Abstract
Glioblastoma 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 EGFRvIII-transformed 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 downregulation 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 (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized 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.
Highlights
Glioblastoma 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)
Changes in Characteristics of Glioma Cells and Their extracellular vesicles (EVs) Following EGFRvIII-Dependent Oncogenic Transformation—As reported in our previous study [12], the expression of the oncogenic EGFRvIII mutant in indolent U373 glioma cells resulted in formation of an aggressive variant (U373vIII) endowed with a highly tumorigenic phenotype in xenograft model systems [3]
We present a comprehensive analysis of the impact exerted by oncogenic EGFRvIII on the protein profiles of EVs released by human GBM cells
Summary
In Vivo Mouse Tumor Model—All in vivo experiments were performed as described earlier [3] according to the Animal Use Protocol (AUP) approved by the Institutional Animal Facility Care Committee and following Guidelines of the Canadian Council of Animal Care (CCAC). From three biologically replicated MS data sets of both proteomes, from U373 and U373vIII EVs, we quantified the relative protein abundance by total ion chromatogram (TIC) and calculated the p value by student’s t test using Scaffold Qϩ software. EV Labeling With Antibodies and Analyses by Nano-flow Cytometry—The conditioned medium from cells grown for 72 h in culture media containing 10% of EV-depleted FBS was centrifuged one time at 400g and filtered through a 0.8 m pore-size mesh. EV Labeling With PKH67 and Uptake Measurement by Flow Cytometry—The conditioned media from cells grown for 72 h in culture media supplemented with 10% of EV-depleted FBS were centrifuged once at 400g, passed through 0.8 m pore-size filter and concentrated as described above. Numerical values were plotted as mean Ϯ S.D
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