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Title: Core pathway mutations induce de-differentiation of murine astrocytes into glioblastoma stem cells that are sensitive to radiation but resistant to temozolomide.      
keywords:
Transcriptome or Gene expression
ID:
PRJNA254456
description:
BACKGROUND: Glioma stem cells (GSCs) from human glioblastomas (GBMs) are resistant to radiation and chemotherapy and may drive recurrence. Treatment efficacy may depend on GSCs, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype. METHODS: To model genetic alterations in human GBM core signaling pathways, we induced Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Neurosphere culture, differentiation, and orthotopic transplantation assays were used to assess whether these mutations induced de-differentiation into GSCs. Genome-wide chromatin landscape alterations and expression profiles were examined by formaldehyde-assisted isolation of regulatory elements (FAIRE) seq and RNA-seq. Radiation and temozolomide efficacy were examined in vitro and in an allograft model in vivo. Effects of radiation on transcriptome subtype were examined by microarray expression profiling. RESULTS: Cultured triple mutant astrocytes gained unlimited self-renewal and multilineage differentiation capacity. These cells harbored significantly altered chromatin landscapes that were associated with downregulation of astrocyte- and upregulation of stem cell-associated genes, particularly the Hoxa locus of embryonic transcription factors. Triple-mutant astrocytes formed serially transplantable glioblastoma allografts that were sensitive to radiation but expressed MGMT and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of GBM allografts from proneural to mesenchymal. CONCLUSION: A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into GSCs with altered chromatin landscapes and transcriptomes. This non-germline genetically engineered mouse model mimics human proneural GBM on histopathological, molecular, and treatment response levels. It may be useful for dissecting the mechanisms of treatment resistance and developing more effective therapies. Overall design: Conditional Rb knockout (T), Kras activation (R), and Pten deletion (P) mutations were induced in cortical murine astrocytes from C57BL/6 mice to model genetic alterations in the core signaling pathways of human GBM. Primary astrocytes were cultured as previously described. Briefly, cells were selectively harvested from the cortices of T, TR, and TRP postnatal day 1-4 pups, dissociated by trituration in trypsin, and incubated at 37°C for 20 minutes. Cells were pelleted, resuspended, and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (complete media). At 50% confluence, cells were infected with a recombinant adenoviral vector expressing Cre recombinase from the constitutive cytomegalovirus promoter (Ad5CMVCre, University of Iowa Gene Transfer Vector Core) at an MOI of 50 for 6 hours in complete media.3 Following infection, cells were rinsed in PBS and serially cultured in complete media at 37°C in 5% CO2 for up to 60 passages. Efficacy of radiation and temozolomide was examined in an orthotopic allograft model of proneural GBM and expression profiling was used to examine the effects of radiation on transcriptomal subtype. To evaluate the effects of treatment, syngeneic C57BL/6 mice were injected with 10^5 TRP astrocytes. Tumors were established for six days and mice (9-12 per group) were randomized to treatment with temozolomide (100 mg/kg q.d. via intraperitoneal injection on days 6-10), radiation (5 Gy on days 6, 8, and 10), or both. Animals were monitored by an observer blinded to treatment group and sacrificed upon development of neurological symptoms, at which point mice were sacrificed, tumors macrodissected, and flash frozen. Survival was evaluated by the Kaplan-Meier method and effects of treatment compared by log-rank tests. Microarray analysis was completed for 37 high-grade astrocytoma allografts that were either untreated or radiation treated.
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA254456
authentication:
none
authorization:
none
ID:
pmid:26826202
name:
Mus musculus
ncbiID:
ncbitax:10090
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject