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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 and conditioned media on E-cadherin and β-catenin (co)localization in PMC42-LA organoids. (a) Using immunocytochemistry and confocal microscopy, localization of E-cadherin was analyzed. Nuclei were visualized using ethidium bromide (red). (Part A) In control PMC42-LA organoids (no fibroblasts or conditioned media), E-cadherin staining was observed at cell junctions, which was confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture (right panel). (Part B) In NMF-conditioned medium E-cadherin label was detected as both junctional and cytoplasmic in PMC42-LA organoids, as confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture containing NMF-conditioned media (right panel). (Part C) In CAF-conditioned media E-cadherin was also detected as junctional and cytoplasmic in PMC42-LA organoids, with more predominant cytoplasmic localization. This was confirmed in 2-dimensional PMC42-LA culture containing CAF-conditioned media by confocal microscopy sectioning (right panel). With (part D) NMFs beneath filter or (part E) CAFs beneath the filter, E-cadherin was again detected at cell junctions and within cytoplasm. (b) E-cadherin and β-catenin are indicated by green and red label, respectively. Areas of colocalization appear yellow. (Part A) In control PMC42-LA cells (no fibroblasts or conditioned media), E-cadherin and β-catenin colocalized at cell junctions with some areas of non-colocalization. (Part B) When in NMF-conditioned medium, colocalization was detected at cell junctions with some independent localization. (Part C) In CAF-conditioned medium, E-cadherin localized to cytoplasm and β-catenin to cytoplasm and nuclei, with some overlap. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.
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Figure 3: Effect of fibroblasts and conditioned media on E-cadherin and β-catenin (co)localization in PMC42-LA organoids. (a) Using immunocytochemistry and confocal microscopy, localization of E-cadherin was analyzed. Nuclei were visualized using ethidium bromide (red). (Part A) In control PMC42-LA organoids (no fibroblasts or conditioned media), E-cadherin staining was observed at cell junctions, which was confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture (right panel). (Part B) In NMF-conditioned medium E-cadherin label was detected as both junctional and cytoplasmic in PMC42-LA organoids, as confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture containing NMF-conditioned media (right panel). (Part C) In CAF-conditioned media E-cadherin was also detected as junctional and cytoplasmic in PMC42-LA organoids, with more predominant cytoplasmic localization. This was confirmed in 2-dimensional PMC42-LA culture containing CAF-conditioned media by confocal microscopy sectioning (right panel). With (part D) NMFs beneath filter or (part E) CAFs beneath the filter, E-cadherin was again detected at cell junctions and within cytoplasm. (b) E-cadherin and β-catenin are indicated by green and red label, respectively. Areas of colocalization appear yellow. (Part A) In control PMC42-LA cells (no fibroblasts or conditioned media), E-cadherin and β-catenin colocalized at cell junctions with some areas of non-colocalization. (Part B) When in NMF-conditioned medium, colocalization was detected at cell junctions with some independent localization. (Part C) In CAF-conditioned medium, E-cadherin localized to cytoplasm and β-catenin to cytoplasm and nuclei, with some overlap. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.

Mentions: In the absence of fibroblast influences, E-cadherin appeared to have a junctional localization in PMC42-LA cells (Figure 3a part A, left and right panels). The presence of either NMFs or CAFs beneath the filter, or their respective conditioned media, induced a change in E-cadherin localization from cell-cell junctions (Figure 3a part A, left and right panels) to cytoplasm, where it adopted a granular distribution (Figure 3a parts B to E). To better visualize this change in localization within individual cells and obtain sections through cells, PMC42-LA monolayers cultured in fibroblast conditioned medium were also immunostained for E-cadherin; confocal sections through these cells clearly demonstrate some junctional and granular cytoplasmic localization in cells cultured in NMF conditioned medium (Figure 3a part B, right panel). Significantly, the E-cadherin localization appeared more cytoplasmic in PMC42-LA cells in the presence of CAF-conditioned medium (Figure 3a part C, right panel).


