RNA-Seq of ALDH positive, ALDH negative and unsorted cells from patient-derived 3D in-vitro models of colon cancer
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500, BD FACSARIA II
ORGANISM(S): Homo sapiens
SUBMITTER: Joseph Regan
PROVIDER: E-MTAB-5209 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Colon cancer patient-derived xenograft (PDX) models were processed to single cells and sorted by FACS (BD FACS Aria II) for ALDH activity (Aldefluor assay) and DAPI. ALDH Negative and ALDH Positive cells from each PDX model were collected and lysed in RLT buffer and processed for RNA using the RNeasy Mini Plus RNA extraction kit (Qiagen). Samples were processed using Illumina’s TrueSeq RNA protocol and sequenced on an Illumina HiSeq 2500 machine as 2x125nt paired-end reads. Reads were mapped to the human reference genome (assembly hg19) using the STAR aligner (version 2.4.2a). Total read counts per gene were computed using the program “featureCounts” (version 1.4.6-p2) in the “subread” package, with the gene annotation taken from Gencode (version 19).
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Project description:The intent of this experiment was to determine the similarities and differences in expression signatures of normal breast cells that express markers associated with an epithelial-like (ALDH+) and mesenchymal-like (CD44+CD24-) stem cells in the normal human breast. Briefly, tissues were collected from women undergoing voluntary reduction mammoplasty. Tissues were dissociated using chemical and enzymatic methods to yield single cells. These cells were sorted by flow cytometry for aldehyde hydrogenase activity (ALDH+), CD44, and CD24, as well as viability, following depletion of hematopoeitc cells, endothelial cells, and fibroblasts.
2018-01-17 | E-MTAB-5585 | biostudies-arrayexpress
Project description:Insulin-producing beta cells become dedifferentiated during diabetes progression. An impaired ability to select substrates for oxidative phosphorylation, or metabolic inflexibility, sets the stage for progression from beta cell dysfunction to beta cell dedifferentiation. In this study, we sought to isolate and functionally characterize failing beta cells, as a preliminary step to identify pathways to reverse dedifferentiation. Using various experimental models of diabetes, we found a striking enrichment in the expression of aldehyde dehydrogenase 1 isoform A3 (ALDH+) as beta cells become dedifferentiated. Flow-sorted ALDH+ islet cells demonstrate impaired glucose-induced insulin secretion, are depleted of Foxo1 and MafA, and include a Neurogenin3-positive subset. RNA sequencing analysis demonstrates that ALDH+ cells are characterized by: (i) impaired oxidative phosphorylation and mitochondrial complex I, IV, and V; (ii) activated RICTOR; and (iii) progenitor cell markers. We propose that impaired mitochondrial function marks the progression from metabolic inflexibility to dedifferentiation in the natural history of beta cell failure. RNA-Sequencing analysis of 2 different cell types in 2 different genotype categories.
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Project description:Transcriptional profiling of cancer stem cells (ALDH-high cells) comparing non-cancer stem cells (ALDH-low cells), sorted out using ALDEFLUOR assay. Goal was to identity cancer stem cell-specific genes.
