Epidermal Growth Factor Plays a Crucial Role in Mitogenic Regulation of Human Brain Tumor Stem Cells
2008; Elsevier BV; Volume: 283; Issue: 16 Linguagem: Inglês
10.1074/jbc.m704205200
ISSN1083-351X
AutoresAkio Soeda, Akihito Inagaki, Naoki Oka, Yuka Ikegame, Hitomi Aoki, Shinichi Yoshimura, Shigeru Nakashima, Takahiro Kunisada, Toru Iwama,
Tópico(s)Microtubule and mitosis dynamics
ResumoA cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells. A cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells. The biology of neural stem cells and their intrinsic properties are now recognized as integral to brain tumorigenesis (1Pardal R. Clarke M.F. Morrison S.J. Nat. Rev. Cancer. 2003; 3: 895-902Crossref PubMed Google Scholar, 2Reya T. Morrison S.J. Clarke M.F. Weissman I.L. Nature. 2001; 414: 105-111Crossref PubMed Scopus (8001) Google Scholar, 3Sanai N. Alvarez-Buylla A. Berger M.S. N. Engl. J. Med. 2005; 353: 811-822Crossref PubMed Scopus (826) Google Scholar). Evidence is presented that brain tumors contain a cancer stem cell (CSC) 2The abbreviations used are: CSC, cancer stem cell; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; EGFRvIII, constitutively active EGFR mutant; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; HGF, hepatocyte growth factor; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; GFAP, glial fibrillary acidic protein; mAb, monoclonal antibody; NOD-SCID, nonobesity diabetic-severe combined immunodeficient. population, capable of self-renewal and multilineage differentiation, which recapitulates the phenotype of the original tumor (4Galli R. Binda E. Orfanelli U. Cipelletti B. Gritti A. de Vitis S. Fiocco R. Foroni C. Dimeco F. Vescovi A. Cancer Res. 2004; 64: 7011-7021Crossref PubMed Scopus (2099) Google Scholar, 5Hemmati H.D. Nakano I. Lazareff J.A. Masterman-Smith M. Geschwind D.H. Bronner-Fraser M. 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Maruyama H. Hara A. Kunisada T. Mori H. Iwama T. Biochem. Biophys. Res. Commun. 2007; 360: 553-559Crossref PubMed Scopus (122) Google Scholar, 12Inagaki A. Soeda A. Oka N. Kitajima H. Nakagawa J. Motohashi T. Kunisada T. Iwama T. Biochem. Biophys. Res. Commun. 2007; 361: 586-592Crossref PubMed Scopus (49) Google Scholar). The concept of CSCs is based on the similarities between stem cells and cancer cells with respect to their self-renewal capacity and multipotential cell fate (13Bonnet D. Dick J.E. Nat. Med. 1997; 3: 730-737Crossref PubMed Scopus (5595) Google Scholar). Since CSCs are thought to play an important role in tumorigenesis and tumor recurrence, effective cancer treatments should be targeted at eliminating this population. The CD133 has been identified as a marker of neural stem cells in the adult central nervous system as well as of brain CSCs (7Singh S.K. Clarke I.D. Terasaki M. Bonn V.E. Hawkins C. Squire J. Dirks P.B. 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A combination of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) promotes their proliferation and differentiation (16Doetsch F. Petreanu L. Caille I. Garcia-Verdugo J.M. Alvarez-Buylla A. Neuron. 2002; 36: 1021-1034Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar, 17Gritti A. Frolichsthal-Schoeller P. Galli R. Parati E.A. Cova L. Pagano S.F. Bjornson C.R. Vescovi A.L. J. Neurosci. 1999; 19: 3287-3297Crossref PubMed Google Scholar, 18Palmer T.D. Markakis E.A. Willhoite A.R. Safar F. Gage F.H. J. Neurosci. 1999; 19: 8487-8497Crossref PubMed Google Scholar). Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are also classified as neurotrophic factors, which play essential roles in the development, maintenance, and regeneration of the central nervous system (19Andrae J. Hansson I. Afink G.B. Nister M. Mol. Cell Neurosci. 2001; 17: 1001-1013Crossref PubMed Scopus (32) Google Scholar, 20Jin K. Zhu Y. Sun Y. Mao X.O. 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However, it remains unknown which of these growth factors play(s) a pivotal role in the proliferation and differentiation of CSCs. In the present study, we have established CSC lines from three brain tumor patients and examined the growth factor dependence of these CSCs. The data obtained indicate that among the growth factors (EGF, FGF-2, PDGF, VEGF, and HGF) tested, only EGF promoted sphere formation and enhanced the self-renewal capacities of CSCs, including the CD133-positive subpopulations. The ability of tyrosine kinase inhibitors selective for EGF receptor (EGFR) kinase to block CSC self-renewal was also tested. The tyrosine kinase inhibitors gefitinib (ZD1839, Iressa; AstraZeneca) and AG1478 quite efficiently inhibited EGF-dependent sphere formation. Gefitinib also significantly decreased the CD133 populations by flow cytometric analysis. Autophosphorylation of EGFR was inhibited by gefitinib in a concentration-dependent manner. Among EGFR downstream signaling molecules (25Woodburn J.R. Pharmacol. Ther. 1999; 82: 241-250Crossref PubMed Scopus (786) Google Scholar, 26Kari C. Chan T.O. de Quadros M.R. Rodeck U. Cancer Res. 2003; 63: 1-5PubMed Google Scholar), phosphorylation of Akt kinase and extracellular signal-regulated kinase 1/2 (ERK1/2), but not STAT3, was suppressed by gefitinib. Blockage of both ERK and Akt pathways by selective inhibitors, PD98059 and LY294002, respectively, resulted in considerable inhibition of EGF-dependent sphere formation. These findings indicate that EGF signaling cascade is essential for the maintenance of brain tumor stem cells. Primary Sphere Formation—Prior informed consent was obtained from all three sample donors. Our study was approved by the Medical Review Board of Gifu University. Tumor sphere cultures were performed as described previously with some modifications in medium containing Dulbecco's modified Eagle's medium/F-12 (Invitrogen), penicillin G, streptomycin sulfate, B-27 (Invitrogen), recombinant human FGF-2 (20 ng/ml; R&D Systems, Minneapolis, MN), recombinant human EGF (20 ng/ml; R&D Systems), and leukemia inhibitory factor (1000 units/ml) (6Ignatova T.N. Kukekov V.G. Laywell E.D. Suslov O.N. Vrionis F.D. Steindler D.A. Glia. 2002; 39: 193-206Crossref PubMed Scopus (786) Google Scholar, 7Singh S.K. Clarke I.D. Terasaki M. Bonn V.E. Hawkins C. Squire J. Dirks P.B. Cancer Res. 2003; 63: 5821-5828PubMed Google Scholar, 11Oka N. Soeda A. Inagaki A. Onodera M. Maruyama H. Hara A. Kunisada T. Mori H. Iwama T. Biochem. Biophys. Res. Commun. 2007; 360: 553-559Crossref PubMed Scopus (122) Google Scholar, 12Inagaki A. Soeda A. Oka N. Kitajima H. Nakagawa J. Motohashi T. Kunisada T. Iwama T. Biochem. Biophys. Res. Commun. 2007; 361: 586-592Crossref PubMed Scopus (49) Google Scholar). Limiting Dilution Assay—Sphere cells were dissociated and seeded in 96-well culture plates in 0.2 ml of medium (7Singh S.K. Clarke I.D. Terasaki M. Bonn V.E. Hawkins C. Squire J. Dirks P.B. Cancer Res. 2003; 63: 5821-5828PubMed Google Scholar, 12Inagaki A. Soeda A. Oka N. Kitajima H. Nakagawa J. Motohashi T. Kunisada T. Iwama T. Biochem. Biophys. Res. Commun. 