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• W3008596935 abstract "•GBM EVs and growth factors promote angiogenesis by distinct pathways•EV-exposed endothelial cells show a footprint of gene regulation by EV miRNAs•The EV miRNA pathway explains failure of anti-angiogenic therapy for GBM•The results suggest an angiogenic pathway and liquid biopsy biomarkers for GBM Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers. Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers. Glioblastoma (GBM), the most common primary brain cancer in adults, is incurable, with 2- and 5-year survival rates of 16% and 5%, respectively (Ostrom et al., 2015Ostrom Q.T. Gittleman H. Fulop J. Liu M. Blanda R. Kromer C. Wolinsky Y. Kruchko C. Barnholtz-Sloan J.S. CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in the United States in 2008-2012.Neuro-oncol. 2015; 17: iv1-iv62Crossref PubMed Scopus (1258) Google Scholar). Aggressive diffuse growth, high tumor heterogeneity, vascular abnormalities, and a population of GBM stem-like cells (GSCs) are major factors that complicate treatment (Ramirez et al., 2013Ramirez Y.P. Weatherbee J.L. Wheelhouse R.T. Ross A.H. Glioblastoma multiforme therapy and mechanisms of resistance.Pharmaceuticals (Basel). 2013; 6: 1475-1506Crossref PubMed Scopus (145) Google Scholar, Harder et al., 2018Harder B.G. Blomquist M.R. Wang J. Kim A.J. Woodworth G.F. Winkles J.A. Loftus J.C. Tran N.L. Developments in Blood-Brain Barrier Penetrance and Drug Repurposing for Improved Treatment of Glioblastoma.Front. Oncol. 2018; 8: 462Crossref PubMed Google Scholar, Rooj et al., 2017Rooj A.K. Ricklefs F. Mineo M. Nakano I. Chiocca E.A. Bronisz A. Godlewski J. MicroRNA-Mediated Dynamic Bidirectional Shift between the Subclasses of Glioblastoma Stem-like Cells.Cell Rep. 2017; 19: 2026-2032Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, Garnier et al., 2019Garnier D. Renoult O. Alves-Guerra M.C. Paris F. Pecqueur C. Glioblastoma stem-like cells, metabolic strategy to kill a challenging target.Front. Oncol. 2019; 9: 118Crossref PubMed Scopus (38) Google Scholar). Diffuse infiltrative growth of GBM, which arises in large part due to the perivascular migration of GSCs, precludes complete resection, sparing the GSCs that resist chemotherapy and radiation and revive the tumor (Hanif et al., 2017Hanif F. Muzaffar K. Perveen K. Malhi S.M. Simjee ShU. Glioblastoma multiforme: a review of its epidemiology and pathogenesis through clinical presentation and treatment.Asian Pac. J. Cancer Prev. 2017; 18: 3-9PubMed Google Scholar, Ramirez et al., 2013Ramirez Y.P. Weatherbee J.L. Wheelhouse R.T. Ross A.H. Glioblastoma multiforme therapy and mechanisms of resistance.Pharmaceuticals (Basel). 2013; 6: 1475-1506Crossref PubMed Scopus (145) Google Scholar). The development of effective targeted therapies is hindered by the heterogeneity and plasticity of GBM cells, which provide the tumor with multiple paths of resistance, while GBM vasculature provides various obstacles to drug delivery (Ramirez et al., 2013Ramirez Y.P. Weatherbee J.L. Wheelhouse R.T. Ross A.H. Glioblastoma multiforme therapy and mechanisms of resistance.Pharmaceuticals (Basel). 2013; 6: 1475-1506Crossref PubMed Scopus (145) Google Scholar, Zanders et al., 2019Zanders E.D. Svensson F. Bailey D.S. Therapy for glioblastoma: is it working?.Drug Discov. Today. 2019; 24: 1193-1201Crossref PubMed Scopus (43) Google Scholar, Perrin et al., 2019Perrin S.L. Samuel M.S. Koszyca B. Brown M.P. Ebert L.M. Oksdath M. Gomez G.A. Glioblastoma heterogeneity and the tumour microenvironment: implications for preclinical research and development of new treatments.Biochem. Soc. Trans. 2019; 47: 625-638Crossref PubMed Scopus (41) Google Scholar, Kane, 2019Kane J.R. The Role of Brain Vasculature in Glioblastoma.Mol. Neurobiol. 