CD44 as a stem cell marker in head and neck squamous cell carcinoma

Abstract

In the recent past, evidence is increasing indicating the existence of a subpopulation of resistant tumor cells in head and neck squamous cell carcinoma (HNSCC) that cannot be eradicated by established antineoplastic treatments. These cancer stem cells (CSCs) have features of somatic stem cells such as selfrenewal, proliferation and differentiation. CD44+ cells in tumors of the head and neck are referred to as CSCs of HNSCC. Expression profiling of CD44 in 29 HNSCC tumors was performed by fluorescence microscopy. ELISA analysis was performed to detect concentration of soluble CD44 in the peripheral blood of 29 HNSCC patients and 11 healthy controls. Expression of CD44 was determined in all HNSCC tissue samples (n=29). In all samples a surface staining pattern was found. The concentration of CD44 in the peripheral blood of HNSCC patients was significantly higher compared to a healthy control group (mHNSCC =13.5 ± 0.5 ng/ ml; mCont = 9.3 ± 0.6 ng/ml; P=0.6 x 10(-12)). The role of CD44 as a marker for CSCs in HNSCC remains to be ascertained. Further experiments might reveal its role as a diagnostic and prognostic factor, and possibly as a therapeutic target.

Full text

ONCOLOGY REPORTS 26: 321-326, 2011
Abstract. In the recent past, evidence is increasing indicating
the existence of a subpopulation of resistant tumor cells
in head and neck squamous cell carcinoma (HNSCC) that
cannot be eradicated by established antineoplastic treatments.
These cancer stem cells (CSCs) have features of somatic stem
cells such as selfrenewal, proliferation and differentiation.
CD44+ cells in tumors of the head and neck are referred to
as CSCs of HNSCC. Expression profiling of CD44 in 29
HNSCC tumors was performed by uorescence microscopy.
ELISA analysis was performed to detect concentration of
soluble CD44 in the peripheral blood of 29 HNSCC patients
and 11 healthy controls. Expression of CD44 was determined
in all HNSCC tissue samples (n=29). In all samples a surface
staining pattern was found. The concentration of CD44 in the
peripheral blood of HNSCC patients was signicantly higher
compared to a healthy control group (mHNSCC=13.5±0.5 ng/ ml;
mCont=9.3±0.6 ng/ml; P= 0.6x10-12). The role of CD44 as
a marker for CSCs in HNSCC remains to be ascertained.
Further experiments might reveal its role as a diagnostic and
prognostic factor, and possibly as a therapeutic target.
Introduction
Head and neck squamous cell carcinoma (HNSCC) is an
aggressive and recurrent malignancy, largely because it is
usually diagnosed at a late stage. Tobacco and alcohol exposure
are the main risk factors and account for ~85% of HNSCC (1).
De spit e adva nces in surgical a nd non -su rgica l th erapy mor tality
from this disease remains high because of the development of
distant metastases and the emergence of therapy-resistant local
and regional recurrences. Antineoplastic treatments such as
chemotherapy or radiation can efciently eradicate a majority
of proliferating malignant cells within malignant tumors.
However, there is increasing evidence that there is a subpopu-
lation of resistant tumor cells that can not be reached by
these regimens. These cancer stem cells (CSCs) have distinct
features of somatic stem cells such as selfrenewal, extensive
proliferation and differentiation. Therefore, these cells are
required and responsible for initiation, but also maintenance
and recurrence of disease. In recent years, the CSC hypothesis
has been coined for HNSCC as well (2,3). Prince at al showed
that CD44+ cancer cells, which typically comprise <10% of
the cells in an HNSCC tumor, but not the CD44- cancer cells,
gave rise to new tumors in vivo (2). Since then, CD44+ cells
in tumors of the head and neck are referred to as CSCs of
HNSCC.
CD44 is an integral cell membrane glycoprotein and it
comprises different isoforms that arise from alternative splicing
of a region of variable exons. They differ in primary amino
acid sequence as well as amount of N- and O-glycosylation
(4), thereby its apparent molecular mass ranges from 85 to
250 kD (5). At least 20 variants of CD44 have been reported
due to the alternative splicing of 10 exons that encode the
membrane's proximal portion of the extracellular domain
(6-8). Originally, it was described as a receptor on circulating
lymphocytes involved in homing, cell adhesion and migration
(9,10).
