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.

Keywords: circulating; microvesicle.

Fig. 1.

Exosome preparations from cancer patient plasma have low miRNA abundance. (A) Differential centrifugation workflow used to prepare exosome fractions. (B) Representative TEM image of exosome preparations. (Scale bars: Left, 0.5 μm; Right, 200 nm.) (C) Relative abundance of selected biomarker (miR-141, -210, and -375) and nonbiomarker control (miR-16) miRNAs in plasma exosome fractions (blue bars) and postultracentrifugation supernatant (gray bars) across prostate cancer patient plasmas (n = 9). (D) Heat map of relative quantity values (relative percentage of abundance) for miRNAs detected by real-time PCR array profiling of exosome and supernatant fractions derived from prostate cancer patient plasmas (n = 3 pools composed of N = 3 individuals each). miRNAs are ranked (top to bottom) according to relative proportion of each miRNA in exosome vs. supernatant fractions. Consistent with our previous study using healthy donor plasma, a small but notable minority of miRNAs was enriched in the exosome fraction, including the hematopoietic-specific miRNA miR-142–3p.

Fig. 2.

Identification of abundant miRNAs in exosome preparations from diverse sources. (A) Schematic of the workflow used to identify (ID) abundant miRNAs. (B–G) TEM images of exosomes prepared from (B) healthy (donor) plasma, (C) prostate cancer (PCa) patient plasma, (D) healthy donor seminal fluid, (E) dendritic cells (in vitro differentiated Langerhans cells), (F) mast cells (HMC-1 cell line), and (G) ovarian carcinoma (OvCa) cells (2008 cell line). Real-time PCR array profiling results for all detected miRNAs [sorted by abundance as represented by cycle threshold (Ct) on a reverse y axis] are displayed in Right for the corresponding samples in Left. Each bar represents a different miRNA. miRNAs selected for absolute copy number quantification from the top five most abundant (as defined by the lowest Ct values) are indicated by arrows. Primary miRNA profiling data are also presented in Table S4. (Scale bars: B, D, and F, 100 nm; C, E, and G, 200 nm.)

Fig. 3.

Quantification of exosome number, size distribution, and miRNA content. Aliquots of exosome preparations described in Fig. 2 were counted and sized by NTA. In parallel, total RNA was extracted from additional exosome aliquots, and the concentrations of abundant exosomal miRNAs identified in Fig. 2 were determined by real-time PCR. (A) Workflow to determine the size distribution, exosome concentration, and absolute quantification of miRNAs in each exosome sample. (B–G) Exosome size distribution histograms (representing the percentage of total counts found within each 1-nm-sized bin), total particle concentrations, and miRNA concentrations in exosome preparations for (B) healthy (donor) plasma (n = 3 donors), (C) prostate cancer (PCa) patient plasma (n = 3 patients), (D) healthy donor seminal fluid (n = 3 donors), (E) dendritic cells (in vitro differentiated Langerhans cells; n = 3 donors), (F) mast cells (HMC-1 cell line; n = 2 preparative replicates: preparation 1 from serum-free conditioned medium and preparation 2 from exosome-depleted serum containing medium), and (G) ovarian carcinoma (OvCa) cells (2008 cell line; n = 3 preparative replicates). Values represent the concentrations in each exosome preparation (i.e., not the concentration in the crude starting specimen).

Fig. 4.

Abundant miRNAs in exosomes are present at much less than one copy per exosome. Dot plot of miRNA copies per exosome determined for each exosome source (based on data collected like in Fig. 3) and sorted by mean value (bar). Numeric values are presented in Table S5. OvCa, ovarian carcinoma; PCa, prostate cancer.

Fig. 5.

Stoichiometric models for exosome miRNA content. (A) High-occupancy/high-miRNA concentration model, in which the number of molecules of an individual miRNA sequence would far exceed the number of exosomes. (B) High-occupancy/low-miRNA concentration model, in which the concentration of miRNA is lower, but most exosomes contain the miRNA. If the number of exosomes exceeds the number of copies of a given miRNA (as observed in our study), the miRNA molecules may be (C) distributed throughout the population in a low-occupancy/low-miRNA concentration distribution or (D) amassed in rare exosomes in a low-occupancy/high-miRNA concentration distribution.