Unknown,Transcriptomics,Genomics,Proteomics

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Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy

ABSTRACT: Objective: The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDHhigh) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDHhigh cells in ovarian cancer. Methods: We compared ALDHhigh to ALDHlow cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. Results: ALDHhigh cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDHhigh cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following drugable targets were consistently expressed in the ALDHhigh cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18 / FGFR3. Conclusions: Based on functional characterization, ALDHhigh ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified drugable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes. FNAR-C1 rat ovarian cancer cells were sorted into Aldefluor high and Aldefluor low populations. Analysis was performed in triplicate. Aldefluor low cells served as the reference cells for fold-change analysis.

ORGANISM(S): Rattus norvegicus

SUBMITTER: Richard Jones

PROVIDER: E-GEOD-82112 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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Characterization of aldehyde dehydrogenase 1 high ovarian cancer cells: Towards targeted stem cell therapy

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