Article

Physiologic Oxygen Concentration Enhances the Stem-Like Properties of CD133(+) Human Glioblastoma Cells In vitro

Moffitt Cancer Center, Tampa, FL 33612, USA.
Molecular Cancer Research (Impact Factor: 4.5). 05/2009; 7(4):489-97. DOI: 10.1158/1541-7786.MCR-08-0360
Source: PubMed

ABSTRACT 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% O(2) 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 1alpha was not affected in CD133(+) TSCs grown at 7% O(2), hypoxia-inducible factor 2alpha 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.

Download full-text

Full-text

Available from: Muhammad Jamal, Aug 25, 2015
0 Followers
 · 
109 Views
  • Source
    • "We assessed the percentages of CD133 positivity, a surrogate marker for GSCs, present in three freshly disaggregated xenolines (GBM- XD456, GBM-X12, and GBM-X6) in tissue culture under normoxic and 1% hypoxic environments. Similar to other reports, CD133 expression increased significantly in all three xenolines under hypoxia (Table 1) [8] [9]. GBM-XD456, which had the lowest percentage of CD133 + cells in normoxia (16.5%), had the greatest relative increase in CD133 expression (nearly four-fold) under hypoxia. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Hypoxia plays a critical role in the tumor microenvironment of high-grade gliomas by promoting the glioma stem cell (GSC)-like phenotype, which displays resistance to standard therapies. We tested three glioblastoma multiforme xenograft lines (xenolines) against γ(1)34.5-deleted recombinant oncolytic herpes simplex virus (oHSV) C101 under 1% (hypoxia) and 20.8% (normoxia) oxygen tension for effects on oHSV infectivity, replication, and cytotoxicity in all tumor cells and CD133(+) GSCs. Expression levels of CD133, a putative GSC marker, and CD111 (nectin-1), an adhesion molecule that is the most efficient method for HSV entry, increased significantly under hypoxia in all three xenolines. Despite increased CD111 expression under hypoxic conditions, oHSV infectivity, cytotoxicity and viral recovery were not improved or were diminished in all three xenolines under hypoxia. In contrast, wild-type HSV-1 equally infected xenoline cells in normoxia and hypoxia, suggesting that the 34.5 mutation plays a role in the decreased C101 infectivity in hypoxia. Importantly, CD133(+) cells were not more resistant to oHSV than CD133(-) tumor cells regardless of oxygen tension. Furthermore, CD133 expression decreased as viral dose increased in two of the xenolines suggesting that up-regulation of CD133 in hypoxia was not the cause of reduced viral efficacy. Our findings that oHSV infectivity and cytotoxicity were diminished under hypoxia in several GBM xenolines likely have important implications for clinical applications of oHSV therapies, especially considering the vital role of hypoxia in the microenvironment of GBM tumors.
    Translational oncology 06/2012; 5(3):200-7. DOI:10.1593/tlo.12115 · 3.40 Impact Factor
  • Source
    • "Neurospheres were maintained in medium consisting of Dulbecco's modified Eagle medium (DMEM)/F-12 (Invitrogen, Carlsbad, CA), B27 supplement (1×; Invitrogen), and human recombinant basic fibroblast growth factor and epidermal growth factor (50 ng/ml each; R&D Systems, Minneapolis, MN). All cultures were maintained at 37°C in an atmosphere of 5% CO 2 /7% O 2 [19]. CD133 + cells were isolated from each neurosphere cultures by FACS [10] and used as a source for the described experiments. