OLFM4 orchestrates the immunosuppressive microenvironment and drives gallbladder cancer progression.

PURPOSE: Gallbladder cancer (GBC) is the most common biliary tract malignancy, characterized by complex microenvironment and poor prognosis. Olfactomedin-4 (OLFM4) has been identified as a key regulator of GBC progression. This study aims to comprehensively elucidate the molecular profiling, tumor microenvironment (TME) and underlying mechanisms through which OLFM4 drives GBC progression. METHODS: Single-cell RNA sequencing (scRNA-seq) was performed on tumor tissues and adjacent non-tumor tissues (ANT) from clinical GBC samples. We analyzed scRNA-seq data to characterize the landscape of heterogeneous TME and intricate intercellular communication networks of the single cells within GBC. Findings were validated through in vitro experiments. RESULTS: Compared with ANT, GBC exhibited a distinct remodeling of the tumor microenvironment. Specifically, epithelial cells were markedly enriched in GBC (18.2% vs. 11.6% in ANT), accompanied by a pronounced reduction in endothelial cells (2.4% vs. 9.7%). In parallel, myeloid cells decreased from 16.8% in ANT to 11.5% in GBC, while fibroblasts remained comparably abundant in both conditions (21.1%). Within the OLFM4+epithelium, there was a significant enrichment of genes and pathways related to malignant progression, including signatures driving cell proliferation, stemness maintenance (WNT/β-catenin), and metastasis, as well as crucial molecules involved in TME remodeling (SPP1, TGF-β). Pseudotemporal trajectory analysis revealed an upregulation of OLFM4 expression correlating with disease progression, suggesting its close association with malignant transition. Cell-cell communication analysis identified OLFM4+epithelial cells as central signaling hubs, primarily communicating with stromal and immune cells via the SPP1-CD44 and SPP1-Integrin axes. Importantly, OLFM4 knockdown inhibited cell proliferation, migration, invasion, and gemcitabine chemoresistance in vitro, underscoring its central role in GBC progression. CONCLUSION: These findings offer a comprehensive insight into the atlas of molecular signatures and TME within GBC, identifying OLFM4 as a potential biomarker and therapeutic target for GBC.