Abstract
The Repressor Element 1 Silencing Transcription factor (REST/NRSF) is a master repressor of neuronal programs in non-neuronal lineages shown to function as a central regulator of developmental programs and stem cell physiology. Aberrant REST function has been associated with a number of pathological conditions. In cancer biology, REST has been shown to play a tumor suppressor activity in epithelial cancers but an oncogenic role in brain childhood malignancies such as neuroblastoma and medulloblastoma. Here we examined REST expression in human glioblastoma multiforme (GBM) specimens and its role in GBM cells carrying self-renewal and tumorigenic competence. We found REST to be expressed in GBM specimens, its presence being particularly enriched in tumor cells in the perivascular compartment. Significantly, REST is highly expressed in self-renewing tumorigenic-competent GBM cells and its knock down strongly reduces their self-renewal in vitro and tumor-initiating capacity in vivo and affects levels of miR-124 and its downstream targets. These results indicate that REST contributes to GBM maintenance by affecting its self-renewing and tumorigenic cellular component and that, hence, a better understanding of these circuitries in these cells might lead to new exploitable therapeutic targets.
Introduction
Glioblastoma multiforme (GBM) represents the most frequent and malignant cerebral neoplasia. Recent research has reported the presence of a population of cells defined as Glioblastoma Stem Cells (GSCs) because of their tumorigenic potential and similarities to the Neural Stem cells (NSCs) (i.e. in vitro growth conditions, self-renewal, expression of stem cell markers and differentiative, although aberrant, multipotency) [1], [2], [3]. These cellular elements have been suggested to display high chemo- and radio-resistance features, so that cells escaping surgical removal can regenerate the tumor despite adjuvant therapies. The elucidation of the molecular circuitries that control their self-renewal and tumorigenic competence can therefore likely foster the development of new targeted therapeutic approaches.
The RE1-silencing transcription factor (REST) [also known as neuron-restrictive silencer factor (NRSF)] has been characterized as a master repressor of neuronal programmes in non-neuronal tissues [4], [5], [6], [7]. REST binds to a highly conserved 21–23 bp DNA sequence called RE1 (repressor element 1) located in many neuronal genes and silences their transcription by recruiting specific co-repressors multicomplexes [8]. REST targets include a large number of genes that encode for neuron-specific proteins. Genome- and transcriptome-wide analyses focused on the RE-1 sequence have revealed a REST-regulated network with over 1000 genes and several microRNAs in humans [9], [10], [11]. REST has been shown to coordinate neural induction and neuronal differentiation programs during both in vivo and in vitro neurodevelopment [12], [13], [14], [15]. Of note interfering with REST levels leads to altered NSCs differentiation and aberrant REST function has been associated with neurological disorders [16], [17], [18], [19]. In human epithelial cells REST has been described as a potent suppressor of malignant transformation [20] and its deregulation has been associated with several non-neural tumors including breast and small cell lung cancers [21], [22]. However, oncogenic roles for REST have been observed in neuroblastoma [23] and medulloblastoma [24]. To date, although a certain level of REST amplification was reported in 36% of GBM specimens [25] and a study indicated expression of REST in GBM cells [26], whether REST levels and functions play critical roles in GBM yet represents a critical issue. Here, we report that REST is expressed in human GBM specimens and that its downregulation strongly impairs self-renewal and tumor-initiating capacity of GBM cells. These results indicate that REST plays critical functions in human GBM.
Results and Discussion
REST Expression is Elevated in Human GBM
To investigate if REST plays a functional role in GBM, we first evaluated, by quantitative real-time PCR assay, REST expression levels in 15 tumor specimens collected from independent primary GBMs patients. In all samples, REST is expressed and upregulated from two- to five-fold as compared to control mRNA obtained from pooled adult normal cerebral cortex tissues (Figure 1A). Interrogation of the National Cancer Institute’s Repository for Molecular Brain Neoplasia Data (REMBRANDT) [27] also indicated a 45% average increase of REST expression in gliomas compared to control (non-tumor) tissue (Figure S1A). As elevated levels of mRNA may reflect an increase in gene copy number, we sought to determine whether genomic changes in REST occurred in glioma patients. Copy number REMBRANDT analysis for REST indicated a 3-fold or greater amplification of the 4q12 chromosomal region which correlated with a slightly worse survival, while a deletion is strongly associated to a better survival (Figure S1B). To confirm the high REST expression in GBM tissues and to extend the analysis to protein level and REST cellular distribution, we performed an immunohistochemistry screening on 96 paraffin-embedded primary human brain tumor surgical biopsy specimens (Table 1). A detailed mapping of REST positive cells in diverse brain tumors has not been reported in the literature. In the control specimens, REST immunoreactivity was detected in the cytoplasm of selected neurons in the cerebral cortex and in dentate gyrus of hippocampus (Figure 1B). Glial cells diffusely showed a nuclear staining (Figure 1C). In neuroepithelial and non-neuronal tumors, REST immunoreactivity variably marked different cell populations, although with variable degrees both in terms of intensity and percentage of positive cells (Table 1; Figure S2A-F). In general, gliomas exhibited a nuclear staining which intensity increased with anaplasia (Figure 1D and E; Figure S2). REST was detected in all GBM specimens (the number of positive tumor cells differed between individual cases, ranging from 10 to 75%; Table 1) in phenotypic areas characterized by high cell density, small and hyperchromatic nuclei (Figure 1F and G). Immunoreactivity appeared as a strong nuclear-restricted staining, particularly intense in the perivascular districts (Figure 1H), areas known to be characterized by the highest malignancy and which also show high expression of Sox2 (Figure 1I) and nestin (not shown). Interrogation of the Human Protein ATLAS database (www.proteinatlas.org/) confirmed the same REST immunoreactive signal and cellular distribution, obtained with different antibodies with respect to the one here employed.
