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Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors.

Lebret SC, Newgreen DF, Thompson EW, Ackland ML - Breast Cancer Res. (2007)

Bottom Line: However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium.Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function.By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media.

View Article: PubMed Central - HTML - PubMed

Affiliation: Deakin University, Burwood Highway, Burwood, Melbourne, 3125, Australia.

ABSTRACT

Background: Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.

Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.

Results: We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of alpha-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active beta-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive beta-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.

Conclusion: We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

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Effect of fibroblasts on major protein expression by PMC42-LA. Western blot analysis for a range of markers on control PMC42-LA organoids (control), and organoids cultured with NMFs beneath the filter (lane 1), with CAFs beneath the filter (lane 2), in medium conditioned by NMFs (lane 3), or in medium conditioned by CAFs (lane 4). Controls had no fibroblasts or fibroblast conditioned-medium. Immunocytochemistry was performed to view organization within organoid structures, and ethidium bromide was used to stain nuclei (red). (a) E-cadherin was not significantly upregulated in any of the fibroblast conditions, with no obvious changes in organization. (b) α-SMA expression was found to be significantly upregulated by all fibroblast conditions, with no obvious changes in organization. (c) Cytokeratin 14 expression was found to be significantly induced by all fibroblast conditions, with no obvious changes in organization. (d) Vimentin expression was also found to be significantly upregulated in all fibroblast conditions, with slight changes in organoid organization detected by immunocytochemistry. In control PMC42-LA cells (control lane in panel d), intermediate filament protein vimentin is localized in the cytoplasm of cells throughout organoids. With NMFs beneath the filter (panel d lane 1), vimentin remains localized in the cytoplasm of PMC42-LA cells and vimentin-positive cells are more organized, visible only on outer layer of organoids. With CAFs beneath the filter (panel d lane 2), the same organizational changes are observed. With NMF-conditioned media (panel d lane 3), the same organizational changes are observed. With CAF-conditioned medium (panel d lane 4), no change in organization is observed, with vimentin expression throughout organoids. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast; SMA, smooth muscle actin.
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Figure 2: Effect of fibroblasts on major protein expression by PMC42-LA. Western blot analysis for a range of markers on control PMC42-LA organoids (control), and organoids cultured with NMFs beneath the filter (lane 1), with CAFs beneath the filter (lane 2), in medium conditioned by NMFs (lane 3), or in medium conditioned by CAFs (lane 4). Controls had no fibroblasts or fibroblast conditioned-medium. Immunocytochemistry was performed to view organization within organoid structures, and ethidium bromide was used to stain nuclei (red). (a) E-cadherin was not significantly upregulated in any of the fibroblast conditions, with no obvious changes in organization. (b) α-SMA expression was found to be significantly upregulated by all fibroblast conditions, with no obvious changes in organization. (c) Cytokeratin 14 expression was found to be significantly induced by all fibroblast conditions, with no obvious changes in organization. (d) Vimentin expression was also found to be significantly upregulated in all fibroblast conditions, with slight changes in organoid organization detected by immunocytochemistry. In control PMC42-LA cells (control lane in panel d), intermediate filament protein vimentin is localized in the cytoplasm of cells throughout organoids. With NMFs beneath the filter (panel d lane 1), vimentin remains localized in the cytoplasm of PMC42-LA cells and vimentin-positive cells are more organized, visible only on outer layer of organoids. With CAFs beneath the filter (panel d lane 2), the same organizational changes are observed. With NMF-conditioned media (panel d lane 3), the same organizational changes are observed. With CAF-conditioned medium (panel d lane 4), no change in organization is observed, with vimentin expression throughout organoids. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast; SMA, smooth muscle actin.

Mentions: Compared with control, expression of the luminal epithelial marker E-cadherin, as measured by Western blot analysis, was not significantly upregulated by PMC42-LA organoids cultured with either NMFs (fold increase: 1.8 ± 1.17) or CAFs (fold increase: 1.8 ± 1.31) beneath the filter (Figure 2a, lanes 1 and 2, respectively). Similar values were seen when fibroblast-conditioned medium was used in place of the fibroblasts (fold increase compared with control: 1.5 ± 2.13 with NMF-conditioned medium and 2.4 ± 1.10 with CAF-conditioned medium; Figure 2a, lanes 3 and 4). Immunocytochemistry confirmed that E-cadherin was expressed by most cells in all conditions tested, with no obvious changes in the organization of E-cadherin expressing cells within organoid structures (Figure 2a, lanes 1 to 4).


Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors.