In 1991 Günthert et al showed that the expression of CD44
gave metastatic potential to a non-metastatic line of cells in a
rat carcinoma model (11,12). Since then, several analysis have
indicated that there is a correlation between the expression
of CD44 variants and progression, metastasis and prognosis
of malignant disease. This has also been shown in different
types of epithelial carcinoma in addition to HNSCC such
as colorectal carcinoma (13,14), breast carcinoma (15) and
certain types of gastric carcinoma (5,16).
The in-depth analysis of expression markers such as CD44
in tissue samples of HNSCC patients may reveal their role
as potential prognostic biomarkers or therapeutic targets,
e.g. for antigen directed immunotherapy. The analysis of
soluble CD44 in peripheral blood of HNSCC patients may
reveal important ndings concerning diagnosis and possible
pathways of metastasis in head and neck cancer.
CD44 as a stem cell marker in head and
neck squamous cell carcinoma
ANNE FABER, CHRISTINE BARTH, KARL HÖRMANN, STEFAN KASSNER, JOHANNES DAVID SCHULTZ,
ULRICH SOMMER, JENS STERN-STRAETER, CARSTEN THORN and ULRICH REINHART GOESSLER
Department of Otorhinolaryngology Head and Neck Surgery, University Medical Centre
Mannheim, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany
Received February 14, 2011; Accepted March 30, 2011
DOI: 10.3892/or.2011.1322
Correspondence to: Dr Anne Faber, Department of Otorhino-
laryngology Head and Neck Surgery, University Medical Centre
Mannheim, Theodor-Kutzer-Ufer 1-3, D-68167 Mannheim, Germany
E-mail: faberanne@gmx.de, anne.faber@umm.de
Key words: CD44, cancer stem cells, head and neck squamous cell
carcinoma

FABER et al: CD44 AS A STEM CELL MARKER IN HEAD AND NECK SCC
322
Materials and methods
Tissue and peripheral blood sample collection. A total of 29
HNSCC tissue samples from tumor patients were selected
out of a tissue data base collected from 1997 to 2010 at the
Department of Otorhinolaryngology, Head and Neck Surgery at
the University of Mannheim. Samples were xated immediately
after excision by freezing in liquid nitrogen. All samples were
conrmed by pathology after H&E staining. In addition to the
tissue collection, peripheral blood samples were taken from
HNSCC patients and healthy donors. Peripheral blood was
collected before, during, but never after tumor surgery. A group
of 11 healty blood donors served as a control. Blood samples
were centrifuged at 2500 rpm for 10 min. Afterwards serum
samples were harvested and xated by freezing at -80˚C. The
histological and clinical characteristics of all tumor samples are
summarized in Table I.
Immunof luorescence labeling. To detect the expression
of CD44 in HNSCC tissue samples, tumors underwent
xation by freezing in liquid nitrogen as mentioned above.
Specimens were prepared in slices of 5-8 µm, air-dried
and xed in acetone for 10 min. Afterwards, slices were
treated with 4% paraformaldehyde (PFA) for 10 min at room
temperature. After 3 washing steps with PBS tumor samples
were treated with 1% ser um (goat) for another 10 min.
Then slices were incubated with CD44 antibody (mouse
monoclonal, 1:100, Abcam, Cambridge, UK) for 1 h at 37˚C
followed by incubation with a second biotinylated antibody
(anti-mouse, 1:100) for 30 min. After further washing steps
with PBS, slices were treated with Streptavidin-Cy3 (1:1000)
for 30 min at room temperature. Subsequently, slices were
stained with DAPI after washing with PBS. Finally, slices
were covered in f luorsave and dried to be evaluated by
uorescence microscopy.
Enzyme-linked immunosorbent assay. Serum levels of CD44
were measured with a human CD44 ELISA Kit (Abcam). A
monoclonal antibody against soluble CD44 was adsorbed to
microwells in 96-well microtiter plates. Samples, including
standards of known CD44 concentrations and samples were
pipetted into these wells. During the rst incubation, the CD44
antigen was added to wells. After washing, a biotinylated
monoclonal antibody specic for CD44 was incubated and the
enzyme (streptavidin-peroxidase) was added. After incubation
and washing to remove a ll unbou nd enzyme, a subst rate solution
was added, which catalyzed a reaction on the bound enzyme
and so induced a coloured reaction product. The intensity of
this product is directly proportional to the concentration of
CD44 present in the samples.
