ABSTRACT: At the cellular level, the malignant characteristics of GBM are largely attributed to GBM stem cells (GSCs). These cells, endowed with stem-like properties, can self-renew, generate diverse cancerous cell populations, and initiate tumors in vivo. The GSC lines, NCH644 and NCH421k, when cultured as neurospheres (NS) — a condition that preserves their stem-like state, as evidenced by the expression of stem cell markers like SOX2, CD133, and Nestin — were found to align with the PN subclass and CL-B/C respectively.
Project description:Tissue from the telencephalon was isolated from E13.5 BALB/C mouse and allowed to culture as neurospheres in the presence of FGF2. These cultures were assessed for undifferentiated neural stem cells by the expression of Nestin and were found to be ~98% Nestin positive. Comparisons of these nestin positive neural stem cells will be made to R1 ES cells to identify the genes that are important in totipotent, self-renewing ES cells vs. commitment to the multipotent, self-renewing neural stem cell phenotype. Keywords: other
Project description:Tissue from the telencephalon was isolated from E13.5 BALB/C mouse and allowed to culture as neurospheres in the presence of FGF2. These cultures were assessed for undifferentiated neural stem cells by the expression of Nestin and were found to be ~98% Nestin positive. Comparisons of these nestin positive neural stem cells will be made to R1 ES cells to identify the genes that are important in totipotent, self-renewing ES cells vs. commitment to the multipotent, self-renewing neural stem cell phenotype. Experiment Overall Design: this experiment include 3 samples and 4 replicates
Project description:Tumor heterogeneity of high-grade glioma (HGG) is recognized by four clinically relevant subtypes based on core gene signatures. However, molecular signaling in glioma stem cells (GSCs) in individual HGG subtypes is poorly characterized. Here we identified and characterized two mutually exclusive GSC subtypes with distinct dysregulated signaling pathways. Analysis of mRNA profiles distinguished proneural (PN) from mesenchymal (Mes) GSCs and revealed a pronounced correlation with the corresponding PN or Mes HGGs. Mes GSCs displayed more aggressive phenotypes in vitro and as intracranial xenografts in mice. Further, Mes GSCs were markedly resistant to radiation compared with PN GSCs. The glycolytic pathway, comprising aldehyde dehydrogenase (ALDH) family genes and in particular ALDH1A3, were enriched in Mes GSCs. Glycolytic activity and ALDH activity were significantly elevated in Mes GSCs but not in PN GSCs. Expression of ALDH1A3 was also increased in clinical HGG compared with low-grade glioma or normal brain tissue. Moreover, inhibition of ALDH1A3 attenuated the growth of Mes but not PN GSCs. Last, radiation treatment of PN GSCs up-regulated Mes-associated markers and downregulated PN-associated markers, whereas inhibition of ALDH1A3 attenuated an irradiation-induced gain of Mes identity in PN GSCs. Taken together, our data suggest that two subtypes of GSCs, harboring distinct metabolic signaling pathways, represent intertumoral glioma heterogeneity and highlight previously unidentified roles of ALDH1A3-associated signaling that promotes aberrant proliferation of Mes HGGs and GSCs. Inhibition of ALDH1A3- mediated pathways therefore might provide a promising therapeutic approach for a subset of HGGs with the Mes signature.
Project description:SUMMARY Despite numerous genome-wide association studies involving glioblastoma (GBM), few therapeutic targets have been identified for this disease. Using patient derived glioma sphere cultures (GSCs), we have found that a subset of the proneural (PN) GSCs undergo transition to a mesenchymal (MES) state in a TNFa/NFkB dependent manner with an associated enrichment of CD44 sub-populations and radio-resistant phenotypes. To the contrary, MES GSCs exhibit constitutive NFkB activation, CD44 enrichment and radio-resistance. Patients whose tumors exhibit a higher MES metagene, increased expression of CD44, or activated NFkB were associated with poor radiation response and shorter survival. Our results indicate that NFkB activation mediated MES differentiation and radiation resistance presents an attractive therapeutic target for GBM. SIGNIFICANCE In this study, we show plasticity between the proneural (PN) and mesenchymal (MES) transcriptome signatures observed in glioblastoma (GBM). Specifically, we show that PN glioma sphere cultures (GSCs) can be induced to a MES state with an associated enrichment of CD44 expressing cells and a gain of radio-resistance, which we implicate as NFkB- dependent. Newly diagnosed GBM samples show a direct correlation between radiation response, higher MES metagene, CD44 expression, and NFkB activation. This correlation is also observed in the subset of GBM samples that do not exhibit IDH1 mutation, a favorable prognostic marker. Our results uncover a previously unknown link between subtype plasticity that is regulated by NFkB. Inhibition of NFkB activation can directly impact radio-resistance and presents an attractive therapeutic target for GBM. Gene expression data for 17 isolated Glioma Stem Cells
Project description:Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples. The significance of its expression was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). We found that an off-target nucleostemin RNAi (shRNA22) abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is severely compromised. Attempts were made to identify the primary target of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways. The use of this shRNA may offer a new therapeutic approach for this incurable type of brain tumors. The transcriptional profile of neurosphere cultures infected with lentiviral particles containing shRNA22 was compared with the profile of neurosphere cultures infected with lentiviral particles containing control shRNA. Experiments were done in triplicate.
Project description:We compared whole genome expression profiles of GSCs with normal human cortex, human neural stem cells (hNSC) from fetal cortex, glioblastoma (GBM) primary, and recurrent tumors to find GSC-specific plasma membrane transcripts. All of the expression profiles were batch normalized by a robust multichip average (RMA) algorithm using Geospiza GeneSifter (PerkinElmer) online microarray database and analysis software. The data was then exported into Microsoft Office Excel 2010 and organized for GSC transcripts with raw intensity values 10 fold or higher over normal brain, hNSCs, GBM primary and recurrent tumor samples. The reverse sorting algorithm was done to obtain downregulated GSC trascripts.
Project description:Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples. The significance of its expression was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). We found that an off-target nucleostemin RNAi (shRNA22) abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is severely compromised. Attempts were made to identify the primary target of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways. The use of this shRNA may offer a new therapeutic approach for this incurable type of brain tumors.
Project description:Genome-wide DNA methylation and trancription profiling of different subtypes in GBM (TCGA) and glioma stem cells (GSCs) were carried out using Illumina BeadChip HumanMethylation 450K array (450K array) to analyse over 485K CpG sites accross each samples. 450K array data for 94 GBM samples comprising 4 different subtypes i.e. Proneural (PN), Mesenchymal (MES), Classical (CL) and Neural (N) were used for GBM analysis. Similarly, 450K array for 23 GSCs and 1NHA, RNA seq for 29 GSCs and affimetrix microarray gene expression array for 12 GSCs were used for GBM data analyses.
Project description:We have developed spontaneous genetically engineered GBM mouse models from two distinct cells of origin: subventricular zone neural stem cells (SVZ; Nestin-creERT2) and oligodendrocyte lineage progenitor cells (OPC; NG2-creERTM). These tumors are biologically separable and are reflective of their lineage of origin.