Multi-omics integration identifies PON2 as a prognostic biomarker and mediator of tumor microenvironment remodeling...

Gallbladder cancer (GBC) is an aggressive biliary tract malignancy with limited therapeutic options and poor prognosis. Tumor microenvironment (TME) remodeling contributes substantially to GBC progression; however, the molecular drivers linking malignant epithelial programs to microenvironmental alterations remain poorly defined. This study aimed to characterize the cellular ecosystem of GBC and identify clinically relevant molecular targets associated with tumor progression and TME remodeling.

Single-cell RNA sequencing (RNA-seq) data from 10 GBC tissues and 4 matched adjacent normal tissues were analyzed to define the cellular landscape of GBC. Unsupervised clustering, cell-type annotation, and copy number variation analysis were used to characterize TME composition and identify malignant epithelial populations. Integrated analyses of single-cell transcriptomic, bulk RNA-seq, and tumor proteomic datasets were performed to screen candidate prognostic biomarkers. The clinical relevance of the candidate gene was evaluated by survival analysis and validated in clinical GBC specimens using immunohistochemistry and quantitative real-time polymerase chain reaction. Functional assays were conducted in GBC cell lines to assess its effects on tumor cell proliferation and migration. Cell-cell communication analysis was further used to explore potential interactions between malignant epithelial cells and non-malignant microenvironmental components.

Single-cell profiling revealed substantial cellular heterogeneity within the GBC microenvironment and identified malignant epithelial clones with distinct transcriptional programs. Multi-omics integration identified paraoxonase 2 (PON2) as a potential prognostic biomarker in GBC. Elevated PON2 expression was validated in clinical GBC specimens and was associated with worse overall survival. In vitro knockdown of PON2 significantly suppressed the proliferation and migration of GBC cells. Further single-cell analysis showed that the PON2-high malignant epithelial state was accompanied by remodeling of endothelial and stromal subpopulations. Cell-cell communication analysis suggested that PON2-high malignant epithelial cells exhibited enhanced interactions with endothelial cells, fibroblasts, and pericytes through specific ligand-receptor networks, indicating a potential role of PON2 in shaping a pro-tumorigenic microenvironment.

This multi-omics study identifies PON2 as a clinically relevant biomarker associated with GBC progression and TME remodeling. PON2 may serve as a prognostic indicator and potential therapeutic target, providing new insights into TME-directed translational strategies for GBC.