(C-F) qRT PCR analysis of EMT genes in MCF 10A mammary epithelial cells exposed to 0.5% CSE for 21 weeks (clones SC1 and SC2) or 0.5-1.0% CSE for 40 weeks (without subcloning). breast malignancy cells using and models of chronic cigarette smoke Tafluprost exposure. Results We show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, impartial of cigarette smoke effects around Rabbit polyclonal to PRKAA1 the host and stromal environment. The extent of transformation and the producing phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by circulation cytometry showed that treatment with CSE prospects to the emergence of a CD44hi/CD24low populace in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis. Conclusions Tafluprost Our results indicate that exposure to cigarette smoke prospects to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state. and models. Our results indicate that exposure to cigarette smoke prospects to a more aggressive and transformed phenotype in human mammary epithelial cells, and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state. Results Cigarette smoke induces anchorage-independent cell growth, migration, invasion and morphological changes in mammary epithelial cells and breast cancer cells It has been shown that the risk of developing cancer increases with the number of years a person has smoked or been exposed to second hand smoke [12,13]. For this reason we developed a model to study the progressive, chronic effects of cigarette smoke exposure. Cells were constantly cultured for 72 weeks with an aqueous cigarette smoke extract (CSE) from main stream smoke prepared in our laboratory (0.25%, 0.5% or 1% CSE) or for approximately 40 weeks with cigarette smoke condensate (CSC) a commercial product based on condensate from second-hand-like smoke (10 g/ml or 25 g/ml CSC). A concentration of 0.5% CSE, or 25 g/ml CSC in the media corresponds to approximately 0.001 cigarettes/ml, which is an amount comparable to, or lower than those used in other studies [9,10,14-16]. The corresponding amount of nicotine in the media (1.30.1 g/ml) approximates the Tafluprost upper limit of the concentrations of cotinine found in the plasma or breast milk of smokers, which has been reported as high as 300C800 ng/ml and 200C500 ng/ml, respectively . Non-tumorigenic MCF 10A cells cultured with either CSE or CSC were transferred to soft agar to assess anchorage-independent growth after 15, 21, 27 and 39 or 37 weeks of treatment. Both CSE and CSC caused a significant increase in colony formation in soft agar (up to 42 fold; Figure?1A) which is a feature typical of malignancy cells. Linear regression analysis indicated that the effect was both dose and time dependent as the number of colonies increased in parallel with the duration of treatment (r2>0.9; results, we hypothesized that treatment with CSE might drive these cells to become more invasive or pre-malignant. To investigate this scenario, we used an intraductal transplantation model originally developed to study ductal carcinoma in situ (DCIS). In this model, malignancy cells are injected through the nipple, into the main mammary duct, which allows in situ analysis of intraductal growth and/or invasion through the basement membrane into the stroma . MCF 10A cells treated with 0.5% CSE for 46 weeks or mock treated were injected into the primary inguinal mammary ducts of 8-week-old female immunodeficient mice (NSG). The.