2019; 56: 6645-6653Crossref PubMed Scopus (11) Google Scholar). Extensive molecular profiling of GBM tissues (Freije et al., 2004Freije W.A. Castro-Vargas F.E. Fang Z. Horvath S. Cloughesy T. Liau L.M. Mischel P.S. Nelson S.F. Gene expression profiling of gliomas strongly predicts survival.Cancer Res. 2004; 64: 6503-6510Crossref PubMed Scopus (537) Google Scholar, Phillips et al., 2006Phillips H.S. Kharbanda S. Chen R. Forrest W.F. Soriano R.H. Wu T.D. Misra A. Nigro J.M. Colman H. 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Martuza R.L. et al.Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma.Science. 2014; 344: 1396-1401Crossref PubMed Scopus (1825) Google Scholar, Rooj et al., 2017Rooj A.K. Ricklefs F. Mineo M. Nakano I. Chiocca E.A. Bronisz A. Godlewski J. MicroRNA-Mediated Dynamic Bidirectional Shift between the Subclasses of Glioblastoma Stem-like Cells.Cell Rep. 2017; 19: 2026-2032Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, Neftel et al., 2019Neftel C. Laffy J. Filbin M.G. Hara T. Shore M.E. Rahme G.J. Richman A.R. Silverbush D. Shaw M.L. Hebert C.M. et al.An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.Cell. 2019; 178: 835-849.e21Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, Ricklefs et al., 2016Ricklefs F. Mineo M. Rooj A.K. Nakano I. Charest A. Weissleder R. Breakefield X.O. Chiocca E.A. Godlewski J. Bronisz A. Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity.Cancer Res. 2016; 76: 2876-2881Crossref PubMed Scopus (61) Google Scholar, Wang et al., 2019bWang L. Babikir H. Müller S. Yagnik G. Shamardani K. Catalan F. Kohanbash G. Alvarado B. Di Lullo E. Kriegstein A. et al.The phenotypes of proliferating glioblastoma cells reside on a single axis of variation.Cancer Discov. 2019; 9: 1708-1719Crossref PubMed Scopus (53) Google Scholar), and secreted nanoparticles (Ricklefs et al., 2016Ricklefs F. Mineo M. Rooj A.K. Nakano I. Charest A. Weissleder R. Breakefield X.O. Chiocca E.A. Godlewski J. Bronisz A. Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity.Cancer Res. 2016; 76: 2876-2881Crossref PubMed Scopus (61) Google Scholar, Wei et al., 2017Wei Z. Batagov A.O. Schinelli S. Wang J. Wang Y. El Fatimy R. Rabinovsky R. Balaj L. Chen C.C. Hochberg F. et al.Coding and noncoding landscape of extracellular RNA released by human glioma stem cells.Nat. Commun. 2017; 8: 1145Crossref PubMed Scopus (187) Google Scholar, Spinelli et al., 2018Spinelli C. Montermini L. Meehan B. Brisson A.R. Tan S. Choi D. Nakano I. Rak J. Molecular subtypes and differentiation programmes of glioma stem cells as determinants of extracellular vesicle profiles and endothelial cell-stimulating activities.J. Extracell. Vesicles. 2018; 7: 1490144Crossref PubMed Scopus (24) Google Scholar, Zhang et al., 2019bZhang Q. Higginbotham J.N. Jeppesen D.K. Yang Y.-P. Li W. McKinley E.T. Graves-Deal R. Ping J. Britain C.M. Dorsett K.A. et al.Transfer of Functional Cargo in Exomeres.Cell Rep. 2019; 27: 940-954.e6Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) has driven recent developments in the identification of GBM tumor subtypes that support a coherent model of GBM heterogeneity. Gene expression-based tumor subtypes have been resolved to sample-specific mixtures of up to 4 dominant single-cell GBM expression signatures with unique underlying functional cell states that are governed by genetic and microenvironmental cues, but appear to be both plastic and commutable, which is consistent with other similarities to neural precursor cells (Neftel et al., 2019Neftel C. Laffy J. Filbin M.G. Hara T. Shore M.E. Rahme G.J. Richman A.R. Silverbush D. Shaw M.L. Hebert C.M. et al.An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.Cell. 2019; 178: 835-849.e21Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). Studies focused on GSCs identified two distinct functional states that match GBM molecular subtypes (Rooj et al., 2017Rooj A.K. Ricklefs F. Mineo M. Nakano I. Chiocca E.A. Bronisz A. Godlewski J. MicroRNA-Mediated Dynamic Bidirectional Shift between the Subclasses of Glioblastoma Stem-like Cells.Cell Rep. 2017; 19: 2026-2032Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, Wang et al., 2019bWang L. Babikir H. Müller S. Yagnik G. Shamardani K. Catalan F. Kohanbash G. Alvarado B. Di Lullo E. Kriegstein A. et al.The phenotypes of proliferating glioblastoma cells reside on a single axis of variation.Cancer Discov. 