Contribution of HIF-1 and drug penetrance to hypoxia- mediated oxaliplatin resistance in colorectal cancer spheroids

A198 Hypoxic tumour cells are often resistant to standard chemotherapeutics yet underlying mechanisms and contribution of HIF-1 remain unclear. Using HCT116 colorectal cancer cells, hypoxic-cell chemoresistance was revealed for Oxaliplatin using short-term viability and/or clonogenic survival assays. Stable expression of dominant negative HIF-1α (DN) ablated hypoxic resistance (compared with isogenic empty vector controls (EV)). To study a more physiologically relevant 3D model of hypoxia, HCT116 cells were grown as spheroids and treated with Oxaliplatin. Oxaliplatin-induced apoptosis (25µM, cleaved caspase 3) was restricted to outer regions of the spheroid. The relative sensitivity of subpopulations within the spheroid was then analysed by disaggregating Hoechst 33342 incubated, Oxaliplatin-treated spheroids. Cells were then sorted by FACS into four fractions, Bright (B), Mid Bright (MB), Mid Dim (MD) and Dim (D) based on their fluorescence intensity. Fluorescence decreased with spheroid depth. Consistent with apoptosis, a progressive decrease in oxaliplatin-sensitivity was observed from outer to inner fractions (D