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

  • Dongsic Choi
    Affiliations
    Research Institute of the McGill University Health Centre, Glen Site, McGill University, Montreal, Quebec, H4A 3J1, Canada
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  • Laura Montermini
    Affiliations
    Research Institute of the McGill University Health Centre, Glen Site, McGill University, Montreal, Quebec, H4A 3J1, Canada
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  • Dae-Kyum Kim
    Affiliations
    Donnelly Centre and Departments of Molecular Genetics and Computer Science, University of Toronto, Toronto, Ontario, M5S 3E1, Canada

    Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada
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  • Brian Meehan
    Affiliations
    Research Institute of the McGill University Health Centre, Glen Site, McGill University, Montreal, Quebec, H4A 3J1, Canada
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  • Frederick P. Roth
    Affiliations
    Donnelly Centre and Departments of Molecular Genetics and Computer Science, University of Toronto, Toronto, Ontario, M5S 3E1, Canada

    Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada

    Canadian Institute for Advanced Research, Toronto, Ontario, M5G 1M1, Canada
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  • Janusz Rak
    Correspondence
    To whom correspondence should be addressed:Department of Pediatrics, McGill University, The Research Institute of the McGill University Health Centre, Montreal Children's Hospital, 1001 Decarie Blvd, Montreal, Quebec, Canada. Tel.:+1-514-412-4400 (ex: 76240); Fax:+1-514-412-4331
    Affiliations
    Research Institute of the McGill University Health Centre, Glen Site, McGill University, Montreal, Quebec, H4A 3J1, Canada
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  • Author Footnotes
    * This work was supported by the operating grants from Canadian Institutes for Health Research (CIHR Foundation Grant, MOP 102736, MOP 111119) and Innovation to Impact Grant from the Canadian Cancer Society Research Institute to J.R., who is also a recipient of the Jack Cole Chair in Pediatric Hematology/Oncology. DSC was supported by a Fellowship from McGill Integrated Cancer Research Training Program (MICRTP, CIHR/FRSQ-FRN53888). DKK was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) through Banting Fellowship. F.P.R. was supported by the Canada Excellence Research Chairs Program, and by NIH Grant HG001715. Infrastructure funds were provided by the Fonds de Recherche en Santé du Quebec (FRSQ), Research Institute of the McGill University Health Centre (RI-MUHC), Montreal Children's Hospital, and McGill University.
    This article contains supplemental Figures and Tables. We declare no conflicts of interest.
Open AccessPublished:July 13, 2018DOI:https://doi.org/10.1074/mcp.RA118.000644
      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.
      Glioblastoma multiforme (GBM)
      The abbreviations used are:
      GBM
      glioblastoma multiforme
      EGFR
      epidermal growth factor receptor
      EGFRvIII
      EGFR variant III
      EVs
      extracellular vesicles
      ECM
      extracellular matrix
      FBS
      fetal bovine serum
      CM
      conditioned medium
      NTA
      nanoparticle tracking analysis
      TIC
      total ion chromatogram
      GO
      gene ontology
      V-SSC
      violet SSC.
      1The abbreviations used are:GBM
      glioblastoma multiforme
      EGFR
      epidermal growth factor receptor
      EGFRvIII
      EGFR variant III
      EVs
      extracellular vesicles
      ECM
      extracellular matrix
      FBS
      fetal bovine serum
      CM
      conditioned medium
      NTA
      nanoparticle tracking analysis
      TIC
      total ion chromatogram
      GO
      gene ontology
      V-SSC
      violet SSC.
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      DISCUSSION

      In this study, we present a comprehensive analysis of the impact exerted by oncogenic EGFRvIII on the protein profiles of EVs released by human GBM cells. We used an isogenic GBM model system where EGFRvIII possesses a strong and well characterized transforming ability and the changes that occur downstream are not obscured by intercellular variability. In this setting, we made several novel observations, which suggest a multifaceted effect of the oncogenic transformation on glioma vesiculation processes, including the molecular regulation of EV biogenesis, as well as their protein composition, molecular heterogeneity, uptake by recipient cells and possible changes in biological activity (
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      ).
