CD133, CD15/SSEA-1, CD34 or side populations do not resume tumor-initiating properties of long-term cultured cancer stem cells from human malignant glio-neuronal tumors
- Cristina Patru1, 9,
- Luciana Romao1, 2,
- Pascale Varlet1, 3,
- Laure Coulombel1,
- Eric Raponi4,
- Josette Cadusseau5,
- François Renault-Mihara1,
- Cécile Thirant1,
- Nadine Leonard1, 3,
- Alain Berhneim6,
- Maria Mihalescu-Maingot1,
- Jacques Haiech7,
- Ivan Bièche8,
- Vivaldo Moura-Neto2,
- Catherine Daumas-Duport1, 3,
- Marie-Pierre Junier1, 3 and
- Hervé Chneiweiss1Email author
© Patru et al; licensee BioMed Central Ltd. 2010
Received: 29 July 2009
Accepted: 24 February 2010
Published: 24 February 2010
Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.
We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas.
A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide.
Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.
Tumor initiating cells (TICs) from various types of cancers have been isolated and characterized. The tumors of origin range from glioblastomas and medulloblastomas [1–6] to epithelial tumors of the breast , lung , colon , and prostate . Gliomas represent the majority of primary tumors from the central nervous system (CNS) . Difficulties in clinical management (e.g. treatment and prognosis) are related to the complex identity of gliomas, which lack reliable morphological and molecular signatures, precluding thus the establishment of a clear cut classification discriminating between different tumor subtypes .
Historically, it has been proposed that gliomas (astrocytomas and oligodendrogliomas) originate respectively from mature astrocytes or oligodendrocytes. The fact that these brain tumors frequently include a mixture of cells expressing neuronal and glial markers, has recently led to the alternative proposal that gliomas arise from neural stem/progenitor cells. Support for this hypothesis comes from mouse models in which changes in the expression of oncogenes or tumor suppressors lead to experimental tumors . Neural progenitor cells are, for example, more sensitive than differentiated astrocytes to the oncogenic effects of combined over-activation of Ras and Akt signaling pathways . It should however be kept in mind that glioblastomas, the most malignant form of gliomas, can be generated in mice by retroviral transduction of oncogenes into mature glial cells [14–16]. In good agreement, the conversion of mature astrocytes toward neural progenitors induced by TGFα , a growth factor overexpressed early in the development of human gliomas sensitizes them to cancerous transformation . The isolation from human glioblastoma biopsies of malignant cells that express markers of neural stem cells supports the existence of tumor stem cells within gliomas [1–3, 6]. Most importantly, some of these cells exhibit the true properties of tumor initiating cells (TICs), including the ability to give rise to a tumor identical to the one observed in the patient upon orthotopic grafting in mouse brains [1, 3, 6]. It remains, however, unknown whether these TICs might help to discriminate between glioma sub-types. Moreover, the design of specific therapies awaits the identification of the molecular pathways presiding over the maintenance of the properties of these tumor stem cells.
Here, we sought for tumor stem-like cells in 47 human adult malignant glial tumors. We identified a subset of glial tumors that contain at high frequency of cells generating long-term self-renewing floating spheres in vitro, and novel tumors in immunodeficient mice. This subset corresponds to malignant glio-neuronal tumors (MGNTs) . MGNTs are World Health Organization grade III and IV tumors that always present numerous glial fibrillary acidic protein (GFAP)- and a few neurofilament protein (NFP)-positive tumor cells. The other neuronal markers tested (NeuN, synaptophysin, and chromogranin) are inconstantly expressed. Distinction of MGNTs from other malignant gliomas is of clinical importance since gross total surgical resection of these tumors is the major prognostic factor predicting long-term survival . Flow cytometry and 2D-SDS-PAGE analyses showed stable and common proteomic profiles of MGNT-derived tumor initiating cells growing as floating spheres. These cells are highly resistant to temozolomide and thus represent a novel tool to screen for more efficient therapies.
All of the samples were classified according to World Health Organization guidelines (grade II, III or IV for gliomas), and the classification of Sainte Anne Hospital (low grade oligodendroglioma or type A, high grade oligodendroglioma or type B, glioblastomas and malignant glio-neuronal tumors, . The biopsies were collected by a pathologist in the surgical room from July 2002 to July 2005. All patients were 18 years old or older, had signed a written agreement for participation to the research project after having being informed of the goals, potential interest of the research and methods. This biomedical research was conducted according to the declaration of Helsinki, to the French laws, and was approved by the institutional review board of Ste Anne Hospital, Paris. Anatomopathological diagnosis classified tumors as glioblastoma multiformis (n = 6), Malignant Glio-Neuronal Tumors (MGNTs, n = 15), medulloblastoma (n = 2), ganglioglioma (n = 1), oligodendroglioma (n = 23). Immunolabeling of formalin-fixed, paraffin-embedded tissue sections was performed as previously described .
