Low dose radiotherapy combined with immune checkpoint inhibitor induced ferroptosis in lung cancer through Nrf2/HO-1/GPX4 axis

Jing Luo^1*Qiongjie Zhi²Dongxia Li³Yue Xu¹Hui Zhu¹Lujun Zhao¹Guibing Ren³Jian Wang¹Ningbo Liu^1,4*

• ¹Tianjin Medical University Cancer Institute and Hospital, Tianjin, Tianjin, China
• ²The Second People's Hospital of Hefei, Hefei, Anhui Province, China
• ³Characteristic Medical Center of Chinese People's Armed Police Force, Tianjin, China
• ⁴Tianjin Medical University, Tianjin, China

Background: The clinical use of immune checkpoint inhibitors (ICI) has revolutionized the current therapeutic direction for lung cancer, however the response rates remain unsatisfactory. Recently, the combination of ICI and the low dose radiotherapy (LDR), a novel kind of radiotherapy that effectively mobilizes innate and adaptive immunity, is gaining interest among scientists. However, the molecular mechanism that determines the anti-tumor effect of LDR plus ICI remains poorly understood. Methods: The in vivo anti-tumor effect of LDR plus ICI was measured in murine tumor models. The immune response and the alterations in the tumor microenvironment (TME) were measured by flow cytometry and ELISA. Cell viability and the cell death type was assessed using CCK-8 assays. Fluorescent probes and ELISA were used to assess ferroptosis induced by combination therapy in vitro and in vivo. Western blotting and qPCR were conducted to detect the alterations of the Nrf2/HO-1/GPX4 pathway. Furthermore, a phase 1 clinical trial with the combined regimen of LDR and anti-PD-1 antibody in lung cancer patients was performed. Results: The combined regimen of LDR and ICI exhibited a great anti-tumor effect in murine tumor models, promoting immune response and increasing the IFN-γ levels. In vitro data showed that LDR plus ICI could induce ferroptosis in cancer cells by increasing reactive oxygen species (ROS) and MDA levels, promoting Fe2+ accumulation and suppressing GSH. Furthermore, the ferroptosis induced by combination therapy was associated with the suppression of the Nrf2/HO-1/GPX4 antioxidant axis. Importantly, a phase 1 clinical trial with the combination therapy showed promising efficacy in lung cancer patients with chemoimmunotherapy resistance. Conclusion: This research demonstrates that LDR plus ICI induce ferroptosis through the Nrf2/HO-1/GPX4 pathway, resulting in a significant anti-tumor effect, providing a combinatorial strategy to overcome lung cancer. This combination strategy merits further clinical translation and investigation.