2007; 361: 586-592Crossref PubMed Scopus (49) Google Scholar). The final cell dilutions ranged from 1 to 1000 cells/well. Cultures were fed 20 μl of medium every 2 days until day 7. The percentage of wells not containing spheres for each cell plating density was calculated and plotted against the number of cells/well. Immunofluorescent Staining—Immunocytochemistry of tumor spheres and differentiated spheres were performed as described (11Oka N. Soeda A. Inagaki A. Onodera M. Maruyama H. Hara A. Kunisada T. Mori H. Iwama T. Biochem. Biophys. Res. Commun. 2007; 360: 553-559Crossref PubMed Scopus (122) Google Scholar, 12Inagaki A. Soeda A. Oka N. Kitajima H. Nakagawa J. Motohashi T. Kunisada T. Iwama T. Biochem. Biophys. Res. Commun. 2007; 361: 586-592Crossref PubMed Scopus (49) Google Scholar). Antibodies used were as follows: anti-EGFR (mouse monoclonal antibody (mAb), 1:100; Upstate, Temecula, CA), anti-phospho-EGFR (mouse mAb, 1:200; Upstate), human anti-nestin (rabbit polyclonal antibody, 1:200; Chemicon, Temecula, CA) and CD133 (mouse mAb, 1:10; Miltenyi Biotec, Auburn, CA) for the detection of neural stem and progenitor cells, anti-βIII-tubulin (Tuj1) (mouse mAb, 1:200; Chemicon) for neurons, anti-glial fibrillary acidic protein (GFAP; rabbit polyclonal antibody, 1:500; DAKO, Glostrup, Denmark) for astrocytes, anti-galactocerebroside (mouse mAb, 1:200; Chemicon) for oligodendrocytes, and anti-cleaved caspase-3 (rabbit polyclonal antibody, 1:500; Cell Signaling Technology, Beverly, MA) for the apoptosis assay. Visualization was performed with Alexa fluorophore-conjugated secondary antibodies (1:1,000; Molecular Probes, Inc., Eugene, OR). Cells were simultaneously stained with Hoechst 33342 for identifying nuclei. Transplantation into Immunodeficient Mice—Our experimental procedures involving animals followed the guidelines of the Animal Experimental Committee of Gifu University. Tumorigenicity was determined by injecting brain tumor-derived CSCs orthotopically into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. After 8-12 weeks in primary culture, 2 μl of a cell suspension (1 × 108 cells/ml) in proliferation medium was injected stereotactically into the right striatum (0.2 μl/min) of anesthetized NOD-SCID mice using a Hamilton syringe. The injection coordinates were 3 mm to the right of the midline and 2 mm anterior to the coronal suture at a depth of 3 mm. The mice were sacrificed at 4-14 weeks postinjection depending on the injected cell line (11Oka N. Soeda A. Inagaki A. Onodera M. Maruyama H. Hara A. Kunisada T. Mori H. Iwama T. Biochem. Biophys. Res. Commun. 2007; 360: 553-559Crossref PubMed Scopus (122) Google Scholar, 12Inagaki A. Soeda A. Oka N. Kitajima H. Nakagawa J. Motohashi T. Kunisada T. Iwama T. Biochem. Biophys. Res. Commun. 2007; 361: 586-592Crossref PubMed Scopus (49) Google Scholar). Histochemical Analysis of Brain Tissues—Tumor samples were fixed with 4% paraformaldehyde. 4-μm sections were cut from paraffinized tissue blocks. They were mounted on silanized slides, dried, deparaffinized with xylene, hydrated with decreasing concentrations of ethanol, and washed with phosphate-buffered saline. For hematoxylin-eosin staining, slides were first stained with Harris hematoxylin (2 min) and then counterstained with alcoholic eosin. For immunohistochemical studies, endogenous peroxidase was neutralized with 3% H2O2 in methanol (15 min). The sections were stained with the Histofine mouse stain kit (Nichirei, Tokyo, Japan) and then with primary antibodies: anti-human nestin (mouse mAb, 5 μg/ml; R&D Systems) for neural stem