Clonal variation in drug and radiation response among glioma-initiating cells is linked to proneural-mesenchymal transition (Methylation BeadChip)
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE89400 | GEO | 2016/12/13
SECONDARY ACCESSION(S): PRJNA352066
REPOSITORIES: GEO
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Project description:Intra-tumor heterogeneity is a hallmark of glioblastoma multiforme, and thought to negatively affect treatment efficacy. Here we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability between clones, including a wide range of responses to radiation and drugs. This widespread variability was observed as a continuum of multitherapy resistance phenotypes linked to a proneural-to-mesenchymal shift in the transcriptome.
2016-12-13 | GSE89398 | GEO
Project description:Intra-tumor heterogeneity is a hallmark of glioblastoma multiforme, and thought to negatively affect treatment efficacy. Here we establish libraries of glioma-initiating cell (GIC) clones from patient samples and find extensive molecular and phenotypic variability between clones, including a wide range of responses to radiation and drugs. This widespread variability was observed as a continuum of multitherapy resistance phenotypes linked to a proneural-to-mesenchymal shift in the transcriptome.
2016-12-13 | GSE89399 | GEO
Project description:Glioblastoma multiforme (GBM) is an aggressive, heterogeneous and highly vascularized brain tumor. GBM is thought to arise from glioblastoma stem-like cells (GSCs) which are characterized as being either proneural or mesenchymal. The former isolates of GSCs are tight sphere forming and slow growing while mesenchymal GSCs are lose sphere forming, fast growing, highly invasive and when dominant yield poorer patient prognosis. GSCs are known to be plastic in nature and can therefore evolve from a proneural to a mesenchymal state. Here, we observed that factors secreted by endothelial cells (which make up the brain vasculature) alter several properties of GSCs resulting in the acquisition of a more mesenchymal and invasive phenotype coupled with changes at the level of secretory and cellular proteome. Thus, using mass spectrometry, quantitative proteomic analysis and GO term filters, we identified several mesenchymal traits in proneural GSCs exposed to endothelial cell secretome. Specifically, proneural cells treated with the conditioned media derived from human umbilical vein endothelial cells (HUVEC) upregulated the expression of mesenchymal proteins such as CD44 and VIM, while downregulating the expression of the proneural proteins such as NOTCH1, activated NOTCH intracellular domain (NICD), SOX2 and NESTIN, which were validated using flow cytometry (FACS) and western blots (WB). Using DAVID analysis tool, we detected the features of cellular proteome indicative of the activation of NFkB, Wnt and several other pathways in the proneural cells treated with HUVEC conditioned media. Using conditioned media fractionation through several centrifugation steps we identified the extracellular vesicles (EV) sedimented at 100,000 x g using ultracentrifugation, as the source of activity in endothelial conditioned media capable of triggering mesenchymal shift in proneural GSCs. EVs are heterogeneous membrane structures containing multiple bioactive macromolecules, which have the ability to carry multiple bioactive proteins, transfer them to recipient cells and alter their function, signalling and biological programmes. We compared the effects of EVs, soluble fraction and unfractionated conditioned media in terms of their ability to trigger mesenchymal changes in the phenotype of proneural GSCs. Once the cultures were established, the culture medium was removed and replaced with HUVEC-derived material (conditioned media, supernatant or EV fraction) and responses evaluated over 7 days by microscopical analysis of sphere structures, and biochemically by following the aforementioned proneural or mesenchymal markers (WB, FACS). We observed an upregulation of mesenchymal proteins, as well as downregulation of the proneural proteins, mentioned above. These effects recapitulated those of unfractionated conditioned media and were absent from target cells exposed to EV-depleted conditioned media. The data analysis of EV proteome included canonical markers and pathways of cellular vesiculation as well as markers and pathways