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The side population of ovarian cancer cells defines a heterogeneous compartment exhibiting stem cell characteristics.

Boesch M, Zeimet AG, Reimer D, Schmidt S, Gastl G, Parson W, Spoeck F, Hatina J, Wolf D, Sopper S - Oncotarget (2014)

Bottom Line: In a systematic screen of 13 ovarian cancer cell lines we show that cells with stem cell properties are reliably detectable as a minor population, characterized by ABC transporter expression resulting in the side population (SP) phenotype.In different cell lines, either ABCG2 or ABCB1 was found to be responsible for this effect.Thus, our study confirms previous results showing that CSC are contained within the SP.

View Article: PubMed Central - PubMed

Affiliation: Internal Medicine V, Innsbruck Medical University, Innsbruck, Austria. Tyrolean Cancer Research Institute, Innsbruck, Austria.

ABSTRACT
Cancer stem cells (CSC) are believed to be involved in tumor evasion of classical antitumor therapies and have thus become an attractive target for further improvement of anticancer strategies. However, the existence and identity of CSC are still a matter of controversy. In a systematic screen of 13 ovarian cancer cell lines we show that cells with stem cell properties are reliably detectable as a minor population, characterized by ABC transporter expression resulting in the side population (SP) phenotype. In different cell lines, either ABCG2 or ABCB1 was found to be responsible for this effect. Purified SP cells featured virtually all characteristics of bona fide CSC, including clonogenicity, asymmetric division and high tumorigenicity in vivo. Using in-depth phenotyping by multicolor flow cytometry, we found that among the investigated ovarian cancer cell lines the SP compartment exhibits tremendous heterogeneity and is composed of multiple phenotypically distinct subpopulations. Thus, our study confirms previous results showing that CSC are contained within the SP. However, the exact identity of the CSC is still disguised by the high complexity of the CSC-containing compartment. Further functional studies are needed to determine whether a single cellular subset can unambiguously be defined as CSC or whether multiple stem cell-like cells with different properties coexist. Moreover, the observed heterogeneity may reflect a high level of plasticity and likely influences tumor progression, escape from immune-surveillance and development of resistance to anticancer therapies and should therefore be considered in the development of new treatment strategies.

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In-depth phenotypic characterization of ovarian cancer cell lines and purified SP/NSP fractionsCells were stained with fluorochrome-conjugated monoclonal antibodies and analyzed by flow cytometry. (A) Illustration of the principal possibilities of marker expression as encompassed in this study. (B) Systematic heatmap analysis of surface marker expression in ovarian cancer cell lines. Shown are MFI above cut-off after normalization to respective controls and log-transformation. (C) Systematic heatmap analysis of surface marker expression in purified SP and NSP fractions. Shown are MFI above cut-off after normalization to respective controls and log-transformation. Data represent the mean of at least two, but typically three, independent experiments. SP, side population; NSP, non-SP; MFI, median fluorescence intensity.
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Figure 4: In-depth phenotypic characterization of ovarian cancer cell lines and purified SP/NSP fractionsCells were stained with fluorochrome-conjugated monoclonal antibodies and analyzed by flow cytometry. (A) Illustration of the principal possibilities of marker expression as encompassed in this study. (B) Systematic heatmap analysis of surface marker expression in ovarian cancer cell lines. Shown are MFI above cut-off after normalization to respective controls and log-transformation. (C) Systematic heatmap analysis of surface marker expression in purified SP and NSP fractions. Shown are MFI above cut-off after normalization to respective controls and log-transformation. Data represent the mean of at least two, but typically three, independent experiments. SP, side population; NSP, non-SP; MFI, median fluorescence intensity.

Mentions: Cells with stem cell properties were enriched but not exclusively found in the SP compartment, and not all SP cells exhibited CSC properties. To further characterize the phenotype and potentially detect a further restricted ovarian CSC identity downstream of the SP denominator [19], we extended the panel to include markers implicated in ovarian cancer progression (e.g., CD140a, CD171) [20, 21], epithelial-to-mesenchymal transition (EMT; e.g., CD325) [22], cell migration/chemotaxis (e.g., chemokine receptors) [23], and interaction with the immune system (e.g., HLA-ABC, CD95) [24, 25] (for complete list see Suppl. Table 3). In these experiments, we observed a broad spectrum of marker expression, ranging from no expression to intermediate and high expression, and expression in distinct subsets (Fig. 4A). More importantly, these analyses showed marked heterogeneity between the various cell lines, with virtually no common pattern in expression levels as determined by median fluorescence intensity (MFI; Fig. 4B). Accordingly, cluster analysis failed to identify marker groups showing relevant clustering (data not shown).