cells, anti-human Ki-67 (mouse mAb, 1:50; DAKO) for proliferation indices, anti-GFAP (mouse mAb, 1:500; DAKO) for astrocytes, and anti-human βIII-tubulin (mouse mAb, 1:500; Chemicon) for neurons. After treatment with the secondary antibody and MAX-PO (Nichirei), color reactions were performed with peroxidase-substrate 3,3′-diaminobenzidine (DAKO). All tissue sections were counterstained with Mayer's hematoxylin. Growth Factor Stimulation—Cultures were initiated in medium containing a combination of EGF and FGF-2 (4Galli R. Binda E. Orfanelli U. Cipelletti B. Gritti A. de Vitis S. Fiocco R. Foroni C. Dimeco F. Vescovi A. Cancer Res. 2004; 64: 7011-7021Crossref PubMed Scopus (2099) Google Scholar, 5Hemmati H.D. Nakano I. Lazareff J.A. Masterman-Smith M. Geschwind D.H. Bronner-Fraser M. Kornblum H.I. 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Then the tumor spheres were washed and dissociated into single cells. They were transferred to medium containing human recombinant FGF-2, EGF, PDGF, VEGF, or HGF alone (all factors were added at 20 ng/ml) and cultured for 7-14 days. Upon sphere formation, we assessed the effects of the growth factors by plating 500 cells/well in 96-well plates and adding various concentrations of a growth factor. After sphere formation, the percentage of wells containing spheres was calculated. Reverse Transcription-PCR—Total RNA was purified from cultures, using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. cDNA was prepared from RNA templates (5 μg) using oligo(dT) primers and Super Script II reverse transcriptase (Invitrogen). The cDNA product (1 μg) was subjected to PCR with rTaq polymerase (TaKaRa Shuzo Co., Ltd., Tokyo, Japan). Each PCR product was electrophoresed on 1% agarose gels and stained with ethidium bromide. Forward and reverse primer sequences for the specific amplification of EGFR and a constitutively active EGFR mutant (EGFRvIII) were 5′-CTT CGG GGA GCA GCG ATG CGA C-3′/5′-ACC AAT ACC TAT TCC GTT ACA C-3′. These primers generate a 1044-bp PCR product for the wild-type EGFR transcript compared with a 243-bp PCR product for the EGFRvIII transcript (27Mellinghoff I.K. Wang M.Y. Vivanco I. Haas-Kogan D.A. Zhu S. Dia E.Q. Lu K.V. Yoshimoto K. Huang J.H. Chute D.J. Riggs B.L. Horvath S. Liau L.M. Cavenee W.K. Rao P.N. Beroukhim R. Peck T.C. Lee J.C. Sellers W.R. Stokoe D. Prados M. Cloughesy T.F. Sawyers C.L. Mischel P.S. N. Engl. J. Med. 2005; 353: 2012-2024Crossref PubMed Scopus (1242) Google Scholar). The sequences of the gene-specific primers (sense and antisense) were 5′-GCA CCA CAC CTT CTA CAA TGA GC-3′/5′-TTG AAG GTA GTT TCG TGG ATG CC-3′ for β-actin. The amplification conditions were 94 °C for 2 min, followed by 42 cycles of denaturation at 95 °C for 30 s, annealing at 56.5 °C for 30 s, and extension at 68 °C for 80 s for (EGFR/EGFRvIII) and 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 60 s for β-actin. Antiproliferative Assays—AG1478 (EMD Biosciences, San Diego, CA), a highly selective inhibitor of EGFR tyrosine kinase, was dissolved in Me2SO to obtain final concentrations of 0.01, 0.1, 1.0, and 10.0 μm. The tyrosine kinase inhibitor gefitinib, a synthetic anilinoquinazoline that targets EGFR, was acquired by dissolving Iressa tablets (250 mg; AstraZeneca, Wilmington, DE) in the above concentrations of Me2SO. For antiproliferative assays, the tumor spheres were washed and dissociated into single cells with phosphate-buffered saline. They were plated at 500 cells/well in 96-well plates in the medium containing Me2SO, AG1478, or