of interest with regards to the biological effects associated with treatment of GSC recipient cells. In this regard we observed several EV related tetraspanin markers, which were validated using western blot including CD9, CD63, CD81 and a purity control, BIP. Although we identified several potential effectors associated with endothelial cell EVs that could impact proneural cell phenotype, we focused on MMPs for at least three reasons: (i) evidence in the literature (see text) indicated that MMPs may induce differentiation programs in neural stem cells; (ii) MMPs in EV cargo were relatively abundant and have been implicated in various biological processes; (iii) MMPs released from endothelial cells could be functionally involved in disrupting proneural cell sphere structures that we observed in the presence of endothelial cell secretome. We noted the expressions of MMP1, MMP2, MMP11 and MP14 in our HUVEC-EV mass spectrometry dataset, the activity of which was validated using the MMP activity assay kit from abcam (ab112146). Using GO terms we also detected a signal for NFkB pathway activation in the proteome of endothelial (HUVEC) conditioned media-treated proneural GSCs. NFkB activation is regarded as hallmark of mesenchymal phenotype in GSCs and GBM cells. We validated that the upregulation of NFkB, was also true for the proneural cells treated with HUVEC derived EVs. Moreover, upon blocking MMP expression in proneural cells treated with endothelial cell EVs, we inhibited the activation of NFkB activity thereby documenting that the initial effects of MMPs trigger a shift in cellular phenotype toward NfkB activation and mesenchymal reprogramming. Briefly, we compared GO terms of GSC157 cells treated with their own or HUVEC-derived EVs. Validation of the NFkB pathway activation was analysed using WB and immunofluorescence for levels of NFkB and phosphor-NFkB.
2022-10-14 | PXD034819 | Pride
Project description:Glioma initiating cells (GICs) are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanism of GIC maintenance/differentiation, we established GIC clones from GBM patient tumors having the potential to differentiate into malignant gliomas in mouse intracranial xenograft, and established an in vitro glioma induction system by using serum stimulation. Upon the serum stimulation, the GIC spheres showed increased cellular proliferation, motility, filopodia/lameripodia formation and adhesion to the culture dishes. Simultaneously, the NSC marker proteins such as CD133 and Sox2 were down-regulated, and the astrocyte/glioma marker GFAP and the malignancy marker CD44 dramatically up-regulated. To identify genes/proteins whose expression changes dynamically during the differentiation of GICs into glioma cells, these GICs were subjected to DNA microarray/iTRAQ based integrated proteomics. Within 4 hours of tumor removal from GBM patients, tissues were subjected to GIC preparation. After successive cloning, total RNA from GIC clones (GIC03A and GIC03U) on day 2 or 7 of subculture in NSC medium with or without 10% FCS was subjected to the analysis with Affymetrix microarrays. Simultaneously, the proteins extracted from the same set of cells were subjected to LC-shot gun analyses using the 8-plex iTRAQ method. We sought to obtain the information of the common molecules that were up- or down-regulated during the GSC differentiation process, and functional targets for the early onset of GIC-associated glioma.
2013-01-26 | E-GEOD-43762 | biostudies-arrayexpress
Project description:Glioma initiating cells (GICs) are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanism of GIC maintenance/differentiation, we established GIC clones from GBM patient tumors having the potential to differentiate into malignant gliomas in mouse intracranial xenograft, and established an in vitro glioma induction system by using serum stimulation. Upon the serum stimulation, the GIC spheres showed increased cellular proliferation, motility, filopodia/lameripodia formation and adhesion to the culture dishes. Simultaneously, the NSC marker proteins such as CD133 and Sox2 were down-regulated, and the astrocyte/glioma marker GFAP and the malignancy marker CD44 dramatically up-regulated. To identify genes/proteins whose expression changes dynamically during the differentiation of GICs into glioma cells, these GICs were subjected to DNA microarray/iTRAQ based integrated proteomics.
