Carbon ion irradiation combined with PD-1 inhibitor trigger abscopal effect in Lewis lung cancer via a threshold dose

Abstract

Background and purpose Carbon ion is radiobiologically more effective than photons and are beneficial for treating radioresistant tumors, several animal experiment with tumor-bearing suggest that carbon ion beam irradiation in combination with immunotherapy yields better results, especially in controlling distant metastases, which suggests that carbon ion beam induces a different anti-tumor immune response than photon beam, more complex molecular mechanisms need to be further explored. We conduct this in vivo and in vitro experiment was to investigate the radio-immune effects and its mechanism of different LET rays combined PD-1 inhibitors.Methods and Materials: Lewis lung adenocarcinoma cells and C57BL/6 mice were stablished tumor-bearing mouse model, the irradiated tumor located on the left back and the non-irradiated tumor located on the right hind limb. The left back tumor was irradiated with 10Gy X-ray or carbon ion beam, combined with or without PD-1 inhibitor. Immunohistochemistry was used to detect the expression of immunogenicity-related molecules and the infiltration of CD8 + T cells in tumor tissues. ELISA was used to detect the changes of IFN-β in mouse serum, and flow cytometry was used to detect the changes of CD8 + T cells in mouse peripheral blood. Lewis cells were irradiated with different doses of X-ray and carbon ion. The changes of TREX1, PD-L1, and IFN-β in mRNA and protein levels were detected with RT-PCR or Western blot respectively. TREX1 knockdown were constructed by siRNA transfection and irradiated with different rays. Changes in cell proliferation, viability, apoptosis rate were detected using CCK8 assay, BrdU assay, and flow cytometry.Results X-ray or carbon ion combined with αPD-1 had a significant inhibitory effect on bilateral tumors, especially carbon ion in non-irradiated side tumor(p < 0.05). 10 Gy carbon ion irradiation enhanced the proportion of infiltrating CD8 + T cells and the expression level of IFN-β in irradiated side tumor, however, it also increased PD-L1 and TREX1 expression level. Lewis cell in vitro experiment further confirmed that both X-ray and carbon ion irradiation can up-regulate the expression levels of PD-L1 and TREX1 with dose-dependent in tumor, especially the trend of up-regulation TREX1 is more obvious at a higher dose in carbon ions, i.e 8 or 10Gy, while the level of IFN-βis decreased. By gene silencing TREX1, IFN-βlevels were also significantly increased under hypofractioned dose with carbon ion irradiation.Conclusions The combination of X-ray or carbon ion irradiation and PD-1 inhibitors produce better anti-tumor effect and trigger abscopal effect on Lewis lung adenocarcinoma bearing mice through enhanced tumor immunogenicity and increased CD8 + T infiltration in TME via a threshold dose. TREX1 could serve as a prognostic marker and potential target of immunotherapy.