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 and conditioned media on E-cadherin and β-catenin (co)localization in PMC42-LA organoids. (a) Using immunocytochemistry and confocal microscopy, localization of E-cadherin was analyzed. Nuclei were visualized using ethidium bromide (red). (Part A) In control PMC42-LA organoids (no fibroblasts or conditioned media), E-cadherin staining was observed at cell junctions, which was confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture (right panel). (Part B) In NMF-conditioned medium E-cadherin label was detected as both junctional and cytoplasmic in PMC42-LA organoids, as confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture containing NMF-conditioned media (right panel). (Part C) In CAF-conditioned media E-cadherin was also detected as junctional and cytoplasmic in PMC42-LA organoids, with more predominant cytoplasmic localization. This was confirmed in 2-dimensional PMC42-LA culture containing CAF-conditioned media by confocal microscopy sectioning (right panel). With (part D) NMFs beneath filter or (part E) CAFs beneath the filter, E-cadherin was again detected at cell junctions and within cytoplasm. (b) E-cadherin and β-catenin are indicated by green and red label, respectively. Areas of colocalization appear yellow. (Part A) In control PMC42-LA cells (no fibroblasts or conditioned media), E-cadherin and β-catenin colocalized at cell junctions with some areas of non-colocalization. (Part B) When in NMF-conditioned medium, colocalization was detected at cell junctions with some independent localization. (Part C) In CAF-conditioned medium, E-cadherin localized to cytoplasm and β-catenin to cytoplasm and nuclei, with some overlap. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.
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Figure 3: Effect of fibroblasts and conditioned media on E-cadherin and β-catenin (co)localization in PMC42-LA organoids. (a) Using immunocytochemistry and confocal microscopy, localization of E-cadherin was analyzed. Nuclei were visualized using ethidium bromide (red). (Part A) In control PMC42-LA organoids (no fibroblasts or conditioned media), E-cadherin staining was observed at cell junctions, which was confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture (right panel). (Part B) In NMF-conditioned medium E-cadherin label was detected as both junctional and cytoplasmic in PMC42-LA organoids, as confirmed by confocal microscopy sectioning of PMC42-LA in 2-dimensional culture containing NMF-conditioned media (right panel). (Part C) In CAF-conditioned media E-cadherin was also detected as junctional and cytoplasmic in PMC42-LA organoids, with more predominant cytoplasmic localization. This was confirmed in 2-dimensional PMC42-LA culture containing CAF-conditioned media by confocal microscopy sectioning (right panel). With (part D) NMFs beneath filter or (part E) CAFs beneath the filter, E-cadherin was again detected at cell junctions and within cytoplasm. (b) E-cadherin and β-catenin are indicated by green and red label, respectively. Areas of colocalization appear yellow. (Part A) In control PMC42-LA cells (no fibroblasts or conditioned media), E-cadherin and β-catenin colocalized at cell junctions with some areas of non-colocalization. (Part B) When in NMF-conditioned medium, colocalization was detected at cell junctions with some independent localization. (Part C) In CAF-conditioned medium, E-cadherin localized to cytoplasm and β-catenin to cytoplasm and nuclei, with some overlap. CAF, cancer-associated fibroblast; NMF, normal mammary fibroblast.
Mentions: In the absence of fibroblast influences, E-cadherin appeared to have a junctional localization in PMC42-LA cells (Figure 3a part A, left and right panels). The presence of either NMFs or CAFs beneath the filter, or their respective conditioned media, induced a change in E-cadherin localization from cell-cell junctions (Figure 3a part A, left and right panels) to cytoplasm, where it adopted a granular distribution (Figure 3a parts B to E). To better visualize this change in localization within individual cells and obtain sections through cells, PMC42-LA monolayers cultured in fibroblast conditioned medium were also immunostained for E-cadherin; confocal sections through these cells clearly demonstrate some junctional and granular cytoplasmic localization in cells cultured in NMF conditioned medium (Figure 3a part B, right panel). Significantly, the E-cadherin localization appeared more cytoplasmic in PMC42-LA cells in the presence of CAF-conditioned medium (Figure 3a part C, right panel).

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