In vitro investigations of tumor stem-like cells (TSC) isolated from human glioblastoma (GB) surgical specimens have been done primarily at an atmospheric oxygen level of 20%. To determine whether an oxygen level more consistent with in situ conditions affects their stem cell–like characteristics, we compared GB TSCs grown under conditions of 20% and 7% oxygen. Growing CD133+ cells sorted from three GB neurosphere cultures at 7% O2 reduced their doubling time and increased the self-renewal potential as reflected by clonogenicity. Furthermore, at 7% oxygen, the cultures exhibited an enhanced capacity to differentiate along both the glial and neuronal pathways. As compared with 20%, growth at 7% oxygen resulted in an increase in the expression levels of the neural stem cell markers CD133 and nestin as well as the stem cell markers Oct4 and Sox2. In addition, whereas hypoxia inducible factor 1α was not affected in CD133+ TSCs grown at 7% O2, hypoxia-inducible factor 2α was expressed at higher levels as compared with 20% oxygen. Gene expression profiles generated by microarray analysis revealed that reducing oxygen level to 7% resulted in the up-regulation and down-regulation of a significant number of genes, with more than 140 being commonly affected among the three CD133+ cultures. Furthermore, Gene Ontology categories up-regulated at 7% oxygen included those associated with stem cells or GB TSCs. Thus, the data presented indicate that growth at the more physiologically relevant oxygen level of 7% enhances the stem cell–like phenotype of CD133+ GB cells.

Purpose: CD133+ glioblastoma (GB) tumor stem-like cells (TSCs) have been defined as radioresistant. However, whereas previously classified relative to CD133− cells, the radiosensitivity of CD133+ TSCs with respect to the standard GB model, established glioma cell lines, has not been determined. Therefore, to better understand the radioresponse of this cancer stem cell, we have used established cell lines as a framework for defining their in vitro radioresponse. Experimental Design: The intrinsic radiosensitivity of CD133+ TSC cultures and established glioma cell lines was determined by clonogenic assay. The TSCs and established cell lines were also compared in terms of DNA double strand break (DSB) repair capacity and cell cycle checkpoint activation. Results: Based on clonogenic analysis, each of the six TSC cultures evaluated was more sensitive to radiation than the established glioma cell lines. Consistent with increased radiosensitivity, the DSB repair capacity as defined by neutral comet assay and γH2AX and Rad51 foci was significantly reduced in TSCs as compared to the cell lines. Whereas G2 checkpoint activation was intact, in contrast to the cell lines, DNA synthesis was not inhibited in TSCs after irradiation indicating the absence of the intra-S phase checkpoint. Conclusions: These data indicate that the mechanisms through which CD133+ TSCs respond to radiation are significantly different from those of the traditional GB in vitro model, established glioma cell lines. If TSCs play a critical role in GB treatment response, then such differences are likely to be of consequence in the development and testing of radiosensitizing agents.