Statistical analysis. All results were plotted as the mean ±
standard deviation. To estimate the probability of differences,
we employed the Student's t-test. P<0.05 denoted statistical
signica nce.
Results
Tissue and peripheral blood sample collection. A total of
29 HNSCC tissue samples and the according blood serum
samples were selected out of a data base collected from 1997
to 2010 at the Department of Otorhinolaryngology Head
and Neck Surgery at the Faculty of Medicine Mannheim,
University of Heidelberg, Germany. Samples entering the
study were derived from 5 female and 24 male patients aged
48-76 (mean age: 60) years. Locations of the primary tumor
were differentiated as larynx, oropharynx, hypopharynx and
oral cavity (10 larynx, 5 oropharynx, 7 hypopharynx, 7 oral
cavity). Patient characteristics are summarized in Table I.
A group of 11 healthy blood donors served as a control for
experiments concerning CD44 concentration in peripheral
blood of HNSCC patients. All studies were approved by the
Ethics Committee of the Faculty of Medicine Mannheim,
University of Heidelberg.
Expression of CD44 in HNSCC tissue samples. Immuno-
uorescence labeling of 29 tissue samples was performed to
detect the expression of CD44 in HNSCC. In all samples,
Table I. Tumor tissue and blood sample collection.
Patient Gender Age Tumor TNM Smoking/
(yrs.) location ETOH
1 M 54 Larynx T4N2xMx +/+
2 F 55 Larynx T1N0 +/-
3 M 62 Larynx T4N0 +/-
4 M 70 Larynx T4N0 1
5 M 68 Larynx T3N2b 1
6 M 68 Larynx 1 1
7 M 55 Larynx T4N2b 1
8 M 74 Larynx T4N0 1
9 M 59 Larynx 1 1
10 M 57 Larynx T4N1 +/+
11 M 55 Oral Cavity T4N1 +/+
12 M 69 Oral Cavity T1N2b 1
13 F 54 Oral Cavity T1N2b 1
14 M 48 Oral Cavity 1 1
15 M 49 Oral Cavity T3N1 1
16 M 51 Oral Cavity 1 1
17 M 66 Oral Cavity T2N0 +/+
18 F 50 Oropharynx T3N2c 1
19 M 64 Oropharynx 1 1
20 M 60 Oropharynx T4N2c 1
21 M 64 Oropharynx T3N2 1
22 F 55 Oropharynx T4N3 1
23 M 61 Hypophyarnx T4N2b +/+
24 M 66 Hypopharynx T2N0 1
25 M 76 Hypopharynx 1 1
26 M 59 Hypopharynx T4N2b 1
27 F 62 Hypopharynx T2N2b 1
28 M 51 Hypopharynx 1 1
29 M 57 Hypopharynx T3N2c +/-
1, information not available.

ONCOLOGY REPORTS 26: 321-326, 2011 323
pathology of HNSCC was conrmed via H&E staining by
pathology during routine diagnostics (Fig. 1A). CD44 was
visualised in red color by immunouorescence labeling via
Cy3. In all 29 HNSCC tissue samples an intense uorescence
signal could be detected (Fig. 1B and C). In HNSCC tissues,
CD44 was mainly expressed on cell surface in all samples
stained (Fig. 1C). Stromal cells did not show any uorescence
by staining of CD44 (Fig. 1B).
Concent ra tio n of CD44 in periph eral bloo d of HNSCC
patients. ELISA experiments were performed to measure
the concentration of soluble CD44 in the peripheral blood
of H NSCC patients compared to healthy cont rols. The
expression of soluble CD44 in serum of HNSCC and healthy
controls is shown in Fig. 2. There was a signicantly higher
concentration of CD44 in peripheral blood of HNSCC
patients compared to the healthy control group (mHNSCC =
13.5±0.5 ng/ml, mCont=9.3±0.6 ng/ml, P=0.6x10-12). Primary
tumor location ( Fig. 3A), T-stadium (B) and N-st adium
(C) did not significa ntly inf luence the concentration of
CD44 in peripheral blood of HNSCC patients. The values
determined and showed in Fig. 3 are: mlarynx=13.9± 0.4 ng/
ml, moroph=13.6±0.5 ng/ml, mhy poph=13.1±0.3 ng/ml, mor alcav
=13.5±0.3 ng/ ml (A); mT1=13.8±0.7 ng/ml, mT2=13.0± 0.3 ng/
ml, mT3=13.5±0.5 ng/ ml, mT4=13. 8 ± 0.5 ng /ml (B);
mN0=13.8±0.5 ng/ml, mN1=13.6±0.3 ng/ml, mN2=13.5±0.6 ng/
ml, mN3=14.1 (C); mCont 9.3±0.6 ng/ml (A-C).
