Acute Hypoxia Does Not Alter Tumor Sensitivity to FLASH Radiation Therapy

Background Tumor hypoxia is a major cause of treatment resistance, especially to radiotherapy at conventional dose rate (CONV), and we wanted to assess whether hypoxia does alter tumor sensitivity to FLASH. Methods We engrafted several tumor types (glioblastoma – GBM, Head & Neck cancer, and lung adenocarcinoma) subcutaneously in mice to provide a reliable and rigorous way to modulate oxygen supply via vascular clamping or carbogen breathing. We irradiated tumors using a single 20 Gy fraction at either CONV or FLASH, measured oxygen tension, monitored tumor growth, and sampled tumors for bulk RNAseq and pimonidazole analysis. Next, we inhibited glycolysis with trametinib in GBM tumors to enhance FLASH efficacy. Results Using various subcutaneous tumor models, and in contrast to CONV, FLASH retained anti-tumor efficacy under acute hypoxia. These findings show that in addition to normal tissue sparing, FLASH could overcome hypoxia-mediated tumor resistance. Follow-up molecular analysis using RNAseq profiling uncovered FLASH-specific profile in human GBM that involved cell cycle arrest, decreased ribosomal biogenesis, and a switch from oxidative phosphorylation to glycolysis. Glycolysis inhibition by trametinib enhanced FLASH efficacy in both normal and clamped conditions. Conclusions These data provide new and specific insights showing the efficacy of FLASH in radiation resistant context providing additional benefit of FLASH over CONV. Tumor hypoxia is a major cause of treatment resistance, especially to radiotherapy at conventional dose rate (CONV), and we wanted to assess whether hypoxia does alter tumor sensitivity to FLASH. We engrafted several tumor types (glioblastoma – GBM, Head & Neck cancer, and lung adenocarcinoma) subcutaneously in mice to provide a reliable and rigorous way to modulate oxygen supply via vascular clamping or carbogen breathing. We irradiated tumors using a single 20 Gy fraction at either CONV or FLASH, measured oxygen tension, monitored tumor growth, and sampled tumors for bulk RNAseq and pimonidazole analysis. Next, we inhibited glycolysis with trametinib in GBM tumors to enhance FLASH efficacy. Using various subcutaneous tumor models, and in contrast to CONV, FLASH retained anti-tumor efficacy under acute hypoxia. These findings show that in addition to normal tissue sparing, FLASH could overcome hypoxia-mediated tumor resistance. Follow-up molecular analysis using RNAseq profiling uncovered FLASH-specific profile in human GBM that involved cell cycle arrest, decreased ribosomal biogenesis, and a switch from oxidative phosphorylation to glycolysis. Glycolysis inhibition by trametinib enhanced FLASH efficacy in both normal and clamped conditions. These data provide new and specific insights showing the efficacy of FLASH in radiation resistant context providing additional benefit of FLASH over CONV.