Figure 1.
Flow cytometry analysis of CD24 and CD44 cell surface markers in NPC cell lines.
Flow cytometry analysis of CD24+ and CD44+ sub-populations of TW02 and TW04 cells. 1×106 cancer cells were collected and stained with anti-human CD24-fluorescein isothiocyanate (FITC) and/or anti-human CD44-phycoerythrin (PE) antibodies. Isotype-matched human antibodies served as control.
Table 1.
Percentage of cells expressing cancer cell and stem cell markers in human NPC cell lines.
Figure 2.
Expression levels of stem cell genes and activation of the Wnt/β-catenin pathway in CD24+ cells.
(A) mRNA expression levels of Sox2, Oct4, Nanog, Bmi-1 and Rex-1 in parental, CD24+ and CD24− cells from the NPC cell lines, TW02 (left) and TW04 (right), was evaluated using quantitative RT-PCR analysis. The results shown represented the average of three independent experiments. *: p<0.05, **: p<0.01. (B) Western blots analysis of phosphorylated-GSK, GSK, phosphorylated-β-catenin, β-catenin and β-actin in whole cell lysates, and of β-catenin and lamin B1 in nuclear fractions of parental, CD24+, and CD24− cells isolated from the TW02 and TW04 cell lines. Quantitative result was calculated by ImageJ software. *: p<0.05, **: p<0.01, ***: p<0.001.
Figure 3.
CD24+ cells show increased proliferation rate and enhanced clone and sphere formation.
(A) Cell proliferation curves of parental, CD24+ and CD24− cells from the TW02 (left) and TW04 (right) NPC cell lines cultured in complete DMEM for 9 days. The results shown represent the average of three independent experiments. *: p<0.05, **: p<0.01, ***: p<0.001. (B) Clone formation efficiency of parental, CD24+, and CD24− cells, and the quantification analysis. *: p<0.05. (C) Sphere formation ability of parental, CD24+ and CD24− cells cultured in DMEM supplemented with 20 ng/ml bFGF and 20 ng/ml EGF for 30 days. (D) Effect of the Wnt inhibitor on sphere formation by CD24+ cells in TW02 and TW04 cells. CD24+ cells were treated or not with the Wnt inhibitor ICG-001 (10 µM) for seven days prior to sphere formation analysis. Scale bars: 100 µm.
Figure 4.
Cell differentiation potential of CD24+ cells.
(A) Ten-day old cell masses originally cultured from CD24+ and CD24− cell cultures in DMEM medium were harvested, and stained with anti-human CD24-fluorescein isothiocyanate (FITC) antibody followed by flow cytometry analysis. The percentages shown represent cells with CD24+ surface markers from either (A) TW02 or (B) TW04 cell lines. Flow cytometry analysis of TW02 and TW04 cells immediately after sub-fractionation (right panels).
Figure 5.
CD24+ cells show enhanced chemoresistance.
(A) Sorted CD24+ and CD24− cells from the TW02 and TW04 NPC cell lines were treated or not with 50 µM verapamil for 30 min prior to processing for the Hoechst 33342 dye exclusion assay as described in Materials and Methods. (B) Parental, CD24+, and CD24− cells from the TW02 or TW04 cell lines were treated with various concentrations of cisplatin and docetaxel for 24 h, and cell viability was determined using the MTT assay. CD24+ cells showed higher viability than parental and CD24− cells after treatment with either cisplatin or docetaxel. The results shown represented the average of three independent experiments. *: p<0.05, **: p<0.01, ***: p<0.001. IC50 values of cisplatin or docetaxel treatment against parental, CD24+, and CD24− cells were shown for (C) TW02 and (D) TW04 cells lines.
Figure 6.
Cellular ABCG2 expression levels in parental, CD24+, and CD24− cells.
(A) Cellular ABCG2 protein levels in parental, CD24+, and CD24− TW02 and TW04 cells were determined by Western blot analysis. Quantitative result was calculated by ImageJ software. *: p<0.05, **: p<0.01. (B) The mRNA expression level of ABCG2 in parental, CD24+, and CD24− TW02 and TW04 cells was determined by quantitative RT-PCR. The results shown represent the average of three independent experiments. *: p<0.05, **: p<0.01.
Figure 7.
A low number of CD24+ NPC cells initiates tumor formation in NOD/SCID mice.
(A) Formation of tumors following injection of CD24+ cells. Groups of six NOD/SCID mice were injected with 100, 500, or 1,000 freshly-sorted CD24+ or CD24− cells from the TW02 (left) or TW04 (right) cell line. Mice injected with PBS were used as a negative control. Tumor formation was assessed 12 weeks after cell inoculation. (B) Mice injected with TW02 (left) or TW04 (right) cells were sacrificed for evaluation of tumor formation twelve weeks after inoculation. The arrows indicate the presence of tumors in mice injected with 500 or 1,000 CD24+ cells. (C) Tissue H&E staining results of TW02 (left) and TW04 (right) mice inoculated with 500 cells. Inoculation of as few as 500 CD24+ cells produced histological signs of tumors at the site of injection.
Figure 8.
CD24+ cells show higher invasion ability and enhanced expression of MMP2 and MMP9.
(A) The cell invasion assay was performed using the transwell chamber assay, as described in Materials and Methods. Matrigel membranes containing invading cells were observed by optical microscopy, and the cells were counted. The number of invading cells from each cell population was quantified. Results shown were obtained from three independent experiments. **: p<0.01, ***: p<0.001 (B) The levels of MMP2 and MMP9 protein produced by parental, CD24+, or CD24− cells from the TW02 and TW04 cell lines were quantified by Western blotting analysis. Quantitative result was calculated by ImageJ software. *: p<0.05. (C) The mRNA expression levels of MMP2 and MMP9 in parental, CD24+, and CD24− cells were determined by quantitative RT-PCR from the TW02 and TW04 cell lines. Results shown represent an average from three independent experiments. *: p<0.05, **: p<0.01.
Table 2.
DNA primers used for q-RT-PCR analysis in this study.