Gene Fusion Discovery through RNA Sequencing of Human Glioblastoma Stem Cell Lines
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ABSTRACT: We used massively parallel, paired-end sequencing of expressed transcripts (RNA-seq) to detect novel gene fusions in short-term cultures of glioma stem-like cells freshly isolated from nine patients carrying primary glioblastoma multiforme (GBM). The culture of primary GBM tumors under serum-free conditions selects cells that retain phenotypes and genotypes closely mirroring primary tumor profiles as compared to serum-cultured glioma cell lines that have largely lost their developmental identities.
SECONDARY ACCESSION(S): PRJNA170222PRJNA170223
REPOSITORIES: dbGaP
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Project description:<p>We used massively parallel, paired-end sequencing of expressed transcripts (RNA-seq) to detect novel gene fusions in short-term cultures of glioma stem-like cells freshly isolated from nine patients carrying primary glioblastoma multiforme (GBM). The culture of primary GBM tumors under serum-free conditions selects cells that retain phenotypes and genotypes closely mirroring primary tumor profiles as compared to serum-cultured glioma cell lines that have largely lost their developmental identities.</p>
| phs000505.v1.p1 | EGA
Project description:We compared a large panel of human glioblastoma stem-like (GS) cell lines, corresponding primary tumors and conventional glioma cell lines to identify cell lines that preserve the transcriptome of human glioblastomas most closely, thereby allowing identification of shared therapeutic targets. We used Affymetrix HG-U133 Plus 2.0 microarrays to compare human glioblastoma stem-like (GS) cell lines, corresponding primary tumors and conventional glioma cell lines. We extracted total RNA from 32 conventional glioma cell lines, 12 GS cell lines (8 in two different passages), 7 clonal sublines derived from two GS lines, 12 original tumors, and 4 monolayer cultures established from the same tumors as GS-lines using standard serum conditions.
2011-02-12 | E-GEOD-23806 | biostudies-arrayexpress
Project description:We compared a large panel of human glioblastoma stem-like (GS) cell lines, corresponding primary tumors and conventional glioma cell lines to identify cell lines that preserve the transcriptome of human glioblastomas most closely, thereby allowing identification of shared therapeutic targets. We used Affymetrix HG-U133 Plus 2.0 microarrays to compare human glioblastoma stem-like (GS) cell lines, corresponding primary tumors and conventional glioma cell lines.
2011-02-12 | GSE23806 | GEO
Project description:Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test. The glioblastoma cell and exosome RNA was analyzed on two dual color arrays.
2008-11-15 | E-GEOD-13470 | biostudies-arrayexpress
Project description:Glioblastoma represents the most common and aggressive primary brain tumor type in adults. Stem cell regulatory pathways have been shown to be activated in gliomas supporting self-renewal, tumor maintenance and survival under stress. Glioblastoma stem-like phenotype, cell motility and tumor cell heterogeneity are considered significant hurdles to overcome for developing new treatment against these tumors. Transcription factor PROX1 has been associated with stem-like-phenotypes. Here, we overexpressed and suppressed PROX1 in glioma cell lines in order understand the gene expression regulated by this transcription factor.
2018-12-29 | E-MTAB-6991 | biostudies-arrayexpress
Project description:Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test.
2008-11-16 | GSE13470 | GEO
Project description:To identify receptors and pathways active in glioblastoma (GBM) stem like cells (GSCs), we generated and screened thousands of monoclonal antibodies (mAbs) for preferential binding to primary cultures enriched in GSCs. This led to the identification of the integrin alpha 7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression was negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses demonstrated a key biological function of ITGA7 in growth and invasion of GSCs. In addition, we showed that targeting ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling and led to a significant delay of tumor engraftment and strong reduction in size and invasion. Our data underline the potential value of ITGA7 as glioma biomarker and therapeutic target.
