Integrated single-cell and spatial transcriptomics reveal immune landscape and NKT–Th1 signatures in colorectal cancer

Background: Colorectal cancer (CRC) is a prevalent malignancy with high morbidity and mortality. Immunotherapy benefits only a subset of patients, highlighting the need to explore immune-related signatures associated with response. Materials and methods: We analyzed a single-cell RNA sequencing dataset of 27,414 cells from tumor core, edge, and matched normal mucosa to investigate NKT cell heterogeneity. A spatial transcriptomics dataset characterized interactions between NKT and Th1 cells in CRC tissues. Integrating nine CRC cohorts from TCGA and GEO, we assessed associations between NKT cell infiltration and clinical outcomes. NKT and Th1 cell-related genes (NTRG) were identified and integrated into an NTRG score, which was systematically validated across multiple independent CRC cohorts to characterize its associations with survival and immunotherapy response. In addition to multi-database validation using several external transcriptomic datasets, we analyzed somatic mutation profiles, GO enrichment, and drug sensitivity patterns associated with different NTRG score groups. Furthermore, key NTRG genes were biologically validated in clinical CRC tissue specimens using immunohistochemistry. Results: Elevated NKT cell infiltration correlated with improved prognosis and enhanced immunotherapy sensitivity. Spatial analysis revealed NKT and Th1 co-localization mediated by COLLAGEN signaling. The NTRG score quantified tumor-immune properties, and high scores were linked with worse overall survival yet stronger immunotherapy response. Validation confirmed differential NTRG expression between normal and cancerous tissues and across immunotherapy responders and non-responders. Conclusion: NTRG is an exploratory multi-gene signature associated with the immune landscape of colorectal cancer, offering a foundation for future investigation into its relationship with immunotherapy response and tumor microenvironmental organization.