Viable fragments were transferred to a beaker containing 0.25% trypsin in 0.1 mM EDTA (4:1), and slowly stirred at 37°C for 30-60 min. Dissociated cells were plated in 75 cm2 tissue culture flasks plated at 2500-5000 cells/cm2 in Dulbecco's modified Eagle's: F-12 medium (1:1) containing the N2, G5 (containing FGF and EGF) and B27 supplements (all from Invitrogen, France). After 2 to 47 days in culture, spheres bloomed from clusters of adherent cells. They were dissociated in single-cell suspension each week with a renewal of two third of their culture medium.
Characterization of sphere-forming cells
Dissociated sphere-derived cells were plated in 18-mm diameter wells in 1 ml volumes at a density of 200 000 cells/well. Cell proliferation/viability was evaluated using the WST1 kit from Roche according to the manufacturer's protocol. We verified that the results were the same as those obtained by counting the absolute number of viable cells using trypan blue exclusion. Clonality was evaluated in 96-well plates seeded at a cell density of 1-200 cells/well/0.1 ml. The number of wells containing at least one secondary sphere was evaluated after 3-4 weeks of culture.
Flow cytometry analysis of cell surface antigens
Single-cell suspensions were incubated with fluorochrome-conjugated monoclonal antibodies: fluorescein (FITC)-coupled CD15, FITC-CD11b, FITC-CD45, phycoerythrin (PE)-coupled CD34, PE-Thy-1 (CD90), PE-CD133 and phycoerythrin cyanin 5 (PC5)-coupled CD34, PC5-CD56, and allophycocyanin (APC)-coupled CD133. CD133 antibodies were from Miltenyi (France), all others from Beckman-Coulter. Dead cells were excluded from the analysis by 7-AAD. Acquisition was performed on a Facscalibur (Becton Dickinson) and viable cells were analyzed with Cellquest software.
Assessment of the neural differentiation potential
Cells were plated at a density of 5.000 cells/cm2 onto polyornithine-coated glass coverslips and onto Lab-Tek™ II Chamber Slide™ System (Nalge Nunc International) http://www.nuncbrand.com/page.aspx?ID=234 in the presence of FCS or B27 (Invitrogen) for 2 to 14 days. Multiple immunofluorescence assays for neural antigens were performed as previously described [17, 20].
The theoretical and practical aspects of real-time quantitative RT-PCR using the ABI Prism 7900 Sequence Detection System (Perkin-Elmer Applied Biosystems) have been described in detail elsewhere . Briefly, total RNA is reverse-transcribed before real-time PCR amplification. Quantitative values are obtained from the threshold cycle (Ct) number at which the increase in the signal associated with exponential growth of PCR products begins to be detected using PE Biosystems analysis software, according to the manufacturer's manuals. We also quantified transcripts of TBP gene, which encodes the TATA box-binding protein as the endogenous RNA control, and each sample was normalized on the basis of its TBP content.
Results, expressed as N-fold differences in target gene expression relative to the TBP gene, termed Ntarget, were determined by the formula: Ntarget = 2ΔCt sample , where ΔCt value of the sample was determined by subtracting the Ct value of the target gene from the Ct value of the TBP gene.
The Ntarget values of the samples were subsequently normalized to a "basal mRNA level", i.e. normalized to the smallest amount of target gene mRNA detectable and quantifiable by real-time quantitative RT-PCR assays (target gene Ct value = 35; Ntarget value = 1).
The nucleotide sequences of primers for TBP and the 4 target genes are available on request. The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min and 50 cycles at 95°C for 15 s and 65°C for 1 min. Experiments were performed with duplicates for each data point.
Chromosome Analysis of neurosphere-derived cells
Cells were treated with medium containing 10 μg/ml Colchicine for 1 to 2 hours, and resuspended in hypotonic 1% sodium citrate at room temperature for 30 minutes. The cells were then washed in a methanol-acetic acid (3:1, v/v) fixative solution for 30 minutes, and spread onto clean dry slides. Q-banding staining was then performed, and 10 metaphases were analyzed for each sample.
Cells were washed three times with PBS for 10 min with gentle shaking, prior to lyses in buffer containing 8 M urea, 4% CHAPS and 40 mM Tris. The protein pellets were dissolved in isoelectric focusing buffer and quantified using the Micro BCA protein assay kit (Pierce). First-dimension isoelectric focusing was performed with the Ettan IPGphor system (Amersham Biosciences) at 20°C with a maximum current setting of 50 mA/strip. Immobiline DryStrip gels (IPG stripes, Amersham Biosciences) with a pH gradient of 3-10 or 4-7 and a length of 13 cm were rehydrated in 250 μl sample solution, containing 80 μg proteins. Prior to SDS-PAGE analysis, IPG stripes were equilibrated in second-dimension equilibration buffer (6 M urea, 2% SDS, 50 mM Tris-HCl, pH 8.8, 30% glycerol) containing 1% DTT (Sigma) to reduce the disulfide bonds of the proteins. The second dimension was carried out using 12.5% gradient polyacrylamide gels at constant 20 mA current per gel. Comparison of the protein maps was achieved using the PD-Quest software according to the manufacturer instructions (BioRad).