      Genetic and epigenetic driver events have already been implicated by us and others as regulators of the EV release and intercellular communication in cancer, including intercellular trafficking of transforming mutant macromolecules, such as HRAS, EGFR, and EGFRvIII (
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      Among biological responses elicited by glioma EVs, such as angiogenesis, altered growth, survival and therapy responses (
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      • Meehan B.
      • Micallef J.
      • Lhotak V.
      • May L.
      • Guha A.
      • Rak J.
      Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells.
      ,
      • Al-Nedawi K.
      • Meehan B.
      • Kerbel R.S.
      • Allison A.C.
      • Rak J.
      Endothelial expression of autocrine VEGF upon the uptake of tumor-derived microvesicles containing oncogenic EGFR.
      ,
      • Skog J.
      • Wurdinger T.
      • van Rijn S.
      • Meijer D.H.
      • Gainche L.
      • Sena-Esteves M.
      • Curry Jr, W.T.
      • Carter B.S.
      • Krichevsky A.M.
      • Breakefield X.O.
      Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.
      ,
      • Arscott W.T.
      • Tandle A.T.
      • Zhao S.
      • Shabason J.E.
      • Gordon I.K.
      • Schlaff C.D.
      • Zhang G.
      • Tofilon P.J.
      • Camphausen K.A.
      Ionizing radiation and glioblastoma exosomes: implications in tumor biology and cell migration.
      ) invasion is relatively less studied. This is paradoxical given the crucial involvement of the infiltrative growth pattern in the biology of GBM (
      • Reifenberger G.
      • Wirsching H.G.
      • Knobbe-Thomsen C.B.
      • Weller M.
      Advances in the molecular genetics of gliomas - implications for classification and therapy.
      ), and the emerging evidence for the role of tumor-derived EVs in invasion related processes, such as directional cell motility (
      • Sung B.H.
      • Ketova T.
      • Hoshino D.
      • Zijlstra A.
      • Weaver A.M.
      Directional cell movement through tissues is controlled by exosome secretion.
      ,
      • Di Vizio D.
      • Kim J.
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      ,
      • Wu D.
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      Pairing of integrins with ECM proteins determines migrasome formation.
      ), proteolysis (
      • Hendrix A.
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      • De W.O.
      An ex(o)citing machinery for invasive tumor growth.
      ), pre-metastatic niche formation (
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      • Shen T.L.
      • Rodrigues G.
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      • Matei I.
      • Peinado H.
      • Bromberg J.
      • Lyden D.
      Tumour exosome integrins determine organotropic metastasis.
      ) and frank tumor dissemination (
      • Hood J.L.
      • San R.S.
      • Wickline S.A.
      Exosomes released by melanoma cells prepare sentinel lymph nodes for tumor metastasis.
      ). Although GBM is normally confined to the brain some of the cellular phenotypes involved could be relevant to the regional and systemic pathogenesis of this aggressive disease.
      In this regard, our study suggests that EGFRvIII mutation may lead to EV-mediated release of several proteins with a documented role in tumor dissemination and interaction with the ECM, including CD44, MCAM, THBS1, and integrin α6β4. Among them CD44 is known to interact with hyaluronic acid, whereas MCAM interacts with laminin, which is also a ligand for exosomal integrin α6β4 implicated in organ specific metastasis (
      • Hoshino A.
      • Costa-Silva B.
      • Shen T.L.
      • Rodrigues G.
      • Hashimoto A.
      • Tesic M.M.
      • Molina H.
      • Kohsaka S.
      • Di G.A.
      • Ceder S.
      • Singh S.
      • Williams C.
      • Soplop N.
      • Uryu K.
      • Pharmer L.
      • King T.
      • Bojmar L.
      • Davies A.E.
      • Ararso Y.
      • Zhang T.
      • Zhang H.
      • Hernandez J.
      • Weiss J.M.
      • Dumont-Cole V.D.
      • Kramer K.
      • Wexler L.H.
      • Narendran A.
      • Schwartz G.K.
      • Healey J.H.
      • Sandstrom P.
      • Jorgen L.K.
      • Kure E.H.
      • Grandgenett P.M.
      • Hollingsworth M.A.
      • de S.M.
      • Kaur S.
      • Jain M.
      • Mallya K.
      • Batra S.K.
      • Jarnagin W.R.
      • Brady M.S.
      • Fodstad O.
      • Muller V.
      • Pantel K.
      • Minn A.J.
      • Bissell M.J.
