Comprehensive Integrated Single‐Cell and Spatial Transcriptomics Unveil the Dynamic Landscape of Betel Nut‐Associated Oral Mucosal Carcinogenesis and Its Tumor Microenvironment

ABSTRACT Betel nut chewing is a major etiological factor for oral squamous cell carcinoma (OSCC), yet its mechanistic underpinnings remain poorly defined. Here, we performed single‐cell RNA sequencing and spatial transcriptomics from six OSCC patients to comprehensively dissect the tumor microenvironment (TME) dynamics and cellular heterogeneity associated with betel nut‐induced oral mucosal carcinogenesis. We identify a fibrotic, immunosuppressive TME characterized by expanded cancer‐associated fibroblasts (CAFs) and B/plasma cells, alongside depletion of cytotoxic T/NK cells and macrophages. CAFs, particularly antigen‐presenting CAFs, are spatially enriched at the invasive front and drive epithelial plasticity and malignant transformation. Notably, we uncover a malignant epithelial subpopulation, LAMC2 + EpiC6, enriched for epithelial–mesenchymal transition (EMT) programs, angiogenesis, and metastasis‐associated pathways, which engaged in extensive crosstalk with CAFs and other nonmalignant components. Clinically, LAMC2 expression was significantly elevated in OSCC tissues from betel nut chewers, and arecoline treatment of OSCC cell lines induced LAMC2 upregulation, EMT, and enhanced migratory and invasive capacities in vitro. Collectively, our study delineates a malignant trajectory of epithelial cell progression, highlighting LAMC2 + EpiC6 as a key aggressive subpopulation orchestrated by EMT‐related transcriptional regulators and extracellular matrix remodeling. These findings offer mechanistic insights and identify potential therapeutic targets to disrupt tumor–stroma interplay and mitigate disease progression.