P06.01 Spatial and single-cell profiling reveals stromal regulation of NK-cell cytotoxicity in gallbladder cancer

Background Gallbladder cancer (GBC) is a highly lethal malignancy with limited responsiveness to immunotherapy. Natural killer (NK) cells are essential innate immune effectors responsible for tumor surveillance; however, how their cytotoxic activity is regulated within the unique tumor microenvironment (TME) of GBC remains largely unknown. Understanding NK-cell dysfunction in GBC may provide new insights into mechanisms of immune escape and therapeutic resistance.Methods We integrated single-cell RNA sequencing and spatial transcriptomic profiling to characterize NK-cell heterogeneity and its cellular and molecular regulation in GBC. CellChat-based cell–cell communication analysis, spatial co-localization assessment, and multiplex immunofluorescence were conducted to identify regulatory interactions and validate spatial associations. Spatial transcriptomics further enabled the identification of an immunosuppressive ecological niche in which NK-cell cytotoxicity was markedly reduced compared with immune-active niches.Results A tumor-associated NK (TaNK) subset exhibiting impaired cytotoxicity was identified in GBC. Myofibroblastic cancer-associated fibroblasts (myCAFs) emerged as dominant regulators of NK-cell dysfunction, interacting strongly with TaNK cells through the COL1A1–LAIR1 ligand–receptor axis. This signaling pathway represents a key mechanistic link connecting stromal remodeling to NK-cell suppression. Spatial transcriptomic mapping and histological validation confirmed the co-localization of myCAFs and TaNK cells, particularly within an immunosuppressive niche enriched in extracellular matrix components and immune-exclusion features. Tumors with high co-infiltration of myCAFs and TaNKs displayed pronounced immune exclusion and were associated with unfavorable immunotherapy outcomes. NK cells residing in the immunosuppressive niche demonstrated markedly reduced cytotoxic gene expression and elevated exhaustion signatures, highlighting niche-specific functional impairment.Conclusions Our study reveals that myCAF-derived COL1A1–LAIR1 signaling plays a crucial role in suppressing NK-cell cytotoxicity and fostering the development of an immunosuppressive ecological niche in GBC. By integrating single-cell and spatial analyses, we delineate a stromal–immune interaction axis that contributes to immune evasion and immunotherapy resistance. These findings provide mechanistic insights into NK-cell dysfunction in GBC and identify potential therapeutic targets for improving immunotherapy efficacy in this highly aggressive cancer. The authors have no relevant financial or non-financial conflicts of interest to disclose.