Cells release extracellular vesicles (EVs) that range in size from small exosomes (< 150 nm) to apoptotic fragments (cell sized). These lipid spheres arise from the cell's plasma membrane and are laden with cell-specific surface antigens. Our group has recently shown that acute myeloid leukemia (AML) cells make EVs that can affect their microenvironments within the bone marrow. We have cultured GFP+AML cells and have shown that we can purify exosomes from the media by differential ultracentrifugation to isolate 100kg pellets rich in exosome sized EVs that have greater biologic activity in the marrow bioassay compared with media alone. The objective of this project was to compare proteomic profiles obtained by various EV preparation methods, including ultracentrifugation, to better understand potential ligands involved with EV bioactivity. We compared standard centrifugation and density gradient (Optiprep) approaches with multiparametric high resolution flow cytometry (HRFC) sorting, which has the advantage of simultaneously sorting targeted EVs and spiked negative control nano-particles like 100nm polystyrene beads. Our pilot data suggest that HRFC provides a more specific profile of exosome-related peptides compared with sorted beads, Optiprep fractions, or 100kg pellets obtained by differential centrifugation. A subset of proteins are shared by all three preparation methods. We conclude that HRFC provides a more specific isolate of cell-specific targeted EVs and the benefit of sorting “negative controls” from the same liquid biopsy. In contrast, centrifugation and gradient fractions produce many more false positive peptide “hitch-hikers” that may confound efforts to identify biologically active ligands and biomarkers.
Dr. Morgan is a scientist and anatomic pathologist at Oregon Health & Science University (OHSU). He completed his MD/PhD in Human Genetics at the University of Utah in 2000 and pathology training at Stanford University in 2005. He is an NIH-funded principal investigator and for the past two years has led the OHSU collaboration with BD Biosciences Advanced Flow Cytometry Research Group in San Jose, California to develop a new approach to extracellular vesicle (EV) imaging, quantitation, and isolation called multiplex high resolution flow cytometry (HRFC). By employing cell surface antigen targeted multiplex antibody panels, in conjunction with 100nm FITC-conjugated liposomes and 100nm polystyrene bead controls, his group can simultaneously isolate cell- and size-specific EVs, as well as sort liposomes and beads from the same liquid biopsy samples (e.g plasma, culture media, etc.). Simultaneous isolation of EV targets, liposome controls, and bead controls by HFRC provides an approach to isolate technical “hitch-hikers” that may be false positive peptides associated with nano-sized particles. This novel approach has led to new insights into cell- and size- specific EV-related peptides versus profiles generated by less specific differential centrifugation, density gradient fractions, or affinity capture technologies.