2013-01-26 | GSE43762 | GEO
Project description:Phenotypic variability among different knockout clones of the same gene is a common problem confounding the establishment of robust genotype-phenotype correlations. Optimized genome editing protocols to enhance reproducibility include measures to reduce off-target effects. However, even if current state-of-the-art protocols are applied phenotypic variability is frequently observed. Here we identify heterogeneity of wild-type cells as an important and often neglected confounding factor in genome-editing experiments. We demonstrate that isolation of individual wild-type clones from an apparently homogenous stable cell line uncovers significant phenotypic differences between clones. Strikingly, we observe hundreds of differentially regulated transcripts when comparing two populations of wild-type cells. Heterogeneity of wild-type cells thus contributes to variability in genome-edited cells when these are generated through isolation of clones. We show that the generation of monoclonal isogenic wild-type cells prior to genomic manipulation reduces phenotypic variability.
2022-02-18 | GSE196762 | GEO
Project description:Asymmetric neuronal expansion is thought to drive evolutionary transitions from lissencephalic to gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes interact to sustain neurogenic continuity and lissencephaly in rodents. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage-restricted due to Neurog2-Ascl1 cross-repression, and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selective killing of double+ NPCs using Neurog2-Ascl1 split-Cre mice and three ‘deletor’ strains breaks neurogenic symmetry by locally disrupting Notch signaling, leading to cortical folding. Consistent with proneural genes driving discontinuous neurogenesis and folding via Notch, NEUROG2, ASCL1 and HES1 transcripts are modular in gyrencephalic macaque cortices. Neurog2/Ascl1 double+ NPCs are thus Notch-ligand expressing ‘niche’ cells that control neurogenic periodicity and cortical gyrification.
2021-07-08 | GSE151775 | GEO
Project description:To identify factors involved in glioma-initiating cells (GICs), we compared gene expressions between GIC-like cells and non-GICs.
2011-06-01 | GSE28091 | GEO
Project description:Fluorescence-activated cell sorting of M4-GFP wing imaginal disc cells was used to recover a purified population of the cells that comprise the “proneural clusters” from which sensory organ precursors of the peripheral nervous system (PNS) arise. Whole-genome microarray analysis and in situ hybridization was then used to identify and verify a set of genes that are preferentially expressed in proneural cluster cells. Genes in this set encode proteins with a diverse array of implied functions, and loss-of-function analysis of two candidate genes shows that they are indeed required for normal PNS development. Keywords = Proneural Keywords = PNS Keywords = PNC Keywords = SOP Keywords: parallel sample
2005-03-05 | GSE2359 | GEO
Project description:Glioma initiating/stem cells (GIC/GSC) are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanism of GIC/GSC maintenance/differentiation, we established GIC/GSC clones, having the potential to differentiate into malignant gliomas, from glioblastoma patient’s tissues, and subjected to DNA microarray/iTRAQ based integrated proteomics. 21,857 mRNAs and 8,471 proteins were identified and integrated into a gene/protein expression analysis chart (iPEACH). The data integration and extraction of global proteins and mRNAs revealed that, during the GIC/GSC differentiation, cell adhesion molecules including integrin aV/ECMs and RAS-MAPK/PI3K signalings were significantly up-regulated, meanwhile, SOX2, CD133, and specific proteoglycans/synthetic-enzymes/metabolic pathways were obviously down-regulated. Among them, we focused proteoglycans and their synthetic-enzymes. GIC/GSC differentiation was significantly associated with the decrease of CSPG (CS-modified form) and dramatically induced by the CS-degradation enzyme which also induces the up-regulation of ERK-AKT signaling and GFAP without any other agents. Importantly, these differentiation processes were also associated with the interaction of CSPG (CS-unmodified form) and integrin-aV, and suppressed by integrin-inhibitors/CS administrations significantly. Combination treatments of a cancer-drug Temozolomide and these GIC/GSC-differentiation inhibitors suppressed glioma progression, increased the chemosensitivities, and led the longer survival of mouse xenograft-models. Functional integrated proteomics for the first time demonstrates that the GIC/GSC induces the specific proteoglycans to regulate GIC/GSC stemness/differentiation via the integlin signalings which may be a clinical target against malignant gliomas.
2018-11-30 | PXD008331 | JPOST Repository