Discussion
Expression of CD44 in HNSCC tissue samples. In this
study, we showed that CD44, which is referred to as a cancer
stem cell (CSC) marker of the head and neck (2), can be
detected in HNSCC tissue samples by immunouorescence
labeling. Others have used flow cytometry analysis (2,17),
immunohistochemistry (18-20), or microarray technology
(18) to verify CD44+ cells in HNSCC tissue samples. Results
consistently indicate the presence of CD44 in HNSCC tumors
on both protein and gene level. In our experiments CD44 was
abundantly expressed in all HNSCC samples tested (n=29)
and therefore conform to data collected by Han et al (18),
who performed immunohistochemical analysis of 16 HNSCC
tumor samples.
Figure 1. Expression of CD44 in HNSCC samples. Pathology of HNSCC
was established for each sample by H&E staining during routine diagnostics
(A). CD44 was visualised in red color by immunouorescence labeling via
Cy3 (B and C). Cell nuclei were stained in blue by DAPI. High uorescence
int en sity for CD44 was found in each of 29 HNSCC samples stained
(B and C). Each sample showed a surface staining patter n of CD44 (C).
Stromal cells did not show any uorescence by staining of CD44 via Cy3 (B).
A
B
C
Figu re 2. CD44 in per ipheral blood of HNSCC patients. Concentration
of soluble CD4 4 in p eripheral blood of HNSCC patients was evaluated
by ELISA and compared to healthy controls. There was a sig nificantly
higher concentration of CD44 in peripheral blood of HNSCC patients in
comparison to healthy controls (✩P=0.6x10-12).

FABER et al: CD44 AS A STEM CELL MARKER IN HEAD AND NECK SCC
324
The staining pattern shown in immunouorescence (Fig. 1B
and C) and immunohistochemistry (18) shows a monotonic
staining of almost all cells except stromal and vascular cells.
This stands in contrast to previous work e.g. by Prince et al,
who first postulated CD44+ cells as CSCs of the head and
neck (2,21). Prince et al reported, that CD44+ cancer cells
typically comprise <10% of the cells in an HNSCC tumor (2).
It is obvious that the percentage of cells of the tumor samples
stained as CD44+ in our experiments is much higher than 10%.
A possible reason might be the different experimental methods
of CD44 detection, e.g. ow cytometry versus immunouores-
cence labeling. It is even more reasonable that the percentage
of <10% considered by Prince et al comes off because of the
exclusion of Lin- cells. Lin- was dened as negative for the
surface markers CD2, CD3, CD10, CD18, CD31, CD64 and
CD140b (2). The ow cytometric analysis done by Pries et al
showed, that CD44 expression in HNSCC tumors differs inter-
individually. In their study, the percentage of cells in HNSCC
tumors varied from ~4 up to over 90% (17). It is possible that
this is correct because Pries et al had a look at the whole of the
tumor and not just at a small specimen cut out of the bulk of
an HNSCC. It has been shown in previous work, that CD44 is
a ubiquitary marker e.g. in the hematopoietic or mesenchymal
system (22).
