SPP1 ⁺ macrophages facilitate the differentiation and maturation of regulatory T cells in tumour-draining lymph nodes of colorectal cancer

Background Tumour-draining lymph nodes (TDLNs) serve as the closest immunological hubs to the primary tumour site in colorectal cancer (CRC). Owing to their unique and irreplaceable anatomical advantage and dynamic immune cell repertoire, TDLNs represent an intensively studied immunological niche and are a potential therapeutic target. Objective This study aimed to map the immune microenvironment of CRC TDLNs to identify targetable immunomodulatory axes. Design Single-cell RNA sequencing (scRNA-seq) was performed on 23 quadruplet-matched samples, including primary tumours, adjacent normal tissues, tumour-free lymph nodes (TFLN) and tumour-invaded lymph nodes (TILN) from seven CRC patients. Mechanistically significant findings were further validated through in vitro functional assays, CRISPR knockout in primary regulatory T cells (Tregs), in vivo murine footpad‐popliteal lymph node metastasis models with lipid nanoparticle-encapsulated siSPP1 (LNP-siSPP1) and/or anti-CD44 mAb, and multiomics analysis of independent CRC cohorts. Results scRNA-seq analysis delineated TILN-specific immunological landscapes dominated by SPP1 + macrophage expansion and active Treg differentiation niches, establishing TILNs as maturation hubs for Tregs versus TFLNs. Mechanistically, SPP1 + macrophages drove Treg differentiation into immunosuppressive CD137 + subsets via the SPP1-CD44 axis, which required NF-κB1 to directly bind the TNFRSF9 promoter. In vivo, LNP-siSPP1 plus anti-CD44 mAb synergistically suppressed lymph node metastasis, reduced CD137 + Tregs and enhanced CD8 + T cell function. Findings were consistently observed across all experimental models and patient-derived datasets. Conclusions SPP1 + macrophages established an immunosuppressive niche in CRC TDLNs by promoting CD137 + Treg maturation via the SPP1-CD44-NF-κB1 axis. Targeting this axis with LNP-siSPP1 and anti-CD44 mAb might overcome Treg-mediated immunosuppression in CRC.