Inhibition of glycolysis: a novel strategy to overcome drug resistance in CD133+ tumor initiating cells under hypoxic microenvironment

LB-94 Increasing evidences suggest that the existence of a subpopulation of drug resistant tumor initiating cells or cancer stem cells likely attributes to the failure of clinical cancer treatment and disease recurrence. Development of new strategies to effectively eliminate the cancer stem cells is critical to improve treatment outcomes. Based on our recent observations, we hypothesize that tumor initiation cells (or stem-like cells) favor hypoxic conditions and maintain the stemness through high glycolytic activity, and that glycolytic inhibition might represent a novel approach to eliminating cancer stem cells. To test the hypothesis, we established a CD133+ enriched cell model (U87-SC) by isolating tumor cells from human malignant glioma U87 xenograft pre-selected in vivo by doxorubicin treatment, and maintaining the cells in a simplified brain tumor stem cell medium. At earlier passages, U87-SC cells readily formed neuroshere-like clusters in hypoxia but not in normoxia, indicating that hypoxia significantly favored the maintenance of neurosphere-forming capacity. There was a 2-fold increase of expression of CD133 in hypoxic U87-SC compared with normoxia as analyzed by flow cytometry. Interestingly, the neurosphere phenotype was eventually lost when cells were cultured in regular DMEM with 10% FBS. The neurosphere-like cells highly expressed nestin, a neural progenitor cell marker, suggesting that the neurosphere-forming capacity may reflect a stemness stage in proper culture conditions. Colony formation and anchorage-independent assay in soft-agar showed a higher colony formation capacity for hypoxic U87-SC cells. Furthermore, in vivo tumorigenecity of the hypoxic U87-SC cells was 100-fold higher than the parental U87 cells as quantitatively tested by by inoculation in nude mice. MTT assay showed that hypoxic U87-SC cells were less sensitive to doxorubicin, vincristin, and BCNU compared with normoxic conditions. The residual U87-SC cells after doxorubicin treatment re-grew when the drug was removed. Importantly, the sphere-forming U87-SC cells exhibited less oxygen consumption, and were highly sensitive to glycolytic inhibition, leading to severe ATP depletion. Combination with glycolytic inhibition with doxorubicin was able to eliminate the residual cells and abolish cell re-growth. Apoptosis induction by the combination treatment was confirmed by PARP cleavage and caspase activation using Western blot. Furthermore, this in vitro drug combination treatment effectively eliminated the tumorigenesic capacity of the hypoxic U87-SC cells to form tumors in vivo. Our data suggest that the chemotherapy resistance phenotype of hypoxic brain tumor cells is likely due to an enrichment of highly glycolytic tumor-initiating cells, which can be eliminated by a combination treatment of chemotherapeutic agents with glycolytic inhibition.