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Lake, NJ, USA), and allophycocyanin-conjugated mouse Assessment of the growth of UP-LN1 cells and
anti-human CD24 (clone ML10, Biolegend, San Diego, subsets following WA treatment
USA) mAbs according to the manufactural instructions.
Labeled cells were then washed 3 times by phosphate- Sulforhodamine B (SRB) dye (Sigma-Aldrich, Chemie
buffered saline (PBS) plus 2% fetal bovine serum (FBS) GmbH, Munich, Germany) was used to test the effects of
followed by fixation with 1% paraformaldehyde. The fixed selective inhibitors on cell growth and viability of SP cells.
samples were then analyzed cytofluorometrically. The WA was dissolved in dimethyl sulfoxide (DMSO) before
diluting with growth medium to a final DMSO concentration
Cell line, subsets and culture conditions of 0.05%. The P, A, and F cells were seeded into 96 well
plates in growth medium at 3,000 cells/well. After 24 h, the
The UP-LN1 cell line and its A and F subsets were used medium was replaced with fresh growth medium containing
in this study. Unless specified, all the cell lines were the WA. The cells were incubated for another 48 h. The cells
maintained in the condition described previously. For the were fixed with trichloroacetic acid (TCA) by gently adding
[8]
separation of A cells, we discarded all floating cells in the 50 µL TCA (50%) to each well to a final TCA concentration
culture supernatant and then harvested only the adherent of 10% with subsequent incubation for 1 h at 4 °C. The
cells by light trypsinization to set up new cultures for A plates were then washed 5 times with tap water and air dried.
cells. To obtain F cells, we only collected the floating The dried plates were stained with 100 µL of 0.4% (w/v)
cells in the culture supernatant of UP-LN1 culture for the SRB prepared in 1% (v/v) acetic acid for 10 min at room
subsequent culture passage. Each of these 2 protocols was temperature. The plates were rinsed quickly 4 times with 1%
used for the enrichment of A or F cells when subculturing acetic acid to remove unbound dye and were then air dried
for 10 consecutive rounds. To maintain the parental (P) UP- until no moisture was visible. The bound dye was solubilized
LN1 cells (termed P cells), floating cells and trypsinized in 20 mmol/L Tris-base (100 µL/well) for 5 min on a shaker.
adherent cells were washed and pooled, then set up in a new Optical densities were read on a microplate reader (Molecular
culture. Trypan blue dye exclusion was used to determine Devices, Sunnyvale, CA, USA) at 562 nm.
cell viability. A and F cells were maintained in Roswell Park
Memorial Institute (RPMI)-1640 medium supplemented Apoptosis assay
with 10% FBS.
Apoptosis was assessed by staining the cells with Annexin
Characterization of UP-LN1 cells by SP analysis V-FITC (BD-Pharmingen, Franklin Lakes, NJ, USA), and
PI and analyzing stained cells via flow cytometry. Briefly,
To examine the existence of CSCs in the UP-LN1 the UP-LN1 P, A, and F cells (1 × 10 cells/mL) were grown in
6
carcinoma cell line, the SP cells were isolated by flow RPMI medium alone or in the same medium supplemented
cytometry and cell sorting techniques. SP cells have been with either WA or DMSO. After 48 h, cells were washed
shown to express an elevated level of ATP-binding cassette twice with ice-cold PBS and then re-suspended in 100 µL
transporters (ABCG2), which enhance their ability to pump binding buffer containing 2 µL of FITC-conjugated Annexin
out Hoechst 33342 dye. This efflux activity of Hoechst dye V and 2 µL of PI for 15 min. Following the incubation,
is similar to many drug-resistant cancer cells and can be without washing the cells with excess reagents, 400 µL
sorted by FACSAria flow cytometry. Therefore, we utilized binding buffer was added. Samples were then analyzed by
SP analysis as one of the characteristics to demonstrate flow cytometry. Data acquisition and analysis were done
and analyze the population of cancer stem-like cells in our using CellQuest™ software (Becton Dickinson, San Jose,
unique UP-LN1 cells. UP-LN1 cells were labeled with 2.5 CA, USA).
µg/mL Hoechst 33342 (Sigma-Aldrich, Chemie GmbH,
Munich, Germany) for 30 min at 37 °C. The control cells Knockdown of CXCR4 by siRNA
were incubated in the presence of 50 µmol/L verapamil
(Sigma-Aldrich, Chemie GmbH, Munich, Germany). UP-LN1 cells were transfected with Validated MISSION®
Propidium iodine (PI) 1 µg/mL was added to identify dead siRNA (SASI_Hs01_00084886, Sigma-Aldrich Taiwan,
cells. Analysis and sorting were performed on FACSAria Linkou, New Taipei City, China) according to the vendor’s
flow cytometry (Becton Dickinson, San Jose, CA, USA), instructions. Transfected cells were lyzed and subjected to
similar to that described by Patrawala et al. After sorting, both total RNA extraction and Western blot analysis 48 h
[20]
SP sphere cells of UP-LN1 were placed at a density of 1,000 post-transfection. CXCR4 expression was confirmed using
cells/mL under stem cell conditions by resuspension in Western blot and anti-CXCR4 antibody (SAB3500383,
tumor sphere medium consisting of serum-free HEScGRO Sigma-Aldrich, China).
medium, N2 supplement (Invitrogen, Carlsbad, CA, USA),
10 ng/mL human recombinant bFGF (Invitrogen, Carlsbad, CDy1 immunofluorescence staining
CA, USA), and 10 ng/mL EGF (Invitrogen, Carlsbad, CA,
USA), followed by culturing in ultra-low attachment plates UP-LN1 P, A, and F cells were cultured in a 60-mm
(Corning, NY, USA) for about 1 week. culture dish for 24 h in the presence of 500 nmol/L

Journal of Cancer Metastasis and Treatment ¦ Volume 2 ¦ Issue 1 ¦ January 15, 2016 ¦ 31