Lebret SC, Newgreen DF, Thompson EW, Ackland ML - Breast Cancer Res. (2007)

Effect of fibroblasts on major protein expression by PMC42-LA. Western blot analysis for a range of markers on control PMC42-LA organoids (control), and organoids cultured with NMFs beneath the filter (lane 1), with CAFs beneath the filter (lane 2), in medium conditioned by NMFs (lane 3), or in medium conditioned by CAFs (lane 4). Controls had no fibroblasts or fibroblast conditioned-medium. Immunocytochemistry was performed to view organization within organoid structures, and ethidium bromide was used to stain nuclei (red). (a) E-cadherin was not significantly upregulated in any of the fibroblast conditions, with no obvious changes in organization. (b) α-SMA expression was found to be significantly upregulated by all fibroblast conditions, with no obvious changes in organization. (c) Cytokeratin 14 expression was found to be significantly induced by all fibroblast conditions, with no obvious changes in organization. (d) Vimentin expression was also found to be significantly upregulated in all fibroblast conditions, with slight changes in organoid organization detected by immunocytochemistry. In control PMC42-LA cells (control lane in panel d), intermediate filament protein vimentin is localized in the cytoplasm of cells throughout organoids. With NMFs beneath the filter (panel d lane 1), vimentin remains localized in the cytoplasm of PMC42-LA cells and vimentin-positive cells are more organized, visible only on outer layer of organoids. With CAFs beneath the filter (panel d lane 2), the same organizational changes are observed. With NMF-conditioned media (panel d lane 3), the same organizational changes are observed. With CAF-conditioned medium (panel d lane 4), no change in organization is observed, with vimentin expression throughout organoids. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast; SMA, smooth muscle actin.
© Copyright Policy - open-access
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Figure 2: Effect of fibroblasts on major protein expression by PMC42-LA. Western blot analysis for a range of markers on control PMC42-LA organoids (control), and organoids cultured with NMFs beneath the filter (lane 1), with CAFs beneath the filter (lane 2), in medium conditioned by NMFs (lane 3), or in medium conditioned by CAFs (lane 4). Controls had no fibroblasts or fibroblast conditioned-medium. Immunocytochemistry was performed to view organization within organoid structures, and ethidium bromide was used to stain nuclei (red). (a) E-cadherin was not significantly upregulated in any of the fibroblast conditions, with no obvious changes in organization. (b) α-SMA expression was found to be significantly upregulated by all fibroblast conditions, with no obvious changes in organization. (c) Cytokeratin 14 expression was found to be significantly induced by all fibroblast conditions, with no obvious changes in organization. (d) Vimentin expression was also found to be significantly upregulated in all fibroblast conditions, with slight changes in organoid organization detected by immunocytochemistry. In control PMC42-LA cells (control lane in panel d), intermediate filament protein vimentin is localized in the cytoplasm of cells throughout organoids. With NMFs beneath the filter (panel d lane 1), vimentin remains localized in the cytoplasm of PMC42-LA cells and vimentin-positive cells are more organized, visible only on outer layer of organoids. With CAFs beneath the filter (panel d lane 2), the same organizational changes are observed. With NMF-conditioned media (panel d lane 3), the same organizational changes are observed. With CAF-conditioned medium (panel d lane 4), no change in organization is observed, with vimentin expression throughout organoids. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast; SMA, smooth muscle actin.
Mentions: Compared with control, expression of the luminal epithelial marker E-cadherin, as measured by Western blot analysis, was not significantly upregulated by PMC42-LA organoids cultured with either NMFs (fold increase: 1.8 ± 1.17) or CAFs (fold increase: 1.8 ± 1.31) beneath the filter (Figure 2a, lanes 1 and 2, respectively). Similar values were seen when fibroblast-conditioned medium was used in place of the fibroblasts (fold increase compared with control: 1.5 ± 2.13 with NMF-conditioned medium and 2.4 ± 1.10 with CAF-conditioned medium; Figure 2a, lanes 3 and 4). Immunocytochemistry confirmed that E-cadherin was expressed by most cells in all conditions tested, with no obvious changes in the organization of E-cadherin expressing cells within organoid structures (Figure 2a, lanes 1 to 4).

Bottom Line: However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium.Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function.By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media.

View Article: PubMed Central - HTML - PubMed

Affiliation: Deakin University, Burwood Highway, Burwood, Melbourne, 3125, Australia.

ABSTRACT

Background: Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.

Methods: We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.

Results: We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of alpha-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active beta-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive beta-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.

Conclusion: We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Show MeSH
Related in: MedlinePlus