Role of VISTA/TIM3 heterodimerization and macrophage metabolic reprogramming in microsatellite stable colorectal cancer.

e15586 Background: Immune checkpoint inhibitors (ICIs), particularly anti-PD-1 antibodies, represent a major paradigm shift in cancer management. However, microsatellite stable (MSS) colorectal cancer (CRC), which accounts for over 95% of CRC cases, exhibits a dismal response rate of less than 5%. Increasing evidence suggests that heterogeneity and complex crosstalk among immune checkpoints limit the efficacy of monotherapy strategies. This study aims to break through current therapeutic bottlenecks by systematically profiling the immune checkpoint landscape of MSS CRC to identify core drivers of immune tolerance and explore novel intervention strategies. Methods: We collected tumor tissues from a multicenter cohort of treatment-naive MSS CRC patients. The immune checkpoint landscape was constructed using flow cytometry, multiplex immunofluorescence, and integrated analysis of public single-cell RNA sequencing and TCGA datasets. To elucidate molecular mechanisms and metabolic characteristics, we employed tumor-bearing mouse models combined with metabolomics, co-immunoprecipitation, and site-directed mutagenesis assays. Results: We identified four distinct immune checkpoint patterns in MSS CRC, among which the VISTA/TIM3 co-expression pattern (14.6%) was most strongly associated with poor prognosis and CD8 + T cell exhaustion. Clinical validation confirmed a specific enrichment of VISTA + TIM3 + tumor-associated macrophages (TAMs) in the tumor microenvironment of approximately 14% of patients (n = 35), where these cells constituted over 50% of the macrophage population. Notably, patients with high VISTA + TIM3 + TAM infiltration exhibited significantly shorter overall survival compared to those with low infiltration ( P = 0.019). Mechanistically, we discovered that VISTA and TIM3 do not function independently on macrophages but physically interact to form a heterodimer complex. This complex induces metabolic rewiring to favor succinate accumulation, thereby promoting protein succinylation and sustaining an immunosuppressive macrophage phenotype. In vivo and in vitro experiments demonstrated that disrupting the VISTA-TIM3 interaction or combining therapy with succinylation inhibitors significantly reversed the immunosuppressive function of VISTA + TIM3 + TAMs and restored sensitivity to anti-PD-1 therapy. Conclusions: This study is the first to reveal that "metabolic-immune" crosstalk mediated by the VISTA-TIM3 heterodimer is a key mechanism driving immune tolerance in MSS CRC. Targeting VISTA + TIM3 + TAMs and their downstream succinate metabolic pathway represents a promising breakthrough strategy to overcome immunotherapy resistance in MSS CRC.