GBM cell lines derived and propagated according to Cambridge protocol
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Watts Colin
PROVIDER: S-ECPF-GEOD-15846 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:We screened the gene expression in GBM cell lines cultured under serum-free conditions. We have special interest in genes associated with glial development such as NG2, Olig2 and PDGFRa. This microarray data gave us an idea about the expression of these genes and made the basis for further investigation.
2009-04-28 | GSE15846 | GEO
Project description:We screened the gene expression in GBM cell lines cultured under serum-free conditions. We have special interest in genes associated with glial development such as NG2, Olig2 and PDGFRa. This microarray data gave us an idea about the expression of these genes and made the basis for further investigation. Seven GBM cells were derived from 7 different patients under serum-free conditions according to Cambridge protocol as previously described (Fael Al-Mayhani et al, 2009). The RNA was extracted and its quality was assessed. The samples were sent for microarray screening using Illumina platform. The gene was considered as present when the detection value was greater than 0.99.
2010-04-09 | E-GEOD-15846 | biostudies-arrayexpress
Project description:Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants.Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling.Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors.We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.
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Project description:Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Hypoxia, low oxygen, has been linked to GBM infiltration and aggressive tumor progression leading to poor patient outcome. Hypoxia-induced pathways are activated by the stabilization of HIF1-α, which upregulates genes related to reprogramming metabolism, angiogenesis, cell trafficking, and cell survival. However, underlying mechanisms of HIF-1α involvement with GBM cell specific pathways is not fully understood. Here, we show that hypoxia can induce specific genes dependent on GBM subtype. In a in vitro migration assay, we screened eight patient-derived GBM stem cell lines. Two lines, UCA and UNB, were found to induce an increased migration ability in response to hypoxia. By RNA bulk sequencing, we identified key specific genes that were upregulated in each cell line that were not shared between them. Furthermore, we found that this data set correlates to previous single-cell RNA sequencing findings of GBM having differentiated transcriptional subtypes. Our results demonstrate how hypoxia induces a specific response in tumor cells by activating genes for survival and tumor progression. Our study forms the basis for future approaches to characterize top candidate genes and elucidate their role in tumor survival in response to hypoxia. Inhibition of these top candidate genes can be a strategy used to dampen hypoxia pro-tumor properties and may lead to better outcomes for GBM patients.
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Project description:The recent incorporation of molecular features into the diagnosis of Glioblastoma Multiforme patients has led to an improved categorisation into different tumour subtypes with different prognosis and disease management. In this work, we have exploited the benefits of genome-wide multiomic approaches to identify potential molecular vulnerabilities existing on GBM patients. We used the Illumina MethylationEPIC Beadchip platform to describe the genome-wide 5mC and 5hmC DNA methylation landscape of a total of 9 patient-derived Glioblastoma Multiforme Cell lines obtained from the human glioblastoma cell culture resource (HGCC) and 4 brain samples obtained from non-tumoral controls
2023-06-01 | E-MTAB-12473 | biostudies-arrayexpress
Project description:Oncopanel testing capture was carried out with Illumina’s TruSight Cancer Kit, which screens 94 genes and 284SNPs (Illumina, Inc). Sequencing was performed using MiSeq sequencer (Illumina) to produce 2 × 150 bp reads. Raw reads were mapped against reference genome hg19 using bwa-mem, de-duplicated using Picard tools and variant calling was performed using GATK best practices pipeline. Quality filtered variants (minimum of × 20 coverage and no more than 10% MAPQ0) were annotated using ANNOVAR with avSNP release of 142, 1000 genomes release of 2014 along with NIH-NHLBI 6500 exome database version 2. MAF filter (<0.01) was set based on ExAC, NIH 6500, gnomAD and 1000 genomes data. Pathogenicity evaluation was performed based on the inheritance mode, database entries (HGMD, ClinVar, CentoMD), in silico prediction tools (SIFT, Polyphen2, MutationTaster) and ACMG recommendations. All intronic variants located outside the boundaries of 10 bp from the exons and synonymous exonic ones were filtered out. Putative splicing variants were analyzed using Human Splicing Finder (HSF).
