Primary and Secondary Bystander Effects of Proton Microbeam Irradiation on Human Lung Cancer Cells under Hypoxic Conditions

Simple Summary: The SPICE-QST proton microbeams (3.4 MeV proton, LET similar to 11.7 keV/similar to m) are powerful tools to investigate the mechanism(s) underlying radiation-induced bystander effects (RIBE) and radiation-induced secondary bystander effect (RISBE) in hypoxic cells. The purpose of this study was to elucidate the RIBE and RISBE in hypoxic lung cancer cells following high-LET proton irradiation. Specifically, the role of intercellular communication through gap-junction intercellular communication (GJIC) and/or soluble factors from irradiated to nonirradiated cells, such as nitric oxide (NO), is the major mechanism responsible for the observed effects. Our results indicate that the propagation of RIBE and RISBE in hypoxic cancer cells depends on the radiation dose and oxygen status. These results also highlight the key role of intercellular communication via GJIC in the modulation of stressful effects in primary and secondary bystander cancer cells under hypoxic conditions, which may contribute to improving the treatment outcomes. Tumor hypoxia is the most common feature of radioresistance to the radiotherapy (RT) of lung cancer and results in poor clinical outcomes. High-linear energy transfer (LET) radiation is a novel RT technique to overcome this problem. However, a limited number of studies have been elucidated on the underlying mechanism(s) of RIBE and RISBE in cancer cells exposed to high-LET radiation under hypoxia. Here, we developed a new method to investigate the RIBE and RISBE under hypoxia using the SPICE-QST proton microbeams and a layered tissue co-culture system. Normal lung fibroblast (WI-38) and lung cancer (A549) cells were exposed in the range of 06 Gy of proton microbeams, wherein only similar to 0.04-0.15% of the cells were traversed by protons. Subsequently, primary bystander A549 cells were co-cultured with secondary bystander A549 cells in the presence or absence of a GJIC and NO inhibitor using co-culture systems. Studies show that there are differences in RIBE in A549 and WI-38 primary bystander cells under normoxia and hypoxia. Interestingly, treatment with a GJIC inhibitor showed an increase in the toxicity of primary bystander WI-38 cells but a decrease in A549 cells under hypoxia. Our results also show the induction of RISBE in secondary bystander A549 cells under hypoxia, where GJIC and NO inhibitors reduced the stressful effects on secondary bystander A549 cells. Together, these preliminary results, for the first time, represented the involvement of intercellular communications through GJIC in propagation of RIBE and RISBE in hypoxic cancer cells.