Microwave hyperthermia enhances radiosensitivity of highly invasive non-small cell lung cancer cells via inhibiting Sonic Hedgehog signaling pathway

Objective Radiotherapy (RT) is the main treatment for lung cancer. However, the presence of highly invasive cell (INV) subgroups in tumors often leads to recurrence and metastasis. We hypothesize that microwave hyperthermia (HT) can significantly enhance the radiosensitivity of these INV subgroups, and further explore the molecular mechanisms involved.Methods Cell proliferation was assessed by colony formation assay and 5-Ethynyl-2’-deoxyuridine (EdU) incorporation assay; cell migration and invasion were evaluated by wound healing and Transwell assays. Western blot (WB) analysis was used to evaluate protein expression associated with epithelial-mesenchymal transition (EMT) and DNA damage repair pathway. Flow cytometry was used to detect cell cycle distribution and apoptosis. TMT proteomics analysis was used to explore radiosensitization mechanism. The radiation sensitization effect of HT was confirmed in in vivo experiments.Results Compared with RT monotherapy, RT+HT treatment significantly inhibited cell proliferation, repressed cell motility, and reversed the EMT. Moreover, a substantial reduction in proteins associated with DNA damage repair was observed. G2/M arrest and apoptosis were induced by RT+HT treatment. The proteomics analysis, WB assay and the animal experiment indicated that Sonic Hedgehog (Shh) pathway might participate the regulation of radiosensitization by HT. Furthermore, SAG (a Shh agonist) partially reversed the suppression of EMT induced by the combination of RT and HT in INV cells.Conclusion Our findings demonstrate that HT enhances radiosensitivity of highly invasive NSCLC cells through Shh pathway-mediated EMT reversal coupled with DNA damage potentiation. This study provides a novel therapeutic strategy to overcome radioresistance in NSCLC.