gefitinib (0-10.0 μm) in the presence of EGF (20 ng/ml). After sphere formation, the percentage of wells containing spheres was calculated. For functional analysis of EGFR downstream signals, a selective inhibitor of the ERK pathway, PD98059 (28Janmaat M.L. Kruyt F.A.E. Rodfiguez J.A. Giaccone G. Clin. Cancer Res. 2003; 9: 2316-2326PubMed Google Scholar) (EMD Biosciences), and LY294002 (29Gibso S. Tu S. Oyer R. Anderson S.M. Johnson G.L. J. Biol. Chem. 1999; 274: 17612-17618Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) (EMD Biosciences) for the phosphatidylinositol 3-kinase (PI3K)/Akt pathway were used. 1000 cells were plated in 24-well plates in the medium containing EGF (20 ng/ml) in the absence or presence of PD98059 (10 μm) and/or LY294002 (10 μm). The number of formed spheres was counted. Western Blotting—Western blot analysis was performed essentially as described previously (30Yoshimura S. Sakai H. Nakashima S. Nozawa Y. Shinoda J. Sakai N. Yamada H. Mol. Brain Res. 1997; 45: 90-98Crossref PubMed Scopus (35) Google Scholar). Inhibition of EGFR signaling by increasing concentrations of gefitinib was assessed by Western blotting for detecting EGFR autophosphorylation with antibodies against EGFR (BD Biosciences, San Jose, CA) and phospho-EGFR (phospho-Tyr1068; Cell Signaling Technology). The activation of EGFR downstream signaling molecules was determined by detecting their phosphorylation. The following antibodies were used: Akt (Cell Signaling Technology), phospho-Akt (phospho-Ser473; Cell Signaling Technology), STAT3 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), phospho-STAT3 (phospho-Ser727; Santa Cruz Biotechnology), ERK1/2 (Cell Signaling Technology), and phospho-ERK1/2 (phospho-Thr202/Tyr204; Cell Signaling Technology). Tumor spheres were lysed in lysis buffer consisting of 20 mm Tris-HCl (pH 7.4, 150 mm NaCl, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). After brief sonication, lysates were clarified by centrifugation at 12,000 × g for 10 min at 4 °C, and protein content in the supernatant was measured according to the Bradford method. An aliquot (30-50 μg of protein/lane) of total protein was separated by 7.5% SDS-polyacrylamide gel electrophoresis and blotted to nitrocellulose transfer membranes (0.2 μm; Amersham Biosciences). The membrane was blocked with 5% nonfat dry milk in TBS-T (20 mm Tris-HCl, pH 7.6, 137 mm NaCl, and 0.01% Tween 20) for 1 h at room temperature, followed by incubation with the appropriate primary antibodies overnight at 4 °C. After extensive washing with TBS-T, the membrane was further incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:1,000) in TBS-T containing 5% nonfat dry milk for 1 h at room temperature. Detection was performed using enhanced chemilumi-nescence reagent (Amersham Biosciences) according to the manufacturer's protocol. Flow Cytometry—To assess the effects of the EGF and gefitinib on the population of the CD133-positive cells within tumor spheres, 1 × 106 cells were placed in proliferation medium containing growth factors. Different EGF and gefitinib concentrations were added every 2 days; control cells were grown in medium containing an equal concentration of Me2SO. After 7 days, aliquots of CD133-positive and -negative cells were evaluated by flow cytometry with a fluorescence-activated cell sorter Aria (BD Biosciences), using anti-CD133/2 (293C3)-allophycocyanin-conjugated antibody (mouse mAb; Miltenyi Biotec) according to the manufacturer's recommendation. All experiments were performed in