The CXCL9-CXCR3 axis mediates tumor progression and immune checkpoint regulation in colorectal cancer

Tumor-immune interactions in the cancer microenvironment have a major influence on the development of colorectal cancer (CRC), as well as immune evasion by the CRC tumor. Chemokine CXCL9 (which signals through the CXCR3 receptor) has been implicated in the immune recruitment of cells and progression of tumors; however, the role of CXCL9 in CRC is poorly understood. To explore the mechanistic role of CXCL9 in CRC cell proliferation, migration, invasion, survival, and immune modulation, we utilized CRC cell lines HCT116 and HT-29. Knocking down CXCL9 expression using siRNA decreased CRC cell proliferation, EdU incorporation, clonogenic survival, and anchorage-independent growth. The restoration of CXCL9 expression partially reversed these observations. In migration and invasion assays, CXCL9 increased motility and epithelial-mesenchymal transition (EMT) of CRC cells via the regulation of E-cadherin, N-cadherin, and vimentin expression. Silencing CXCL9 expression (due to the CXCL9 siRNA) resulted in increased apoptosis and G0/G1 cell cycle arrest, whereas restoring CXCL9 expression allowed CRC cells to transition to S-phase. Mechanistically, CXCL9 maintained redox homeostasis by preventing accumulation of reactive oxygen species (ROS), and activating pro-survival signaling pathways (AKT, ERK1/2, and NF-κB). Importantly, CXCL9 positively regulated immune checkpoint molecules (PD-L1 and IDO1) at both mRNA and protein levels. In co-culture experiments, CXCL9 promoted CD8⁺ T-cell chemotaxis through CXCR3 and enhanced T-cell-mediated cytotoxicity, effector molecule expression, and pro-inflammatory cytokine secretion under the tested in vitro conditions. Together, these in vitro findings suggest that CXCL9 contributes to CRC cell proliferation, migration, invasion, redox regulation, immune checkpoint-associated signaling, and CD8⁺ T cell responses in simplified co-culture models. The novelty of this study lies in the combined evaluation of tumor-cell phenotypes, PD-L1/IDO1-associated signaling, and CD8⁺ T cell responses within the same CXCL9-modulated CRC cell-line system. Further validation using patient-derived samples, in vivo models, and clinically annotated cohorts is required before broader conclusions regarding the CRC tumor microenvironment or therapeutic targeting can be made.