Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome profiling of s-SHIP/GFP+ drug-resistant cells of the MAM326 murine mammary cancer cell line

ABSTRACT: The MAM326 cancer cell line was derived from a mammary tumor of bitransgenic FVB/N s-SHIP-GFP X C(3)1-Tag mice. The MAM326 cell line contained approximatively 10% of s-SHIP/GFP+ cells. This cell subpopulation was more resistant to drugs. To investigate the molecular basis of this chemoresistance, GFP+ cells (GFPpos files) and GFP-negative (GFPneg files) cells were isolated by fluorescence-activated cell sorting and analyzed for gene expression.

ORGANISM(S): Mus musculus

SUBMITTER: frederic lepretre

PROVIDER: E-MTAB-14967 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Isolation of prostate stem cells is crucial for understanding their biology during normal development and tumorigenesis. In this aim, we used a transgenic mouse model expressing GFP from the stem cell-specific s-SHIP promoter to mark putative stem cells during postnatal prostate development. We showed that cells identified by s-SHIP/GFP expression are present transiently during early prostate development and localize to the basal cell layer of the epithelium. These prostate s-SHIP/GFP-positive cells represent a subpopulation of the lineage-negative / CD24-positive / Sca-1-positive / CD49f-positive (LSC) cells and are capable of selfârenewal together with enhanced growth potential in sphereâforming assay in vitro, a phenotype consistent with that of a prostate stem cell population. Transplantation assays of these prostate GFP-positive cells demonstrate reconstitution of prostate ducts containing both basal and luminal cells in renal grafts. Altogether, these results demonstrate that s-SHIP promoter expression is a new marker for neonatal basal prostate cells exhibiting stem cell properties that enables prostate stem cells in situ identification and isolation via a single consistent parameter. Since the GFP-positive cell population is a small subset of basal LSC cells and is most responsible for stem-like activity, we performed transcriptional profiling of GFP-negative LSC and GFP-positive LSC cells to distinguish a basal cell profile from a tissue stem cell profile. Prostate tissue was collected from 6-day-old male mice, minced into small fragment, digested with 200 U/ml collagenase IAâS (Sigma; C5894) in Dulbeccoâs modified Eagles medium (DME, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) (DME-10% FBS) at 37Â°C for 60 min with gentle agitation. The digested cells were filtered through a 40-Î¼m cell strainer (BD Biosciences) washed, and resuspended in DME-10% FBS., filtered and labeled with antibodies against lineage (Ter119, CD31, CD45), CD49f and Sca-1 cell surface markers (Affymetrix ebiosciences). Labeled cells were analyzed and the GFP-negative LSC and GFP-positive-LSC populations were sorted by FACS. For each cell population, 3 independent samples were collected and analysed.

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Transcriptome profiling of s-SHIP/GFP+ drug-resistant cells of the MAM326 murine mammary cancer cell line

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