Fig. 3.

Belinostat inhibited chemotherapy-promoted CSL state in ER+ cell lines and patient cancer cell samples. (A) Cell culture strategy for CSL reversal drug screening with cancer breast cell lines after chemotherapy promotion. The FACS mean values of ALDH, CD44, and CSL cells/live were shown in (B) for CAMA-1 and (C) for MDA-MB-231 with time-course curves. CAMA-1 cells and MDA-MB-231 cells were cultured in medium (3D) and treated for 72 h with doxorubicin (0.1 µM for CAMA-1, 0.5 µM for MDA-MB-231), carboplatin (50 µM), and paclitaxel (1 µM), and then replaced with medium containing belinostat (5 µM) for 72 h incubation. Samples were collected every day from Day 1 to Day 6, and followed by ALDEFLUOR/CD44/DAPI staining and FACS analysis. The differences between chemo plus DMSO (9 replicates from three chemo plus DSMO) and chemo plus belinostat (9 replicates from three chemo plus belinostat) at Day 6 were evaluated by student's T tests. The ALDH (D), CD44 (E), CSL cells/live (F) were expressed with curves for Patient#5, #6, and #7. Patient cells were cultured in Renaissance medium (3D) and treated with chemotherapy (doxorubicin 0.1 µM, carboplatin 0.1 mM, paclitaxel 1 µM) for 72 h, and then treated with DMSO/belinostat (1 µM or 2.5 µM) for another 72 h, followed by ALDEFLUOR/CD44/DAPI staining and FACS analysis. Column graphs were generated with averages from three patients for each treatment to evaluate the changes in ALDH (G), CD44 (H), and CSL/Live (I). In Figures B-I, DMSO treatment only in each day served as controls and was set as fold one. All the other treatments were expressed as the fold values relative to the controls. Each spot represents the mean values of triplicate tests. The statistical analysis was performed with student's t-tests between the indicated groups. Significance is marked with * for P<0.05, ** for P<0.01, *** for P<0.001, **** for P<0.0001. .