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Project description:GBM cell lines were plated onto 10 cm dishes at 100,000-200,000 cells per dish and allowed to grow for 3-4 days until confluency reached 50-70%. Media for all cell lines was DMEM with 10% FBS including 25 mM glucose, 6.2 mM glutamine and 200 uM Oleic Acid (conjugated to BSA). 1-2 hours prior to tracer incubation, media was aspirated and fresh media was used. Immediately prior to tracer incubation, media was again aspirated and then cells were washed with warm PBS to remove unlabeled metabolites. Cells were then incubated with labeled tracer (DMEM with 10% FBS including 25 mM Uniformly labeled 13C-6 glucose, 6.2 mM unlabeled glutamine and 200 uM unlabeled oleic acid). After 2 hours of incubation with tracer, media was aspirated, cells were quickly washed with 15 mL DI water, quenched with liquid nitrogen and then stored at -80oC until analysis.
2015-03-17 | ST000301 | MetabolomicsWorkbench
Project description:Ribosomal (r)DNA undergoes concerted evolution, the mechanisms of which are unequal crossing over and gene conversion. Despite the fundamental importance of these mechanisms to the evolution of rDNA, their rates have been estimated only in a few model species. We estimated recombination rate in rDNA by quantifying the relative frequency of intraindividual length variants in an expansion segment of the 18S rRNA gene of the cladoceran crustacean, Daphnia obtusa, in four apomictically propagated lines. We also used quantitative PCR to estimate rDNA copy number. The apomictic lines were sampled every 5 generations for 90 generations, and we considered each significant change in the frequency distribution of length variants between time intervals to be the result of a recombination event. Using this method, we calculated the recombination rate for this region to be 0.02-0.06 events/generation on the basis of three different estimates of rDNA copy number. In addition, we observed substantial changes in rDNA copy number within and between lines. Estimates of haploid copy number varied from 53 to 233, with a mean of 150. We also measured the relative frequency of length variants in 30 lines at generations 5, 50, and 90. Although length variant frequencies changed significantly within and between lines, the overall average frequency of each length variant did not change significantly between the three generations sampled, suggesting that there is little or no bias in the direction of change due to recombination.
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Project description:TMZ-resistance remains a main limitation in glioblastoma (GBM) treatment. TMZ is an alkylating agent whose cytotoxicity is modulated by O6-methylguanine-DNA methyltransferase (MGMT), whose expression is determined by MGMT gene promoter methylation status. The inflammatory marker COX-2 has been implicated in GBM tumorigenesis, progression, and stemness. COX-2 inhibitors are considered a GBM add-on treatment due to their ability to increase TMZ-sensitivity. We investigated the effect of TMZ on COX-2 expression in GBM cell lines showing different COX-2 levels and TMZ sensitivity (T98G and U251MG). β-catenin, MGMT, and SOX-2 expression was analyzed. The effects of NS398, COX-2 inhibitor, alone or TMZ-combined, were studied evaluating cell proliferation by the IncuCyte® system, cell cycle/apoptosis, and clonogenic potential. COX-2, β-catenin, MGMT, and SOX-2 expression was evaluated by RT-PCR, Western blotting, and immunofluorescence and PGE2 by ELISA. Our findings, sustaining the role of COX-2/PGE2 system in TMZ-resistance of GBM, show, for the first time, a relevant, dose-dependent up-regulation of COX-2 expression and activity in TMZ-treated T98G that, in turn, correlated with chemoresistance. Similarly, all the COX-2-dependent signaling pathways involved in TMZ-resistance also resulted in being up-modulated after treatment with TMZ. NS398+TMZ was able to reduce cell proliferation and induce cell cycle arrest and apoptosis. Moreover, NS398+TMZ counteracted the resistance in T98G preventing the TMZ-induced COX-2, β-catenin, MGMT, and SOX-2 up-regulation.
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Project description:Distinct responses of patient-derived GBM cell lines to hypoxia
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