High-Throughput High-Content Nanoscale Extracellular Vesicle Analysis - PROJECT SUMMARY
There is growing recognition that extracellular vesicles (EVs)¾micrometer- or nanometer-sized lipid
particles containing protein and nucleic acid cargoes (information)¾are highly promising for new
diagnostics/prognostics and experimental therapies. There is, however, a key unmet need for technical
platforms able to solve a fundamental problem confounding the exploitation of EV information in biology and
medicine. That is, because EV populations are naturally complex and thus heterogeneous, it can be
challenging to detect and characterize unique subpopulations-of-interest present only at low abundance in a
sample containing other (normal) EVs from, e.g., a host. In this Phase I project, we will apply a novel
technology platform designed to address the problem of resolving multiple EV subpopulations at the single-
particle level by directly identifying specific surface proteins of dispersed particles, through rapid highly-
multiplexed fluorescence imaging analysis We have chosen to focus on analyzing medically important
categories of EVs called exosomes (here from human cancer cells) and outer membrane vesicles (OMVs; from
bacterial cells). Our goal ultimately is to develop a unique imaging platform for the high-content, high-
throughput analysis of the surface proteins of single exosomes or OMVs present in clinically relevant
specimens.
Briefly, our innovative platform will simultaneously analyze, in one pass, up to 10 known or potential
exosome/OMV surface markers in up to 107 dispersed particles obtained from a biofluid such as blood, saliva,
or stool. Unlike conventional methods, our platform can rapidly: a) analyze diverse EVs; b) simultaneously read
multiple protein surface markers (cargoes) and thus detect subpopulations-of-interest; c) read single particle
protein surface cargoes, greatly raising information yield compared to typical bulk methods producing pooled
cargo data; and d) identify exosome/OMV subpopulations based on unique combinations of biomarkers from
points a-c. We propose three stepwise aims in development of a highly multiplexed, high throughput platform
for nano-scale EV detection: (1) construction of the instrument based on an existing high speed hyperspectral
designed specifically for cells and tissue sections; (2) testing and calibration of the platform using fluorescent
nanoparticles and fluorescently labeled liposomes; and (3) application of the platform to detecting exosomes
from human cancer cell lines or OMVs from Gram-negative and Gram-positive bacteria (represented by E. coli
and S. aureus, respectively).
We anticipate that our platform could become a new tool for EV research; a new diagnostic/prognostic tool
for studying and managing pathologies in which EV analysis is clinically informative; and a novel method for
monitoring normal or aberrant microbiome status.
