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Analysis of epithelial and mesenchymal markers in ovarian cancer reveals phenotypic heterogeneity and plasticity.

Strauss R, Li ZY, Liu Y, Beyer I, Persson J, Sova P, Möller T, Pesonen S, Hemminki A, Hamerlik P, Drescher C, Urban N, Bartek J, Lieber A - PLoS ONE (2011)

Bottom Line: Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity.In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition.We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Genetics, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherin(low)/cytoplasmic E-cadherin(high)/CD133(high), CD44(high), Tie2(low)) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.

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Differentiation potential of CD133+ cells.A) Viability of different subsets. Low passage ovc316-XC cells contain high numbers of CD133+ and Tie2+ cells. Tie2+/CD133+ double positive cells have lower levels of CD133+ than CD133+/Tie2− cells. The vast majority of Tie2+ cells stains positive for the dead cell marker 7-AAD after treatment with versen. Viable cells show higher mean fluorescence of CD44 and CD133. All attached cells are positive for the cell proliferation marker Ki-67. B) CD133+ cells isolated from ovc316-XC tumor xenografts adapt to their environment. When cultured at low cell densities, sorted CD133+/E-cadherinhigh and CD133+/E-cadherinlow cells differentiate into similar cell populations with an epithelial phenotype. When co-cultured with clonal ovc316-XC epithelial or mesenchymal cultures (ratio 1∶10), CD133+ cells differentiate into epithelial or mesenchymal cells, depending on the environment. Cell membrane dye PHK26 was used to discriminate between clonal cells (PHK26 positive) and cells derived from the CD133+ population (PHK26 negative). Cells were cultured for 7 days. C and D). Similarities between primary ovarian cancer cells and normal ovarian surface epithelial cells (OSE). Early passage OSE cells show high similarities with early passage ovarian cancer cultures. OSE cultures are low in E-cadherin and are CD133+. CD133+ cells within OSE and ovarian cancer cultures are enriched in cells positive for E-cadherin and CD44. Tie2+ cells in OSE and ovarian cancer cultures are enriched for E-cadherin and show lower mean fluorescence for CD44 than CD133+ cells.
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pone-0016186-g007: Differentiation potential of CD133+ cells.A) Viability of different subsets. Low passage ovc316-XC cells contain high numbers of CD133+ and Tie2+ cells. Tie2+/CD133+ double positive cells have lower levels of CD133+ than CD133+/Tie2− cells. The vast majority of Tie2+ cells stains positive for the dead cell marker 7-AAD after treatment with versen. Viable cells show higher mean fluorescence of CD44 and CD133. All attached cells are positive for the cell proliferation marker Ki-67. B) CD133+ cells isolated from ovc316-XC tumor xenografts adapt to their environment. When cultured at low cell densities, sorted CD133+/E-cadherinhigh and CD133+/E-cadherinlow cells differentiate into similar cell populations with an epithelial phenotype. When co-cultured with clonal ovc316-XC epithelial or mesenchymal cultures (ratio 1∶10), CD133+ cells differentiate into epithelial or mesenchymal cells, depending on the environment. Cell membrane dye PHK26 was used to discriminate between clonal cells (PHK26 positive) and cells derived from the CD133+ population (PHK26 negative). Cells were cultured for 7 days. C and D). Similarities between primary ovarian cancer cells and normal ovarian surface epithelial cells (OSE). Early passage OSE cells show high similarities with early passage ovarian cancer cultures. OSE cultures are low in E-cadherin and are CD133+. CD133+ cells within OSE and ovarian cancer cultures are enriched in cells positive for E-cadherin and CD44. Tie2+ cells in OSE and ovarian cancer cultures are enriched for E-cadherin and show lower mean fluorescence for CD44 than CD133+ cells.

