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. 2014 Aug 30;5(16):7027-39.
doi: 10.18632/oncotarget.2053.

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

Maximilian Boesch  1 , Alain G Zeimet  2 , Daniel Reimer  2 , Stefan Schmidt  3 , Guenther Gastl  3 , Walther Parson  4 , Franziska Spoeck  1 , Jiri Hatina  5 , Dominik Wolf  6 , Sieghart Sopper  7
Affiliations

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

Maximilian Boesch et al. Oncotarget. .

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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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors declare that there is no conflict of interest. There is also no non-author involvement in the preparation of the manuscript.

Figures

Figure 1
Figure 1. Screening of various ovarian cancer cell lines for ALDH+ subsets
Cell lines were stained for ALDH enzymatic activity and analyzed by flow cytometry. ALDH+ subsets are indicated by rectangular gates, and the percentage of cells within these gates is given (upper row). Corresponding DEAB inhibition controls are shown in the lower row. Data are representative examples of at least three independent experiments. ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde.
Figure 2
Figure 2. Screening of various ovarian cancer cell lines for SP subsets
Cell 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.
Figure 3
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.
Figure 4
Figure 4. In-depth phenotypic characterization of ovarian cancer cell lines and purified SP/NSP fractions
Cells 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.
Figure 5
Figure 5. SPICE analysis of ovarian cancer heterogeneity
Defined mixtures of corresponding SP and NSP fractions were stained for different markers (staining 1 of Suppl. Table 4) and analyzed by multicolor flow cytometry. (A) Definition of populations positive for individual markers (cell line: A2780). These gates were combined by Boolean operations to obtain proportions of cells within all possible combinations, which were then imported into SPICE for final data analysis. (B) SPICE analysis of ovarian cancer heterogeneity after class-division into SP (upper panel) and NSP (lower panel). Subset distributions are presented in the weighted category mode, and only subsets above a threshold of ≥0.1% are shown. Data are representative examples of at least two independent experiments. SP, side population; NSP, non-SP.
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