Effect of irradiation on the killing effect of NK cells in colon cancer through MYB/TIM3 axis.

3133 Background: Natural killer (NK) cells play a crucial role in tumor progression and anti-tumor immunity. However, they often exhibit an exhausted phenotype within the tumor microenvironment (TME), limiting their full cytotoxic potential. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) has emerged as a novel immune checkpoint that is highly expressed on NK cells and suppresses their cytotoxic function. TIM-3 is closely associated with immune evasion and anti-tumor immune tolerance. This study aims to investigate the effects and mechanisms by which radiation modulates NK cell function, providing a foundation for developing strategies that specifically target TIM-3 on NK cells. Methods: First, mRNA high-throughput sequencing, RT-qPCR, and Western blot experiments were used to analyze changes in the expression of related genes in NK92 cells after radiotherapy. The LDH release assay was employed to evaluate the effect of radiation on the viability of NK92 cells. ELISA was conducted to detect changes in the release levels of tumor necrosis factor TNF-α and other factors after radiotherapy. Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) experiments confirmed that the transcription factor MYB mediates radiation-induced regulation of NK cell activation by targeting and binding to the TIM-3 promoter region. A non-contact co-culture system was established, and flow cytometry demonstrated that radiation combined with MYB overexpression enhanced the cytotoxicity of NK cells against tumor cells. A colon cancer mouse model was constructed to evaluate the anti-tumor effect of combining anti-TIM-3 antibodies with radiotherapy. Results: We found that radiation can activate NK92 cells in vitro and enhance TIM-3 expression,promoting the secretion of granzyme B, perforin, TNF-α, IFN-γ, and other cytokines and chemokines that modulate the TME and enhance anti-tumor immune responses. Moreover, the transcription factor MYB inhibits TIM-3 expression by directly binding to the TIM-3 promoter region, mediating the effects of radiation on the TME through NK cell activation. In vivo, the combination of radiotherapy and anti-TIM-3 antibodies effectively controlled the growth of subcutaneously transplanted colon cancer tumors in C57BL/6 mice. However, this combined treatment effect was significantly diminished after NK cells were depleted by the anti-NK1.1 antibody. Conclusions: This study elucidates a novel mechanism by which radiation activates NK cells in the tumor microenvironment through the MYB/TIM-3 pathway. It provides new insights for enhancing the efficacy of radiotherapy and offers a theoretical basis for the potential clinical application of these cells in future research.