Decoding Hypoxia-Induced Metabolomic Changes in Breast Cancer EVs and Their Functional Effects on Cancer Cells

This study utilized an untargeted metabolomic approach using LC-MS/MS to analyze the metabolic alterations in extracellular vesicles (EVs) secreted by breast cancer cells under hypoxic conditions. EVs were isolated using an established ultracentrifugation protocol, and their homogeneity was assessed through TEM, SEM, NTA, and immunoblotting. Metabolic extracts from hypoxic EVs were found to enhance breast cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT), underscoring their role in tumor progression. Metabolomic analysis identified over 400 annotated metabolites, with univariate statistical analysis (p < 0.05, Log₂FC > 0.58/< -0.58) revealing 48 significantly altered metabolites in MDA-MB-231- and MCF-7-derived EVs, respectively, indicating substantial metabolic reprogramming under hypoxia. Integrated univariate and multivariate analyses (p < 0.05, Log₂FC > 0.58/< -0.58, VIP > 1) identified key upregulated metabolites, including riboflavin, 5,6-epoxy-8,11,14-eicosatrienoic acid, and furandicarboxylic acid, in hypoxic EVs from both cell lines. Additionally, N-acetyl-l-methionine, 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine, and 5,10-methenyltetrahydrofolic acid were identified as critical metabolites potentially influencing cell proliferation, DNA damage, and epigenetic regulation. Metabolic pathway analysis highlighted heightened activity in aromatic amino acid and riboflavin metabolism. These findings reveal significant metabolic alterations in hypoxic EVs, providing insights into their potential role in driving tumor progression.