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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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E/M hybrid cells differentiate into mesenchymal cells in vitro.A) Triple-color flow cytometry analysis of xenograft/biopsy cell suspensions and cultured cells in vitro at different passages. Left panel: EpCAM/CD44/CD133. Right panel: EpCAM/vimentin/CD133. B) Passage 1 and 5 cells were subjected to 14 days of growth factor/FCS starvation and then analyzed by flow cytometry. Shown are data from ovc316-X and ovc316-XC. The findings were confirmed on cell suspensions from biopsies and primary cultures (ovc100506-biopsy, ovc100506-PC, ovc100728-biopsy, and 100914). C) Immunofluorescence analysis of passage 3 and 18 cultures of ovc316-XC. Cells in early passages (passage 3) have elevated levels of E-cadherin and laminin when compared to passage 18. Cells sizes are increased and sphere-growth in high cell densities is markedly reduced in high passages. Percentage of side population (SP) positive cells are shown below. The scale bar is 40µm. D) Tumor formation after transplantation of ovc316-XC passage 3 and 18 cells into SCID-beige mice (evaluated 4 months after inoculation). TIC p3: 1/110, TIC p18: 1/744. Chi-square: 0.245.
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pone-0016186-g003: E/M hybrid cells differentiate into mesenchymal cells in vitro.A) Triple-color flow cytometry analysis of xenograft/biopsy cell suspensions and cultured cells in vitro at different passages. Left panel: EpCAM/CD44/CD133. Right panel: EpCAM/vimentin/CD133. B) Passage 1 and 5 cells were subjected to 14 days of growth factor/FCS starvation and then analyzed by flow cytometry. Shown are data from ovc316-X and ovc316-XC. The findings were confirmed on cell suspensions from biopsies and primary cultures (ovc100506-biopsy, ovc100506-PC, ovc100728-biopsy, and 100914). C) Immunofluorescence analysis of passage 3 and 18 cultures of ovc316-XC. Cells in early passages (passage 3) have elevated levels of E-cadherin and laminin when compared to passage 18. Cells sizes are increased and sphere-growth in high cell densities is markedly reduced in high passages. Percentage of side population (SP) positive cells are shown below. The scale bar is 40µm. D) Tumor formation after transplantation of ovc316-XC passage 3 and 18 cells into SCID-beige mice (evaluated 4 months after inoculation). TIC p3: 1/110, TIC p18: 1/744. Chi-square: 0.245.

Mentions: To quantify numbers of cells with epithelial and mesenchymal phenotypes in cultured cells, we employed flow cytometry and started by monitoring cells for the epithelial marker EpCAM as well as monitoring for vimentin or CD44 marking cells with mesenchymal attributes. Furthermore, to delineate phenotypic changes overtime such as initiation of EMT, we analyzed different passages of ovc316-XC cells (Figure 3). These studies revealed a number of interesting observations. i) In tumor cell suspensions that were freshly isolated from xenografts, the majority of cells were either E/M cells (EpCAMhigh/Vimentinhigh, EpCAMhigh/CD44high) or E-cells (EpCAMhigh/Vimentinlow, EpCAMhigh/CD44low), however, at passage 1 only E/M cells could be detected, indicating that the majority of cells obtained from xenografts that adapt to tissue culture are E/M (Figure 3A). This implies that, even at early passage, cultures do not adequately reflect the cellular composition of the tumor in situ. Passage 1 contained EpCAMhigh/CD44high/Vimentinhigh and EpCAMhigh/CD44low/Vimentinlow cells. ii) Importantly during culture of cells in MEGM containing growth factors/FBS (see passages 1, 5, 7, 20), both the EpCAMhigh/CD44high/Vimentinhigh or EpCAMhigh/CD44low/Vimentinlow cell types disappeared and were replaced by EpCAMlow/CD44high/Vimentinhigh cells. This demonstrates that E/M cells differentiate into mesenchymal cells in vitro in an EMT-like manner. iii) The majority of CD133+ cells are E/M hybrid cells. After passaging in culture ovc316-XC cells rapidly lose CD133 expression concomitantly with the loss of epithelial features.


