Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human MDA-MB-231 breast cancer cells in vitro and in vivo as xenografts to assess the effect of ectopic expression of sFRP1

ABSTRACT: RNA was isolated from ectopically sFRP1-expressing MDA-MB-231 cells and control MDA-MB-231 cells and as well from tumor lysates arising from these cells as nude mouse xenograft. Gene expression profiles for these samples were investigated using Affymetrix arrays. Experiment Overall Design: MDA-MB-231 human breast cancer cells were stably transfected with human sFRP1 encoding vector or empty vector as control. After the selection with antibiotics, three clones of MDA-MB-231/sFRP1 and three clones of MDA-MB-231/control were selected. These six clones were cultured individually in DMEM 10% FCS with 1mg/ml G-418. When cells reached 70-80% confluence, RNA was isolated from the cells. In parallel, the three clones of MDA-MB-231/sFRP1 and the three clones of MDA-MB-231/control were pooled respectively. One million of cells from each pool were suspended in 100ul PBS and injected to fat pads of female balb/c nude mice (6 mice were injected with MDA-MB-231/sFRP1 and 5 mice were injected with MDA-MB-231/control) to do a xenograft experiment. A few - several weeks after, mice were sacrificed when tumor reached a certain size, tumors were taken and RNA was isolated using trizol reagent.

ORGANISM(S): Homo sapiens

SUBMITTER: Edward Oakeley

PROVIDER: E-GEOD-13806 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Vascular endothelial growth factor is a multifunctional cytokine playing important roles in angiogenesis, tumor progression and metastasis. Alternative splicing results in the production of several different isoforms of VEGF. We have previously generated human breast cancer cells overexpressing VEGF165 or VEGF189 isoforms (referred to as the V165 and V189 clones, respectively) and showed that VEGF189-transfected cells were less tumorigenic. In this study, we used bioluminescence imaging to analyze the metastasis capacity of breast cancer cell lines (MDA-MB-321) overexpressing VEGF isoforms in nude mice. V165, V189 and control cV clones were transfected with a luciferase plasmid to generate bioluminescent clones (the V165-B, V189-B and cV clones, respectively). These clones were then injected into the left heart ventricle of nude mice. Analysis of the location of metastases shows that V189-B cells induced fewer metastases in lung and bone than V165-B and cV-B cells. Moreover, there was a delay on metastasis appearance in mice receiving VEGF V189-B clone, consistent with increase survival in these mice. We investigated the possible mechanisms underlying these effects and found that VEGF189 increased adhesion and decreased cell invasion and survival in vitro. In the other side, transcriptomic analyses shown expression of genes, implicated in breast cancer and metastatic dissemination, in VEGF165 overexpressing cells. After in silico analyses of genes differentially expressed between V189 and V165 cells,we used Q-PCR assays to quantify mRNA levels of some gene of interest in 120 breast tumors from patients with or without metastasis and/or specially lung metastasis. We found that genes have prognostical significance and /or showed an influence on relapse-free survival. These data provide the first evidence that different VEGF isoforms have different effects on breast cancer cell line invasion and colonization. Human MDA-MB-231 breast cancer cells were used to generate stable transfected clones overexpressing VEGF165 isoform, VEGF189 isoform, or control vector (referred to as 42ctl8, 13ctl3 and PCIctl3, respectively). Analysis of the metastasis capacity of these clones in nude mice.

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Transcription profiling of human MDA-MB-231 breast cancer cells in vitro and in vivo as xenografts to assess the effect of ectopic expression of sFRP1

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