Table 1. Intensity and distribution of REST expression and demographic data (male:female ratio, mean age, age range) of the tumor specimens analysed in this study.
Tumor diagnosis | Total cases | Male: female ratio | Mean age at diagnosis (years) | Age range(years) | REST+veCells (%) | Intensity |
Glioblastoma Multiforme | 60 | 37∶23 | 62 | 23–81 | 10–75% | ++/+++ |
Diffuse astrocytoma | 3 | 2∶1 | 47 | 38–56 | 10–20% | + |
Anaplastic astrocytoma | 6 | 4∶6 | 66 | 51–75 | 30–50% | + |
Pilocytic astrocytoma | 6 | 2∶4 | 45 | 19–65 | 100% | ++ |
Oligodendroglioma | 10 | 5∶5 | 55 | 45–67 | 10–20% | + |
Anaplastic oligodendroglioma | 7 | 4∶3 | 55 | 31–80 | 30–40% | +/++ |
Medulloblastoma | 3 | 2∶1 | 35 | 32–39 | 10–50% | +/++ |
Central neurocytoma | 1 | Female | 22 | / | positive | + |
Normal nervous tissue | 2 | 1∶1 | 46 | 43–49 | / | / |
Elevated REST Expression is Maintained in Human Tumorigenic-competent GBM Cells
Since previous studies suggested that the perivascular region is enriched for cellular elements defined as Glioblastoma Stem Cells (GSCs) because of their tumorigenic potential and some similarities to the Neural Stem cells (NSCs) [28], we assayed REST expression levels in self-renewing and tumorigenic competent cell populations that we have established from primary GBMs. Quantitative real-time PCR assay confirmed high level of REST mRNA expression, comparable to those observed in normal human fetal brain-derived NSCs and consistently higher compared to adult normal (non-pathological) brain tissue, in all the analyzed GBM-derived cells cultured in mitogen-supplemented Serum Free (SFM) conditions typically employed for growing NSCs (Figure 2A), both as adherent monolayer [29] (here named “GB cells”; for a characterization of GB cells see Figures S3 and S4) or as neurospheres. Immunocytochemistry confirmed the presence of REST nuclear signal in GB cells (Figure 2B). Noteworthy, a strong dotted staining could be observed in the nuclei, likely corresponding to nuclear foci, where co-expression of REST and telomere repeat factor 2 (TRF-2) has been previously reported [26].
