Single-cell dissection of remodeled inflammatory ecosystem in primary and metastatic gallbladder carcinoma

Gallbladder carcinoma (GBC) is the most common biliary tract malignancy with the lowest survival rate, primarily arising from chronic inflammation. To better characterize the progression from inflammation to cancer to metastasis, we performed single-cell RNA sequencing across samples of 6 chronic cholecystitis, 12 treatment-naive GBCs, and 6 matched metastases. Benign epithelial cells from inflamed gallbladders displayed resting, immune-regulating, and gastrointestinal metaplastic phenotypes. A small amount of PLA2G2A+ epithelial cells with copy number variation were identified from a histologically benign sample. We validated significant overexpression of PLA2G2A across in situ GBCs, together with increased proliferation and cancer stemness in PLA2G2A-overexpressing GBC cells, indicating an important role for PLA2G2A during early carcinogenesis. Malignant epithelial cells displayed pervasive cancer hallmarks and cellular plasticity, differentiating into metaplastic, inflammatory, and mesenchymal subtypes with distinct transcriptomic, genomic, and prognostic patterns. Chronic cholecystitis led to an adapted microenvironment characterized by MDSC-like macrophages, CD8+ TRM cells, and CCL2+ immunity-regulating fibroblasts. By contrast, GBC instigated an aggressive and immunosuppressive microenvironment, featured by tumor-associated macrophages, Treg cells, CD8+ TEX cells, and STMN1+ tumor-promoting fibroblasts. Single-cell and bulk RNA-seq profiles consistently showed a more suppressive immune milieu for GBCs with inflammatory epithelial signatures, coupled with strengthened epithelial-immune crosstalk. We further pinpointed a subset of senescence-like fibroblasts (FN1+TGM2+) preferentially enriched in metastatic lesions, which promoted GBC migration and invasion via their secretory phenotype. Collectively, this study provides comprehensive insights into epithelial and microenvironmental reprogramming throughout cholecystitis-propelled carcinogenesis and metastasis, laying a new foundation for the precision therapy of GBC.