2019; 9: 1708-1719Crossref PubMed Scopus (53) Google Scholar), as well as a microRNA (miRNA)-driven, possibly extracellular vesicle (EV)-mediated, bidirectional transition between distinct GSC subpopulations within the tumor (Ricklefs et al., 2016Ricklefs F. Mineo M. Rooj A.K. Nakano I. Charest A. Weissleder R. Breakefield X.O. Chiocca E.A. Godlewski J. Bronisz A. Extracellular Vesicles from High-Grade Glioma Exchange Diverse Pro-oncogenic Signals That Maintain Intratumoral Heterogeneity.Cancer Res. 2016; 76: 2876-2881Crossref PubMed Scopus (61) Google Scholar, Rooj et al., 2017Rooj A.K. Ricklefs F. Mineo M. Nakano I. Chiocca E.A. Bronisz A. Godlewski J. MicroRNA-Mediated Dynamic Bidirectional Shift between the Subclasses of Glioblastoma Stem-like Cells.Cell Rep. 2017; 19: 2026-2032Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Molecular profiles of GSC-EVs suggest that their effects within the tumor may depend on the molecular subtype and functional state of the donor cells (Spinelli et al., 2018Spinelli C. Montermini L. Meehan B. Brisson A.R. Tan S. Choi D. Nakano I. Rak J. Molecular subtypes and differentiation programmes of glioma stem cells as determinants of extracellular vesicle profiles and endothelial cell-stimulating activities.J. Extracell. Vesicles. 2018; 7: 1490144Crossref PubMed Scopus (24) Google Scholar, Wei et al., 2017Wei Z. Batagov A.O. Schinelli S. Wang J. Wang Y. El Fatimy R. Rabinovsky R. Balaj L. Chen C.C. Hochberg F. et al.Coding and noncoding landscape of extracellular RNA released by human glioma stem cells.Nat. Commun. 2017; 8: 1145Crossref PubMed Scopus (187) Google Scholar). Heterotypic interactions of GSCs with immune, endothelial, and other cells within the tumor microenvironment (TME) are implicated in generating histological heterogeneity of GBM tumors, recurrent treatment failures, and high lethality (Perrin et al., 2019Perrin S.L. Samuel M.S. Koszyca B. Brown M.P. Ebert L.M. Oksdath M. Gomez G.A. Glioblastoma heterogeneity and the tumour microenvironment: implications for preclinical research and development of new treatments.Biochem. Soc. Trans. 2019; 47: 625-638Crossref PubMed Scopus (41) Google Scholar, Schiffer et al., 2018Schiffer D. Annovazzi L. Casalone C. Corona C. Mellai M. Glioblastoma: microenvironment and niche concept.Cancers (Basel). 2018; 11: 5Crossref Scopus (58) Google Scholar). Molecular profiles capturing the effects of GBM on these non-malignant cells are emerging (Sankowski et al., 2019Sankowski R. Böttcher C. Masuda T. Geirsdottir L. Sagar Sindram E. Seredenina T. Muhs A. Scheiwe C. Shah M.J. et al.Mapping microglia states in the human brain through the integration of high-dimensional techniques.Nat. Neurosci. 2019; 22: 2098-2110Crossref PubMed Scopus (84) Google Scholar, Venteicher et al., 2017Venteicher A. Tirosh I. Hebert C. Yizhak K. Neftel C. Filbin M. Hovestadt V. Escalante L. Shaw M. Rodman C. et al.Decoupling genetics, lineages, and microenvironment in IDH-mutant gliomas by single-cell RNA-seq.Science. 2017; 355: eaai8478Crossref PubMed Scopus (293) Google Scholar), but the predominant intercellular agents are not known. GSCs communicate with the TME through several modes, including exchange of soluble molecules and EVs, as well as by cell-to-cell contacts (Broekman et al., 2018Broekman M.L. Maas S.L.N. Abels E.R. Mempel T.R. Krichevsky A.M. Breakefield X.O. Multidimensional communication in the microenvirons of glioblastoma.Nat. Rev. Neurol. 