To label metabolites, proteins and lipids in CAFs-secreted exosomes, CAFs were cultured in RPMI with labeled 13C3 pyruvate (pyr), 13C5 glutamine (gln), 13C6 leucine (leu), 13C6 lysine (lys), 13C9-phenylalanine (phe) and U-13C6 glucose. After 72h of CAFs cultures, sufficient labeling was observed in metabolites, proteins and lipids contained in exosomes. Supply of metabolites to prostate cancer cells from labeled CDEs were measured under complete or deprivation medium cultures in culture media without labeling. (A) Percentage labeling (mean enrichment) observed in metabolites inside PC3 cells cultured with labeled CDEs. (n=4). Mean enrichment is calculated as . where is number of carbons in the metabolite and is abundance of (M+i) isotopologue (B) Mass fraction of heaviest labeled isotopologues of TCA cycle metabolites enriched by labeled CDEs, in prostate cancer cells cultured under complete or nutrient-deprived (without lys, phe, gln, pyr, leu) unlabeled medium (n=4). (C) Effect of CDEs on PC3 cell viability under deprivation (without lys, phe, gln, pyr, leu) conditions and exosome uptake inhibitors. CDEs rescue loss of PC3 cell proliferation under deprivation medium. CytoD, (1.5 μg/ml), heparin(50 μg/ml), and CQ (chloroquine, 20 μM) inhibited this rescue of viability under deprivation conditions n=10. (D) EIPA(25 μM) inhibited rescue of PC3 viability by CDEs under deprivation conditions, (n≥7). Data information: data are expressed as mean ± SEM,*p<0.05,**p<0.01, ***p<0.001.