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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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The effect of concentrated NMF-conditioned media on two-dimensional scratch wound closure. PMC42-LA cells were cultured on glass in 1×, 2×, 4×, or 10× NMF-conditioned medium. Using scratch tests, PMC42-LA cells were analyzed for the extent of wound closure 24 hours after scratch. Scratches were measured and averaged, and the extent of wound closure calculated and averaged. (a) Cells in 1× NMF-conditioned media (control) had average wound sizes of 307 ± 24.3 μm 24 hours post-scratch. (b) Cells in 2× NMF-conditioned medium had average wound sizes of 319 ± 23.2 μm 24 hours post-scratch. (c) Cells in 4× NMF-conditioned medium had average wound widths of 232 ± 49.7 μm 24 hours post-scratch, and (d) cells in 10× NMF-conditioned medium had average wound widths of 160 ± 62.4 μm 24 hours post-scratch. Vimentin localization appeared unchanged in all cultures; some change in cell morphology was noted in 10× NMF-conditioned media cultures. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.
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Figure 6: The effect of concentrated NMF-conditioned media on two-dimensional scratch wound closure. PMC42-LA cells were cultured on glass in 1×, 2×, 4×, or 10× NMF-conditioned medium. Using scratch tests, PMC42-LA cells were analyzed for the extent of wound closure 24 hours after scratch. Scratches were measured and averaged, and the extent of wound closure calculated and averaged. (a) Cells in 1× NMF-conditioned media (control) had average wound sizes of 307 ± 24.3 μm 24 hours post-scratch. (b) Cells in 2× NMF-conditioned medium had average wound sizes of 319 ± 23.2 μm 24 hours post-scratch. (c) Cells in 4× NMF-conditioned medium had average wound widths of 232 ± 49.7 μm 24 hours post-scratch, and (d) cells in 10× NMF-conditioned medium had average wound widths of 160 ± 62.4 μm 24 hours post-scratch. Vimentin localization appeared unchanged in all cultures; some change in cell morphology was noted in 10× NMF-conditioned media cultures. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.

Mentions: PMC42-LA cells were cultured on glass to enable the scratch removal of cells and the measurement of wound closure 24 hours after scratch. In the presence of NMF-conditioned medium at a 1× concentration/control, PMC42-LA cell wounds at 24 hours averaged 307 ± 24.29 μm (Figure 6a). Cells cultured in a 2× NMF-conditioned medium had an average wound width of 319 ± 23.15 μm (Figure 6b) at 24 hours. Cells cultured in a 4× NMF-conditioned medium had wounds of 232 ± 49.65 μm (Figure 6c) at 24 hours and cells cultured in a 10× NMF-conditioned medium had wounds of 160 ± 62.43 μm (Figure 6d) 24 hours post-scratch. Vimentin localization did not appear changed in any of the concentrated media compared with control, but PMC42-LA cells cultured in 10× NMF-conditioned medium cultures exhibited an elongated/spindle shape, with some detached cells, neither of which were seen in the less concentrated NMF-conditioned media cultures (Figure 6d versus a to c). Average wound widths of these cultures are displayed graphically (Figure 6e).


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)

The effect of concentrated NMF-conditioned media on two-dimensional scratch wound closure. PMC42-LA cells were cultured on glass in 1×, 2×, 4×, or 10× NMF-conditioned medium. Using scratch tests, PMC42-LA cells were analyzed for the extent of wound closure 24 hours after scratch. Scratches were measured and averaged, and the extent of wound closure calculated and averaged. (a) Cells in 1× NMF-conditioned media (control) had average wound sizes of 307 ± 24.3 μm 24 hours post-scratch. (b) Cells in 2× NMF-conditioned medium had average wound sizes of 319 ± 23.2 μm 24 hours post-scratch. (c) Cells in 4× NMF-conditioned medium had average wound widths of 232 ± 49.7 μm 24 hours post-scratch, and (d) cells in 10× NMF-conditioned medium had average wound widths of 160 ± 62.4 μm 24 hours post-scratch. Vimentin localization appeared unchanged in all cultures; some change in cell morphology was noted in 10× NMF-conditioned media cultures. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851381&req=5

Figure 6: The effect of concentrated NMF-conditioned media on two-dimensional scratch wound closure. PMC42-LA cells were cultured on glass in 1×, 2×, 4×, or 10× NMF-conditioned medium. Using scratch tests, PMC42-LA cells were analyzed for the extent of wound closure 24 hours after scratch. Scratches were measured and averaged, and the extent of wound closure calculated and averaged. (a) Cells in 1× NMF-conditioned media (control) had average wound sizes of 307 ± 24.3 μm 24 hours post-scratch. (b) Cells in 2× NMF-conditioned medium had average wound sizes of 319 ± 23.2 μm 24 hours post-scratch. (c) Cells in 4× NMF-conditioned medium had average wound widths of 232 ± 49.7 μm 24 hours post-scratch, and (d) cells in 10× NMF-conditioned medium had average wound widths of 160 ± 62.4 μm 24 hours post-scratch. Vimentin localization appeared unchanged in all cultures; some change in cell morphology was noted in 10× NMF-conditioned media cultures. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.
Mentions: PMC42-LA cells were cultured on glass to enable the scratch removal of cells and the measurement of wound closure 24 hours after scratch. In the presence of NMF-conditioned medium at a 1× concentration/control, PMC42-LA cell wounds at 24 hours averaged 307 ± 24.29 μm (Figure 6a). Cells cultured in a 2× NMF-conditioned medium had an average wound width of 319 ± 23.15 μm (Figure 6b) at 24 hours. Cells cultured in a 4× NMF-conditioned medium had wounds of 232 ± 49.65 μm (Figure 6c) at 24 hours and cells cultured in a 10× NMF-conditioned medium had wounds of 160 ± 62.43 μm (Figure 6d) 24 hours post-scratch. Vimentin localization did not appear changed in any of the concentrated media compared with control, but PMC42-LA cells cultured in 10× NMF-conditioned medium cultures exhibited an elongated/spindle shape, with some detached cells, neither of which were seen in the less concentrated NMF-conditioned media cultures (Figure 6d versus a to c). Average wound widths of these cultures are displayed graphically (Figure 6e).

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