Fig. 3
EVs derived from chemotherapy-treated NSCCs enhance CSC chemoresistance and progression. a–d Chemotherapy-induced F1-EVs promote the acquisition of chemoresistance in CSCs. S2 cells were pre-conditioned with naïve F1-EVs, F1/CDDP-EVs, or F1/Gem-EV for 10 days. Cells were subjected to chemotoxicity tests using CDDP (DMSO, 2.5, 5, 10, 20, 40, 80, 160 µM), and gemcitabine (ddH2O, 2.5, 5, 10, 20, 40, 80, 160 µM) for 48 h, after which MTT assays (a and c) and clonogenic assays (b and d) were performed. e Chemotherapy-induced F1-EVs enhance CSC invasion and mobility. S2 cells maintained in CSC medium were treated with F1/CDDP-EV, F1/Gem-EV, or PBS for 10 days. Cells were then collected and subjected to transwell migration and invasion assays. Representative images of cells in the lower chambers are shown, and cell numbers were quantified and plotted. f Chemotherapy-induced F1-EVs promote the self-renewal of CSCs. S2 cells maintained in CSC medium were treated with F1/CDDP-EV, F1/Gem-EV, or PBS control for 10 days. Single suspended cells (1000 cells/ml) were seeded in CSC culture medium without any additional treatment for 10 days. Spheroid forming ability was determined by counting the number of spheres. Representative images on day 10 were taken, and sphere numbers were counted and plotted. g Chemotherapy-induced F1-EVs alter CSC marker expression. CSC markers in the spheroids described in (f) were measured by quantitative real-time PCR. Data were normalized to PBS controls, and the 2−ΔΔCt method was used to calculate relative gene expression. Experiments were repeated independently at least three times. Data were compared via Student’s t-tests or one-way ANOVAs with Tukey’s multiple comparisons test, as appropriate. *P < 0.05; **P < 0.01; ***P < 0.001