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Cigarette smoke induces epithelial to mesenchymal transition and increases the metastatic ability of breast cancer cells.

Di Cello F, Flowers VL, Li H, Vecchio-Pagán B, Gordon B, Harbom K, Shin J, Beaty R, Wang W, Brayton C, Baylin SB, Zahnow CA - Mol. Cancer (2013)

Bottom Line: 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, independent of cigarette smoke effects on the host and stromal environment.Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21287, USA.

ABSTRACT

Background: Recent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke 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, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population 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: Our results indicate that exposure to cigarette smoke leads 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.

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Cigarette smoke facilitates intraductal colonization of MCF 10A cells and distant metastasis of MCF7 cells. (A-B) H&E and FITC/DAPI staining of mammary gland sections from NSG immunodeficient mice injected intraductally with 100,000 control or 0.5% CSE-treated MCF 10A or MCF7 cells. Each mouse was injected at two sites. MCF10 A cells were injected into 3 mice and MCF7 cells into 2 mice for a total of 6 and 4 sites respectively. Two magnifications are shown. The xenografts were analyzed via immunoflourescence using a FITC antibody specific for human cytokeratin-18. (C) Growth of subcutaneous xenograft tumors from control and CSE-treated (0.5%) MCF7 cells. Each mouse (n=5) was injected at one site. Mean ± SD of five replicates are shown. (D) H&E staining of sections of lung and liver containing metastatic colonies from three representative NSG immunodeficient mice subcutaneously injected with 750,000 MCF7 cells treated with 0.5% CSE. Two additional mice were analyzed by gross pathology for a total n=5.
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Figure 3: Cigarette smoke facilitates intraductal colonization of MCF 10A cells and distant metastasis of MCF7 cells. (A-B) H&E and FITC/DAPI staining of mammary gland sections from NSG immunodeficient mice injected intraductally with 100,000 control or 0.5% CSE-treated MCF 10A or MCF7 cells. Each mouse was injected at two sites. MCF10 A cells were injected into 3 mice and MCF7 cells into 2 mice for a total of 6 and 4 sites respectively. Two magnifications are shown. The xenografts were analyzed via immunoflourescence using a FITC antibody specific for human cytokeratin-18. (C) Growth of subcutaneous xenograft tumors from control and CSE-treated (0.5%) MCF7 cells. Each mouse (n=5) was injected at one site. Mean ± SD of five replicates are shown. (D) H&E staining of sections of lung and liver containing metastatic colonies from three representative NSG immunodeficient mice subcutaneously injected with 750,000 MCF7 cells treated with 0.5% CSE. Two additional mice were analyzed by gross pathology for a total n=5.

Mentions: MCF 10A cells are not able to form tumors in immunodeficient mice, and are thus neither malignant nor tumorigenic. Based on our in vitro 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, cancer cells are injected through the nipple, into the primary mammary duct, which allows in situ analysis of intraductal growth and/or invasion through the basement membrane into the stroma [18]. 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 mammary fat pads were harvested after three months and labeled with an antibody for human cytokeratin-18 to identify the injected human cells. Untreated MCF 10A cells did not appear to colonize or grow in the ducts; however, colonies of CSE-treated MCF 10A cells were found within the ducts up to 3 months post-injection (Figure 3A). We then investigated if CSE could further increase the invasiveness of MCF7 breast cancer cells. Because these cells are tumorigenic, and grow much faster than MCF 10A, we harvested the mammary glands seven days after intraductal injection. At that time, untreated MCF7 cells had formed several intraductal masses that appeared to remain within the confines of the ducts. In contrast, MCF7 cells treated with CSE for 21 weeks had invaded through the duct and had formed colonies in the stroma (Figure 3B). Since treatment with CSE had clearly increased the invasiveness of MCF7 cells, we investigated if tumorigenesis and metastasis of MCF7 cells were also affected using a subcutaneous xenograft model. Transplanted MCF7 cells treated with 0.5% CSE for 18 weeks resulted in smaller tumors than mock-treated cells (Figure 3C); however, these smaller tumors were associated with metastases in the lungs of all animals and isolated` cells were found in the liver of at least one animal (Figure 3D). The three mice shown are representative, and two additional mice injected with CSE treated cells were analyzed by gross pathology for a total of 5. In contrast, we did not observe metastases from untreated MCF7 cells, suggesting that cigarette smoke may have favored the expansion of a highly metastatic subpopulation of MCF7 cells. Although MCF10A cells had exhibited increased intraductal survival and colonization after treatment with CSE, these cells did not produce subcutaneous tumors even after 43 weeks of exposure to CSE.