      • Garcia B.A.
      • Kang Y.
      • Rajasekhar V.K.
      • Ghajar C.M.
      • Matei I.
      • Peinado H.
      • Bromberg J.
      • Lyden D.
      Tumour exosome integrins determine organotropic metastasis.
      ,
      • Mu W.
      • Rana S.
      • Zoller M.
      Host matrix modulation by tumor exosomes promotes motility and invasiveness.
      ). Hyaluronic acid is highly overexpressed in patients with malignant glioma (
      • Cha J.
      • Kang S.G.
      • Kim P.
      Strategies of Mesenchymal Invasion of Patient-derived Brain Tumors: Microenvironmental Adaptation.
      ) and laminins are major constituents of blood vessel basement membranes in brain tumor microenvironment (
      • Ljubimova J.Y.
      • Fujita M.
      • Khazenzon N.M.
      • Ljubimov A.V.
      • Black K.L.
      Changes in laminin isoforms associated with brain tumor invasion and angiogenesis.
      ). Thus, EGFRvIII may reprogram glioma related EVs to interact with ECM and structures within the brain microenvironment and thereby modulate tumor invasiveness. Because GBM-related EVs are also detected systemically, changes in their surface properties may contribute to distant paraneoplastic effects of these intracranial tumors (
      • Magnus N.
      • Garnier D.
      • Meehan B.
      • McGraw S.
      • Lee T.H.
      • Caron M.
      • Bourque G.
      • Milsom C.
      • Jabado N.
      • Trasler J.
      • Pawlinski R.
      • Mackman N.
      • Rak J.
      Tissue factor expression provokes escape from tumor dormancy and leads to genomic alterations.
      ).
      Different activating EGFR mutations may impact EV mediated processes in cancer. For example constitutively active EGFR mutant (E746 - A750 deletion in tyrosine kinase domain) expressed in HCC827 lung cancer cells, was reported to stimulate protein sorting and the resulting enrichment of the EV compartment in EGFR, SRC, GRB2, RALA, RAC1, and KRAS, relative to EVs produced by immortalized bronchial epithelial cell HBE4 (
      • Clark D.J.
      • Fondrie W.E.
      • Yang A.
      • Mao L.
      Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.
      ). Notably, BSG and CD151 were upregulated whereas CD81 was downregulated in HCC827-derived EVs (
      • Clark D.J.
      • Fondrie W.E.
      • Yang A.
      • Mao L.
      Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.
      ), which is similar to our observations involving EGFRvIII.
      Other oncogenic events within the EGFR signaling pathway have also been implicated in proinvasive reprogramming of the EV cargo. For example, integrins and metalloproteases associated with EVs are upregulated by HRAS (
      • Tauro B.J.
      • Mathias R.A.
      • Greening D.W.
      • Gopal S.K.
      • Ji H.
      • Kapp E.A.
      • Coleman B.M.
      • Hill A.F.
      • Kusebauch U.
      • Hallows J.L.
      • Shteynberg D.
      • Moritz R.L.
      • Zhu H.J.
      • Simpson R.J.
      Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition.
      ) EGFR, KRAS, and SRC (
      • Demory Beckler M.
      • Higginbotham J.N.
      • Franklin J.L.
      • Ham A.J.
      • Halvey P.J.
      • Imasuen I.E.
      • Whitwell C.
      • Li M.
      • Liebler D.C.
      • Coffey R.J.
      Proteomic analysis of exosomes from mutant KRAS colon cancer cells identifies intercellular transfer of mutant KRAS.
      ). Overexpression of the EGFR-related proto-oncogene, ERBB2/HER2 in C5.2 breast cancer cells leads to increased content of CD44 in tumor EVs (
      • Amorim M.
      • Fernandes G.
      • Oliveira P.
      • Martins-de-Souza D.
      • Dias-Neto E.
      • Nunes D.
      The overexpression of a single oncogene (ERBB2/HER2) alters the proteomic landscape of extracellular vesicles.
      ). Such changes may be influenced by both oncogenic mutations and cellular contexts in which they occur. For example, integrin beta-1 in EVs is upregulated in the case of EGFR-driven HCC827 cells (
      • Clark D.J.
      • Fondrie W.E.
      • Yang A.
      • Mao L.
      Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer-derived exosomes.
