Journal
JOURNAL OF EXTRACELLULAR VESICLES
Volume 9, Issue 1, Pages -Publisher
TAYLOR & FRANCIS LTD
DOI: 10.1080/20013078.2019.1689784
Keywords
Extracellular vesicles; large extracellular vesicles; small extracellular vesicles; biomarkers; glioma
Categories
Funding
- NCI [CA232103, CA069246, U01 CA230697, UH3 TR000931, P01 CA069246, R00CA131472, R01CA218526]
- DoD [PC150836, CA230697, TR000931, R01CA229777, U01CA233360]
- MGH Scholar Fund
- NIH [P01 HI36028]
- Inflammatory Bowel Disease Grant [DK043351]
- Boston Area Diabetes and Endocrinology Research Center (BADERC) Award [DK057521]
- Defense Advanced Research Projects Agency [PC150836]
- Division of Cancer Prevention, National Cancer Institute [CA131472, CA218526]
- Massachusetts General Hospital
- National Cancer Institute [CA232103, CA069246, CA230697, R01CA229777]
- National Institutes of Health [P01 HI36028]
- National Center for Advancing Translational Sciences (US) [TR000931]
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Tumour cells release diverse populations of extracellular vesicles (EVs) ranging in size, molecular cargo, and function. We sought to characterize mRNA and protein content of EV subpopulations released by human glioblastoma (GBM) cells expressing a mutant form of epidermal growth factor receptor (U87(EGFRvIII)) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87(EGFRvIII) cancer cells, non-cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87(EGFRvIII) glioma-bearing mice using differential centrifugation (EVs that sediment at 10,000 x g or 100,000 x g are termed large EVs and small EVs, respectively) were characterized using transmission electron microscopy (TEM), confocal microscopy, nanoparticle tracking analysis (NTA), flow cytometry, immunofluorescence (IF), quantitative-polymerase chain reaction (qPCR), droplet digital polymerase chain reaction (ddPCR) and micro-nuclear magnetic resonance (mu NMR). We report that both U87(EGFRvIII) and HUVEC release a similar number of small EVs, but U87(EGFRvIII) glioma cells alone release a higher number of large EVs compared to non-cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87(EGFRvIII) glioma cells was comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Similarly, EGFRvIII mRNA in large and small EVs isolated from the serum of U87(EGFRvIII) glioma-bearing mice is comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Here we report for the first time a direct comparison of large and small EVs released by glioma U87(EGFRvIII) cells and from serum of U87(EGFRvIII) glioma-bearing mice. Both large and small EVs contain tumour-specific EGFRvIII mRNA and proteins and combining these platforms may be beneficial in detecting rare mutant events in circulating biofluids.
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