Fingerprinting Cancer via Multiparameter Extracellular Vesicle Analysis

Project Summary Electronic BioSciences (EBS) proposes, in collaboration with Distinguished Professor Henry White at the University of Utah, to develop a nanopore-based extracellular vesicle (EV) characterization system capable of high-resolution, single-vesicle, solution-based characterization, sorting, and isolation. EVs are nanometer-scale, cell-derived vesicles that are produced by nearly all mammalian cells under normal physiological conditions and with increased production rates for various disease states (e.g. cancers, neurological diseases, psychiatric disorders, etc.). EVs can be found in most biological fluids and contain proteins, nucleic acids, and lipid molecules that are distinctly dissimilar to the parent cell’s cytoplasmic contents, indicating that exosome loading is not a diffusive or unregulated process. Furthermore, EVs facilitate intercellular communication by transferring these contents into recipient cells to alter the target cells’ phenotype and function. Thus, in addition to their general physiological roles, EVs have important implications in disease pathogenesis, and significant potential as therapeutic targets and drug carriers. Consequently, this has spurred a great deal of interest in new methods to not only characterize the physical properties of EVs (size, zeta potential, surface markers, nucleic acid cargo, and concentration), but increased interest in developing new techniques that have the ability to efficiently and accurately sort/isolate EVs such that the contents/cargo can be profiled in a subpopulation-specific manner. Advances in the areas of EV characterization and sorting/isolation would greatly assist the efforts to understand the general role of EVs in human health, and also benefit efforts aimed at utilizing EVs in molecular diagnostics, biomarker identification, and targeted therapies for cancer. The EV characterization and sorting system that EBS and Professor Henry White will be developing during this program will specifically enable single-particle assessments of an EV sample’s true distribution of size, zeta potential, density of fluorescently tagged surface markers, and nucleic acid cargo, along with the ability to isolate/collect specific EV subpopulations with tightly defined biophysical parameters, all on a single platform. The feasibility of the proposed system will be demonstrated by building a prototype platform and showing its ability to characterize, sort and isolate EV samples. The resulting system will have an ease-of-use, throughput, and price point similar to flow cytometry, dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), or single-pass resistive-pulse techniques, with a resolution comparable to microscopy techniques, e.g. scanning or tunneling electron microscopy (SEM or TEM, respectively) or atomic force microscopy (AFM), yielding a state-of-the-art instrument which will directly enable and advance the field of EV diagnostics.Project Narrative Extracellular vesicles (a subset called exosomes in particular) are emerging as powerful agents in altering cell phenotypes, especially in tumorigenesis and cancer progression, via their protein, lipid, and nucleic acid cargo. They are widely distributed and rich in biomarkers making them ideal targets for “liquid biopsy” diagnostics, but their small size and heterogeneity places them at the limits of current characterization and sorting technologies. In response to this urgent need, EBS proposes to develop a nanopore-based, high-resolution extracellular vesicle characterization system capable of isolating subpopulations of extracellular vesicles based upon multiple biophysical properties simultaneously to enable cancer diagnostics and monitoring.