2018-01-10 | PXD006069 | Pride
Project description:Malignant glioma is the most common type of primary brain tumor diagnosed annually in 16,000 individuals in the United States. We performed a systematic large-scale transcriptomics data mining study of 9,783 Affymetrix samples from the GeneSapiens database in order to identify those genes that are most glioma-specific as compared to other cancers and normal tissues. We searched for genes that are highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared to 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (Hairy and Enhancer of Split 6) emerged as one of the most glioma-specific genes. In immunostaining of a tissue microarray series, HES6 was expressed in 335 (98.8%) out of the 339 clinical glioma samples. Recurrent grade 2 astrocytomas and grade 2-3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. Functional studies implied a critical role for HES6 in supporting survival of glioma cells, as evidenced by 60% reduced cancer cell viability and induction of Caspase 3/7 activity after HES6 silencing by RNA interference in A172 and LN405 cells. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which indicated a key role for HES6 in e.g. p53, c-myc, and NF-?B transcriptional networks. We conclude that HES6 has a critical role in sustaining glioma cell growth, survival, migration and possibly angiogenesis. HES6 is a potential therapeutic target and biomarker for glioma.
2011-06-30 | GSE22692 | GEO
Project description:Hypoxia is negatively associated with glioblastoma patient survival and contributes strongly to tumor resistance. Unfortunately novel anti-angiogenic therapy increases hypoxia and activates survival pathways in tumor cells leading to inevitable tumor relapse. Here we demonstrate that primary glioma cultures and cell lines depend on autophagy to survive severe hypoxia but also at normal oxygen levels. Positive regulators of autophagy are expressed at higher levels in tumor cells and induction in severe hypoxia is more prominent compared to normal brain cells. We demonstrate that autophagy is an essential/critical target for novel treatment in glioblastoma. We show ATG9A is induced by severe hypoxia and could be a novel player of the autophagic response in glioma cells. ATG9A targeting inhibited tumor growth, suggesting an essential role in glioma cell survival in vivo. While autophagy induction was also observed in normal astrocytes, glioma cells displayed a higher sensitivity towards autophagy inhibitors. Importantly, patient-derived cultures exhibited varying sensitivity towards anti-autophagy treatment, where certain cultures were dependent on autophagy already in normoxic conditions. The treatment of tumor bearing mice with the autophagy inhibitor chloroquine significantly increased mice survival, but combination treatment of the agent with bevacizumab did not reveal additive or synergistic effect. The present study demonstrates that inhibition of autophagy using chloroquine as a single agent provides a novel treatment strategy against glioblastoma. It remains to be seen whether autophagy inhibition will improve current standard of care treatment of newly diagnosed glioblastoma patients and whether more specific inhibitors will lead to stronger therapeutic outcome. (Provisional)
2015-11-10 | E-MTAB-3085 | biostudies-arrayexpress
Project description:Malignant glioma is the most common type of primary brain tumor diagnosed annually in 16,000 individuals in the United States. We performed a systematic large-scale transcriptomics data mining study of 9,783 Affymetrix samples from the GeneSapiens database in order to identify those genes that are most glioma-specific as compared to other cancers and normal tissues. We searched for genes that are highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared to 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (Hairy and Enhancer of Split 6) emerged as one of the most glioma-specific genes. In immunostaining of a tissue microarray series, HES6 was expressed in 335 (98.8%) out of the 339 clinical glioma samples. Recurrent grade 2 astrocytomas and grade 2-3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. Functional studies implied a critical role for HES6 in supporting survival of glioma cells, as evidenced by 60% reduced cancer cell viability and induction of Caspase 3/7 activity after HES6 silencing by RNA interference in A172 and LN405 cells. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which indicated a key role for HES6 in e.g. p53, c-myc, and NF-?B transcriptional networks. We conclude that HES6 has a critical role in sustaining glioma cell growth, survival, migration and possibly angiogenesis. HES6 is a potential therapeutic target and biomarker for glioma. A172 and LN405 cells (240 000 per well on 6-well plates) were transfected for 12-24 h with siHES6 pool, or three individual siRNAs (HES6_1, HES6_2, HES6_3 from Qiagen) or AllStars negative control siRNA (Qiagen) at 30 nM using SiLentFect (Bio-Rad Laboratories, Hercules, CA). One to two biological replicate transfections were performed. LN405 cells were also used for creating stable cell lines by transfecting either pEYFP-C1-mock or pEYFP-HES6 with Fugene6 (Roche) and selecting the positive cells with 600ug/ml of G418 (Sigma). The cells were kept in culture in the presence of 400ug/ml of G418. Total RNA was isolated with RNeasy (Qiagen) or MiRVana? Total RNA Isolation kit (Ambion). RNA quality was evaluated by using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).
2011-06-30 | E-GEOD-22692 | biostudies-arrayexpress