A comprehensive analysis of supermere, exomere, and extracellular vesicle isolation and cargo in colorectal cancer

Biofluids contain a heterogeneous mixture of extracellular vesicles and non-vesicular nanoparticles (including exomeres and supermeres) that transport a diverse array of proteins, RNA, and lipids. Our previous efforts to characterize the contents of these carriers in colorectal cancer relied on 2D culture systems requiring large-scale setups and time-consuming ultracentrifugation-based isolation. To streamline this process, we have combined 3D hollow-fiber bioreactor production and fast-protein liquid chromatography-based size-exclusion chromatography. Here, we compare the impact of culture methods and purification strategies on small extracellular vesicle, exomere and supermere cargo. Proteomic analyses show distinct profiles for extracellular vesicles, exomeres, and supermeres consistent regardless of culture conditions or isolation method. In contrast, these two variables influence small RNAs, their base modifications, and lipidomic profiles. We present an online tool to query these and future secretome datasets (https:/superomics.shinyapps.io/browse). Overall design: small RNAseq of the extracellular vesicles, exomeres, and supermeres from 2D or 3D cell culturing conditions isolated by either ultracentrifugation-based method, straight cushion-density gradient ultracentrifugation or FPLC-SEC Gradient samples: They represent two classes of extracellular vesicles, heavy (hEV in our paper) and light (EV in our paper), as well as non-vesicular material (NV in our paper); NV is a mixture of different entities, mostly dense material that may include histones and other non-lipid containing protein complexes and some exomeres. All the EVs are small as we prepare the media by gravity filtering through a 0.22um filter.