Mentions: Our finding that, during passaging, E/M cells give rise to cultures that predominantly contain mesenchymal cells and lost surface CD133 indicates differentiation (Figure 3). Notably, during this process, a small subset of CD133+ cells is maintained and this subset expresses low to high levels of surface E-cadherin and high levels of CD44. The maintenance of potential cancer stem cells also explains the development of tumors after transplantation of passage 18 cells (see Figure 3D). Interestingly, when passage 18 cultures were subjected to stress in the form of serum/growth factor starvation, E/M-cells regain features of stem cells, particularly the expression of nuclear Nanog, indicating a potential reversion of E/M-E to E/M-MP cells (Figure S8). To gather further evidence for differentiation of E/M subsets, we analyzed early passage ovc316-CX cells for cell viability after detachment using versene (Figure 7A). In agreement with earlier experiments, we found high numbers of CD133+ and Tie2+ cells, whereby the majority of CD133+ and Tie2+ cells are mutually exclusive. Tie2+/CD133+ double positive cells have lower levels of CD133+ than CD133+/Tie2− cells. The vast majority of Tie2+ cells stain positive for the dead cell marker 7AAD after treatment with versene, indicating their loss of resistance to anoikis after detachment, and therefore a higher degree of differentiation. Viable cells show higher mean fluorescence of CD44 and CD133. All attached cells are positive for the cell proliferation marker Ki-67.


Analysis of epithelial and mesenchymal markers in ovarian cancer reveals phenotypic heterogeneity and plasticity.

Strauss R, Li ZY, Liu Y, Beyer I, Persson J, Sova P, Möller T, Pesonen S, Hemminki A, Hamerlik P, Drescher C, Urban N, Bartek J, Lieber A - PLoS ONE (2011)

Differentiation potential of CD133+ cells.A) Viability of different subsets. Low passage ovc316-XC cells contain high numbers of CD133+ and Tie2+ cells. Tie2+/CD133+ double positive cells have lower levels of CD133+ than CD133+/Tie2− cells. The vast majority of Tie2+ cells stains positive for the dead cell marker 7-AAD after treatment with versen. Viable cells show higher mean fluorescence of CD44 and CD133. All attached cells are positive for the cell proliferation marker Ki-67. B) CD133+ cells isolated from ovc316-XC tumor xenografts adapt to their environment. When cultured at low cell densities, sorted CD133+/E-cadherinhigh and CD133+/E-cadherinlow cells differentiate into similar cell populations with an epithelial phenotype. When co-cultured with clonal ovc316-XC epithelial or mesenchymal cultures (ratio 1∶10), CD133+ cells differentiate into epithelial or mesenchymal cells, depending on the environment. Cell membrane dye PHK26 was used to discriminate between clonal cells (PHK26 positive) and cells derived from the CD133+ population (PHK26 negative). Cells were cultured for 7 days. C and D). Similarities between primary ovarian cancer cells and normal ovarian surface epithelial cells (OSE). Early passage OSE cells show high similarities with early passage ovarian cancer cultures. OSE cultures are low in E-cadherin and are CD133+. CD133+ cells within OSE and ovarian cancer cultures are enriched in cells positive for E-cadherin and CD44. Tie2+ cells in OSE and ovarian cancer cultures are enriched for E-cadherin and show lower mean fluorescence for CD44 than CD133+ cells.
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Related In: Results  -  Collection