      ) and mutant HRAS overexpressing MDCK cells (
      • Tauro B.J.
      • Mathias R.A.
      • Greening D.W.
      • Gopal S.K.
      • Ji H.
      • Kapp E.A.
      • Coleman B.M.
      • Hill A.F.
      • Kusebauch U.
      • Hallows J.L.
      • Shteynberg D.
      • Moritz R.L.
      • Zhu H.J.
      • Simpson R.J.
      Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition.
      ), but not in U373vIII cells. Thus, a better definition of signaling cues and cellular modifiers (
      • Bobrie A.
      • Krumeich S.
      • Reyal F.
      • Recchi C.
      • Moita L.F.
      • Seabra M.C.
      • Ostrowski M.
      • Thery C.
      Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression.
      ) operative in different tumor subtypes (
      • Nakano I.
      • Garnier D.
      • Minata M.
      • Rak J.
      Extracellular vesicles in the biology of brain tumour stem cells - Implications for inter-cellular communication, therapy and biomarker development.
      ) may be needed to fully understand the regulation of EV biogenesis and cargo assembly in cancer.
      Cancer-related EVs have been implicated as carriers of proteolytic activity. Indeed, we observed a trend for enrichment in U373vIII EVs (as compared with their U373-derived counterparts) of several proteases, including ADAM9, ADAM10, cathepsin Z (CTSZ), and MMP14 involved in the degradation of ECM components, including laminins and collagens (supplemental Table S2). Moreover, two elements of the fibrinolytic system (PLAT and PLAU) are significantly overexpressed in U373vIII EVs. This is consistent with the notion that activated EGFR upregulates PLAU and its receptor (PLAUR) resulting in an increase of cellular invasiveness (
      • Mori T.
      • Abe T.
      • Wakabayashi Y.
      • Hikawa T.
      • Matsuo K.
      • Yamada Y.
      • Kuwano M.
      • Hori S.
      Upregulation of urokinase-type plasminogen activator and its receptor correlates with enhanced invasion activity of human glioma cells mediated by transforming growth factor-alpha or basic fibroblast growth factor.
      ), as well as signaling interactions (
      • Hu J.
      • Jo M.
      • Cavenee W.K.
      • Furnari F.
      • VandenBerg S.R.
      • Gonias S.L.
      Crosstalk between the urokinase-type plasminogen activator receptor and EGF receptor variant III supports survival and growth of glioblastoma cells.
      ) in which the role of EVs remains to be studied. Collectively, our observations suggest that EGFRvIII expression may enable GBM cells to emit EVs endowed with the enhanced capacity to selectively bind and degrade specific ECM proteins (
      • McCready J.
      • Sims J.D.
      • Chan D.
      • Jay D.G.
      Secretion of extracellular hsp90alpha via exosomes increases cancer cell motility: a role for plasminogen activation.
      ), thereby possibly contributing to migratory, signaling and stromal responses (
      • Charles N.A.
      • Holland E.C.
      • Gilbertson R.
      • Glass R.
      • Kettenmann H.
      The brain tumor microenvironment.
      ).
      Another interesting finding is the role of EGFRvIII in differential EV uptake. Quantitative proteomics and GO term analysis indicated that EVs released by glioma cells expressing this potent oncogene are enriched in adhesion molecules of which some are known for homophilic interactions, including CD151 (
      • Hong I.K.
      • Jin Y.J.
      • Byun H.J.
      • Jeoung D.I.
      • Kim Y.M.
      • Lee H.
      Homophilic interactions of Tetraspanin CD151 up-regulate motility and matrix metalloproteinase-9 expression of human melanoma cells through adhesion-dependent c-Jun activation signaling pathways.
      ), BSG (
      • Grass G.D.
      • Toole B.P.
      How, with whom and when: an overview of CD147-mediated regulatory networks influencing matrix metalloproteinase activity.
      ), NPTN (
      • Owczarek S.
      • Berezin V.
      Neuroplastin: cell adhesion molecule and signaling receptor.
      ), and PCDH10 (
      • Kim S.Y.
      • Yasuda S.
      • Tanaka H.
      • Yamagata K.
      • Kim H.
      Non-clustered protocadherin.
      ). We have also observed a greater homophilic EV uptake by U373vIII cells relative to their U373 counterparts, suggesting the existence of an autocrine or a homotypic ('homocrine') interaction between GBM cell subsets and their own EVs. Such interactions have been implicated in directional cell migration (
      • Sung B.H.