2018; 14: 482-495Crossref PubMed Scopus (125) Google Scholar, Spinelli et al., 2019Spinelli C. Adnani L. Choi D. Rak J. Extracellular Vesicles as Conduits of Non-Coding RNA Emission and Intercellular Transfer in Brain Tumors.Noncoding RNA. 2019; 5: 1Google Scholar). The exchange of molecules and genetic information via EVs plays a critical role in GBM progression and tumor angiogenesis, which may involve reprogramming the epigenome and transcriptome of endothelial cells (ECs) (Aslan et al., 2019Aslan C. Maralbashi S. Salari F. Kahroba H. Sigaroodi F. Kazemi T. Kharaziha P. Tumor-derived exosomes: implication in angiogenesis and antiangiogenesis cancer therapy.J. 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To further elucidate non-conventional angiogenic pathways in GBM, we here examine the GSC-EV-mediated transfer of extracellular RNAs (exRNAs) from human GSCs to human brain microvascular ECs (HBMVECs) in vitro by molecular profiling and to ECs in vivo via histoepigenetic analysis by computational deconvolution. EV-derived miRNAs are known to convey growth-promoting and angiogenic signaling in GBM (Beyer et al., 2017Beyer S. Fleming J. Meng W. Singh R. Haque S.J. Chakravarti A. The Role of miRNAs in angiogenesis, invasion and metabolism and their therapeutic implications in gliomas.Cancers (Basel). 2017; 9: 85Crossref PubMed Scopus (37) Google Scholar, Chen et al., 2019Chen X. Yang F. Zhang T. Wang W. Xi W. Li Y. Zhang D. Huo Y. Zhang J. Yang A. Wang T. MiR-9 promotes tumorigenesis and angiogenesis and is activated by MYC and OCT4 in human glioma.J. Exp. Clin. Cancer Res. 2019; 38: 99Crossref PubMed Scopus (48) Google Scholar, Todorova et al., 2017Todorova D. Simoncini S. Lacroix R. Sabatier F. Dignat-George F. Extracellular vesicles in angiogenesis.Circ. Res. 2017; 120: 1658-1673Crossref PubMed Scopus (218) Google Scholar, Wong et al., 2015Wong H.A. Fatimy R.E. Onodera C. Wei Z. Yi M. Mohan A. Gowrisankaran S. Karmali P. Marcusson E. Wakimoto H. et al.The Cancer Genome Atlas analysis predicts microRNA for targeting cancer growth and vascularization in glioblastoma.Mol. Ther. 2015; 23: 1234-1247Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar); however, the molecular events controlling this process in HBMVECs are largely unknown. We hypothesized that GSC-derived exRNAs, along with more conventional vascular GFs, jointly modulate the gene-expression landscape of ECs to promote angiogenesis. To this end, we compared the effects of GFs and GSC-EVs on angiogenic pathways elicited in cultured HBMVECs, by associating changes in DNA methylome and total RNA profiles in ECs with microRNA (miRNA) content of GSC-EVs. The expression profiles obtained from ECs by histoepigenetic analysis of GBM molecular profiles in the The Cancer Genome Atlas (TCGA) collection (Cancer Genome Atlas Research Network, 2008Cancer Genome Atlas Research NetworkComprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (5160) Google Scholar) revealed a concordance of effects in vitro and in vivo. Finally, we identified candidate proangiogenic miRNAs that are transferred via GSC-EVs into HBMVECs. To investigate the potential of GSC-EVs to elicit an angiogenic response from brain microvasculature, we isolated EVs from the conditioned media of GBM8 human primary GBM stem-like cells (Wakimoto et al., 2012Wakimoto H. Mohapatra G. Kanai R. Curry Jr., W.T. Yip S. Nitta M. Patel A.P. Barnard Z.R. Stemmer-Rachamimov A.O. Louis D.N. et al.Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells.Neuro-oncol. 