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|Title:||Extracellular Vesicles as Modifiers of Phenotypic Behavior of Co-cultured Recipient Breast Cancer Cells||Authors:||Amenah Jassem AlRabeea||Supervisor:||Prof. Yunus Luqmani||Keywords:||Phenotypic : Breast Cancer||Issue Date:||2018||Publisher:||Kuwait university - college of graduate studies||Abstract:||Microvesicles (MVs) are a subclass of extracellular vesicles (EVs) shed by numerous cell types and thought to be involved in inter-cellular communication. We hypothesized that MVs derived from estrogen receptor positive (ER+) breast epithelial cells (EII) could be used to deliver cellular content to the aggressive ER silenced (ER-) mesenchymal-like cells (pII), to induce reversion back to their original less invasive epithelial phenotype. MVs were isolated from EII cell cultures by differential ultracentrifugation and analysed by atomic force and confocal microscopy. Three preparations of conditioned media from EII cell cultures were co-cultured with pII cells: complete media with all extracellular vesicles (CM1), depleted of MVs (CM2), and MVs alone. Modifications in cell behavior were assessed by morphological assessments, invasion and motility assays, gene and protein expression of E-cadherin (epithelial marker) and vimentin (mesenchymal marker) using qPCR and fluorescence immunocytochemistry. MVs of mean dia 250nm were isolated from EII culture supernatants and shown to contain cellular components by virtue of the presence of green fluorescent protein expressed by these cells. The MVs were endocytosed by pII cells when co-cultured. Alone MVs had no discernible effect on pII, neither did CM2 but CMI significantly reduced motility and invasive capacity of pII cells following two days of co-culture, without altering expression levels of E-cadherin and vimentin. However, prolonged exposure of CM1 for 14 days resulted in complete loss of the suppressive effect seen after the shorter 48h exposure. EII cells shed cytoplasm containing MVs. However, only the complete EII conditioned medium containing all EVs was effective in reducing the aggressive properties of pII cells but without altering its mesenchymal-like properties. This suggests that there may be suppressive factors released from EII cells which actively prevent them from becoming invasive and these may be absent from pII cultures. The loss of effect after 14 days of CM1 exposure could be due to resistance to the derived influence coming from EII or opposing factors released from the pII cells that decreased/nullified the former; contributory factors may be highlighted by analyzing their respective proteomic profiles in future studies.||URI:||http://hdl.handle.net/123456789/746|
|Appears in Programs:||2050 Molecular Biology|
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