Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse model of glioma reveals cancer stem cells are enriched in the side-population cells

ABSTRACT: The recent identification of cancer stem cells (CSCs) in multiple human cancers provides a new inroad to understanding tumorigenesis at the cellular level. CSCs are defined by their characteristics of self-renewal, multipotentiality, and tumor initiation upon transplantation. By testing for these defining characteristics, we provide evidence for the existence of CSCs in a transgenic mouse model of glioma, S100Ã-verbB;Trp53. In this glioma model, CSCs are enriched in the side-population (SP) cells. These SP cells have enhanced tumor-initiating capacity, self-renewal, and multipotentiality compared to non-SP cells from the same tumors. Furthermore, gene expression analysis comparing FACS-sorted cancer SP cells to non-SP cancer cells and normal neural SP cells identified 45 candidate genes that are differentially expressed in glioma stem cells. We validated the expression of two genes from this list (S100a4 and S100a6) in primary mouse gliomas and human glioma samples. Analyses of xenografted human GBM (glioblatoma multiforme) cell lines and primary human glioma tissues show that S100A4 and S100A6 are expressed in a small subset of cancer cells and that their abundance is positively correlated to tumor grade. In conclusion, this study shows that CSCs exist in a mouse glioma model, suggesting that this model can be used to study the molecular and cellular characteristics of CSCs in vivo and to further test the cancer stem cell hypothesis. Experiment Overall Design: This study features two factors, injected cell origin (either tumorsphere or neurosphere) and FACS cell population (either side population or non-side population cells). There were two different tumorspheres, labeled 3447 and 4346 that were isolated from brain tumors in S100beta-verbB;p53-/- or S100beta-verbB;p53+/- mice. The tumorspheres were injected separately into the brains of NOD.Cg-PrkdcIl2rg/SzJ mice to generate biological triplicates of each primary tumor. Tumorspheres were isolated and cultured before FACS sorting to obtain side population and non-side population cells. As a control, untransformed neurospheres from three independent S100beta-verbB;p53-/- or S100beta-verbB;p53+/- mice were isolated, cultured, and FACS sorted to obtain side population and non-side population cells. Side population and non-side population cells cultured from three mice injected with the 3447 cultured tumorspheres were assayed for gene expression (six samples). Side-population stem cells cultured from three mice injected with the 4346 cultured tumorspheres were assayed for gene expression (three samples). Side-population and non-side population cells cultured from three mice injected with the neurospheres were assayed for gene expression (six samples).

ORGANISM(S): Mus musculus

SUBMITTER: Kyuson Yun

PROVIDER: E-GEOD-13490 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Novel therapies targeting cancer stem cells (CSCs), which play critical roles in chemo- and radio-resistance, metastasis, and possibly resistance against cancer immunotherapy including granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccines, may provide beneficial clinical outcomes. Here, we used syngeneic immunocompetent mice that allowed precise evaluation of the immunogenicity of the side population (SP) isolated from 4T1 murine breast carcinoma (4T1-SP) cells as putative CSCs. 4T1-SP cells showed various stem cell properties including high capacities for colony formation and tumorigenicity as well as high expression of phosphorylated signal transducer and activator of transcription-3 and vascular endothelial growth factor that are inductive of immune tolerance. Despite these progressive malignant characteristics of 4T1-SP cells, subcutaneous injection of non-transmissible Sendai virus-mediated GM-CSF gene-transduced 4T1-SP (4T1-SP/GM) cells remarkably impaired their tumorigenicity compared with that of the controls. This impairment of tumorigenicity was partially dependent on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Notably, therapeutic vaccinations using irradiated 4T1-SP/GM cells markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with that of the controls including irradiated 4T1-non-SP/GM cells. Tumor suppression was accompanied by robust accumulation of mature dendritic cells at vaccination sites and systemic Th1-based cellular immunity. Moreover, vaccinations comprising primary 4T1-SP cells isolated from transplanted 4T1-SP tumors elicited antitumor effects. cDNA microarray analysis showed that 4T1-SP cells predominantly expressed genes of cancer-related antigens including cancer/testis antigens. Collectively, we demonstrate that SP cell-based vaccinations induce effective antitumor immunity that may improve the efficacy of SP cell-based immunotherapy. Gene expression profiles were compared between sorted 4T1-SP and 4T1-NSP cells.

