(510a) Fractionation of Extracellular Vesicles into Molecularly Homogeneous Subpopulations

The heterogeneity of extracellular
vesicles (EVs) in biofluids is, both, the basis of
liquid biopsy, in which the molecular composition of exosomes secreted by afflicted
cells is used in disease detection and characterization, and the fundamental
challenge. At an early stage of a disease, exosomes secreted by few afflicted
cells form an exceedingly small subpopulation that is difficult to detect and
characterize by downstream molecular analysis. At all conditions, the molecular
signaling intermediated by heterogeneous exosomes is complex. Molecules
transferred by molecularly distinct exosomes may transmit competing signals.
For example, within the population of the exosomes isolated from blood of a
cancer patient, some may contain microRNAs associated with tumor proliferation,
while others may include tumor-suppressing microRNAs. By separating the sample
into subpopulations with the reduced molecular heterogeneity, it will become
possible to repeatably study signaling interactions and identify biologically
active compound associated with specific functional outcomes.

For a specific
case of microRNA miR21 cargo in MCF-7 exosomes, we demonstrate a widely
different abundance of miR21 in MCF7 exosomes along different biophysical
vectors. We show the feasibility of obtaining subpopulations of intact exosomes
with higher uniformity in miR21, including the capability to enrich or deplete
the miR21 expression from the original population.

EVs were
isolated from the growth medium of MCF-7 cells by precipitation. The exosomal
biomarkers confirming the enrichment of exosomes in the isolated population of
EVs was confirmed by the protein biomarker array, Figure 1(c). The nanoparticle
tracking analysis (NTA) was used to verifu that the
isolated vesicles are in the range of hydrodynamic sizes expected for the
exosomes, Figure 1(a). The bilayer morphology
of the vesicles was confirmed by cryo-TEM imaging
which shows the expected 3-4 nm thickness of the membrane, Fig. 1(b).

The
fractionation of EVs by their sizes and buoyant density was performed by
asymmetric and centrifugal field flow fractionations (AFFF
and cFFF), respectively. Figure 2 summarizes the fractionation and the
quantification workflow used in this study. The absolute quantification of the
miR21 copies per vesicle was obtained by digital droplet PCR (ddPCR). The ddPCR
results for the miR21 abundance in different factions are shown in Figure 3. The
same results expressed in miR21 copy number per vesicle units is shown in
Figure 4 for the size fractions and in Figure 5 for mass fractions.

Overall, exosomes with smaller size and mass were found to
be more abundant in miR21. A 10-fold difference was observed between the first
and last fraction after cFFF and after AFFF. A 4-5-fold
difference was observed when comparing the original sample used for field flow
fractionation with the fraction being most enriched with exosomes containing
miR21. The revealed molecular heterogeneity in EVs prior to mass-size
fractionation explains the significant complexity in understanding the
signaling role of EVs in health and disease.