      • Ketova T.
      • Hoshino D.
      • Zijlstra A.
      • Weaver A.M.
      Directional cell movement through tissues is controlled by exosome secretion.
      ) and may represent programs linked to oncogenic transformation, as documented in cells harboring mutant KRAS or HRAS (
      • Lee T.H.
      • Chennakrishnaiah S.
      • Meehan B.
      • Montermini L.
      • Garnier D.
      • D'Asti E.
      • Hou W.
      • Magnus N.
      • Gayden T.
      • Jabado N.
      • Eppert K.
      • Majewska L.
      • Rak J.
      Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras.
      ,
      • Kamerkar S.
      • LeBleu V.S.
      • Sugimoto H.
      • Yang S.
      • Ruivo C.F.
      • Melo S.A.
      • Lee J.J.
      • Kalluri R.
      Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.
      ). Further studies are needed to understand the role of such processes in GBM.
      Our study also revealed novel aspects of EV heterogeneity in the context of EGFRvIII-dependent oncogenic transformation. Although traditionally EVs were analyzed in bulk, or according to their physical properties, recent technologies enabled greater insights into the properties of EV subsets or even single EVs. This includes Raman spectroscopy (
      • Smith Z.J.
      • Lee C.
      • Rojalin T.
      • Carney R.P.
      • Hazari S.
      • Knudson A.
      • Lam K.
      • Saari H.
      • Ibanez E.L.
      • Viitala T.
      • Laaksonen T.
      • Yliperttula M.
      • Wachsmann-Hogiu S.
      Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content.
      ), microfluidic platforms (
      • Olcum S.
      • Cermak N.
      • Wasserman S.C.
      • Christine K.S.
      • Atsumi H.
      • Payer K.R.
      • Shen W.
      • Lee J.
      • Belcher A.M.
      • Bhatia S.N.
      • Manalis S.R.
      Weighing nanoparticles in solution at the attogram scale.
      ) and high-resolution flow cytometry (
      • van der Vlist E.J.
      • Nolte-'t Hoen E.N.
      • Stoorvogel W.
      • Arkesteijn G.J.
      • Wauben M.H.
      Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.
      ,
      • Pospichalova V.
      • Svoboda J.
      • Dave Z.
      • Kotrbova A.
      • Kaiser K.
      • Klemova D.
      • Ilkovics L.
      • Hampl A.
      • Crha I.
      • Jandakova E.
      • Minar L.
      • Weinberger V.
      • Bryja V.
      Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer.
      ,
      • Higginbotham J.N.
      • Zhang Q.
      • Jeppesen D.K.
      • Scott A.M.
      • Manning H.C.
      • Ochieng J.
      • Franklin J.L.
      • Coffey R.J.
      Identification and characterization of EGF receptor in individual exosomes by fluorescence-activated vesicle sorting.
      ). Indeed, EVs originating from the same parental cell population may exhibit a remarkable spectrum of characteristics (
      • Smith Z.J.
      • Lee C.
      • Rojalin T.
      • Carney R.P.
      • Hazari S.
      • Knudson A.
      • Lam K.
      • Saari H.
      • Ibanez E.L.
      • Viitala T.
      • Laaksonen T.
      • Yliperttula M.
      • Wachsmann-Hogiu S.
      Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content.
      ,
      • Laulagnier K.
      • Vincent-Schneider H.
      • Hamdi S.
      • Subra C.
      • Lankar D.
      • Record M.
      Characterization of exosome subpopulations from RBL-2H3 cells using fluorescent lipids.
      ,
      • Oksvold M.P.
      • Kullmann A.
      • Forfang L.
      • Kierulf B.
      • Li M.
      • Brech A.
      • Vlassov A.V.
      • Smeland E.B.
      • Neurauter A.
      • Pedersen K.W.
      Expression of B-cell surface antigens in subpopulations of exosomes released from B-cell lymphoma cells.
      ), which may change dynamically with the state of donor cells (
      • Tauro B.J.
      • Greening D.W.
      • Mathias R.A.
      • Mathivanan S.
      • Ji H.
      • Simpson R.J.
      Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids.
      ,
      • Roucourt B.
      • Meeussen S.
