Figure 3. Effect of metformin treatment of GBM TIC survival. (A) Metformin inhibits GBM1–4 TIC viability, evaluated using cells grown as spheres (black dots) or as monolayers on matrigel (white dots) by MTT assay. Both culture methods showed similar sensitivity to metformin that induced in all GBM cells a statistically significant inhibition. (p < 0.01 vs. untreated cells reported as 100%, n = 4.) (B) Antiproliferative effects of metformin evaluated as percentage of Ki-67-positive cells in GBM1–4 TIC spheres. White dots represent Ki-67-expressing cells, while gray fluorescence corresponds to DAPI nuclear counterstain. In the upper picture, a representative sphere from GBM1 TICs untreated or metformin-treated (20 mM, 48 h), while in the lower graph, an average of experiments pooling data from all the cultures is reported. (n = 3; ** = p < 0.01 vs. untreated cell.) (C) Cell growth recovery experiments evaluated by MTT assay. GBM1–4 TICs were treated for 48 h with increasing concentrations of metformin (black bars, untreated cells are represented as white bars), followed by 48 h culture in drug-free medium (gray bars). Low metformin concentrations were mainly cytostatic, as shown by cell growth recovery after drug withdrawal, while concentrations higher than the calculated IC₅₀ (about 10 mM) resulted in a cytotoxic effect. (n = 3; * = p < 0.05 and ** = p < 0.01 vs. respective metformin-treated experimental points.)