We next tested the suitability of the generated platforms for the testing of chemicals with potential anti-cancer activities. First we investigated the GTIC dependence for hyperactive PI3K/MAPK signalling and their causative role in the acquisition of stem cell properties and metabolic reprogramming. Notably, only MAPK inhibition with PD098059 restored normal glycolytic activities, whereas inhibition of both, PI3K (LY294002) and MAPK signalling, reduced oxidative phosphorylation to the levels observed in WTiNPCs (Fig. 3b,c). In addition, chemical inhibition of PI3K and MAPK compromised stem cell self-renewal properties (Supplementary Fig. 7). Next, we focused on metabolic modulation by using: (i) Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), a compound dissipating mitochondrial membrane potential; (ii) rotenone in conjunction with antimycin A (Rot/AA), both compounds targeting complexes I and III and leading to the massive production of ROS; and (iii) 2-DG, an analogue glucose acting as a suicide inhibitor. Among all metabolic modulators, inhibition of glycolysis by 2-DG, a compound previously reported for the targeting of gliomas and currently in clinical testing, significantly compromised the self-renewal properties of transformed iNPCs (Fig. 3d). Then, we performed MTS assays to
