Supplementary materials and methods
Transfection of plasmids
Using a retroviral transfection system with the PT67 amphotropic packaging cell line,
pBabeSOX2-neo and pBabe control-neo plasmids were stably transfected into SNU1041 cells
and FaDu cells to generate SOX2-overexpressing and control HNSCC cell clones,
respectively. To implement a gene knockdown of SOX2, the cells were infected with a
retrovirus carrying SOX2-shRNA, which was cloned into the pSuperRetro plasmid,
according to the manufacturer’s protocols (Oligoengine). The target sequences were SOX2shRNA: 5’-aaccaagacgctcatgaagaatt-3’. Small interfering RNA for ABCG2 and Snail was
purchased from
(Korea) and transfected into HNSCC CSCs using Lipofectamine 2000 (Life
Technologies, USA).
Patient samples and immunohistochemistry
The immunohistochemical (IHC) analysis of HNSCC patients’ specimens was approved by
the local institutional review board. Formalin-fixed and paraffin-embedded tissues from 69
HNSCC lesions were retrieved from the archives of the Pathology Department of Kanguk
Samsung Hospital, Korea. Main clinical and pathological characteristics are listed in Table I.
Four-micrometer-thick sections of paraffin-embedded tissue blocks were used for
immunohistochemical analysis according to the methods described in the Vectastain ABC kit
(Vector, Burlingame, CA, USA). All slides were scored by at least 2 pathologists who were
blinded to the clinical status of the patients. A 4-point scale based on the percentage of
positive cells was used: 0 when no positive cells were identified (no expression), 1: weak
staining (<25% positive cells), 2: moderate staining (25–50% positive cells), and 3: strong
staining (>50% positive cells). For practical reasons, we defined positive expression as >25%
cells positive for SOX2.
Tissue immunofluorescence
The human HNSCC tissues embedded in paraffin were sliced into 4-μm-thick sections and
stained with primary antibodies against human CD44 (1:200) and SOX2 (1:200) for 12 hr at
4°C. After that, the slides were incubated with an appropriate secondary antibody (1:1000)
for 12 hr at 4C. Some slides were stained with 6-diamidino-2-phenylindole (DAPI; 1μg/ml)
for 5 min. Slides were photographed using an optical microscope (Carl Zeiss).
The proliferation assay
Cells (2 × 104) were plated and grown in media with the FBS supplement. Every 2 days, the
cells were harvested by trypsinization and the total number of cells was counted.
Quantitative RT-PCR and western blot
Quantitative RT-PCR and western blot experiments were carried out as described previously
(4). The sequences of human specific primers used are;
Cyclin A: forward 5’-GCAAATGGGCAGTACAGGAG-3’,
reverse 5’-CCCCTGCTCTAGTTCATCCA-3’
Cyclin B1: forward 5’- CAGCTCTTGGGGACATTGGT-3’,
reverse 5’- AGGCTCAGGTTCTGGCTCAG-3’
Cyclin D1: forward 5’- GGAGCTGCTGCAAATGGAGC-3’,
reverse 5’- CAGAGGGCAACGAAGGTCTG-3’
Cyclin E: forward 5’- TCAACGTGCAAGCCTCGGAT-3’,
reverse 5’- CTGCAGAAGAGGGTGTTGCT-3’
SOX2: forward 5’- CCTCCGGGACATGATCAG-3’, reverse 5’TTCTCCCCCCTCCAGTTC-3’;
The sphere formation assay
To assess self-renewal in vitro, the cells were dissociated into a single-cell suspension,
seeded in a 24-well plate at the density of 200 cells/well, and cultured in serum-free media
with EGF and bFGF added every other day. Spheres with a diameter exceeding 10 µm were
counted after 14 days.
FACS analysis for detection of CD44 expression
Spheroid cells were dissociated into a single-cell suspension, washed, and resuspended in
PBS. The cells were labeled with an anti-CD44 primary antibody and a FITC-labeled
secondary antibody, and then subjected to flow cytometric analysis on a FACSCalibur
instrument (BD Biosciences).
The chemosensitivity assay
Primary spheroid cells were dissociated into a single-cell suspension and then plated in a 96well plate at the density of 7  103 cells per well under serum-free culture conditions. The
cells were treated with cisplatin at the indicated concentrations and then cultured at 37C in a
humidified 5% CO2 atmosphere. Twenty-four hours later, 20 μL of 3-(4,4-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL in PBS) was added to each
well, and the plate was incubated at room temperature for 3 hr. Absorbance was measured on
a SpectraMax 190 device (Molecular Devices) at the wavelength of 570 nm.
Isolation of ALDHhigh and ALDHlow cells from primary spheres
Cells were prepared from freshly dissociated spheres and were analyzed using an Aldefluor
assay kit (Stem Cell Technologies, Durham, NC, USA). The cells were suspended in an
Aldefluor assay solution containing an ALDH substrate (BAAA, 1 mol/L per 106 cells). A
sample of cells was stained using the specific ALDH inhibitor diethylaminobenzaldehyde
(DEAB) as a negative control. Flow cytometric sorting was conducted on a FACS Aria
instrument (Becton Dickinson, CA). The sorting gates were established using the cells stained
with PI only as negative controls, for viability, and with the antibody alone.
Subcutaneous and orthotopic implantation for a tumorigenicity assay
All procedures were approved by the Konkuk University Institutional Animal Care and Use
Committee (IACUC). For a limiting-dilution xenograft assay, groups of mice were inoculated
with HNSCC stemlike cells transfected with scrambled-sequence shRNA and with HNSCC
stemlike cells transfected with short hairpin (sh) RNA shSOX2 at 104 and 105 cells per
injection, respectively. Engrafted mice were inspected weekly for tumor development by
visual observation and palpation until 8 weeks postinjection. In the orthotopic experiment,
HNSCC stemlike cells transfected with scrambled-sequence shRNA and HNSCC stemlike
cells transfected with shSOX2 (1  104 per animal) were engrafted directly into the anterior
tongue using a 50 L Hamilton syringe with a 30-gauge needle (Hamilton Co., Reno, NV,
USA). The mice were then examined every other day for the development of tongue tumors.
Statistical analysis
Experimental data were statistically assessed using either 2-tailed Student’s t test or ANOVA.
Correlation between SOX2 expression and clinical parameters was analyzed using Fisher’s
exact test. The survival analysis in the human sample and in mice was performed using the
Kaplan-Meier survival curve and the logrank test. Differences with p < 0.05 were considered
statistically significant.