Expression data from SP and non-SP sorted anti-EpCAM treated A2C12 cells
ABSTRACT: Targeted therapies against cancer stem cells which are enriched in side populations (SP) involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/β-catenin signalling was studied. SP of the murine lung adenocarcinoma cell line A2C12 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment. Anti-EpCAM treated and untreated A2C12 cells were subjected to Hoechst 33342 dye exclusion assay and sorted to SP and non-SP fractions by FACS. Three biological replicates.
Project description:Targeted therapies against cancer stem cells, which are enriched in side populations (SP), involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/β-catenin signalling was studied. SP of the human lung adenocarcinoma cell line A549 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment. Anti-EpCAM treated and untreated A549 cells were subjected to Hoechst 33342 dye exclusion assay and sorted to SP and non-SP fractions by FACS. Three biological replicates.
Project description:This SuperSeries is composed of the following subset Series: GSE31246: Expression data from SP and non-SP sorted anti-EpCAM treated A2C12 cells GSE31313: Expression data from anti-EpCAM treated and untreated SP cells compared to lung tissue GSE31315: Expression data from SP and non-SP sorted anti-EpCAM treated A549 cells Refer to individual Series
Project description:Earlier studies had shown that side population cells isolated from established non-small cell lung cancer (NSCLC) cell lines exhibit cancer stem cell properties. Microarray data from side population (SP) and main population (MP) cells isolated from 4 NSCLC lines (A549, H1650, H460, H1975) were used to examine gene expression profiles associated with stemness. Total RNA extracted from SP and MP samples were used to generate cRNA targets, which were hybridized to Human Genome U133 Plus 2.0 probe arrays. Raw data was processed and the mean center expression level for each gene was determined. Four cell lines (A549, H1650, H460, H1975), each having 1 SP and 1 MP sample.
Project description:Malignant pleural mesothelioma (MPM), which is associated with occupational asbestos exposure, is a deadly disease with no effective treatments due mainly to its high resistance to anti-cancer drugs. The molecular mechanisms responsible for its chemotherapeutic resistance are complicated and undefined. However, the presence of side population cells (SP cells) in tumors is a well-accepted explanation for their anti-cancer drug resistance. To identify SP cell-specific gene expression signature, microarray technique has been employed. Our data show differential gene expression profiles between SP and non-SP cells of H2714 mesothelioma cells. SP cells over-expressed genes associated with cancer stem cell (CSC) and drug resistance: DUSP6, SPRY2 and IL6, as well as multi-pathways, including the cancer stem cell-associated pathways Notch and c-Kit. Therefore, we believe that targeting CSC-specific genes and pathways in SP cells may hold the key to the discovery of effective treatments for reversing chemotherapeutic resistance to MPM treatment. 4 samples
Project description:Intratumoral heterogeneity is a major barrier against effective cancer therapy. Human malignant mesothelioma (HMM) that is closely associated with asbestos exposure is extremely heterogeneous in morphology and molecular phenotype. Contrast to genetic mutations, the role of epigenetic modifications in the generation and maintenance of heterogeneous populations in cancers remains largely undetermined. The present study was performed to investigate underlying molecular mechanisms for the emergence of intratumoral heterogeneity by identifying the global microRNA expression profile of distinct subpopulations of MS1 cell line, a HMM cell line. More aggressive cancer cells could be enriched by side population (SP) assay in HMM . The sorted SP and NSP subpopulations were subjected to the microarray analysis of miRNA expression to investigate differentially altered miRNA genes defining tumor heterogeneity in HMM. Total RNAs were isolated from the sorted subpopulations of HMM cells, SP and non-SP fractions. The expression profile of miRNAs was evaluated using Affymetrix GeneChip miRNA Arrays. After data extraction and normalization, the microRNAs defining the cell subpopulations were determined using bioinformatics softwares. A total of 95 miRNAs including 42 up-regulated and 53 down-regulated were identified based on the criteria of 2 fold difference and a p-value < 0.05. Functional ontology of the dysregulated miRNAs revealed that a large number of target genes were categorized into the regulation of various cellular processes, including cell proliferation, programmed cell death, cell migration, cellular response to stress, and stem cell maintenance. The data show that microRNAs are significantly involved in the generation and maintenance of intratumoral heterogeneity and their regulation could be an effective strategy to eradicate a more aggressive cancer cell subpopulation. This is the first to report the profile of miRNA expression in CSCs in HMM by using side population assay assisted with flow cytometry. It will be valuable to understand the regulatory function of HMM CSC miRNAs in generation and maintenance of intratumoral heterogeneity. Total RNAs were isolated from the sorted subpopulations of HMM cells, SP and non-SP fractions. (no replicates)
Project description:Targeted therapies against cancer stem cells which are enriched in side populations (SP) involves interruption of Wnt-signalling. Furthermore, EpCAM is a SP marker and modulator of Wnt-signalling. Therefore, the effects of an anti-EpCAM treatment on SP-cells and WNT/β-catenin signalling was studied. SP of the murine lung adenocarcinoma cell line A2C12 was obtained by fluorescence activated cell sorting and whole genome scans helped to define their molecular phenotype after anti-EpCAM antibody treatment. Overall design: Anti-EpCAM treated and untreated A2C12 cells were subjected to Hoechst 33342 dye exclusion assay and sorted to SP fractions by FACS. Gene expression of SP cells was compared to non-transgenic lung tissue.
