{
  "abstract": "Background Because tumor-derived extracellular vesicles (EVs) demonstrate highly conserved functional attributes (especially EMT and immune modulation), we hypothesized that we could identify small EV (sEV) cargo features expressed proportionately with tumor metastatic potential and across tumor histologies. We evaluated tumor-derived sEVs at the level of subsets and individual particles, and identified a set of ‘hallmarks’ that are shared by EVs from tumors with high metastatic potential. EVs from the 4T1-derived series of syngeneic mouse breast cancer cell lines with a spectrum of metastatic potential served as a discovery set, and we validated the EV hallmarks of metastatic potential using three additional series of ‘low metastatic potential’ to ‘high metastatic potential’ cell series, representing three additional tumor types.Methods Tumor cell EV-production was performed in serum- and phenol red-free OptiMEM. EV productions were harvested after 36-48 hours, concentrated with Omega 100K JumboSep centrifugal ultrafiltration, and sEVs were further separated from soluble proteins with Izon qEV10 size exclusion chromatography. sEV particle and protein concentrations were estimated with Nanoparticle Tracking Analysis and micro-BCA assays, respectively. Surface marker repertoires of EV preparations were then characterized by multiplex exosome flow cytometry, analyzed with MPA-PASS software. Protein identification mass spectrometry was performed with DIA LC/MS, and EVs were further characterized with negative stain TEM and Western blots.Results We identified a striking proportional relationship between each series’ tumor metastatic potential and sEV concentration, in absolute and normalized datasets. Similarly, we observed higher EV protein yields in each EV preparation with higher metastatic potential. Multiplex flow cytometric analysis across more than 50 EV surface proteins identified tetraspanins, adhesion proteins, and stemness markers as having patterns of conservation across the tumor types (such as CD44), as well as including markers distinctive to tumor type (such as EpCAM). In-depth proteomic analyses identified >1000 proteins from the sEV protein preparations. Among the most significantly associated with metastatic potential were histone proteins, including H2A.Z (P<0.001) and macroH2A (P<0.00001).Conclusions Our results confirm the hypothesis that sEVs from tumors with higher metastatic potential share common features which may relate to their pathogenesis and mediation of changes in the tumor microenvironment. Specifically, we found that higher metastatic tumor cells produced more sEVs and more sEV-associated protein than their lower metastatic potential counterparts. Moreover, those proteins are enriched for markers of stemness and DNA damage responses. Further study is required to identify functional roles for the sEV cargo in modulating tumor metastases and tumor immune responses.",
  "authors": [
    {
      "affiliations": [
        "National Cancer Institute, National Institutes of Health, Bethesda, MD, USA"
      ],
      "name": "Stephanie Chidester"
    },
    {
      "affiliations": [
        "National Cancer Institute, National Institutes of Health, Bethesda, MD, USA"
      ],
      "name": "Jubin George"
    },
    {
      "affiliations": [
        "National Cancer Institute, National Institutes of Health, Bethesda, MD, USA"
      ],
      "name": "Michelle Pleet"
    },
    {
      "affiliations": [
        "National Cancer Institute, National Institutes of Health, Bethesda, MD, USA"
      ],
      "name": "Jennifer Clare Jones"
    }
  ],
  "title": "32 Extracellular vesicle determinants of metastatic potential",
  "uid": "d9a0021a-90f9-55cc-afff-1e70cefa4baf"
}