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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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Screening of various ovarian cancer cell lines for SP subsetsCell lines were stained using DCV and analyzed by flow cytometry. SP subsets are indicated by polygonal gates, and the percentage of cells within these gates is given (first row). Corresponding FTC (second row) and verapamil (Vera; third row) inhibition controls are shown. DCV-stained cells were subsequently stained using fluorochrome-conjugated monoclonal antibodies directed against ABC drug transporters. Rectangular gates indicating positive staining for ABCG2 (fourth row) and ABCB1 (fifth row) are shown, and the percentage of cells within these gates is given. Data are representative examples of at least three independent experiments. SP, side population; DCV, Vybrant® DyeCycleTM Violet; FTC, fumitremorgin C.
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Figure 2: Screening of various ovarian cancer cell lines for SP subsetsCell lines were stained using DCV and analyzed by flow cytometry. SP subsets are indicated by polygonal gates, and the percentage of cells within these gates is given (first row). Corresponding FTC (second row) and verapamil (Vera; third row) inhibition controls are shown. DCV-stained cells were subsequently stained using fluorochrome-conjugated monoclonal antibodies directed against ABC drug transporters. Rectangular gates indicating positive staining for ABCG2 (fourth row) and ABCB1 (fifth row) are shown, and the percentage of cells within these gates is given. Data are representative examples of at least three independent experiments. SP, side population; DCV, Vybrant® DyeCycleTM Violet; FTC, fumitremorgin C.

Mentions: To characterize the relevance of various CSC markers in ovarian cancer cells, we performed systematic phenotyping of a panel of ovarian cancer cell lines (A2780, A2780V, B2/92, B16/92, B17/92, IGROV1; see Suppl. Table 1 for cell line characteristics) using flow cytometry. Strikingly, the surface markers CD24, CD44, CD90, CD133 and CD326 all failed to consistently identify distinct small subpopulations in the different cell lines. These markers are either not expressed at all or bulk-expressed in many of the cell lines (Suppl. Fig. 1, Suppl. Table 2). In contrast, both ALDH (Fig. 1, Suppl. Table 2) and the SP phenotype (Fig. 2, Suppl. Table 2) robustly identified distinct small subsets (typically ≤2%) in each of the six cell lines. Moreover, distinct small subsets of ALDH+ and SP cells could also be detected in 3/7 (Suppl. Fig. 2) and 6/7 (Suppl. Fig. 3) additional ovarian cancer cell lines, respectively (see again Suppl. Table 1 for cell line characteristics). Furthermore in a proof-of-concept study, SP subsets could also be detected in freshly isolated primary ovarian tumor cells (Suppl. Fig. 4A). Of note, the response pattern of SP cells to fumitremorgin C (FTC) (which selectively blocks ABCG2 [16, 17]) and verapamil (which blocks several ABC drug transporters including ABCG2 and ABCB1 [18]) suggested differential drug transporter expression among different SP fractions. In line with these pharmacological drug transporter inhibition data, we found that FTC/verapamil double-sensitive SP (i.e., A2780, B16/92, B17/92) expressed high amounts of ABCG2, whereas verapamil-sensitive and FTC-insensitive SP (i.e., A2780V, B2/92, IGROV1) highly expressed ABCB1 (co-expression of both transporters was not observed) (Fig. 2). Taken together, the presence of distinct minority populations of ALDH+ and SP cells, but not of subpopulations defined by other CSC markers, is a common feature of ovarian cancer cell lines (total prevalence: ALDH 9/13 cell lines, SP phenotype 12/13 cell lines).


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)

Screening of various ovarian cancer cell lines for SP subsetsCell lines were stained using DCV and analyzed by flow cytometry. SP subsets are indicated by polygonal gates, and the percentage of cells within these gates is given (first row). Corresponding FTC (second row) and verapamil (Vera; third row) inhibition controls are shown. DCV-stained cells were subsequently stained using fluorochrome-conjugated monoclonal antibodies directed against ABC drug transporters. Rectangular gates indicating positive staining for ABCG2 (fourth row) and ABCB1 (fifth row) are shown, and the percentage of cells within these gates is given. Data are representative examples of at least three independent experiments. SP, side population; DCV, Vybrant® DyeCycleTM Violet; FTC, fumitremorgin C.
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Figure 2: Screening of various ovarian cancer cell lines for SP subsetsCell lines were stained using DCV and analyzed by flow cytometry. SP subsets are indicated by polygonal gates, and the percentage of cells within these gates is given (first row). Corresponding FTC (second row) and verapamil (Vera; third row) inhibition controls are shown. DCV-stained cells were subsequently stained using fluorochrome-conjugated monoclonal antibodies directed against ABC drug transporters. Rectangular gates indicating positive staining for ABCG2 (fourth row) and ABCB1 (fifth row) are shown, and the percentage of cells within these gates is given. Data are representative examples of at least three independent experiments. SP, side population; DCV, Vybrant® DyeCycleTM Violet; FTC, fumitremorgin C.
Mentions: To characterize the relevance of various CSC markers in ovarian cancer cells, we performed systematic phenotyping of a panel of ovarian cancer cell lines (A2780, A2780V, B2/92, B16/92, B17/92, IGROV1; see Suppl. Table 1 for cell line characteristics) using flow cytometry. Strikingly, the surface markers CD24, CD44, CD90, CD133 and CD326 all failed to consistently identify distinct small subpopulations in the different cell lines. These markers are either not expressed at all or bulk-expressed in many of the cell lines (Suppl. Fig. 1, Suppl. Table 2). In contrast, both ALDH (Fig. 1, Suppl. Table 2) and the SP phenotype (Fig. 2, Suppl. Table 2) robustly identified distinct small subsets (typically ≤2%) in each of the six cell lines. Moreover, distinct small subsets of ALDH+ and SP cells could also be detected in 3/7 (Suppl. Fig. 2) and 6/7 (Suppl. Fig. 3) additional ovarian cancer cell lines, respectively (see again Suppl. Table 1 for cell line characteristics). Furthermore in a proof-of-concept study, SP subsets could also be detected in freshly isolated primary ovarian tumor cells (Suppl. Fig. 4A). Of note, the response pattern of SP cells to fumitremorgin C (FTC) (which selectively blocks ABCG2 [16, 17]) and verapamil (which blocks several ABC drug transporters including ABCG2 and ABCB1 [18]) suggested differential drug transporter expression among different SP fractions. In line with these pharmacological drug transporter inhibition data, we found that FTC/verapamil double-sensitive SP (i.e., A2780, B16/92, B17/92) expressed high amounts of ABCG2, whereas verapamil-sensitive and FTC-insensitive SP (i.e., A2780V, B2/92, IGROV1) highly expressed ABCB1 (co-expression of both transporters was not observed) (Fig. 2). Taken together, the presence of distinct minority populations of ALDH+ and SP cells, but not of subpopulations defined by other CSC markers, is a common feature of ovarian cancer cell lines (total prevalence: ALDH 9/13 cell lines, SP phenotype 12/13 cell lines).

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