Spatial organization of stromal subtypes stratifies colorectal cancer patients and predicts clinical outcomes

Tumor-associated stromal (TAS) cells within the tumor microenvironment (TME) exhibit marked transcriptional heterogeneity. However, the spatial organization of TAS subpopulations and their clinical relevance in human colorectal cancer (CRC) are incompletely understood. Using single-cell RNA sequencing (scRNA-seq) and multiplex imaging, we identified multiple TAS subpopulations with distinct intratumoral and peritumoral abundance, and found that CRC patients enriched in FAP⁺ TAS cells and CD73⁺ tumor cells had the lowest disease-free survival (DFS). NGFR⁺ stromal cells localized predominantly within tertiary lymphoid structures (TLS), which could be further classified into NGFR⁺ organized TLS, NGFR⁺ unorganized TLS and NGFR^- TLS based on the spatial distribution of NGFR⁺ stromal cells. CRC patients enriched for NGFR⁺ organized TLS exhibited improved DFS. SCENIC analysis revealed HAND2, IKZF2 and SOX10 as transcriptional regulators of human NGFR⁺ antigen-presenting TAS cells, and they frequently expressed enteric glial cell markers SOX10, PLP1, CDH19 and NCAM1. In summary, our study demonstrates that the frequency and spatial distribution of TAS subpopulations, particularly NGFR⁺ TAS organization within TLS, varies between CRC patients and correlates with DFS and distinct changes in the TME.