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Project description:Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) has a poor prognosis with less than 1-year median survival. Platinum-based chemotherapy (cisplatin or carboplatin) remains the first-line treatment for HNSCC. The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self renewing cell populations that constitute the bulk of the tumor. A small population of CSCs exists within HNSCC that are relatively resistant to chemotherapy and clinically predicted to mediate tumor recurrence. These CSCs are identified by high cell-surface expression of CD44 and high intracellular activity of aldehyde dehydrogenase (ALDH) and termed ALDHhighCD44high. We investigated the molecular pathways active in ALDHhighCD44high cells, which remain poorly studied. Additionally, we performed a molecular examination of cisplatin-resistant ALDHhighCD44high cells, which has not been reported. Two HNSCC cell lines, UM-SCC-1 and UM-SCC-22b, were utilized in this study. For microarray analysis, UM-SCC-22b cells were treated for 5 days in vitro with 2uM cisplatin and analyzed by flow cytometry, sorted and submitted for microarray analysis of ALDHhighCD44high and ALDHlowCD44low cells from untreated and cisplatin treated cells. Four separate flow cytometry experiments were performed using Affymetrix Human Gene ST 2.1 microarrays. Microarray data was analyzed using R/Bioconductor. Files were preprocessed by Robust Multiarray Average (RMA) with background substraction, quantile normalization, and median polish (oligo package). Data was fitted with robust probe level linear models to all the probesets (oligo package). Experiment and processing batch differences were accounted for using 'ComBat' within the SVA package. Differentially expressed genes were identified using univariate comparisons after fitting data to a linear model (limma package). Initial statistics were determined using an empirical Bayesian model. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant. Unsupervised hierarchical clustering with complete linkage and Euclidean distance was performed on only statistically significant probes. In four separate experiments, the head and neck squamous cell carcinoma cell line UM-SCC-22b were cultured for 5 days with or without 2uM (micromolar) cisplatin in 6-well plates. Media was replaced every other day. Control and cisplatin treated cells were trypsinized, procesed, and stained for CD44 cell-surface expression and intracellular aldehyde dehydrogenase (ALDH) activity to identify cancer stem cells (ALDH+CD44+). CSCs and non-CSCs (ALDH-CD44-) were collected by flow cytometry from both groups. Total RNA was collected from each fraction (ALDH+CD44+, ALDH-CD44-), treatment (control, cisplatin), and experiment (#1-4). A total of 16 samples were analyzed. One set of 4 (experiment #4) were analyzed on a Human Gene ST 2.1 strip and the rest on a Human Gene ST 2.1 plate. Differential gene expression was determined with R/Bioconductor with Robust Multiarray Average (RMA) and fitting the data to linear models (limma). Experimental and processing batch effects were accounted for using ComBat. Four sets of univariate comparisons were made: 1) Cisplatin ALDH+CD44+ vs Control ALDH+CD44+; 2) Control ALDH+CD44+ vs Control ALDH-CD44-; 3) Cisplatin ALDH+CD44+ vs Cisplatin ALDH-CD44-; 4) Cisplatin ALDH-CD44- vs Control ALDH-CD44-. Multiple testing comparisons were adjusted using Benjamini and Hochberg (aka FDR). Probes with an adjusted p-value <0.05 were considered statistically significant.
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Project description:Transcriptional profiling of cancer stem cells (ALDH-high cells) comparing non-cancer stem cells (ALDH-low cells), sorted out using ALDEFLUOR assay. Goal was to identity cancer stem cell-specific genes. Two-condition experiment, sphere-cultured cells vs. adherent-cultured cells: 1 ALDH-high replicate and 1 ALDH-low replicate.
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Project description:We used an Aldefluor assay system to study the signaling pathways that regulate the frequency and maintenance of ALDH-positive cell population in MCF-7 cells as a CSC model. Through microarray analysis, we investigated a possible receptor that increases the proportion of the ALDH-positive cell population in MCF-7 cells.
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Project description:The hilum region of the mouse ovary, the transitional/junction area between OSE, mesothelium and tubal (oviductal) epithelium is identified as a previously unrecognized stem cell niche of the OSE. OSE cells with high ALDH1 activity have been predominantly detected in the hilum region by immunohistochemical staining. For additional phenotypical characterization, we used microarrays to detail the gene expression profiling between the ALDH1 positive and ALDH1 negative OSE cells. Three independent pools (10 mice each) were analyzed. ALDH1 positive and ALDH negative OSE cells were isolated by FACS and their mRNA used for hybridization onto Affymetrix Mouse Genome 430 2.0 array. Microarray data were analyzed with GeneSifter software. To identify genes significantly altered in ALDH positive population, we performed paired two group analysis with Significance Analysis of Microarrays (SAM) software and visualized with Treeview software.
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Project description:Several human cancers contain a small subpopulation of cells called cancer stem-like cells (CSCs)/cancer initiating cells (CICs), which are defined by the ability of self- renewal, multi-differentiation potential, and tumorigenesis. In current study, we focused on epithelioid sarcoma, which is very rare caner, and isolated CSCs/CICs from epitheloid sarcoma cell line ESX based on ALDH activity using ALDEFLUOR assay towards identification of a new CIC/CSC marker. We performed gene profiling between ALDHhigh and ALDH low cells using cDNA microarray for the identification of good markers of CSCs/CICs of epithelioid sarcoma.
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