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Side population (SP) analyses and CD133 expression have identified cells with stem-like potential in normal and cancerous tissue. Whether stem-like cells exist in cancer cell lines is hotly debated. We have interrogated the DAOY medulloblastoma cell line with respect to stem-like potential. Vital staining for Hoechst 33342 efflux capacity and CD133 immunophenotyping were performed on DAOY cells to assess the presence of the SP and the CD133 stem cell markers, respectively. SP/non-SP and CD133+/CD133− DAOY cells were sorted into separate fractions for limiting dilution analysis (tumor sphere assay) and asymmetric division assessment. SP/non-SP cells were also sorted separately for viability (XTT assay), cell size, cell cycle status, and proliferative capacity (carboxyfluorescein succinimidyl ester (CFSE)) evaluation. A minor proportion of cells displayed either the SP or the CD133+ phenotypes. CD133 expression mapped to both the SP and non-SP compartments, with CD133+ cells being enriched almost fourfold within the non-SP gate. The SP, non-SP, CD133+, and CD133− fractions were all capable of reconstituting the original parental DAOY population. Slight clonogenic enrichment was observed in only the SP fraction; however, both CD133+ and CD133− cells displayed equivalent stem cell-like frequencies. SP cells were resistant to Hoechst 33342-mediated toxicity relative to the parental population and differed from the non-SP cells with respect to increased cell size, decreased S-phase, and slightly decreased proliferative capacity. The multiparametric strategy described in this study revealed that the SP and CD133+ subset may be two independent compartments. Our results highlight the need for new reliable specific cancer stem cell marker(s) as Hoechst 33342 efflux and CD133 expression might not be suitable for selectively isolating cancer stem-like cells from cell lines, as shown for the DAOY cells. As such, care must be used in interpreting therapeutic studies targeting the stem cell compartment of cancer cell lines. © 2008 International Society for Advancement of Cytometry
DESPITE the observation that malignant cellular characteristics closely phenocopy the stem cell-like features of self-renewal and multilineage differentiation, the outstanding feature separating the malignant cell from the normal stem cell is the lack of homeostatic balance between self-renewal and differentiation (1). Furthermore, cancer tissue, much like normal tissue, is hierarchically organized according to stage of differentiation and proliferative potential (2). Such close phenotypic parallels imply that the normal stem cell could transform malignantly; however, it is also possible that lineage-restricted or differentiated progeny transform, through a process of de-differentiation, into malignant cells displaying stem-like features (2). Yet, the target stem cell may require fewer alterations to initiate tumorigenesis compared with its downstream progeny (3). This so-called “cancer stem cell” has been isolated and characterized from a variety of haematological malignancies and solid tumors including medulloblastomas (4).
However, it must be understood that the term “cancer stem cell” is an operational term for a tumor cell that displays the ability to self-renew and also divide to generate another stem cell and a progenitor cell, which generates the multitude of cell types comprising the bulk of the tumor (5). Definitive proof for the existence of cancer stem cells was first elucidated in acute myeloid leukaemia (AML) whereby xenotransplantation of human AML cells into immunodeficient mice revealed the frequency of leukaemic stem cells to be about 0.1–1% of all the tumor cells; specifically, only the CD34+CD38− cells were able to reconstitute the leukaemic phenotype whereas the CD34+CD38+ and CD34− cell were unable to do so (6). More recently, using the neural stem cell marker CD133, a putative brain tumor stem cell has been isolated from medulloblastoma, that is capable of self-renewal and multilineage differentiation (4). Although immunophenotyping has demonstrated that many malignancies may be organized hierarchically, functional assays using vital fluorescent dyes have also revealed that the cancer stem cell phenotype may be defined by high expression of various types of ATP-binding cassette (ABC) transporters. Indeed, seminal experiments using the Hoechst 33342 dye, which can be effluxed by ABCG2 transporters, have identified an unlabeled side population (SP) that is enriched for stem cells in the bone marrow (7). These observations have been extended by an increasing number of studies that have identified the SP phenotype in a variety of primary tumor tissues and cancer cell lines.
Remarkably, the presence of stem-like cells has also been reported in long-established cultured cell lines (8–10). Thus, the major objective of this study was to characterize the established DAOY medulloblastoma cell line for cancer stem-like cells using both immunophenotypic (CD133) and physiological substrate (Hoechst 33342) markers and to determine the cellular properties of the isolated putative stem-like fraction with respect to proliferative capacity, cell cycle status, and reconstitution of the original phenotype. Our results demonstrate a weak relationship between the expression of these stem cell markers and clonogenicity, indicating that these markers may not target the stem cell compartment in established cancer cell lines.
