Single-cell transcriptomics uncovers malignant potential of gallbladder adenomyomatosis and identifies PRDX1+ immunosuppressive macrophages in gallbladder carcinoma

Background: While the inflammation-to-carcinoma transition in gallbladder carcinogenesis is well recognized, the molecular pathogenesis of gallbladder adenomyomatosis (GBA), which is clinically associated with both chronic cholecystitis (CCS) and gallbladder carcinoma (GBC), remains poorly understood. Hence, we aimed to evaluate the premalignant potential of GBA and to identify previously unrecognized molecular pathways involved in gallbladder carcinogenesis.

Methods: We first conducted single-cell RNA Sequencing and downstream bioinformatics analyses on samples from nine patients with CCS, GBA, and GBC. Key molecular findings were subsequently validated using publicly available bulk RNA-seq datasets (n = 80) and multiplex immunohistochemistry in an independent cohort (n = 62). Functional validation of tumor microenvironment-associated molecules was performed through a series of in vitro and in vivo assays.

Results: We profiled the transcriptomic landscape of 89 428 single cells and uncovered distinct epithelial and immune ecosystems across different disease stages. A transitional epithelial subset (SPP1-CCL20⁺ EpiC3) and PTGER4+CCL5⁺ CD8⁺T cells were identified in GBA tissues, indicating malignant potential. Notably, PRDX1 was selectively upregulated in the GBC-derived Macro04 macrophages, which displayed strong interactions with exhausted T cells and malignant epithelial cells. Functionally, PRDX1 knockdown in macrophages disrupted mitochondrial oxidative metabolism, leading to metabolic reprogramming and diminished M2 polarization, thereby attenuating tumor cell proliferation, migration, and invasion both in vitro and in vivo. Mechanistically, PRDX1 loss downregulated PPAR-δ and CPT1A, whereas PPAR-δ agonist GW1516 restored CPT1A expression as well as the associated metabolic and polarization defects, supporting a PRDX1/PPAR-δ/CPT1A regulatory axis governing macrophage immunometabolism in GBC.

Conclusions: Our study reveals that GBA harbors malignant potential at both epithelial and immune microenvironmental levels. The expression of CCL20, CCL5, and PRDX1 may serve as molecular markers for stratifying high-risk GBA, while PRDX1 represents a promising therapeutic target for reprogramming the tumor immune microenvironment in GBC.

PRDX1; gallbladder adenomyomatosis; gallbladder carcinoma; macrophage; single-cell RNA sequencing; tumor microenvironment.