Radiation-Induced Senescence-Metabolic Reprogramming In Oral Cancer-Associated Fibroblasts

Radiation therapy is a cornerstone treatment for oral squamous cell carcinoma (OSCC), but it can induce senescence in cancer-associated fibroblasts (CAFs), potentially altering the tumor microenvironment. This study aimed to establish a radiation-induced senescent fibroblast model and investigate its metabolic reprogramming features using untargeted metabolomics to identify potential therapeutic targets. Primary oral CAFs were isolated from clinical specimens using the tissue block adhesion method and characterized by immunocytochemistry. Cells were irradiated with a cumulative 60Gy dose simulating clinical fractionation to induce senescence, which was validated through multiple markers including DNA damage, SA-β-galactosidase activity, cell cycle arrest, and heterochromatin foci. Untargeted metabolomics was performed on senescent (sCAF) and non-irradiated CAFs, followed by bioinformatics analysis. Irradiated CAFs exhibited classic senescence features, including enlarged morphology, persistent DNA damage as positive γ-H2AX and 53BP1, increased expression of p16INK4A and p21CIP1, elevated SA-β-galactosidase activity, and H3K9me3-positive heterochromatin foci. Metabolomics revealed 125 differentially expressed metabolites (41.6% lipids/lipid-like molecules). KEGG enrichment analysis identified palmitoylcarnitine as a key metabolite involved in 5/ Top20 enriched pathways in sCAFs. MetaboAnalyst further highlighted the significant enrichment of long-chain saturated fatty acid β-oxidation in mitochondria of sCAFs. Correlation analysis between differential metabolites and KEGG pathways revealed that palmitoylcarnitine, an acylcarnitine, was among the top six metabolites most strongly associated with altered metabolic pathways. Radiation-induced senescent CAFs undergo metabolic reprogramming dominated by altered lipid metabolism, notably palmitoylcarnitine-associated pathways, suggesting lipid modulation as a potential target to improve radiotherapy efficacy.