Figure 7.
Ovarian cancer cells in ascites acquire CSC characteristics. (A) Heatmap showing expression of CSC markers in omental ID8 cells (ID8-OM) and ID8 cells from ascites (ID8-A11); see Table S1 for details. (B) Flow cytometry analysis of CSC surface markers on ascitic ID8-A11 cells compared with ID8 cells in omentum (ID8-OM). (C and D) Spheroid formation assay comparing cultured ID8 and ID8-A11 cells; 4 × 104 cells were seeded in ultra-low-adherence 96-well plates, and formation of spheroids was assessed with a wide-field microscope. (E) Analysis of ALDH activity in ID8 and ID8-A11 cells by flow cytometry using the Aldefluor assay. ALDH+ cells were gated using DEAB-treated cells as negative control (left), and relative ALDH activity was calculated by measuring the median fluorescence intensity of Aldefluor. Data are represented as mean ± SEM of n = 5 (ID8) or n = 8 (ID8-A11), and statistically significant difference was calculated using Mann-Whitney U test; *, P < 0.05; **, P < 0.01. (F) Analysis of tumorigenic potential of ID8 and ID8-A11 cells in vivo; total tumor burden was monitored by in vivo bioluminescence imaging. Data are represented as mean ± SEM of n = 6, and statistically significant difference was calculated using two-way ANOVA followed by Tukey post hoc test; **, P < 0.01; ****, P < 0.0001. (G) Representative images of tumor localization at 30 d after injection of parental ID8-Luc and ID8-A11 cells; scale bar: 1 cm. (H and I) Ex vivo analysis of tumor burden in omentum (H) and ascites (I) at 30 d after transplantation of ID8-Luc or ID8-A11 cells. Data are represented as mean ± SEM of n = 6 (ID8) or n = 5 (ID8-A11), and statistically significant difference was calculated using Mann-Whitney U test; **, P < 0.01. All data are representative of two independent experiments.