Quantitative and stoichiometric analysis of the microRNA content of exosomes

Abstract

Exosomes have been proposed as vehicles for microRNA (miRNA) -based intercellular communication and a source of miRNA biomarkers in bodily fluids. Although exosome preparations contain miRNAs, a quantitative analysis of their abundance and stoichiometry is lacking. In the course of studying cancer-associated extracellular miRNAs in patient blood samples, we found that exosome fractions contained a small minority of the miRNA content of plasma. This low yield prompted us to perform a more quantitative assessment of the relationship between miRNAs and exosomes using a stoichiometric approach. We quantified both the number of exosomes and the number of miRNA molecules in replicate samples that were isolated from five diverse sources (i.e., plasma, seminal fluid, dendritic cells, mast cells, and ovarian cancer cells). Regardless of the source, on average, there was far less than one molecule of a given miRNA per exosome, even for the most abundant miRNAs in exosome preparations (mean ± SD across six exosome sources: 0.00825 ± 0.02 miRNA molecules/exosome). Thus, if miRNAs were distributed homogenously across the exosome population, on average, over 100 exosomes would need to be examined to observe one copy of a given abundant miRNA. This stoichiometry of miRNAs and exosomes suggests that most individual exosomes in standard preparations do not carry biologically significant numbers of miRNAs and are, therefore, individually unlikely to be functional as vehicles for miRNA-based communication. We propose revised models to reconcile the exosome-mediated, miRNA-based intercellular communication hypothesis with the observed stoichiometry of miRNAs associated with exosomes.microvesicle | circulating

Therapeutic and diagnostic applications of extracellular vesicles

“…Unfortunately, the EVs used in this study were not able to escape the endolysosomal degradation pathway and hence failed to functionally deliver the siRNA in contrast to anionic fusogenic liposomes that were equally loaded with chol-siRNA. Moreover, the endogenously present miRNAs were not able to silence their respective target proteins which is in accordance with recent reports describing that (1) even the most abundant miRNAs found in EVs are secreted at a (low) ratio of 1 molecule per 100 vesicles [46,177] and (2) internalized EVs are typically trafficked toward the lysosomes [140,178]. Although this particular combination of EVs and recipient cells did not lead to successful EV-mediated drug delivery [176], it does not invalidate the concept of EVs as drug carriers as their interaction with cells might be highly specific.…”

Selective release of circRNAs in platelet‐derived extracellular vesicles

“…These low read numbers might reflect the fact that circRNAs exist in vesicles only in very low copy numbers, as reported for miRNAs in exosomes [32]. …”

Potential relevance of microRNAs in inter-species epigenetic communication, and implications for disease pathogenesis

“…However, both miR-375 and miR-200c might also affect exosome formation via exocytotic/endocytotic pathways, 42,48,49 and the required amount of miRNAs for biological activity is also intensively debated. 32,50 It would be necessary to develop models that are more physiological, e.g. by studying the transfer of labeled milk miRNAs in healthy animals.…”

“…23,31 Moreover, it is debated whether miRNAs are really included in membrane vesicles, as this might also be related to analytic problems. 23 In very recent studies, most exosomes have turned out to lack miRNA, 23,32,33 and the miRNA copy number in individual exosomes appears to be very low. 32 These findings argue against the biological activity of extracellular miRNA.…”

“…23 In very recent studies, most exosomes have turned out to lack miRNA, 23,32,33 and the miRNA copy number in individual exosomes appears to be very low. 32 These findings argue against the biological activity of extracellular miRNA. From a diagnostic point of view, however, even if only representing cellular debris, extracellular miRNAs are relevant as they can be exploited as minimally invasive biomarkers in different pathological conditions.…”