Prolonged hypoxia switched on cancer stem cell-like plasticity in HepG2 tumourspheres cultured in serum-free media

Tumour hypoxia drives resistance and aggressiveness, and in large part, contributes to treatment failure thereby causing cancer-related deaths. The rapid and uncontrolled tumour growth develops not only a hypoxic niche but also a nutrient-deprived condition due to insufficient blood supply; together, these create a stressful tumour niche, further promoting higher aggressiveness and resistance features of cancer. However, how cellular responses in the prolonged stress is associated with cancer stem cells (CSCs), which is linked to these features, remains unclear. Here, we established HepG2 tumoursphere culture in a hypoxic and serum-free condition that recapitulated differential responses to prolonged tumour growth pressures, evident by their progressive changes in the morphology of tumoursphere formation over a course of 15-day culture. HepG2 tumourspheres formed larger sphere sizes of > 200 μm in hypoxic conditions, concomitant with higher cell yield and upregulation of PCNA marker at day 7, corresponding with higher self-renewal capacity when cultured in SFM compared to SM. Notably, prolonged growth of HepG2 tumourspheres for 15 days under hypoxic and SFM condition increased their sphere counts, yet significantly reduced their cell yield along with downregulation of PCNA expression. Gene expression analysis showed that HepG2 tumourspheres on day 15 exhibited enhanced expression of markers of quiescence, stemness, EMT, and chemoresistance. Interestingly, analysis of HIF1α and HIF2α and their target gene expression indicated complementary HIF expression with preferential upregulation of HIF2α was observed in HepG2 tumourspheres in prolonged hypoxic and serum-free conditions, suggesting HIF2α-dependency and plausibility of the HIF1α-HIF2α switch that govern their survival by promoting CSC-like programmes. Altogether, these findings suggest the implication of prolonged hypoxia and nutrient deprivation stress in promoting CSC-like programmes in cancer cells recapitulating their plasticity, hence having opened many research directions that enable development of effective targeting of CSCs and precision medicine for treating cancer.