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Brain tumor xenografts initiated from glioblastoma (GBM) CD133(+) tumor stem-like cells (TSCs) are composed of TSC and non-TSC subpopulations, simulating the phenotypic heterogeneity of GBMs in situ. Given that the discrepancies between the radiosensitivity of GBM cells in vitro and the treatment response of patients suggest a role for the microenvironment in GBM radioresistance, we compared the response of TSCs and non-TSCs irradiated under in vitro and orthotopic conditions. As a measure of radioresponse determined at the individual cell level, γH2AX and 53BP1 foci were quantified in CD133(+) cells and their differentiated (CD133(-)) progeny. Under in vitro conditions, no difference was detected between CD133(+) and CD133(-) cells in foci induction or dispersal after irradiation. However, irradiation of orthotopic xenografts initiated from TSCs resulted in the induction of fewer γH2AX and 53BP1 foci in CD133(+) cells compared to their CD133(-) counterparts within the same tumor. Xenograft irradiation resulted in a tumor growth delay of approximately 7 days with a corresponding increase in the percentage of CD133(+) cells at 7 days after radiation, which persisted to the onset of neurologic symptoms. These results suggest that, although the radioresponse of TSCs and non-TSCs does not differ under in vitro growth conditions, CD133(+) cells are relatively radioresistant under intracerebral growth conditions. Whereas these findings are consistent with the suspected role for TSCs as a determinant of GBM radioresistance, these data also illustrate the dependence of the cellular radioresistance on the brain microenvironment.
    Neoplasia (New York, N.Y.) 02/2012; 14(2):150-8. DOI:10.1593/neo.111794 · 5.40 Impact Factor
  • Source
    • "In contrast, these regions in Nf1+/− GFAP CKO mice appeared as gross fusiform enlargements involving the pre-chiasmatic optic nerves and optic chiasm (Fig. 1D). Previous studies on human glioma have demonstrated that the tumoral cells often express proteins typically found in neural stem or astroglial progenitor cells (sox2 and nestin) (Almqvist et al., 2002, Schmitz et al., 2007, Zhang et al., 2008, Gangemi et al., 2009, McCord et al., 2009). To determine whether the GFAP-immunoreactive cells in pre-chiasmatic optic nerves and chiasm of Nf1+/ − GFAP CKO mice were astrocytoma cells, we employed nestin and sox2 double labeling immunohistochemistry. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The purpose of this study was to investigate the progression of changes in retinal ganglion cells and optic nerve glia in neurofibromatosis-1 (NF1) genetically-engineered mice with optic glioma. Optic glioma tumors were generated in Nf1+/- mice lacking Nf1 expression in GFAP+ cells (astrocytes). Standard immunohistochemistry methods were employed to identify astrocytes (GFAP, S100beta), proliferating progenitor cells (sox2, nestin), microglia (Iba1), endothelial cells (CD31) and retinal ganglion cell (RGC) axons (Neurofilament 68k) in Nf1+/-, Nf1(GFAP)CKO (wild-type mice with Nf1 loss in glial cells), and Nf1+/-(GFAP)CKO (Nf1+/- mice with Nf1 loss in glial cells) mice. Ultrastructural changes in the optic chiasm and nerve were assessed by electron microscopy (EM). RGC were counted in whole retina preparations using high-resolution, mosaic confocal microscopy following their delineation by retrograde FluoroGold labeling. We found that only Nf1+/-(GFAP)CKO mice exhibited gross pre-chiasmatic optic nerve and chiasm enlargements containing aggregated GFAP+/nestin+ and S100beta+/sox2+ cells (neoplastic glia) as well as increased numbers of blood vessels and microglia. Optic gliomas in Nf1+/-(GFAP)CKO mice contained axon fiber irregularities and multilamellar bodies of degenerated myelin. EM and EM tomographic analyses showed increased glial disorganization, disoriented axonal projections, profiles of degenerating myelin and structural alterations at nodes of Ranvier. Lastly, we found reduced RGC numbers in Nf1+/-(GFAP)CKO mice, supporting a model in which the combination of optic nerve Nf1 heterozygosity and glial cell Nf1 loss results in disrupted axonal-glial relationships, subsequently culminating in the degeneration of optic nerve axons and loss of their parent RGC neurons.
    Neuroscience 09/2010; 170(1):178-88. DOI:10.1016/j.neuroscience.2010.06.017 · 3.33 Impact Factor
Show more