The side population of ovarian cancer cells defines a heterogeneous compartment exhibiting stem cell characteristics.

Boesch M, Zeimet AG, Reimer D, Schmidt S, Gastl G, Parson W, Spoeck F, Hatina J, Wolf D, Sopper S - Oncotarget (2014)

In-depth phenotypic characterization of ovarian cancer cell lines and purified SP/NSP fractionsCells were stained with fluorochrome-conjugated monoclonal antibodies and analyzed by flow cytometry. (A) Illustration of the principal possibilities of marker expression as encompassed in this study. (B) Systematic heatmap analysis of surface marker expression in ovarian cancer cell lines. Shown are MFI above cut-off after normalization to respective controls and log-transformation. (C) Systematic heatmap analysis of surface marker expression in purified SP and NSP fractions. Shown are MFI above cut-off after normalization to respective controls and log-transformation. Data represent the mean of at least two, but typically three, independent experiments. SP, side population; NSP, non-SP; MFI, median fluorescence intensity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196181&req=5

Figure 4: In-depth phenotypic characterization of ovarian cancer cell lines and purified SP/NSP fractionsCells were stained with fluorochrome-conjugated monoclonal antibodies and analyzed by flow cytometry. (A) Illustration of the principal possibilities of marker expression as encompassed in this study. (B) Systematic heatmap analysis of surface marker expression in ovarian cancer cell lines. Shown are MFI above cut-off after normalization to respective controls and log-transformation. (C) Systematic heatmap analysis of surface marker expression in purified SP and NSP fractions. Shown are MFI above cut-off after normalization to respective controls and log-transformation. Data represent the mean of at least two, but typically three, independent experiments. SP, side population; NSP, non-SP; MFI, median fluorescence intensity.
Mentions: Cells with stem cell properties were enriched but not exclusively found in the SP compartment, and not all SP cells exhibited CSC properties. To further characterize the phenotype and potentially detect a further restricted ovarian CSC identity downstream of the SP denominator [19], we extended the panel to include markers implicated in ovarian cancer progression (e.g., CD140a, CD171) [20, 21], epithelial-to-mesenchymal transition (EMT; e.g., CD325) [22], cell migration/chemotaxis (e.g., chemokine receptors) [23], and interaction with the immune system (e.g., HLA-ABC, CD95) [24, 25] (for complete list see Suppl. Table 3). In these experiments, we observed a broad spectrum of marker expression, ranging from no expression to intermediate and high expression, and expression in distinct subsets (Fig. 4A). More importantly, these analyses showed marked heterogeneity between the various cell lines, with virtually no common pattern in expression levels as determined by median fluorescence intensity (MFI; Fig. 4B). Accordingly, cluster analysis failed to identify marker groups showing relevant clustering (data not shown).

Bottom Line: In a systematic screen of 13 ovarian cancer cell lines we show that cells with stem cell properties are reliably detectable as a minor population, characterized by ABC transporter expression resulting in the side population (SP) phenotype.In different cell lines, either ABCG2 or ABCB1 was found to be responsible for this effect.Thus, our study confirms previous results showing that CSC are contained within the SP.

View Article: PubMed Central - PubMed

Affiliation: Internal Medicine V, Innsbruck Medical University, Innsbruck, Austria. Tyrolean Cancer Research Institute, Innsbruck, Austria.

ABSTRACT
Cancer stem cells (CSC) are believed to be involved in tumor evasion of classical antitumor therapies and have thus become an attractive target for further improvement of anticancer strategies. However, the existence and identity of CSC are still a matter of controversy. In a systematic screen of 13 ovarian cancer cell lines we show that cells with stem cell properties are reliably detectable as a minor population, characterized by ABC transporter expression resulting in the side population (SP) phenotype. In different cell lines, either ABCG2 or ABCB1 was found to be responsible for this effect. Purified SP cells featured virtually all characteristics of bona fide CSC, including clonogenicity, asymmetric division and high tumorigenicity in vivo. Using in-depth phenotyping by multicolor flow cytometry, we found that among the investigated ovarian cancer cell lines the SP compartment exhibits tremendous heterogeneity and is composed of multiple phenotypically distinct subpopulations. Thus, our study confirms previous results showing that CSC are contained within the SP. However, the exact identity of the CSC is still disguised by the high complexity of the CSC-containing compartment. Further functional studies are needed to determine whether a single cellular subset can unambiguously be defined as CSC or whether multiple stem cell-like cells with different properties coexist. Moreover, the observed heterogeneity may reflect a high level of plasticity and likely influences tumor progression, escape from immune-surveillance and development of resistance to anticancer therapies and should therefore be considered in the development of new treatment strategies.

Show MeSH
Related in: MedlinePlus