Selection and expansion of cell-forming spheres from adult human brain tumors
In order to preserve tissue architecture and improve sample handling as well as the versatility of the tests that may be applied, we developed a two-step strategy for the 49 surgical samples of our survey. Tumor samples were first sliced in the surgical room in fragments smaller than 1 mm3, and placed on surgical sponges bathed with culture medium. Tumor fragments were either dissociated within 2 hours after surgery (28 out of 49, 58%), or incubated in organotypic conditions for a maximal time of 2 weeks prior dissociation. Tumor fragments cultured on surgical sponges are viable and conserve the tumor architecture for up to 6 weeks (additional file 1). In both cases, the dissociated cells were plated at low density (<5 × 103 cells/cm2) in a serum-free medium containing EGF and FGF2 (G5 supplement).
Summary of the brain tumor cultures. See text for details.
Number of cases
Number of cultures containing floating cellular spheres
Self-renewal length ≤ 6 months
Self-renewal length > 6 months
These data extended previous reports of absence of EGF/FGF2-responsive clonogenic precursors in oligodendrogliomas [2–4, 22]. They revealed the unique ability of MGNTs to yield cellular spheres that could be amplified for very long periods.
MGNT contain cells endowed with long-term self-renewal and clonal properties
Clonal properties of MGNT derived glioma stem cells
28.9 ± 8
9 ± 3
15.5 ± 7.0
7 ± 1
15.6 ± 5.0
7 ± 2
MGNT-derived cells forming spheres exhibit a neural progenitor phenotype in vitro
When bFGF and EGF were removed and replaced by serum, most but not all spheres underwent changes characteristic of differentiation: they adhered rapidly to the plastic substrate, the cells flattened and acquired a fusiform shape. Cellular heterogeneity both between spheres and within each sphere was maintained in these conditions, as shown by immunocytochemical analysis (Figure 1E-G and 1I). When treated with 0.5% FCS for 14 days, all cells exhibited nuclear Olig2-immunolabeling (Figure 1G and 1I). In the presence of B27, a cocktail designed to promote neuron survival and development in cultures of normal nervous tissue, 30% of the cells within the spheres acquired the appearance of neuron-like cells with a rich neurite-like branching, and ß3-tubulin expression (Figure 1H and 1I). Interestingly, a fraction of the differentiated cells co-expressed neuronal and glial markers as in the original tumor (Figure 1F and 1I).
Cell surface phenotype and the functional state of sphere-derived cells
Phenotypic characterization of cell-forming spheres.
% of wells containing spheres
Number of sphere/well
11/11 = 100%
15 ± 2.2 (day 21)
9/13 = 70%
5.9 ± 2.6 (day 21)
14/14 = 100%
22 ± 4.1 (day 36)
12/15 = 80%
20 ± 3.6 (day 36)
10/10 = 100%
18.6 ± 0.5 (day 36)
10/10 = 100%
19.1 ± 0.2 (day 36)
Expression of stemness markers by MGNT-derived cells forming spheres.
MALDI MS/MS characterization of proteins overexpressed in MGNT-derived cells forming spheres.
(P09455) Retinol-binding protein I
(P30626) Sorcin (22 kDa protein) (CP-22) (V19)
(P09211) Glutathione S-transferase P (EC 126.96.36.199)
(P30041) Peroxiredoxin 6 (EC 188.8.131.52)
(P30084) Enoyl-CoA hydratase
(P04792) Heat-shock protein beta-1 (HspB1)
(O60664) Mannose-6-phosphate receptor binding protein 1
(Q96E67) ACTB protein (Fragment)
Altogether, these results demonstrate that cells forming cells derived from MGNT exhibit a common and stable surface antigen and proteomic profile, as well as some features of stem/progenitor cells in long-term cultures.
MGNT-derived spheres contain tumor-initiating cells
Karyotype analysis and in vivo behavior upon orthotopic grafting were performed in order to determine whether the cell-forming spheres derived from MGNTs were indeed tumoral and behaved as tumor-initiating cells (TICs).
Taken altogether these data demonstrate that long-term cultured MGNT-derived floating spheres contain tumor cells with neural stem/progenitor phenotype, thus fulfilling the criteria of tumor initiating cells (TICs).
