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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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Analysis of SP and ALDH+ cells for functional stem cell characteristics(A,B) Analysis of single cell clonogenicity. SP/NSP (A) or ALDH+/ALDH− (B) cells were single cell-sorted and subsequently cultured for two weeks. Wells with outgrowing clones were counted and the proportion of cells capable of colony formation was calculated. (C) Analysis of spheroid formation was performed by seeding 1×103 SP or NSP cells on primary mesothelial cell monolayers pre-established in 8-well chamber slides, followed by culturing for one week. The number of spheroids per chamber is depicted. (D) Representative photomicrographs of SP and NSP spheroids at the indicated magnifications (cell line: B17/92). (E) Analysis of asymmetric cell division was performed by single cell sorting of SP or NSP cells. After three weeks of culture, viable clones were analyzed for the SP fraction to determine the reciprocal cell population present in the sample. (F-H) Kaplan-Meier survival curves of female NOD/SCID mice. Mice were intra-peritoneally inoculated with 5×104 SP or NSP cells. Tumor development was regularly monitored and mice with severe tumor burden were euthanized. SP, side population; NSP, non-SP; ALDH, aldehyde dehydrogenase; * p<0.05, ** p<0.01; n.a., not applicable.
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Figure 3: Analysis of SP and ALDH+ cells for functional stem cell characteristics(A,B) Analysis of single cell clonogenicity. SP/NSP (A) or ALDH+/ALDH− (B) cells were single cell-sorted and subsequently cultured for two weeks. Wells with outgrowing clones were counted and the proportion of cells capable of colony formation was calculated. (C) Analysis of spheroid formation was performed by seeding 1×103 SP or NSP cells on primary mesothelial cell monolayers pre-established in 8-well chamber slides, followed by culturing for one week. The number of spheroids per chamber is depicted. (D) Representative photomicrographs of SP and NSP spheroids at the indicated magnifications (cell line: B17/92). (E) Analysis of asymmetric cell division was performed by single cell sorting of SP or NSP cells. After three weeks of culture, viable clones were analyzed for the SP fraction to determine the reciprocal cell population present in the sample. (F-H) Kaplan-Meier survival curves of female NOD/SCID mice. Mice were intra-peritoneally inoculated with 5×104 SP or NSP cells. Tumor development was regularly monitored and mice with severe tumor burden were euthanized. SP, side population; NSP, non-SP; ALDH, aldehyde dehydrogenase; * p<0.05, ** p<0.01; n.a., not applicable.

Mentions: To investigate whether ALDH+ cells or SP cells (or both) exhibited stem cell traits the clonogenic potential at the single cell level was determined. In all six cell lines tested we found that single cells of the SP had a significantly higher clonogenic potential when compared to their NSP counterparts (Fig. 3A). Conversely, in comparison with ALDH− cells, clonogenicity of ALDH+ cells was significantly increased only in one cell line (Fig. 3B). These functional data suggest that SP cells, rather than ALDH+ cells, are characterized by an increased colony-forming capacity. This prompted us to focus in more detail on the SP phenotype.


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)

Analysis of SP and ALDH+ cells for functional stem cell characteristics(A,B) Analysis of single cell clonogenicity. SP/NSP (A) or ALDH+/ALDH− (B) cells were single cell-sorted and subsequently cultured for two weeks. Wells with outgrowing clones were counted and the proportion of cells capable of colony formation was calculated. (C) Analysis of spheroid formation was performed by seeding 1×103 SP or NSP cells on primary mesothelial cell monolayers pre-established in 8-well chamber slides, followed by culturing for one week. The number of spheroids per chamber is depicted. (D) Representative photomicrographs of SP and NSP spheroids at the indicated magnifications (cell line: B17/92). (E) Analysis of asymmetric cell division was performed by single cell sorting of SP or NSP cells. After three weeks of culture, viable clones were analyzed for the SP fraction to determine the reciprocal cell population present in the sample. (F-H) Kaplan-Meier survival curves of female NOD/SCID mice. Mice were intra-peritoneally inoculated with 5×104 SP or NSP cells. Tumor development was regularly monitored and mice with severe tumor burden were euthanized. SP, side population; NSP, non-SP; ALDH, aldehyde dehydrogenase; * p<0.05, ** p<0.01; n.a., not applicable.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Analysis of SP and ALDH+ cells for functional stem cell characteristics(A,B) Analysis of single cell clonogenicity. SP/NSP (A) or ALDH+/ALDH− (B) cells were single cell-sorted and subsequently cultured for two weeks. Wells with outgrowing clones were counted and the proportion of cells capable of colony formation was calculated. (C) Analysis of spheroid formation was performed by seeding 1×103 SP or NSP cells on primary mesothelial cell monolayers pre-established in 8-well chamber slides, followed by culturing for one week. The number of spheroids per chamber is depicted. (D) Representative photomicrographs of SP and NSP spheroids at the indicated magnifications (cell line: B17/92). (E) Analysis of asymmetric cell division was performed by single cell sorting of SP or NSP cells. After three weeks of culture, viable clones were analyzed for the SP fraction to determine the reciprocal cell population present in the sample. (F-H) Kaplan-Meier survival curves of female NOD/SCID mice. Mice were intra-peritoneally inoculated with 5×104 SP or NSP cells. Tumor development was regularly monitored and mice with severe tumor burden were euthanized. SP, side population; NSP, non-SP; ALDH, aldehyde dehydrogenase; * p<0.05, ** p<0.01; n.a., not applicable.
Mentions: To investigate whether ALDH+ cells or SP cells (or both) exhibited stem cell traits the clonogenic potential at the single cell level was determined. In all six cell lines tested we found that single cells of the SP had a significantly higher clonogenic potential when compared to their NSP counterparts (Fig. 3A). Conversely, in comparison with ALDH− cells, clonogenicity of ALDH+ cells was significantly increased only in one cell line (Fig. 3B). These functional data suggest that SP cells, rather than ALDH+ cells, are characterized by an increased colony-forming capacity. This prompted us to focus in more detail on the SP phenotype.

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