Multiomic and functional validation of ACSL3, a regulator of fatty acid metabolism, as a lymph node metastasis-associated gene in lung adenocarcinoma

This study investigates the role of fatty acid metabolism (FAM)-related genes in lymph node metastasis (LNM) and prognosis of lung adenocarcinoma (LUAD) and elucidates the underlying mechanisms. Transcriptomic and single-cell RNA-seq data from TCGA and GEO were integrated to identify FAM-related genes. Non-negative matrix factorization clustering and univariate Cox regression were applied to develop a FAM-based prognostic risk model (FScore). Associations of FScore with gene mutations and tumor microenvironment features were analyzed. Immunohistochemistry, and functional assays were performed to alidate the role of ACSL3 in LUAD malignancy and lymphangiogenesis. A five-gene FAM-related risk signature (ACSL3, MCAT, NDUFAB1, OLAH, ACSL4) was identified. The derived FScore stratified patient prognosis across multiple independent cohorts, with high FScore linked to significantly worse overall survival. FScore increased progressively with nodal stage (N0 < N1 < N2) and correlated with an immunosuppressive “cold” tumor microenvironment and specific mutation patterns (e.g., low FLG mutation). Single-cell and spatial transcriptomics revealed cell-type–specific FAM activity, predominantly in epithelial, mast, and myeloid cells. ACSL3 was overexpressed in LUAD tissues and served as an independent poor prognostic factor. ACSL3 overexpression elevated intracellular triglyceride and phospholipid levels, upregulated key FAM enzymes (FASN, ACC, ACLY) and the c-Myc/VEGFC axis, promoted proliferation, migration, invasion, and lymphangiogenesis, while suppressing apoptosis. The FScore serves as a robust predictor of LNM and poor prognosis in LUAD. ACSL3 drives lymphatic metastasis via the c-Myc/VEGFC axis, positioning ACSL3 as a potential therapeutic target to suppress LNM in LUAD.