Colorectal cancer (CRC), the third most common cancer globally, presents challenges like metastasis, recurrence, and therapy resistance. Natural Killer (NK) cells are vital for anti-tumor immunity but become dysfunctional in the tumor microenvironment (TME). This study explored radiation’s mechanism regulating NK cell function to support combined radiotherapy (RT) and T cell immunoglobulin domain and mucin domain-3 (TIM-3) targeting in CRC. We examined the effects of radiation on NK cell activation, cytokine secretion, and modulation of the TME. The expression of TIM-3 and MYB was analyzed in NK-92 cells, patient-derived peripheral blood mononuclear cells (PBMCs), and clinical tumor specimens using flow cytometry, immunohistochemistry (IHC) and multiplex immunofluorescence (mIF) staining. TIM-3 knockdown and MYB overexpression were performed to assess their regulatory roles in NK cell function, and the direct binding of MYB to the TIM-3 promoter was validated by dual-luciferase reporter and ChIP assays. In vivo efficacy was tested using a subcutaneous CRC mouse model treated with RT, anti-TIM-3 antibody, and/or NK cell depletion, followed by IHC and mIF analysis of mouse and human CRC tissues. Radiation augmented NK cell activity, enhancing cytokine secretion and modulating the TME. However, radiation paradoxically upregulated the inhibitory receptor TIM-3 while suppressing MYB expression in NK cells, as confirmed in NK-92 cells, primary human NK cells, and clinical tumor specimens. TIM-3 knockdown enhanced NK cell activation and cytokine production following radiation, whereas MYB overexpression potentiated radiation-induced NK cell activation by directly binding to the TIM-3 promoter and suppressing its expression. In vivo, combining RT with anti-TIM-3 antibody synergistically suppressed tumor growth in an NK cell-dependent manner and enhanced intratumoral infiltration of NK and CD8+ T cells. mIF of mouse and human tumor tissues confirmed decreased MYB, increased TIM-3, and enhanced NK cell infiltration following RT. Flow cytometry of irradiated human PBMCs further recapitulated the TIM-3 upregulation and MYB downregulation in primary NK cells. Irradiation activates NK cells but concurrently induces TIM-3 via MYB suppression. Blocking TIM-3 synergizes with RT to promote NK cell immune activation and inhibit CRC progression. These findings provide a mechanistic basis for combined RT and TIM-3 immunotherapy.
Tao et al. (Fri,) studied this question.