Cigarette smoke induces epithelial to mesenchymal transition and increases the metastatic ability of breast cancer cells.

Di Cello F, Flowers VL, Li H, Vecchio-Pagán B, Gordon B, Harbom K, Shin J, Beaty R, Wang W, Brayton C, Baylin SB, Zahnow CA - Mol. Cancer (2013)

Cigarette smoke facilitates intraductal colonization of MCF 10A cells and distant metastasis of MCF7 cells. (A-B) H&E and FITC/DAPI staining of mammary gland sections from NSG immunodeficient mice injected intraductally with 100,000 control or 0.5% CSE-treated MCF 10A or MCF7 cells. Each mouse was injected at two sites. MCF10 A cells were injected into 3 mice and MCF7 cells into 2 mice for a total of 6 and 4 sites respectively. Two magnifications are shown. The xenografts were analyzed via immunoflourescence using a FITC antibody specific for human cytokeratin-18. (C) Growth of subcutaneous xenograft tumors from control and CSE-treated (0.5%) MCF7 cells. Each mouse (n=5) was injected at one site. Mean ± SD of five replicates are shown. (D) H&E staining of sections of lung and liver containing metastatic colonies from three representative NSG immunodeficient mice subcutaneously injected with 750,000 MCF7 cells treated with 0.5% CSE. Two additional mice were analyzed by gross pathology for a total n=5.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 3: Cigarette smoke facilitates intraductal colonization of MCF 10A cells and distant metastasis of MCF7 cells. (A-B) H&E and FITC/DAPI staining of mammary gland sections from NSG immunodeficient mice injected intraductally with 100,000 control or 0.5% CSE-treated MCF 10A or MCF7 cells. Each mouse was injected at two sites. MCF10 A cells were injected into 3 mice and MCF7 cells into 2 mice for a total of 6 and 4 sites respectively. Two magnifications are shown. The xenografts were analyzed via immunoflourescence using a FITC antibody specific for human cytokeratin-18. (C) Growth of subcutaneous xenograft tumors from control and CSE-treated (0.5%) MCF7 cells. Each mouse (n=5) was injected at one site. Mean ± SD of five replicates are shown. (D) H&E staining of sections of lung and liver containing metastatic colonies from three representative NSG immunodeficient mice subcutaneously injected with 750,000 MCF7 cells treated with 0.5% CSE. Two additional mice were analyzed by gross pathology for a total n=5.
Mentions: MCF 10A cells are not able to form tumors in immunodeficient mice, and are thus neither malignant nor tumorigenic. Based on our in vitro 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, cancer cells are injected through the nipple, into the primary mammary duct, which allows in situ analysis of intraductal growth and/or invasion through the basement membrane into the stroma [18]. 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 mammary fat pads were harvested after three months and labeled with an antibody for human cytokeratin-18 to identify the injected human cells. Untreated MCF 10A cells did not appear to colonize or grow in the ducts; however, colonies of CSE-treated MCF 10A cells were found within the ducts up to 3 months post-injection (Figure 3A). We then investigated if CSE could further increase the invasiveness of MCF7 breast cancer cells. Because these cells are tumorigenic, and grow much faster than MCF 10A, we harvested the mammary glands seven days after intraductal injection. At that time, untreated MCF7 cells had formed several intraductal masses that appeared to remain within the confines of the ducts. In contrast, MCF7 cells treated with CSE for 21 weeks had invaded through the duct and had formed colonies in the stroma (Figure 3B). Since treatment with CSE had clearly increased the invasiveness of MCF7 cells, we investigated if tumorigenesis and metastasis of MCF7 cells were also affected using a subcutaneous xenograft model. Transplanted MCF7 cells treated with 0.5% CSE for 18 weeks resulted in smaller tumors than mock-treated cells (Figure 3C); however, these smaller tumors were associated with metastases in the lungs of all animals and isolated` cells were found in the liver of at least one animal (Figure 3D). The three mice shown are representative, and two additional mice injected with CSE treated cells were analyzed by gross pathology for a total of 5. In contrast, we did not observe metastases from untreated MCF7 cells, suggesting that cigarette smoke may have favored the expansion of a highly metastatic subpopulation of MCF7 cells. Although MCF10A cells had exhibited increased intraductal survival and colonization after treatment with CSE, these cells did not produce subcutaneous tumors even after 43 weeks of exposure to CSE.

Bottom Line: 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, independent of cigarette smoke effects on the host and stromal environment.Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21287, USA.

ABSTRACT

Background: Recent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke 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, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population 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: Our results indicate that exposure to cigarette smoke leads 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.

Show MeSH
Related in: MedlinePlus