Radiation and hypoxia-induced non-targeted effects in normoxic and hypoxic conditions in human lung cancer cells

Purpose: Many cell lines with anaerobic metabolism do not show cytotoxic abscopal effect (AE) following irradiation. Further, there is no existing data on the radiation- and hypoxia (H)-induced AE. The purpose of this study was to investigate and compare the status of radiation-induced abscopal effect (RIAE) in normoxic and hypoxic conditions.Methods: Lung cancer cells (A549, H460) were exposed either to hypoxia or normoxia and then irradiated (2 or 10Gy). After 24h, unirradiated hypoxic (H-CM) or normoxic (N-CM) conditioned media (CM) and irradiated hypoxic (H-RCM) or normoxic (N-RCM) CM was collected. Hypoxia-resistant clones (HR: A549/HR, H460/HR) were generated by continuous exposure of the cells to hypoxia. Unirradiated parental cells or HR were exposed to H-CM, N-CM, H-RCM or N-RCM. In some groups, 24h after exposure to CM, cells were directly irradiated with 2Gy. Cell growth was monitored using real-time cell electronic sensing system. Further, levels of hypoxia and HIF1 regulated angiogenesis related growth factors, basic fibroblast growth factor (bFGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase (sFlt-1) and vascular endothelial growth factor (VEGF) were assessed in CM.Results: In the radio-resistant A549 cells, H-RCM was much more effective in inducing growth delay compared to N-RCM. In the radio-sensitive H460 cells, both N-RCM and H-RCM induced growth delay. Interestingly, effects of N-RCM were completely reversed in HR cells. Exposure of cells to direct irradiation (2Gy) 24h after incubation with CM resulted in 50-60% reduction in cell proliferation in A549/HR cells and a very significant induction of death (>95%) in H460/HR cells. Direct irradiation of parental or HR clones of A549 and H460 cells exposed to H-CM 24h with 2Gy induced significant reduction in cell proliferation (from 40% to >95%) in all the cells. Further, levels of sFlt-1 correlated with growth delay in all the cells.Conclusions: These results for the first time demonstrate that irradiation of hypoxic cells and exposing the cells to acute hypoxia lead to significant AE.