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)

E/M hybrid cells differentiate into mesenchymal cells in vitro.A) Triple-color flow cytometry analysis of xenograft/biopsy cell suspensions and cultured cells in vitro at different passages. Left panel: EpCAM/CD44/CD133. Right panel: EpCAM/vimentin/CD133. B) Passage 1 and 5 cells were subjected to 14 days of growth factor/FCS starvation and then analyzed by flow cytometry. Shown are data from ovc316-X and ovc316-XC. The findings were confirmed on cell suspensions from biopsies and primary cultures (ovc100506-biopsy, ovc100506-PC, ovc100728-biopsy, and 100914). C) Immunofluorescence analysis of passage 3 and 18 cultures of ovc316-XC. Cells in early passages (passage 3) have elevated levels of E-cadherin and laminin when compared to passage 18. Cells sizes are increased and sphere-growth in high cell densities is markedly reduced in high passages. Percentage of side population (SP) positive cells are shown below. The scale bar is 40µm. D) Tumor formation after transplantation of ovc316-XC passage 3 and 18 cells into SCID-beige mice (evaluated 4 months after inoculation). TIC p3: 1/110, TIC p18: 1/744. Chi-square: 0.245.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3021543&req=5

pone-0016186-g003: E/M hybrid cells differentiate into mesenchymal cells in vitro.A) Triple-color flow cytometry analysis of xenograft/biopsy cell suspensions and cultured cells in vitro at different passages. Left panel: EpCAM/CD44/CD133. Right panel: EpCAM/vimentin/CD133. B) Passage 1 and 5 cells were subjected to 14 days of growth factor/FCS starvation and then analyzed by flow cytometry. Shown are data from ovc316-X and ovc316-XC. The findings were confirmed on cell suspensions from biopsies and primary cultures (ovc100506-biopsy, ovc100506-PC, ovc100728-biopsy, and 100914). C) Immunofluorescence analysis of passage 3 and 18 cultures of ovc316-XC. Cells in early passages (passage 3) have elevated levels of E-cadherin and laminin when compared to passage 18. Cells sizes are increased and sphere-growth in high cell densities is markedly reduced in high passages. Percentage of side population (SP) positive cells are shown below. The scale bar is 40µm. D) Tumor formation after transplantation of ovc316-XC passage 3 and 18 cells into SCID-beige mice (evaluated 4 months after inoculation). TIC p3: 1/110, TIC p18: 1/744. Chi-square: 0.245.
Mentions: To quantify numbers of cells with epithelial and mesenchymal phenotypes in cultured cells, we employed flow cytometry and started by monitoring cells for the epithelial marker EpCAM as well as monitoring for vimentin or CD44 marking cells with mesenchymal attributes. Furthermore, to delineate phenotypic changes overtime such as initiation of EMT, we analyzed different passages of ovc316-XC cells (Figure 3). These studies revealed a number of interesting observations. i) In tumor cell suspensions that were freshly isolated from xenografts, the majority of cells were either E/M cells (EpCAMhigh/Vimentinhigh, EpCAMhigh/CD44high) or E-cells (EpCAMhigh/Vimentinlow, EpCAMhigh/CD44low), however, at passage 1 only E/M cells could be detected, indicating that the majority of cells obtained from xenografts that adapt to tissue culture are E/M (Figure 3A). This implies that, even at early passage, cultures do not adequately reflect the cellular composition of the tumor in situ. Passage 1 contained EpCAMhigh/CD44high/Vimentinhigh and EpCAMhigh/CD44low/Vimentinlow cells. ii) Importantly during culture of cells in MEGM containing growth factors/FBS (see passages 1, 5, 7, 20), both the EpCAMhigh/CD44high/Vimentinhigh or EpCAMhigh/CD44low/Vimentinlow cell types disappeared and were replaced by EpCAMlow/CD44high/Vimentinhigh cells. This demonstrates that E/M cells differentiate into mesenchymal cells in vitro in an EMT-like manner. iii) The majority of CD133+ cells are E/M hybrid cells. After passaging in culture ovc316-XC cells rapidly lose CD133 expression concomitantly with the loss of epithelial features.

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