiating cells and maintaining the ability to engraft and reproduce
the same morphological and antigenic patterns of the original
tumor by culturing in SFM.11 On the other hand, Lee et al. have
reported that glioblastomas cultured in SFM contain many of the
self-renewal and differentiation characteristics of neural stem cells,
whereas serum-cultured cells do not.12 Similar findings have been
also demonstrated in studies using permanent cancer cell lines.
Kondo et al. have reported that the side population (SP), which
has the ability to exclude the DNA binding dye, Hoechst33342,
was maintained, and that their proportion increased by culturing
in SFM with growth factors in the C6 glioma cell line.13 We were
able to detect a small population of CD44+ cells in a permanent can-
cer cell line. Moreover, the proportion of CD44+ cells dramatically
increased by culturing in SFM with EGF and bFGF. These findings
suggest that even long-term cultured cancer cells may retain CSCs
and the CSC population may reemerge through SFM selection.
 A. Okamoto et al. / Oral Oncology 45 (2009) 633–639 637

Figure 4 Proliferative (A), migratory (B), and invasive (C) capacity of CD44+ and CD44 cells. Cell proliferation assays were performed on days 1, 3, 5, and 7 using MTS. For
migration and invasion assays, 2.5  104 cells were plated onto 8-lm Transwell filters in a 24-well plate without (for migration assay) or with (for invasion assay) Matrigel.
Medium containing 10% FBS was added to the bottom wells as a chemoattractant. p-values were less than 0.01 and 0.05 for migration and invasion assays, respectively.

The second point suggests that expanded CD44+ cells isolated
from the permanent cell line possess intrinsic stem cell proper-
ties. Flow cytometry analysis demonstrated that the CD44+ cell
population showed a higher expression of both CD133 and
ABCG2 than the CD44 cell population. Alternatively, a Gun-1
cell line cultured in SFM had sphere-forming ability, and the de-
rived tumor spheres also expressed CD44, CD133, and ABCG2,
suggesting that tumor spheres might be enriched in CSCs. In-
deed, Chiou et al. have enriched a subpopulation of cancer
stem-like cells from oral squamous cell carcinoma by sphere for-
mation.17 Currently, various markers and/or methodologies are
used to identify and enrich CSCs; however, no single surface
marker or method can provide unequivocal identification of CSCs
in SCCHN. Therefore, the combinations of these markers and/or
methods would allow the clear definition of CSCs. Furthermore,
we found that purified CD44+ cells were capable of extensive
proliferation, migration, and invasion. So far, CD44 expression
on cancer cells has been shown to regulate multiple aspects of
cancer cell phenotypes, modulating tumor proliferation, migra-
tion, invasion, and anginogenesis.18,19 Moreover, Patrawala
et al. revealed that CD44+ prostate cancer cells were more pro-
liferative, clonogenic, tumorigenic, and metastatic than the corre-
sponding CD44- prostate cells.20 Thus, our data and those of
others suggest that CD44+ cells may play pivotal roles in tumor
progression and metastasis. On the other hand, several genes re-
lated to stem cell-specific markers were up-regulated in the
CD44+ cell population. These results confirmed that CD44+ cells
are likely to share many of the properties of normal stem cells
that provide for a long lifespan.
The last point suggests that CD44+ cells should be considered as
targets in future therapies. As expected, CD44+ cells were more
resistant to chemotherapeutic agents than CD44 cells. Indeed,
the treatment of tumor cells with chemotherapeutic agents has
been shown to enrich the CSC population relative to untreated cul-
tures in several tumors.21–23 Moreover, our data indicated that
CD44+ cells expressed a higher level of ABCB1, ABCG2, CYP2C8,
and TERT than CD44 cells. In various malignancies, ABC trans-
porters have been shown to be up-regulated in cancer stem-like
cells,24–26 indicating that ABC transporters could represent impor-
tant markers defining CSCs. On the other hand, CYP2C8 catalyzes
6-hydroxylation on the taxane ring of paclitaxel, and enhancement
of CYP2C8 expression in paclitaxel-resistant cells has been
638 A. Okamoto et al. / Oral Oncology 45 (2009) 633–639

Figure 5 Drug sensitivity of CD44+ and CD44 cells derived from the Gun-1 cell line. After magnetic cell sorting, CD44+ and CD44 cells were plated at a density of
5  103 cells/well in 96-well plates and treated with various concentrations of the indicated chemotherapeutic agents for 48 h. Asterisk (*) indicates significant difference of
cell viability between CD44+ and CD44 cells.

shown.27 Another molecule up-regulated in our study, TERT, is the
catalytic subunit protein of telomerase. TERT expression has been
shown to be higher in the CSC population in melanoma26 and lung
cancer.28 There is a striking correlation between the presence of
TERT mRNA and telomerase activity,29,30 and previous reports sug-
gest that high telomerase activity maintains and/or increases che-
moresistance in tumor cells;31–33 therefore, a high expression of
TERT in the CD44+ cell population might also contribute to chemo-
resistance. Thus, various molecular mechanisms appear to be in-
volved in the chemoresistance of CD44+ cells, and such
 A. Okamoto et al. / Oral Oncology 45 (2009) 633–639 639

Table 1
Up-regulated genes in CD44+ cells

Gene symbol Gene description GenBank Fold change

ABCB1 ATP-binding cassette, sub-family B NM_ 000927 2.88
(MDR/TAP), member 1
ABCG2 ATP-binding cassette, sub-family G NM_004827 2.49
(WHITE), member 2
BMP2 Bone morphogenetic protein 2 NM_001200 2.05
COL2A1 Collagen, type II, alpha 1 NM_001844 2.51
CYP2C8 Cytochrome P450, family 2, subfamily C, NM_000770 2.13
polypeptide 8
FGF3 Fibroblast growth factor 3 NM_005247 2.25
FGF4 Fibroblast growth factor 4 NM_002007 8.62
GJA1 Gap junction protein, alpha 1, 43kDa NM_000165 2.67
NCAM1 Neural cell adhesion molecule 1 NM_ 000615 2.35
NEUROG2 Neurogenin 2 NM_ 024019 2.44
TERT Telomerase reverse transcriptase NM_198255 2.51

subpopulations with the potential to survive conventional chemo-
therapy should be targeted for effective treatment.
Taken together, the CD44+ cells have not only stem cell proper-
ties, but also the ability to resist chemotherapeutic agents. The
presence of such CSCs has important clinical implications for head
and neck cancer treatment. Further characterization of CSCs may
provide new insights for novel therapeutic targets and prognostic
markers.
Conflicts of interest statement
None declared.
Acknowledgments
This work was supported in part by grants-in-aid (19791201 to
AO, 20592013 to KC, 20592014 to KM) from the Ministry of Educa-
tion, Cultures, Sports, Science and Technology, Japan.
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