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pone-0016186-g007: Differentiation potential of CD133+ cells.A) Viability of different subsets. Low passage ovc316-XC cells contain high numbers of CD133+ and Tie2+ cells. Tie2+/CD133+ double positive cells have lower levels of CD133+ than CD133+/Tie2− cells. The vast majority of Tie2+ cells stains positive for the dead cell marker 7-AAD after treatment with versen. Viable cells show higher mean fluorescence of CD44 and CD133. All attached cells are positive for the cell proliferation marker Ki-67. B) CD133+ cells isolated from ovc316-XC tumor xenografts adapt to their environment. When cultured at low cell densities, sorted CD133+/E-cadherinhigh and CD133+/E-cadherinlow cells differentiate into similar cell populations with an epithelial phenotype. When co-cultured with clonal ovc316-XC epithelial or mesenchymal cultures (ratio 1∶10), CD133+ cells differentiate into epithelial or mesenchymal cells, depending on the environment. Cell membrane dye PHK26 was used to discriminate between clonal cells (PHK26 positive) and cells derived from the CD133+ population (PHK26 negative). Cells were cultured for 7 days. C and D). Similarities between primary ovarian cancer cells and normal ovarian surface epithelial cells (OSE). Early passage OSE cells show high similarities with early passage ovarian cancer cultures. OSE cultures are low in E-cadherin and are CD133+. CD133+ cells within OSE and ovarian cancer cultures are enriched in cells positive for E-cadherin and CD44. Tie2+ cells in OSE and ovarian cancer cultures are enriched for E-cadherin and show lower mean fluorescence for CD44 than CD133+ cells.
Mentions: Our finding that, during passaging, E/M cells give rise to cultures that predominantly contain mesenchymal cells and lost surface CD133 indicates differentiation (Figure 3). Notably, during this process, a small subset of CD133+ cells is maintained and this subset expresses low to high levels of surface E-cadherin and high levels of CD44. The maintenance of potential cancer stem cells also explains the development of tumors after transplantation of passage 18 cells (see Figure 3D). Interestingly, when passage 18 cultures were subjected to stress in the form of serum/growth factor starvation, E/M-cells regain features of stem cells, particularly the expression of nuclear Nanog, indicating a potential reversion of E/M-E to E/M-MP cells (Figure S8). To gather further evidence for differentiation of E/M subsets, we analyzed early passage ovc316-CX cells for cell viability after detachment using versene (Figure 7A). In agreement with earlier experiments, we found high numbers of CD133+ and Tie2+ cells, whereby the majority of CD133+ and Tie2+ cells are mutually exclusive. Tie2+/CD133+ double positive cells have lower levels of CD133+ than CD133+/Tie2− cells. The vast majority of Tie2+ cells stain positive for the dead cell marker 7AAD after treatment with versene, indicating their loss of resistance to anoikis after detachment, and therefore a higher degree of differentiation. Viable cells show higher mean fluorescence of CD44 and CD133. All attached cells are positive for the cell proliferation marker Ki-67.

Bottom Line: Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity.In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition.We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures.

View Article: PubMed Central - PubMed

Affiliation: Division of Medical Genetics, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
In our studies of ovarian cancer cells we have identified subpopulations of cells that are in a transitory E/M hybrid stage, i.e. cells that simultaneously express epithelial and mesenchymal markers. E/M cells are not homogenous but, in vitro and in vivo, contain subsets that can be distinguished based on a number of phenotypic features, including the subcellular localization of E-cadherin, and the expression levels of Tie2, CD133, and CD44. A cellular subset (E/M-MP) (membrane E-cadherin(low)/cytoplasmic E-cadherin(high)/CD133(high), CD44(high), Tie2(low)) is highly enriched for tumor-forming cells and displays features which are generally associated with cancer stem cells. Our data suggest that E/M-MP cells are able to differentiate into different lineages under certain conditions, and have the capacity for self-renewal, i.e. to maintain a subset of undifferentiated E/M-MP cells during differentiation. Trans-differentiation of E/M-MP cells into mesenchymal or epithelial cells is associated with a loss of stem cell markers and tumorigenicity. In vivo xenograft tumor growth is driven by E/M-MP cells, which give rise to epithelial ovarian cancer cells. In contrast, in vitro, we found that E/M-MP cells differentiate into mesenchymal cells, in a process that involves pathways associated with an epithelial-to-mesenchymal transition. We also detected phenotypic plasticity that was dependent on external factors such as stress created by starvation or contact with either epithelial or mesenchymal cells in co-cultures. Our study provides a better understanding of the phenotypic complexity of ovarian cancer and has implications for ovarian cancer therapy.

Show MeSH
Related in: MedlinePlus