Project description:Side population (SP) cells are highly enriched in stem and progenitor cells. CD45+and CD45- SP cells were found at all developmental lung stages with the highest frequency of cells present at embryonic day 17.5 (E17.5), In order to clarify the role of these cells in lung development, we used oligonucleotide microarrays to evaluate their gene expression profiles. To do this, oligonucleotide gene arrays were performed in triplicate on RNA derived from CD45+ and CD45-SP cells, and CD45+ and CD45- non-SP populations (main population; MP) isolated from the E17.5 lung.
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
Project description:Side population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival side population cells by global differential gene expression to identify markers and signaling pathways associated with this cell phenotype. Four overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 3342 were cytometrically sorted into SP and non-SP cohorts. RNA was isolated and processed for microarray analysis using a commercial oligonucleotide array representing more than 55,000 transcripts derived from about 30,000 different genes. Selected microarray results were validated at the gene and protein levels by quantitative PCR, and immunological methods. Data mining methods were used to identify cellular processes relevant for stem cell function. Comparative analysis of transcripts expression based on expression levels and present/absent software calls across 4 replicate experiments identified 16993 expressed conjunctival epithelial transcripts including 10,266 unique known genes. Of those genes, 1254 and 363 were over expressed (> 2-fold ) or under expressed (< 0.5-fold), respectively, in the SP. The overexpressed set included genes coding for non-epithelial genes (e.g., CD62E/E-selectin and CD93), genes that have been associated with stem cell function in other cellular systems, including several homeodomain genes and genes whose over- or under-expression may underpin the stem cell slow cycling phenotype ( e.g., dual specificity phosphatases and cyclin kinases). Experiment Overall Design: Whole human conjunctivae from unidentifiable cadaver donors, aged 55 to 65, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) within 48 hours of collection. No donor details apart from age, sex, and cause of death were released. The use of human tissue in the study was in accordance with the provisions of the Declaration of Helsinki and sanctioned by the Institutional Review Board. Fresh rabbit tissue was obtained from local abattoirs within one hour of sacrifice. Unless stated otherwise all reagents were procured from Sigma (St. Louis, Mo). Experiment Overall Design: Eight microarray experiments using side population (Hoechst 33342-transporting cells; SP) or non side population (Hoechst 33342-stained cells; nSP) cells from four cadavers.
Project description:Side populations have recently been identified in ovarian cancers and may play an important role in post treatment relapse and resistance to chemotherapeutic drugs. In this study, we aimed to identify the differential expression between IGROV1 SP and NSP on Affymetrix HG-U133plus2 microarrays. We found ovarian tumour SP cells frequently over-express the multi-drug resistance associated P-glycoprotein (ABCB1) by Rank Product (FDR<0.05), and by geneset enrichment analysis, embryonic stem cell-associated ‘NOS’ signature (Notch/Oct4/Sox2 regulated genes) and Polycomb Repressive Complex 2 (PRC2) genes were over-expressed, while PRC2-repressed target genes were significantly under-expressed in the SP from ovarian cell lines compared to non-SP (FDR<10-4). Cells were isolated using Hoechst 33342 cell sorting without other treatment. The experiment was carried out in triplicates: 3 SP samples and 3 non-SP samples