2012; 14: 132-144Crossref PubMed Scopus (154) Google Scholar) and added them to the basal medium of HBMVECs cultured on a Matrigel substrate (Figure 1A, top panel). A standardized cocktail of angiogenic GFs added to HBMVECs in identical conditions served as a positive control. Cells cultured identically but without added stimulus served as the baseline for comparison. Vascularization metrics were quantified 16 h after application of the stimuli. GSC-EV treatment (+EV) stimulated vascularization similar to that of the GF treatment (+GF), as indicated by increases in total tubule length and total counts of tubules, branch points, and meshes (Figure 1A, bar plot). No meaningful vascularization was observed when HBMVECs were treated with supernatant from the EV isolation procedure (+GBM sup), nor with the pellet or supernatant from a mock isolation of EVs from unconditioned endothelial basal medium (+EBM pellet, +EBM sup) (Figure 1A, bar plot). The responses obtained from GBM8-conditioned media fractions (+EV, +GBM sup) and GFs could not be compared quantitatively because the concentrations in the conditioned media are not normalized to one another nor are they calibrated to physiologically relevant concentrations. These in vitro experiments were designed to detect broad qualitative differences in the EC response to EV and GF stimuli obtained according to well-established (+EV; Zaborowski et al., 2015Zaborowski M.P. Balaj L. Breakefield X.O. Lai C.P. Extracellular vesicles: composition, biological relevance, and methods of study.Bioscience. 2015; 65: 783-797Crossref PubMed Scopus (299) Google Scholar) or standardized (+GF; tube-formation assay) protocols. Specifically, we asked whether the similar vascularization phenotypes of +EV and +GF were associated with similar or divergent transcriptional and epigenomic changes in HBMVECs. Over the set of synergic transcriptional changes (>2-fold), we detected, for +EV and +GF, respectively, the upregulation of 229 and 2 genes (Figure 1B, quadrant I, top right) and the downregulation of 18 and 8 genes (Figure 1B, quadrant III, bottom left). Only 1 gene (SELE: E-Selectin) showed a large concordant change (>2-fold decrease) in both treatments, indicative of divergent transcriptional responses. We therefore focused on the 78 genes showing opposite changes in transcript levels, 72 of which showed larger perturbations in +EV as compared to +GF (Figure 1B, quadrants II—top left and IV—bottom right). Specifically, +EV decreased the abundance of 29 genes, with just 4 genes reduced by +GF. DNA methylation over gene bodies and promoters also diverged (Figure 1C), with +GF increasing and +EV decreasing on average, which is consistent with the upregulation of more genes in +EV versus +GF. Methylation over 100-kb tiles taken genome-wide was less divergent, with demethylation dominating for both treatments, although the methylation gain was more pronounced with +GF, in accordance with the signal from promoters and gene bodies. The highly divergent transcriptomic and epigenomic responses to +GF and +EV belie the similar vascularization phenotypes in vitro and hint at different primary pathways of action. To examine the relevance of our cell line experiments for tumor biology in vivo, we compared the transcriptomic and epigenomic signatures observed in vitro to those observed in ECs of human GBM tumors in vivo. Specifically, we exploited the transcriptomic divergence to determine whether changes in ECs in vivo correlated primarily with the in vitro responses of HBMVECs to +GF or +EV. GBM-associated changes in ECs in vivo were identified by the histoepigenetic analysis of glioma tumors from the TCGA collection (Brennan et al., 2013Brennan C.W. Verhaak R.G. McKenna A. Campos B. Noushmehr H. Salama S.R. Zheng S. Chakravarty D. Sanborn J.Z. Berman S.H. et al.TCGA Research NetworkThe somatic genomic landscape of glioblastoma.Cell. 2013; 155: 462-477Abstract Full Text Full Text PDF PubMed Scopus (2403) Google Scholar) using the Epigenomic Deconvolution (EDec) method (Onuchic et al., 2016Onuchic V. Hartmaier R.J. Boone D.N. Samuels M.L. Patel R.Y. White W.M. Garovic V.D. Oesterreich S. Roth M.E. Lee A.V. Milosavljevic A. Epigenomic deconvolution of breast tumors reveals metabolic coupling between constituent cell types.Cell Rep. 2016; 17: 2075-2086Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The TCGA GBM collection