TIM3 + tumor associated M2 macrophages impair antitumor T cell immunity and promote gastric cancer progression and peritoneal metastasis

Background : Peritoneal metastases (PM) are the leading cause of cancer-related death in gastric cancer (GC), with typical survival of <9 months and no effective targeted or immune therapies available. T-cell immunoglobulin and mucin domain-3 (TIM3) was initially identified on the surface of T helper 1 (Th1) cells and cytotoxic (CTL) lymphocytes as an immunosuppressive molecule that interacts with its ligands (GAL-9, CEACAM1, HMGB1, and PtdSer), and its expression is associated with advanced cancer stage. However, its functional role in promoting GC-associated PM (GCPM) and its clinical relevance in advanced GC remain unclear. Methods: multiple profiling and functional assays including scRNA-seq, CyTOF, qRT-PCR, cytokine arrays, kinase arrays, ELISA, and Western blotting were used to evaluate gene expression and secretion. TIM3 was genetically ablated using CRISPR/Cas9 in in macrophages to determine its functional role in tumor cells and T cells by co-culture system. In vivo , genetic and pharmacologic inhibition of TIM3 in macrophages co-innovation with tumor cells were tested in TIM3 high vs TIM3 KO macrophages on tumor growth in patient-derived xenograft (PDX) and patient-derived orthotopic (PDO) models and in the KP-luc2 syngeneic GC model. Results: We demonstrate that TIM3 and its ligands increased along the GC continuum and are associated with poor survival. Integrated omics analyses, multi-flow and co-immunofluorescent staining revealed that TIM3 is highly enriched in CD163⁺ tumor associated M2 immunosuppressive macrophages, which significantly increased tumor cell invasion in vitro , and promoted tumor growth in vivo. TIM3 depletion in macrophages reduced tumor cell malignant phenotypes and enhanced T-cell immunity from PBMCs or CD45⁺ immune cells isolated from malignant ascites in co-culture assays. Cytokine and kinase array analyses showed that TIM3 depletion in macrophages reduced production of key cytokines and chemokines from M2 macrophages. We further identified the RSK/CCL20/CCR6 axis as essential for the pro-tumor activity of TIM3⁺ macrophages. Finally, we show that TIM3 blockade or genetic knockout exhibits strong antitumor effects, especially when combined with anti-PD-1/PD-L1 immunotherapy or mitomycin C (MMC) chemotherapy. Together, these findings reveal a critical role for TIM3 in tumor associated macrophages and highlight the potential of TIM3 targeted immune therapy in GC patients with PM. Conclusion: TIM3 is highly enriched in tumor associated macrophages in GC with PM and plays a pivotal role in promoting GC progression and suppressing T-cell function. Thus, targeting TIM3 especially in combination with anti-PD1/PD-L1 or chemotherapy MMC may provide a novel therapeutic strategy for advanced GC patients with PM.