Through Author Manuscript Author Manuscript Author Manuscript Author Manuscript Stem Cells. Author manuscript; available in PMC 2017 May 01. Aguilar et al. Page 6 knockdown of the pluripotency factors SOX2, KLF4 and MYC. Both genetic manipulations caused a reduced glycolytic phenotype and diminished the sensitivity of PC-3M cells to 2-DG. Therefore, the marked glycolytic phenotype and glucose dependence observed in PC-3M cells requires the maintenance of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19857367 robust epithelial and self-renewal gene programs. Mesenchymal-like non-CSCs are more dependent on mitochondrial function than e-CSCs The above results demonstrate a marked Warburg effect and a higher dependence on AMI-1 web glycolysis in PC-3M cells 
than PC-3S cells. We further investigated the reliance of these cells on mitochondrial respiration using four mitochondrial drugs. Oligomycin, an ATP synthase inhibitor, reduced more oxygen consumption rate and had a more significant dose-dependent proliferation inhibitory effect in PC-3S cells than PC-3M cells, suggesting a greater dependence on mitochondrial metabolism for cell growth of the non-CSC subpopulation. However, this drug did not significantly affect ATP levels in either subpopulation. Exposure of cells to trifluorocarbonylcyanide phenylhydrazone, a mitochondrial uncoupling agent, boosted OCR more in PC-3S cells than PC-3M cells, indicative of a significantly higher mitochondrial respiratory capacity of the non-CSC subpopulation. Moreover, exposure to the mitochondrial AGI-5198 web complex I and III inhibitors rotenone and antimycin reduced OCR in both cell subpopulations, although significantly more in PC-3S cells. Consistent with mitochondrial metabolic activity being the main source of reactive oxygen species , PC-3S cells exhibited higher basal levels of ROS than PC-3M cells. In support of the importance of the epithelial and pluripotency gene programs for these metabolic readouts, PC-3M/Snai1 and PC-3M/SKMkd variants were more sensitive than parental PC-3M cells to all mitochondrial drugs. We next stained cells with MitoTracker to investigate the possible association of mitochondrial morphology with the above bioenergetics phenotypes. PC-3M cells exhibited fragmented mitochondrial networks consistent with extensive mitochondrial fission, which has been associated with less efficient OXPHOS and a higher reliance on anaerobic glycolysis for bioenergetics. In contrast, the mitochondrial morphologies in PC-3S cells were more compact, consistent with active fusion events, a process coupled to a robust mitochondrial function and bioenergetics. Thus, the nonCSC subpopulation displays a mitochondrial organization predicted to support a better coupled mitochondrial respiratory chain for the generation of ATP. A higher flexibility of mitochondrial metabolism is required for spheroid growth of e-CSCs To investigate the degree of metabolic flexibility of these cells in the use of alternative carbon substrates, we evaluated the consumption of glutamine under glucose deprivation and with 2-DG treatment. These conditions caused enhanced glutamine consumption in PC-3M cells but not PC-3S cells, suggesting that 
PC-3M cells are more adept at using alternative carbon substrates. This was further explored by evaluating baseline OCR in full media, in restricted media or minimal media. PC-3M cells showed higher baseline OCR values than PC-3S cells in all restricted media conditions, while no differences were Author Manuscript Author Manuscript Author Manuscript Author.Through Author Manuscript Author Manuscript Author Manuscript Author Manuscript Stem Cells. Author manuscript; available in PMC 2017 May 01. Aguilar et al. Page 6 knockdown of the pluripotency factors SOX2, KLF4 and MYC. Both genetic manipulations caused a reduced glycolytic phenotype and diminished the sensitivity of PC-3M cells to 2-DG. Therefore, the marked glycolytic phenotype and glucose dependence observed in PC-3M cells requires the maintenance of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19857367 robust epithelial and self-renewal gene programs. Mesenchymal-like non-CSCs are more dependent on mitochondrial function than e-CSCs The above results demonstrate a marked Warburg effect and a higher dependence on glycolysis in PC-3M cells than PC-3S cells. We further investigated the reliance of these cells on mitochondrial respiration using four mitochondrial drugs. Oligomycin, an ATP synthase inhibitor, reduced more oxygen consumption rate and had a more significant dose-dependent proliferation inhibitory effect in PC-3S cells than PC-3M cells, suggesting a greater dependence on mitochondrial metabolism for cell growth of the non-CSC subpopulation. However, this drug did not significantly affect ATP levels in either subpopulation. Exposure of cells to trifluorocarbonylcyanide phenylhydrazone, a mitochondrial uncoupling agent, boosted OCR more in PC-3S cells than PC-3M cells, indicative of a significantly higher mitochondrial respiratory capacity of the non-CSC subpopulation. Moreover, exposure to the mitochondrial complex I and III inhibitors rotenone and antimycin reduced OCR in both cell subpopulations, although significantly more in PC-3S cells. Consistent with mitochondrial metabolic activity being the main source of reactive oxygen species , PC-3S cells exhibited higher basal levels of ROS than PC-3M cells. In support of the importance of the epithelial and pluripotency gene programs for these metabolic readouts, PC-3M/Snai1 and PC-3M/SKMkd variants were more sensitive than parental PC-3M cells to all mitochondrial drugs. We next stained cells with MitoTracker to investigate the possible association of mitochondrial morphology with the above bioenergetics phenotypes. PC-3M cells exhibited fragmented mitochondrial networks consistent with extensive mitochondrial fission, which has been associated with less efficient OXPHOS and a higher reliance on anaerobic glycolysis for bioenergetics. In contrast, the mitochondrial morphologies in PC-3S cells were more compact, consistent with active fusion events, a process coupled to a robust mitochondrial function and bioenergetics. Thus, the nonCSC subpopulation displays a mitochondrial organization predicted to support a better coupled mitochondrial respiratory chain for the generation of ATP. A higher flexibility of mitochondrial metabolism is required for spheroid growth of e-CSCs To investigate the degree of metabolic flexibility of these cells in the use of alternative carbon substrates, we evaluated the consumption of glutamine under glucose deprivation and with 2-DG treatment. These conditions caused enhanced glutamine consumption in PC-3M cells but not PC-3S cells, suggesting that PC-3M cells are more adept at using alternative carbon substrates. This was further explored by evaluating baseline OCR in full media, in restricted media or minimal media. PC-3M cells showed higher baseline OCR values than PC-3S cells in all restricted media conditions, while no differences were Author Manuscript Author Manuscript Author Manuscript Author.