Integrated spatial and single-cell transcriptomics uncover IFI6⁺ regulatory T-cells and SPP1⁺ macrophages immunosuppressive niches in colorectal cancer

Abstract Background In colorectal cancer (CRC), tumor-immune interactions are critical drivers of tumorigenesis, immune evasion, and therapeutic response. Therefore, a deeper spatially resolved understanding of these interactions is urgently needed. Methods Spatial transcriptomics (ST) of CRC and matched normal samples was used to characterize cell populations and gene expression patterns associated with tumorigenesis and immune infiltration. An additional 41 publicly available ST slides, single-cell transcriptomics and bulk transcriptomics datasets from 472 and 617 CRC patients, respectively, were analyzed to validate the findings. Results Cancer regions exhibited profound molecular alterations with increased oncogenic pathways, immunosuppressors, and tryptophan deprivation via KYNU-AHR axis, inducing the macrophage attractant CXCL5, contributing to immune evasion. In tumor boundaries, SPP1⁺ macrophages fostered an immunosuppressive microenvironment with regulatory T-cells (Tregs) recruited via CCL18-CCR8 and CCL22-CCR4. SIT1 emerged as a potential anti-tumor T-cells suppressor within tertiary lymphoid structures, suggesting a novel immune regulator. We also uncovered a previously uncharacterized population of tumor-infiltrating IFI6⁺ Tregs associated with unfavorable prognosis and therapy response, defined by elevated interferon-related genes and LGALS9, a potential immune checkpoint target. Conclusion This study uncovered previously unrecognized immunosuppressive cell populations and key immune regulators that advance understanding of CRC immune evasion and highlight potential targets for personalized immunotherapy.