Figure 1.
The sphere formation from human OSCC cell lines.
(A) Phase-contrast photomicrographs of the spheres cultured from SAS (top) and OECM-1 (bottom) cell lines using a nonadhesive design (four leftmost upper and lower panels: from day 0 to day 7, magnification, 200×; and rightmost upper lower panels: day 10, magnification, 100×). (B) Immunohistochemistry results showing diverse expression patterns of representative epithelial–mesenchymal transition (EMT) markers in OECM1 parental cells and spheres (magnification, 200×).
Figure 2.
Comparison of the expression of specific surface markers of CSCs between parental cells and spheres.
(A) The parental cells and spheres were either stained with a negative-control IgG antibody (open space) or anti-CD133 experimental antibodies (solid space). (B) Comparison of the expression of ALDH1 between parental cells and spheres; DEAB, an inhibitor of ALDH1, was used as a negative control. (C) Quantitative and statistical comparisons of the percentage of positive signals for CD133 and ALDH1 between parental cells and spheres (*P<0.05).
Figure 3.
Comparison of the expression of CSC markers between parental cells and spheres.
(A) A RT-PCR analysis showed that the expression of the SOX2, Oct4, and NANOG genes was upregulated in spheres compared with parental cells. (B) Western blotting analysis showed that the expression of SOX2, Oct4, and NANOG was upregulated in spheres compared with parental cells. (C) Immunofluorescence analysis of CSC markers in spheres demonstrated the presence of CSCs with variable levels of expression of CD133, ALDH1, SOX2, Oct4, and NANOG, as indicated by the arrows (magnification, 200×).
Figure 4.
Comparison of radio- and chemosensitivity between parental cells and spheres of the two cell lines.
Significant differences in (A) radiosensitivity and (B) chemosensitivity were observed between parental cells and spheres. (C) Combined chemo- and radiotherapy (CCRT) with initial chemotherapy for 24 h followed by radiation. (D) CCRT with initial radiation followed by chemotherapy for 24 h. The two CCRT regimens were more effective in reducing the survival rate of parental cells and spheres compared with single treatment using either radiation or chemotherapy (*P<0.05).
Figure 5.
Comparison of newly generated tumors between OECM-1-derived parental cells and spheres in NOD/SCID mice.
(A) Gross appearance of a representative tumor formed by inoculation of parental cells and dissociated spheres into NOD/SCID mice (n = 3 in each group). (B) Corresponding histological findings and immunohistochemical results for representative EMT markers in NOD/SCID mice (magnification, 100×). (C) Primary culture of dissociated cells from OECM-1-induced spheres originally isolated from NOD/SCID mice demonstrated a gradual transformation of primary (1st) and secondary (2nd) spheres (magnification, 100×).
Table 1.
Tumorgenicity of the parental cells and spheres.
Table 2.
Comparison of the techniques in terms of isolation of CSCs related to time, cost, quantity and morphology.