Metabolomic Profiling of Extracellular Vesicles and Alternative Normalization Methods Reveal Enriched Metabolites and Strategies to Study Prostate Cancer-Related Changes

Abstract

Body fluids are a rich source of extracellular vesicles (EVs), which carry cargo derived from the secreting cells. So far, biomarkers for pathological conditions have been mainly searched from their protein, (mi)RNA, DNA and lipid cargo. Here, we explored the small molecule metabolites from urinary and platelet EVs relative to their matched source samples. As a proof-of-concept study of intra-EV metabolites, we compared alternative normalization methods to profile urinary EVs from prostate cancer patients before and after prostatectomy and from healthy controls.Methods: We employed targeted ultra-performance liquid chromatography-tandem mass spectrometry to profile over 100 metabolites in the isolated EVs, original urine samples and platelets. We determined the enrichment of the metabolites in the EVs and analyzed their subcellular origin, pathways and relevant enzymes or transporters through data base searches. EV- and urine-derived factors and ratios between metabolites were tested for normalization of the metabolomics data.Results: Approximately 1 x 1010 EVs were sufficient for detection of metabolite profiles from EVs. The profiles of the urinary and platelet EVs overlapped with each other and with those of the source materials, but they also contained unique metabolites. The EVs enriched a selection of cytosolic metabolites including members from the nucleotide and spermidine pathways, which linked to a number of EV-resident enzymes or transporters. Analysis of the urinary EVs from the patients indicated that the levels of glucuronate, D-ribose 5-phosphate and isobutyryl-L-carnitine were 2-26-fold lower in all pre-prostatectomy samples compared to the healthy control and post-prostatectomy samples (p < 0.05). These changes were only detected from EVs by normalization to EV-derived factors or with metabolite ratios, and not from the original urine samples.Conclusions: Our results suggest that metabolite analysis of EVs from different samples is feasible using a high-throughput platform and relatively small amount of sample material. With the knowledge about the specific enrichment of metabolites and normalization methods, EV metabolomics could be used to gain novel biomarker data not revealed by the analysis of the original EV source materials.

Metabolic alterations in urine extracellular vesicles are associated to prostate cancer pathogenesis and progression

“…The composition of the urine EVs analysed in this study varies in the abundance of phosphatidylcholine species that are major constituents of membranes. In particular we found reduced abundance of PCs in the EVs from PCa samples, in agreement with previously reported by Puhka and coworkers [33]. This result along with studies reporting increased abundance of PC in PCa tissue [34] could suggest that less PC-containing structures, like membrane vesicles are secreted to the extracellular environment.…”

“…In agreement with this alteration, we found increased levels of acyl carnitines in the urinary EVs from PCa patients. This association of differential levels of carnitines on PCa EVs with a metabolic shifting towards β-oxidation of fatty acids has already been proposed by Puhka and coworkers [33]. …”

Urinary biomarkers of prostate cancer

“…The sensitivity and specificity for the detection of prostate cancer by combining these metabolites were 88% and 92%, respectively, although validation studies in independent cohorts were necessary. Other groups also reported that the levels of glucuronate, d ‐ribose 5‐phosphate and isobutyryl‐ l ‐carnitine were significantly lower in urinary exosomes from three patients with prostate cancer compared with three healthy men aged <35 years …”

A new role for extracellular vesicles: how small vesicles can feed tumors' big appetite

“…EVs also contain miRNA that are known to target proteins implicated in metabolism (81) and recent studies point out the presence of metabolites in EVs (13,82,83). Moreover, enzymes carried by hepatocyte vesicles can modify serum metabolome, and especially metabolites involved in oxidative stress (84,85).…”