Abstract

Cancer-associated fibroblasts (CAFs) constitute a predominant cellular component of the tumor microenvironment. Radiotherapy (RT) induces tumor cell death and modulates CAF biology, with irradiated CAFs potentially influencing tumor cell malignant behavior. In this study, primary CAFs were irradiated, and their supernatant was applied to A549 lung cancer cells to assess proliferation and migration. Supernatant metabolomics and CAF transcriptomics were performed to uncover paracrine mechanisms. Results showed that supernatant from irradiated CAFs significantly enhanced A549 cell proliferation and migration. Metabolomics identified 53 differentially expressed metabolites (DEMs), including glycocholic acid, which exhibited dose-dependent biphasic effects on cell viability. Pathway enrichment analysis revealed 15 (positive-mode) and 9 (negative-mode) enriched pathways. Transcriptomics identified 1,485 upregulated and 1,662 downregulated differentially expressed genes (DEGs) in irradiated CAFs. Upregulated DEGs were enriched in p53, Hippo and Wnt pathways, while downregulated DEGs were enriched in extracellular matrix-receptor interaction, focal adhesion and cell adhesion molecules. Collectively, RT reprograms the CAF secretome and transcriptome, enhancing pro-tumorigenic paracrine activity toward lung cancer cells. Limitations include lack of in vivo validation, and future work will focus on functional validation in preclinical models.