Large and small extracellular vesicles released by glioma cells in vitro and in vivo

Abstract

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 (U87EGFRvIII) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87EGFRvIII cancer cells, non-cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87EGFRvIII glioma-bearing mice using differential centrifugation (EVs that sediment at 10,000 × g or 100,000 × 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 (μNMR). We report that both U87EGFRvIII and HUVEC release a similar number of small EVs, but U87EGFRvIII glioma cells alone release a higher number of large EVs compared to non-cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87EGFRvIII 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 U87EGFRvIII 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 U87EGFRvIII cells and from serum of U87EGFRvIII 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.

Peptidylarginine Deiminase Isozyme-Specific PAD2, PAD3 and PAD4 Inhibitors Differentially Modulate Extracellular Vesicle Signatures and Cell Invasion in Two Glioblastoma Multiforme Cell Lines

“…GBM-derived EVs have furthermore been shown to induce tumourpromoting transformation of subventricular zone resident neural stem cells, possibly contributing to GBM recurrence [64]. Differences in EV release profiles between GBM cell lines, including EV subpopulations and during normal growth conditions, have previously been reported by us and others [9,38,65]. Several studies have assessed various types of EV cargo in GBM, including protein and genetic material [9,20,38,[66][67][68].…”

Metabolic characteristics of large and small extracellular vesicles from pleural effusion reveal biomarker candidates for the diagnosis of tuberculosis and malignancy

“…Small EVs (sEVs) and large EVs (lEVs) are commonly studied subpopulations of EVs [24,25]. sEVs, such as exosomes (~100 nm diameter), are demonstrated to carry bioactive molecules from parental cells, and modulate the metabolism of recipient cells [13,26].…”

“…sEVs, such as exosomes (~100 nm diameter), are demonstrated to carry bioactive molecules from parental cells, and modulate the metabolism of recipient cells [13,26]. lEVs, including microvesicles, ectosomes and/ or large oncosomes (~100-1,000 nm and/or >1,000 nm diameter), have been found contain specific proteins, lipids and RNA, which are associated with disease progression, and used as potential biomarkers for disease diagnosis and monitoring [24,25,27]. While the molecular content of sEVs and lEVs remains poor understood, particularly for the EVs from pleural effusion.…”

“…Therefore, a detailed understanding of the metabolite composition of EV subgroups is necessary to further develop diagnostics and therapeutics. In our study, differential centrifugation, a widely used separation technique for EVs of different size and intensity [24,25,34], was used to obtain the lEVs (sediment at 20,000 × g spin) and sEVs (sediment at 110,000 × g spin). Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics and lipidomics analysis were utilized to assess the metabolite characteristics of sEVs and lEVs from pleural effusion, and further explore the metabolic reprogramming of tuberculosis and malignancy from the level of EVs metabolites, to provide new insights into the mechanism and diagnosis of pleural effusion.…”

Extracellular Vesicles in Glioblastoma Tumor Microenvironment

“…Small EV cargo on the other hand contained higher amounts of proteins clustered toward glucose and glutamine metabolism and gluconeogenesis (70). Although both small and large EV subfractions are shown to contain tumor derived cargo useful for tumor biomarking, they subpopulations could be mediating separate wings of metabolic and other functional reprogramming (71). In summary, studies from several cancers hint that tumor EVs drive glycolysis and other tumor supporting energy generating pathways and thus potentiate tumor growth by metabolic remodeling (72) but the exact contribution of EVs in these phenomenon is still unclear.…”