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. 2025 Jul 14;26(14):6735.
doi: 10.3390/ijms26146735.

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

Błażej Ochman  1 , Anna Kot  1 , Sylwia Mielcarska  1 , Agnieszka Kula  2 , Miriam Dawidowicz  2 , Dorota Hudy  1 , Monika Szrot  3 , Jerzy Piecuch  3 , Dariusz Waniczek  2 , Zenon Czuba  4 , Elżbieta Świętochowska  1
Affiliations

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

Błażej Ochman et al. Int J Mol Sci. .

Abstract

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 (p < 0.05 and p < 0.001, respectively). TIM-3 protein concentration was notably higher in PIK3CA-mutated tumors (p < 0.05), while no associations were found with KRAS, NRAS, BRAF, AKT1, 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.

Keywords: BRAF; Galectin-9; KRAS; NRAS; PIK3CA; TIM-3; colorectal neoplasms; microsatellite instability (MSI); tumor immune microenvironment.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(A) TIM-3 protein expression in CRC tissues versus adjacent surgical margins. Wilcoxon signed-rank test (p < 0.001). (B) Gal-9 protein expression in CRC tissues and matched margins. Wilcoxon signed-rank test indicated a highly significant difference, with a p-value < 0.001. All data were subjected to log10 transformation prior to analysis.
Figure 2
Figure 2
TIM-3 protein concentration according to PIK3CA mutation. Density plot shows case counts (n) and significance level determined by the U–Mann–Whitney U test.
Figure 3
Figure 3
Hallmark gene set enrichment analysis (GSEA) comparing high vs. low expression of TIM-3 in CRC tissues.
Figure 4
Figure 4
Hallmark gene set enrichment analysis (GSEA) comparing high vs. low expression of TIM-3 in CRC tissues.
See this image and copyright information in PMC

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