title: Flow cytometric characterization of the DAOY medulloblastoma cell line for the cancer stem-like phenotype.
abstract: 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.
authors.affiliation: Department of Neurology and Neurosurgery, McGill University and Montreal Neurological Institute, Montreal, Canada H3A 2B4.
authors.forename: Vinit Krishna
authors.initials: VK
authors.lastname: Srivastava
authors.forename: Josephine
authors.initials: J
authors.lastname: Nalbantoglu
mesh_terms: D000071916:AC133 Antigen; D015703:Antigens, CD / Q000032:analysis* / Q000096:biosynthesis; D001562:Benzimidazoles / Q000032:analysis; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000096:biosynthesis; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D005452:Fluoresceins / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis* / Q000096:biosynthesis; D006801:Humans; D008527:Medulloblastoma / Q000378:metabolism / Q000473:pathology*; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D010455:Peptides / Q000032:analysis*; D018874:Spheroids, Cellular / Q000378:metabolism / Q000473:pathology; D013388:Succinimides / Q000378:metabolism
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000071916:AC133 Antigen; D015703:Antigens, CD; D001562:Benzimidazoles; D014408:Biomarkers, Tumor; D005452:Fluoresceins; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; D013388:Succinimides; C017807:bisbenzimide ethoxide trihydrochloride