Consistent with the work of others (18,19,23) CD44
showed a surface staining in immunouorescence labeling
in our study. The location of CD44 on the cell surface of
HNSCC might represent its role as an adhesion molecule
in tumor survival and progression. There is evidence that
CD44 proteins anchor the cells to the extracellular matrix
(ECM) and that adhesive properties of malignant cells have
to be altered to detach from the primary tumor in order to
build metastases (24). It has been shown that the extracellular
portion of CD44 serves as a substrate for proteolytic cleavage
by matrix metalloproteinases (MMPs) on several cancer cell
lines and in human tumors. This indicates that CD44 proteins
can serve as ‘platforms’ for enzymes such as MMPs, which
are required for their effective functions. The inhibition of
the reactions catalyzed by these MMPs can block tumor cell
migration (25,26). As tumor cells get in contact with the ECM
via several cell surface adhesion molecules such as CD44,
integrins and cadherins it is likely that these proteins are also
needed for metastatic processes (27). As CD44 obviously gets
in contact with the ECM it is imaginable that there is a CD44-
dependent assembly and organization of the ECM, which
might be useful to protect tumor cells from immune defence
(28,29). Pre-treatment of chondrocytes with an anti-CD44
antibody blocked their interaction with the ECM (30).
Concent ra tio n of CD44 in p eriph eral bloo d of HNSCC
patients. Soluble CD44 has been evaluated in blood serum
of patients with different primary cancers (5,21,31) as well as
in oral rinses of patients with HNSCC (4,32). Elevated CD44
serum levels have been found for several tumor types using
ELISA analysis (31,33,34).
In this study, we showed that the concentration of soluble
CD44 is signicantly higher in peripheral blood of HNSCC
patients compared to healthy controls. This is in contrast to
the ndings reported by Van Hal et al (21), who did not nd
signicant differences between the CD44v6 plasma levels of
HNSCC patients, healthy controls and non-cancer patients.
A reason might be the measurement of different isoforms of
CD44. Van Hal et al focussed on the detection of CD44v6, the
total quantity of CD44 including all isoforms was measured
A
B
C
Figure 3. CD44 in peripheral blood of HNSCC patients depending on
location of primary tumor, T-stage and N-stage. There are no sign ica nt
differenc es in the concentration of s oluble CD44 in HNSCC patient s
according to primary tumor location (A), T-stage (B) or N-stage (C). Note
the signica ntly higher concentr ation of CD44 in the periphe ra l blood
of HNSCC patients compared to healthy controls (A) ✩P= 0.6x10-1 2, (B)
✩P=0,7x10-13, (C) ✩P= 0.7x10-13.

ONCOLOGY REPORTS 26: 321-326, 2011 325
in our experiments. A n explanation for the findings of
Van Hal et al might be given by Herold-Mende et al (19).
They found that the variant exons of CD44 v5, v6, v7, v7-8
and v10 are expressed in epithelia of healthy donors and that
variant exons v7, v8 and v10 were signicantly downregulated
in primary squamous cell carcinoma and were not detected
at all in the majority of metastasis-derived specimens (19).
Expression of CD44v5 and CD44v6, on the other hand, was
mainly unaltered. However, this might give an explanation for
the ndings of Van Hal et al (21). In contrast to this Mack et al
found a slight increase of CD44s and CD44v6 levels in oral
leukoplakia and in moderately differentiated carcinomas (20).
For other types of cancer CD44v6 has already been shown to
be a potential marker of prognosis. Saito et al postulated that
the serum concentration of sCD44v6 and its expression in
tumors were associated signicantly with the depth of invasion
of the tumor, lymph node metastasis and clinical stage in
patients with diffuse type gastric carcinoma (5).
In our experiments, a highly signicant difference between
concentration of CD44 in serum of HNSCC patients was
found in comparison to healthy controls. In contrast to Saito et
al (5), there was no correlation to the stage of primary tumor
expansion, lymph node metastasis or distant metastasis in our
results. It is not the stage of disease, but rather the mass of the
tumor or the specic number of tumor cells that should be
seen in congruence with the concentration of CD44 in periph-
eral blood in HNSCC patients. In any case, the tissue samples
available for experimental analysis are small specimens out
of the bulk of a tumor. This is to guarantee benet of surgery
to patients and optimal terms and conditions of pathological
assessment to ensure the best possible treatment and follow-up
for the patient.
Acknowledgements
We gratefully thank Petra Prohaska for excellent technical
suppor t. Special thanks to Dr C. Barth, Dr U. Gössler,
Dr K. Götte, Dr J.T. Maurer, Dr H. Sadick, Dr A. Sauter,
Dr C. Schubotz-Mitgau, Professor B.A. Stuck and all the
colleagues in anaesthetics for their assistance in obtaining
tumor samples at the University Hospital of Mannheim,
Dep a rtment of Ot o r h i nola r yngology Hea d and Ne ck
S u rg e r y.
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