Integrated spatial, single-cell, and in vivo dissection of matrix CAF-driven immune exclusion in colorectal cancer

Background: Fibrotic colorectal cancers (CRCs) are largely microsatellite stable and characterized by desmoplastic stroma that excludes cytotoxic T cells. Cancer-associated fibroblasts (CAFs) are major stromal constituents, yet how specific CAF subsets regulate immune exclusion remains poorly understood. Methods: We integrated orthotopic in vivo tumor models with spatial and single-cell transcriptomic analyses of human and murine CRCs to elucidate the mechanisms by which matrix CAFs modulate the tumor immune microenvironment. Results: Matrix CAFs highly expressed THBS2 , a matricellular protein enriched at the invasive tumor front. In vivo, global or fibroblast-specific Thbs2 deletion disrupted the fibrotic barrier and markedly increased intratumoral CD8⁺ T-cell infiltration. Mechanistically, THBS2 restrained CXCL9/10 production by dendritic cells and macrophages, limiting recruitment of CXCR3⁺ CD8⁺ T cells. Neutralization of CXCL9/10 or blockade of CXCR3 reversed these effects, confirming the central role of the CXCL9/10-CXCR3 chemokine axis in CAF-mediated immune exclusion. Spatial transcriptomic profiling of human CRC further demonstrated that loss of THBS2 in matrix CAF-rich regions correlated with increased proximity between CD8⁺ T cells and myeloid cells and elevated chemokine expression. Despite enhanced infiltration, these CD8⁺ T cells displayed features of exhaustion, rendering tumors more responsive to immune checkpoint blockade. Conclusion: This integrated spatial, single-cell, and in vivo dissection reveals that matrix CAF-derived THBS2 orchestrates immune exclusion in fibrotic CRC via suppression of the CXCL9/10-CXCR3 axis. Targeting THBS2 or its downstream stromal pathways may represent a promising therapeutic strategy to overcome stromal-driven immunotherapy resistance in microsatellite-stable colorectal cancer.