      • Bao J.
      • Zimmermann P.
      • David G.
      Heparanase activates the syndecan-syntenin-ALIX exosome pathway.
      ). Our analysis revealed several important characteristics associated with EV subsets generated by EGFRvIII-transformed glioma cells. First, we observed that only a fraction of EVs carries the EGFRvIII oncoprotein, mostly in the context of CD9 tetraspanin. However, only subsets of these EVs carried traditional exosomal markers such as CD9 and CD81. Notably, EGFRvIII expression suppressed some of the exosomal features (
      • Kowal J.
      • Arras G.
      • Colombo M.
      • Jouve M.
      • Morath J.P.
      • Primdal-Bengtson B.
      • Dingli F.
      • Loew D.
      • Tkach M.
      • Thery C.
      Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.
      ) in glioma EVs, including downregulation of CD81 and a complete loss of CD82, as reported earlier (
      • Yang C.H.
      • Chou H.C.
      • Fu Y.N.
      • Yeh C.L.
      • Cheng H.W.
      • Chang I.C.
      • Liu K.J.
      • Chang G.C.
      • Tsai T.F.
      • Tsai S.F.
      • Liu H.P.
      • Wu Y.C.
      • Chen Y.T.
      • Huang S.F.
      • Chen Y.R.
      EGFR over-expression in non-small cell lung cancers harboring EGFR mutations is associated with marked down-regulation of CD82.
      ). This may reflect antagonistic interactions between the activated EGFR pathway and CD82 (
      • Wang X.Q.
      • Yan Q.
      • Sun P.
      • Liu J.W.
      • Go L.
      • McDaniel S.M.
      • Paller A.S.
      Suppression of epidermal growth factor receptor signaling by protein kinase C-alpha activation requires CD82, caveolin-1, and ganglioside.
      ), worthy of further exploration in GBM. EGFRvIII transformation also resulted in the enrichment for BSG+/CD44+ double positive EVs, a pattern reminiscent of the colocalization these proteins near active filopodia of U373vIII cells. Interestingly, EGFR-Ras-ERK signaling cascade may promote the assembly of BSG, CD44, and EGFR to form invadopodia (
      • Grass G.D.
      • Tolliver L.B.
      • Bratoeva M.
      • Toole B.P.
      CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness.
      ). The pro-invasive role of this complex is underscored by the recruitment of MMP14 (MT1-MMP) by BSG (
      • Grass G.D.
      • Toole B.P.
      How, with whom and when: an overview of CD147-mediated regulatory networks influencing matrix metalloproteinase activity.
      ), all of which may contribute to the pathogenetic effects of glioma EVs.
      Overall, our results suggest that EV mediated communication between glioma cells and their microenvironment are regulated by oncogenic transformation. Consequently, the impact of EGFRvIII and other transforming events associated with GBM (IDH1, PTEN) (
      • Reifenberger G.
      • Wirsching H.G.
      • Knobbe-Thomsen C.B.
      • Weller M.
      Advances in the molecular genetics of gliomas - implications for classification and therapy.
      ) on the proteome of tumor EVs may have meaningful and underappreciated implications for EV-mediated biological effects, biomarker development and therapeutic applications.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE (
      • Vizcaino J.A.
      • Csordas A.
      • del-Toro N.
      • Dianes J.A.
      • Griss J.
      • Lavidas I.
      • Mayer G.
      • Perez-Riverol Y.
      • Reisinger F.
      • Ternent T.
      • Xu Q.W.
      • Wang R.
      • Hermjakob H.
      2016 update of the PRIDE database and its related tools.
      ) partner repository with the dataset identifier PXD008311 and 10.6019/PXD008311. Summary of ProteomeXchange accession: PXD008311; PubMed ID: 30006486; Project Webpage: http://www.ebi.ac.uk/pride/archive/projects/PXD008311; FTP Download: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2018/07/PXD008311.

      Acknowledgments

      We are grateful to the Montreal Children's Hospital Foundation and Donors for supporting our purchase of Nano-Flow Cytometry Instrument. We thank the Clinical Proteomics Platform for mass spectrometry analyses, Immunophenotyping Platform for nano-flow cytometry, and Molecular Imaging Platform for confocal analyses at RI-MUHC. We would also like to thank our families for their patient and unwavering support for our efforts.

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