triplicate. Cell Colony Formation Assay—Overall survival of the CD133-positive cells in response to a tyrosine kinase inhibitor, gefitinib, was assessed by colony formation. In a colony formation assay, CD133-positive cells dissociated by a fluorescence-activated cell sorter were seeded (5 × 104) in 6-well dishes in medium containing 10% fetal bovine serum with or without gefitinib and cultured for 5 days. Cell colonies were fixed and stained with 0.04% Crystal Violet in 4% paraformaldehyde solution. Statistical Analysis—Parametric comparisons used analysis of variance. The analyses of variance were followed by Tukey's honestly significant difference adjustment for multiple comparisons. Isolation of Tumorigenic CSCs from Human Gliomas—We analyzed fresh samples from three human brain tumors (two glioblastomas (X01GB and X02GB) and one anaplastic oligoas-trocytoma (X03AOA)) (12Inagaki A. Soeda A. Oka N. Kitajima H. Nakagawa J. Motohashi T. Kunisada T. Iwama T. Biochem. Biophys. Res. Commun. 2007; 361: 586-592Crossref PubMed Scopus (49) Google Scholar). Dissociated tumor cells, mostly in single-cell suspension, formed neuronal spherelike aggregates within 7 days of culture. The sphere-forming cells resembled previously reported cells from tumor-derived spheres (Fig. 1A) (4Galli R. Binda E. Orfanelli U. Cipelletti B. Gritti A. de Vitis S. Fiocco R. Foroni C. Dimeco F. Vescovi A. Cancer Res. 2004; 64: 7011-7021Crossref PubMed Scopus (2099) Google Scholar, 5Hemmati H.D. Nakano I. Lazareff J.A. Masterman-Smith M. Geschwind D.H. Bronner-Fraser M. 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The spheres from three CSC lines continuously proliferated, and all lines could be maintained for more than 100 weeks by refeeding fresh medium twice a week (Fig. 2A). X03AOA cells manifested higher proliferative capacity than X01GB and X02GB cells derived from high grade gliomas. The proliferative activity of these cells increased during the late subculture stages compared with earlier passages (data not shown). This phenomenon was previously reported by Galli et al. (4Galli R. Binda E. Orfanelli U. Cipelletti B. Gritti A. de Vitis S. Fiocco R. Foroni C. Dimeco F. Vescovi A. Cancer Res. 2004; 64: 7011-7021Crossref PubMed Scopus (2099) Google Scholar). Limiting dilution analysis revealed that the number of cells required to form at least 1 tumor sphere/well was significantly less in X03AOA compared with both X01GB and X02GB (Fig. 2B), indicating that X03AOA had the highest clonogenic sphere-forming capacity. X03AOA cells displayed higher proliferative and self-renewal capacity than X01GB and X02GB cells.FIGURE 2Proliferation of cultured brain tumor stem cells. A, time-dependent sphere formation of three brain tumor stem cell lines, X01GB, X02GB, and X03AOA. Cells were cultured in medium containing FGF-2 (20 ng/ml), EGF (20 ng/ml), and leukemia inhibitory factor (1000 units/ml). B, limiting dilution analysis. Sphere cells were dissociated and plated in 96-well culture dishes. The final cell dilutions ranged from 1 to 1000 cells/well. The percentage of wells not containing spheres for each cell plating density was calculated and plotted against the number of cells/well. Data are from representative experiments repeated at least five times.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cells grown in proliferation medium formed spheres. Cells in spheres were immunoreactive with the neural progenitor markers, nestin (31Lendahl U. Zimmerman L.B. McKay R.D. 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