TICs resist to high concentrations of temozolomide
TICs-derived from MGNTs are resistant to Temozolomide
TMZ 62,5 μM
TMZ 250 μM
TMZ 500 μM
TMZ 1000 μM
95 ± 7
94 ± 4
95 ± 5
67 ± 4
95 ± 5
87 ± 5
83 ± 4
66 ± 6
96 ± 5
87 ± 5
78 ± 7
58 ± 7
98 ± 6
97 ± 5
80 ± 6
59 ± 4
90 ± 5
80 ± 7
75 ± 5
62 ± 6
92 ± 5
81 ± 7
74 ± 5
67 ± 8
91 ± 6
75 ± 6
71 ± 5
60 ± 6
TG1 without GF
92 ± 6
85 ± 6
78 ± 6
62 ± 7
TG1 10% SVF
90 ± 5
80 ± 5
82 ± 6
79 ± 5
Tumor stem cells or tumor-initiating cells with stem-like properties (TICs) name a small subpopulation of tumor cells that are clonogenic and are capable of forming a tumor mass mimicking the original one. The present work reports on the characterization of tumor initiating cells from 12 cases of a newly characterized form of human adult glioma. These cells exhibit long-term stability in culture, and properties that support their capacity to establish a novel brain tumor from a few cells at least up to two years of continuous passages.
Within the array of brain tumors studied in this work, only MGNTs  yielded at a high frequency cells forming spheres in culture (12 of 15 tumors). In contrast, cells proliferating as spheres from glioblastomas, adult medulloblastoma and ganglioglioma were rapidly lost upon serial passages after 4 to 6 months in accordance with previous studies [3, 4]. Most studies of glioblastomas have identified tumor stem cells in only half or less of the glioblastomas studied [3, 4]. Our results raise the possibility that some of the glioblastomas assayed in these studies belong to the MGNT sub-class. Review of 200 glioblastomas showing that 50% of them contain cells expressing NFP and GFAP supports such a possibility (PV and CDD, unpublished observations). The presence of a few tumor cells co-expressing neuronal and glial antigens is one MGNT characteristic, which can be easily looked for . In addition, our large set of cultured oligodendrogliomas (WHO grade II or III) never yielded any sphere and/or cells expressing a stem-like phenotype. In good agreement with this, no long-term culture from human oligodendroglioma has yet been reported. Determination of the presence or absence of tumor stem cells is, however, insufficient in the present state of knowledge to conclude that MGNTs, medulloblastomas and some glioblastomas result from the targeting of transforming mutations to an early progenitor/stem-like cell, whereas oligodendrogliomas result from mutations accumulating in a more differentiated cell.
CD133 expression, because expressed also by normal neural stem cells, has been proposed to identify cells at the top of the hierarchy formed by tumor stem cells and their more differentiated progeny, and to be therefore the paramount of glioma stem cells. Its pertinence as a glioma stem cell marker is now highly controversial, several groups having demonstrated the tumor initiating properties of CD133- cells . In our hands, CD133 was rapidly down-regulated in cell spheres grown in vitro, whereas expression of other stem cell markers such as CD15 or CD34 was maintained. CD133- cells were also able to form floating cellular spheres with properties undistinguishable from those of CD133+ cells. Similarly, long-term cultured sphere forming cells, whatever established from CD133+ or CD133- tumor cells were equally resistant to temozolomide. CD133 expression most likely reflects the bioenergetics stress of the cells rather than their stem-like properties . CD15, also known as SSEA1 or Lewis X, has also been recently proposed as an enrichment marker of glioma TICs . Although we observed some differences in sphere sizes and proliferation rates between CD15 positive and negative cells during the first week after sorting, these variations disappeared with obtaining the secondary spheres, which in both cases contained a mixture of CD15+ and CD15- cells.
We previously reported that MGNTs represent a clinical entity with distinct clinical, anatomo-pathological and radiological behaviors. We now show that they have also a distinct biological behavior among high-grade glial tumors. The glioma initiating cells described here establish a novel model that may be used to routinely establish adult human tumoral cell lines stable in long-term cultures in a define medium in a reproducible fashion. This may open new ways to identify novel tumor cell markers and surface receptor profiles for therapeutic and diagnostic purposes and to develop patient-tailored pharmacologic approaches for the cure of gliomas.
List of abbreviations used
tumor initiating cells
Malignant glio-neuronal tumors
We thank Dr. Alain Klause for his expert technical assistance for karyotype analysis, Jocelyne Cordier for help to maintain cell cultures, Jean-Philippe Deloulme for help in grafting, Edith Demettre and Philippe Marin for their help in proteomic analysis. This work was supported by Inserm and the following grants: ARC 3952 (HC), Ligue National contre le Cancer (La Ligue 2007, HC), Cancéropole (INCa/Région Ile de France) (CT), Cancer Stem Cell Network Ile de France (HC, MPJ).
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