The Impact of Oncogenic EGFRvIII on the Proteome of Extracellular Vesicles Released from Glioblastoma Cells

Abstract

Epidermal growth factor receptor (EGFR)EGFR SummaryGlioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIIItransformed cells were enriched for extracellular exosome and focal adhesion related proteins.Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and down-regulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins. Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also co-localized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.

Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity

“…Since cellular vesicles of near‐exosomal size could contain up to 200 proteins each, the predicted number of such non‐overlapping protein combinations (vesicle subsets) could be calculated to be on the order of 5–25 . Considering a marked overlap in the protein content between different known EV classes already characterized by differential centrifugation, nano‐flow cytometry, and other methods the possible number of EV subsets could be much greater than presently recognized. This combinatorial diversity could be further amplified by the heterogeneity of nucleic acids contained within known EV subpopulations and through superimposed distribution of other molecular species such as lipids and metabolites.…”

“…For example, the expression of mutant RAS oncogene enhances the uptake of EVs by intestinal and pancreatic cancer cells, mainly through macropinocytosis . Spontaneously transformed human astrocytes expressing hTERT take up EVs more avidly than their non‐transformed counterparts, while EGFRvIII expressing glioma cells preferentially internalize their own EVs …”

“…For example, studies involving the isogenic series of U373 glioma‐derived cell lines revealed a dramatic effect of the EGFRvIII oncogene on both cellular aggressiveness and vesiculation profile, including changes in the global EV proteome and configuration of single EV immunophenotypes, as revealed by nano‐flow cytometry. Notably, EGFRvIII impacted the expression of several vesiculation‐related genes, as well as levels of EV marker proteins such as CD81, CD82, and BSG . While both indolent and EGFRvIII‐expressing glioma cells produced ample EVs, their respective proteomes were markedly different beyond the content of the EGFRvIII oncoprotein itself, with nearly 200 proteins being uniquely deregulated in EVs of the aggressive (U373vIII) cell line.…”

A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

“…While high-throughput nextgeneration-sequencing technologies have allowed thorough characterisation of nucleic acids in blood-derived EVs [30,57], the proteome of circulating-EVs remains largely unexplored. Shot-gun proteomic methods have been primarily used to investigate glioma-EV proteomes [58,59]. However, such approaches achieve limited proteomic coverage, especially for complex blood-derived specimens [60].…”

The critical role of exosomes in tumor biology

“…Brain miR-301a, miR-221, lncRNA-HOTAIR EGFRvIII, PTRF Choi et al, 73 Huang et al, 74 Lan et al, 75 Tan et al, 76 Yang et al 77 Lung miR-451a, miR-126,miR-146a-5p, miR-96, lncRNA-MALAT-1 ADAM10sa, Tim-3, Galectin-9…”