TIM-3/Galectin-9 Immune Axis in Colorectal Cancer in Relation to KRAS, NRAS, BRAF, PIK3CA, AKT1 Mutations, MSI Status, and the Cytokine Milieu

In this study, we investigated the expression of TIM-3 and Galectin-9 (Gal-9) in colorectal cancer (CRC) and their associations with oncogenic mutations, MSI status, cytokine profiles, and transcriptional data. TIM-3 and Gal-9 protein levels were significantly increased in CRC tissues compared to matched non-tumor margins (<i>p</i> < 0.05 and <i>p</i> < 0.001, respectively). TIM-3 protein concentration was notably higher in PIK3CA-mutated tumors (<i>p</i> < 0.05), while no associations were found with <i>KRAS</i>, <i>NRAS</i>, <i>BRAF</i>, <i>AKT1</i>, or MSI status. Multiplex cytokine profiling revealed strong correlations between TIM-3 and Gal-9 levels and key immunomodulatory pathways, including IL-10, IL-17, and chemokine signaling. We also observed significant associations with cytokine subsets involved in protumor activity and immune regulation. Gene set enrichment analysis (GSEA) demonstrated that high TIM-3 and Gal-9 expression was associated with upregulation of cell cycle-related pathways, and downregulation of immune signatures, such as interferon responses and TNF-α/NFκB signaling. These findings suggest that increased TIM-3 and Gal-9 expression reflects a shift toward proliferative activity and immune suppression in the CRC tumor microenvironment, highlighting their potential as biomarkers of immunoevasive tumor phenotypes, especially in PIK3CA-mutant CRC tumors.