generally lacks molecular profiling data for matched normal non-cancerous samples, so we included lower-grade glioma (LGG) samples as a control group, given that microvascular structures of GBM and LGG are characteristically disparate (Guarnaccia et al., 2018Guarnaccia L. Navone S.E. Trombetta E. Cordiglieri C. Cherubini A. Crisà F.M. Rampini P. Miozzo M. Fontana L. Caroli M. et al.Angiogenesis in human brain tumors: screening of drug response through a patient-specific cell platform for personalized therapy.Sci. Rep. 2018; 8: 8748Crossref PubMed Scopus (18) Google Scholar, Louis et al., 2007Louis D.N. Ohgaki H. Wiestler O.D. Cavenee W.K. Burger P.C. Jouvet A. Scheithauer B.W. Kleihues P. The 2007 WHO classification of tumours of the central nervous system.Acta Neuropathol. 2007; 114: 97-109Crossref PubMed Scopus (7353) Google Scholar, Bergers and Benjamin, 2003Bergers G. Benjamin L.E. Tumorigenesis and the angiogenic switch.Nat. Rev. Cancer. 2003; 3: 401-410Crossref PubMed Scopus (2657) Google Scholar). EDec estimated 5 cancer-cell epigenome profiles, all of which correspond to previously defined LGG and GBM molecular subtypes. In GBM tumors, 3 of the cancer-cell profiles (GBM 1, 2, and 3) were found in appropriately high proportions within tumors of the Proneural+G-CIMP (glioma-CpG island methylator phenotype), classical, and proneural subtypes (Figure 2A). The remaining profiles (LGG1 and LGG2) were enriched within LGG tumors (Figure 2A). EDec also estimated proportions of 4 non-cancer cell types: neuronal, glial, immune, and endothelial. Normal adjacent tissue samples collected by TCGA were highly enriched for non-cancer profiles, although some cancer profiles could be detected in certain samples, consistent with the diffuse growth of gliomas (Figure 2A). The GBM8 epigenome revealed the greatest similarity to that of the estimated Proneural cancer epigenome (Figure 2B, GBM.3), consistent with the previous characterization of the GBM8 cell line as a Proneural-like, stem-like cell type with wild-type IDH1 (Teng et al., 2017Teng J. da Hora C.C. Kantar R.S. Nakano I. Wakimoto H. Batchelor T.T. Chiocca E.A. Badr C.E. Tannous B.A. Dissecting inherent intratumor heterogeneity in patient-derived glioblastoma culture models.Neuro-oncol. 2017; 19: 820-832PubMed Google Scholar). The results indicate successful deconvolution, warrant confidence in the inferred gene expression profiles, and validate the GBM8 cell line as an in vitro model for GBM in the TCGA collection. The differences in deconvoluted EC gene expression and methylation profiles between GBM and LGG should therefore reflect GBM-associated differences of the microvasculature in vivo. Differential expression analysis of GBM versus LGG ECs revealed GBM-associated perturbations (>2-fold change, false discovery rate [FDR] < 0.05) of 1,632 genes. To determine whether GFs or EVs play a dominant role in vivo, we asked whether the GBM-associated transcriptomic perturbations in vivo mostly reflected the transcriptomic response of HBMVECs to +GF or +EV in vitro (Figure 2C). The treatments elicited divergently trending changes from 597 (42+243)+(284+28) of the 1,632 genes (Figure 2C, quadrants II—top left and IV—bottom right), and the directions of +GF-induced expression changes were concordant with 54.6% [(42 + 284)/597] (p = 0.0023, binomial test) of the GBM-associated perturbations (Figure 2C, orange dots). This suggests a somewhat larger trans" @default.
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• W3008596935 date "2020-02-01" @default.
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• W3008596935 title "Glioma-Derived miRNA-Containing Extracellular Vesicles Induce Angiogenesis by Reprogramming Brain Endothelial Cells" @default.
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• W3008596935 doi "https://doi.org/10.1016/j.celrep.2020.01.073" @default.
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