Project description:Tumours contain heterogeneous cell populations. A population enriched in tumour-initiating potential has been identified in soft-tissue sarcoma (STS) by the isolation of side population (SP) cells. In this study, we compared the gene expression profiles of SP and non-SP cells in STS and identified Hedgehog (Hh) and Notch pathways as potential candidates for the targeting of SP cells. Upon verification of the activation of these pathways in SP cells, using primary tumor xenografts in NOD-SCID mice as our experimental model, we used the Hh blocker Triparanol and the Notch blocker DAPT to demonstrate that the suppression of these pathways effectively depleted the abundance of SP cells, reduced tumour growth, and inhibited the tumour-initiating potential of the treated sarcoma cells upon secondary transplantation. The data provide additional evidence that SP cells act as tumour initiating cells and points to Hh and Notch pathways as enticing targets for developing potential cancer therapies. We used microarrays to detail the difference in gene expressions between the side population cells in soft-tissue sarcoma in comparison to the bulk non-side-population cells. To examine whether certain pathways may be differentially regulated in SP cells versus non-SP cells, which represent the bulk of tumour cells, we compared the gene expression profiles of SP and non-SP cells in four primary STS tumours each from different patient. Upon surgical excision, these tumours were dissociated mechanically and enzymatically into individual cells. Via Hoechst dye staining and flow-cytometry, these primary tumour cells were sorted into distinct side population and non-side population fractions. Total RNA is extracted from an equal number of SP and NSP cell from each primary tumour. cDNA from each sample was generated from the isolated total RNA and hybridized onto Affymetrix Human Genome EukGE-WS2v4 gene chips against the same reference cDNA library. After initial processing of the raw data, using the Genespring® GX software, the expression of SP cells from each tumour was normalized against the expression of the corresponding non-SP cells. A gene list was constructed by selecting genes that were regulated in the same direction (SP vs. NSP) in all sample pairs with a fold change greater than 1.25. This list was examined using Genespring® GX significant pathway function to identify differentially regulated pathways.

Project description:The side population (SP), recently identified in several normal tissues and in a variety of tumors, may comprise cells endowed with stem cell features. In this study, we investigated the presence of SP in epithelial ovarian cancer (EOC) and found it in 4 out of 6 primary cultures from xenotransplants, as well as in 9 out of 25 clinical samples analyzed. SP cells from one xenograft bearing a large SP fraction were characterized in detail and they were capable of recreate the full repertoire of cancer cell populations observed in the parent tumor. Moreover, SP cells had higher proliferation rates, were much less apoptotic compared to non-SP cells, and generated tumors more rapidly than non-SP cells. We also investigated the effects of interferon-alfa (IFN-alpha), a cytokine which has been widely used to treat solid tumors, on EOC cells and observed that IFN-alpha exerts marked anti-proliferative and pro-apoptotic effects on primary cultures containing SP cells. IFN-alfa-treatment invariably caused a dramatic reduction in SP size in tumor cell lines of different origin and in normal bone-marrow SP cells, associated with a distinctive transcriptional profile. Gene therapy with human IFN-alpha resulted in regression of established tumors bearing a large SP fraction, which was not observed when tumors bearing low SP levels were treated. These findings could have relevant clinical implications since they imply that tumors bearing large SP numbers - albeit rare - could be sensitive to IFN-alpha treatment. Experiment Overall Design: 4 Affymetrix microarrays: Experiment Overall Design: 2 technical replicates: Ovarian cancer, untreated Side Population, rep1 Experiment Overall Design: Ovarian cancer, untreated Side Population, rep2 Experiment Overall Design: + Experiment Overall Design: 2 technical replicates: Ovarian cancer, IFN-alpha treated Side Population, rep1 Experiment Overall Design: Ovarian cancer, IFN-alpha treated Side Population, rep2

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Transcription profiling of mouse model of glioma reveals cancer stem cells are enriched in the side-population cells

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