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- LITERATURE CITED
The technique for stem cell labeling and sorting based on cells displaying low red and blue fluorescence subsequent to incubation with the Hoechst 33342 dye has been appreciated for quite some time (7). With resurgence of the cancer stem cell hypothesis, the Hoechst-based technique has now been recently applied to assessing the stem cell frequency of cancer cell lines and primary tumors. However, a recent study has demonstrated that not all cancer cell lines display an SP phenotype (10). An explanation for this discrepancy may lie in re-visiting the basic premise or assumptions underlying the association between the SP phenotype and stem cell activity. In addition to displaying the classical features of self-renewal and quiescence, stem cells also express high levels of certain members of the ABC transporter family, which includes ABCB1 (P-glycoprotein), ABCC1 (multidrug-associated protein 1 or MRP1), and ABCG2 (breast cancer-related protein or BCRP) (13). Interestingly, integrity of the stem cell compartment is not affected in mice with disruptions in Abcb1, Abcc1, Abcg2; however, there is increased sensitivity to chemotherapeutics such as mitoxantrone, vinblastine, and topotecan suggesting that drug transporters may not play an essential role in stem cell development but only subserve xenobiotic resistance (14–16). Clinically, the SP phenotype may reflect selection of drug transporter expression leading to evolution of treatment-resistant cells in a variety of cancers (13). Previous studies have marked putative stem cells with fluorescent dyes (e.g. Hoechst 33342, rhodamine 123) and cytotoxic compounds (e.g. mitoxantrone, methotrexate) and characterized self-renewal properties (7, 15, 17). However, our results have demonstrated Hoechst 33342-mediated toxicity of DAOY cells and that only SP cells display resistance to this toxicity (Fig. 4), presumably attributed to Hoechst 33342 efflux. Furthermore, DNA-binding affinity of Hoechst 33342 may interfere with cellular replication and differentiation thus confounding the ability to detect relevant biological differences between the SP and non-SP fractions. Indeed, an early study revealed that Hoechst 33342 can induce the F9 embryonal carcinoma cell line to differentiate along the endodermal pathway (18). Likewise, a recent study reported that nuclear Hoechst 33342 staining can have a dramatic impact on C2C12 myogenic differentiation and PC12 neuritic differentiation (19). Regarding our results, it is possible that Hoechst 33342 may interfere with differentiation and thus affect non-SP parameters; interestingly, this may explain the slight stem-like cell enrichment in the SP fraction whereas both CD133+ and CD133− fractions have equivalent stem-like cell frequencies. The tumor sphere limiting dilution assay revealed that SP cells were more clonogenic when compared with the parental unlabeled tumor cell population (Fig. 6). Although not presented here, preliminary experiments did reveal that non-SP cells were also able to generate tumor spheres. As well, the cancer stem cell hypothesis would predict that the SP fraction should be able to regenerate both the SP and non-SP fractions whereas the non-SP fraction should only be able to regenerate itself. Interestingly, the results presented here demonstrate that both the SP and the non-SP fraction have the capacity to completely regenerate both fractions (Fig. 5). A recent article has also observed this same finding in the C6 glioma cell line where either fraction was capable of reconstituting the parental cellular population (20).
Immunophenotyping is an alternative method for assessing the existence of stem-like cells. Indeed, this method may bypass the limitations of current physiological-based dye efflux assays by marking stem-like cells extracellularly thereby potentially avoiding toxicity associated with DNA-dye interacalation. The CD133 extracellular receptor is an established primitive stem cell marker that has now found application for cancer stem cell analysis in a variety of cancer cell lines and primary tumors. A recent report has also shown CD133 expression in all tested medulloblastoma cell lines (8). In the present study, CD133+ cells displayed tumor sphere-like growth (Fig. 6) thereby initially confirming previous findings that CD133+ cells display stem-like activity (4, 21, 22). However, CD133− DAOY cells were able to form tumor spheres with a calculated stem-like frequency comparable to that of CD133+ cells (Fig. 6). Also, similar to the SP and non-SP fractions, either the CD133+ or the CD133− fraction was capable of reconstituting both fractions (Fig. 6). A similar study has also reported that in the C6 glioma cell line CD133− cells displayed self-renewal and tumorigenic features (23). Limiting dilution analysis of the tumor sphere assay revealed that not all cells were capable of clonogenic expansion. Thus, rather than the entire culture being made up of stem cells, it appears that a relatively large minority of DAOY cells display clonogenicity. The present study also suggests that plasticity or stem-like potential of tumor cells, as defined by Hoechst 33342 efflux or CD133 expression, is variable and dependent on environmental factors; indeed, the Hoechst 33342 technique appeared to be highly variable with the percentage of SP cells varying significantly with tumor cell density.
Another novel aspect of this study was the finding that CD133 expression mapped to both the SP and non-SP gates (Fig. 1). Initially, the anticipation was that CD133 expression would be localized primarily to the SP gate; however, CD133 expression appears to be enriched almost fourfold within the non-SP gate. Moreover, given both the very low abundance of CD133 expression in both gates and that non-SP cells constitute the vast majority of the parental cell population, this may explain the similar tumor sphere formation efficiencies that were observed between both the CD133+ and the CD133− fractions (Fig. 6).
In conclusion, this study highlights the novel application of flow cytometric methods in assessing the extension of the cancer stem cell hypothesis into cancer cell lines. Using the established DAOY medulloblastoma cell line, our results do not demonstrate a strong relationship between stem cell marker expression and clonogenicity; both non-SP and CD133− cells also display stem-like characteristics. The DAOY cell line contains a relatively large minority of cells that display clonogenicity independent of stem cell marker expression. Thus, Hoechst 33342 and CD133 expression may not be suitable in selectively isolating stem-like cells in the DAOY cell line. This may be of concern when studies begin to utilize these markers to demonstrate efficacy of targeting the cancer stem cell compartment in cancer cell lines such as DAOY (9). This study provides evidence that cancer cell lines may not recapitulate the hierarchical model of stemness observed in vivo. This may be of fundamental clinical importance as many nonclinical testing strategies utilize established cancer cell lines in modeling tumor biology and in screening anticancer therapies.