REST Knockdown Reduces Self-renewal Potential of Human Tumorigenic-competent GBM Cells and Induces Neuronal Differentiation and Cell Death Programs
To uncover the functional role for REST in tumorigenic-competent GBM-derived cells, we assessed its ability to control self-renewal and neuronal differentiation, both programs known to be regulated by REST in normal NSCs [12], [13], [14], [15], [30], [31], [32]. REST targeting in GB cells by using lentiviral transduced short hairpin RNAs (shREST), resulted in a 75% silencing rate at the mRNA level (Figure S5A and B) and approx. 90% at the protein level (Figure S5C and D). In these cultures, REST targeting induced a marked morphological change in the cells, exhibiting a flatter appearance with respect to mock infected cells (CTRL) and cultures receiving non-targeting shRNA (NT shRNA) (Figure 3A). Furthermore, REST downregulation impaired self-renewal capacity, resulting in the inability to stably propagate shREST GB cells (Figure 3B and Figure S6). Similar effects were observed when performing a neurosphere assay on malignant GBM cells growing in suspension. In this assay, shREST cultures exhibited a decrease both in spheres’ number and size with respect to CTRL and NT shRNA cultures (Figure S7A-C). Antigenic analysis of the shREST cultures indicated a reduction of the number of nestin positive cells (CTRL: 90.9% ±12.4; NT shRNA: 91.9% ±9.7; shREST: 29.1% ±7.3; P<0.001) (Figure 3C and D). Of note, although the number of Olig2 positive cells did not substantially vary upon REST knockdown (P<0.05), we did observe a significant effect on the subcellular localization of Olig2 in nestin negative cells, where it exhibited a diffuse cytoplasmic and nuclear distribution, different from the typical nuclear-restricted expression (see arrows in Figure 3C). Concurrently, REST targeting induced a dramatic increase in the number of β3-tubulin positive cells (CTRL: 5.7% ±0.3; NT shRNA: 4.1% ±0.4; shREST: 76.0% ±9.5; P<0.001) (Figure 3C and D), as expected since it is among the REST-regulated genes, accompanied by a four-fold and ten-fold reduction of GFAP positive and phospho-H3 positive cells, respectively (P<0.001) (Figure 3C and D). Together these results indicated that REST knockdown induces cell-cycle arrest, impairs self-renewal and instructs the cells toward a neuronal fate. Similar results were obtained in three independent GB cell lines and in normal human NSCs and were further strengthened in GB cell cultures exposed to differentiating conditions (Figure S8). We also found that REST de-repression in GB cells stimulated an apoptotic program as shown by increased active-Caspase 3 immunoreactivity in REST-targeted cultures both in self-renewal (Figure 3C and D) and in differentiating conditions (Figure S8). To test the specificity of REST targeting, we transfected GB cells with REST-specific (siREST) or non-targeting (NT siRNA) siRNAs (Figure S9A and B). siREST transfection caused a relaxation of REST transcriptional repression activity on BDNF and SNAP25, two well-characterized REST-controlled genes (Figure S9C). Furthermore, siREST transfection resulted in increased apoptosis, as determined with Annexin V staining (CTRL: 36.3% ±3.5; NT siRNA: 38.4% ±4.7; siREST: 65.1% ±6.3; P<0.001) (Figure 3E) and luminometric assay for Caspases 3/7 activation (P<0.001) (Figure 3F).
REST Knockdown Abolishes Tumor Formation by Human Tumorigenic-competent GBM Cells Xenograft and Impairs in vivo Tumor Growth of Established Xenograft Tumors
According to the in vitro requirements for REST in the survival and self-renewal capacity of GB cells demonstrated so far, it would be expected that delivery of REST-specific shRNA could affect their tumorigenic capability in vivo. To address this issue, GB cells were transduced with shREST or NT shRNA followed by a 3 days of puromycin selection to completely eliminate non transduced cells and afterwards cells were transplanted into the brains of immunocompromised SCID mice. Animals bearing shREST GB cells displayed no tumor formation and complete survival when compared to the ones receiving NT shRNA GB cells or not transduced GB cells (CTRL), up to 6 months post-transplantation, the latest time point we have examined (P<0.001) (Figure 4A). Histological analyses confirmed that NT shRNA transduced GB cells developed into tumors which invaded the brains, whereas no tumor developed in the brains receiving shREST transduced GB cells (Figure S10). To further confirm this result, mock transfected cells (CTRL) or NT siRNA or siREST transfected GB cells were heterotopically injected in immunocompromised SCID mice and tumor formation and growth was monitored. Within 12 days post-injection CTRL and NT siRNA injected animals developed tumor masses whose volume progressively increased with time, while in the same period no tumors could be detected in animals injected with REST targeted cells up to 50 days (P<0.001) (Figure 4B). However, these animals showed a delayed formation of tumors (80 days after the injection), likely because of the presence of unsuccessfully targeted cells. We conclude that targeting REST in GB cells is sufficient to impair their in vivo tumorigenic competence.
This was further verified by directly interfering with REST activity in vivo. For this purpose, NT shRNA or shREST lentiviral particles were directly delivered in GB-derived heterotopic tumors. Prior injection of lentiviral particles carrying eGFP reporter was used to determine the feasibility of the intratumoral delivery procedure and to set up the appropriate lentiviral injection dose (Figure S11A). Two intratumoral injections in seven days interval were performed in order to target a wider tumor area. In all experiments, animals receiving NT shRNA treatments exhibited the typical progressive growth of the tumor masses, whereas mice receiving shREST particles displayed a partial but significant reduction of the tumor growth rate, already after 10 days (day 50 after cell injection in Figure 4C) from the first lentiviral particles injection (P<0.001). Analysis of these tumor samples indicated the presence of areas exhibiting β3-tubulin and active Caspase 3 immunoreactivities, likely corresponding to the sites of shREST lentiviral particles injection (Figure S11B). Collectively, these data indicate that direct REST inhibition in vivo can interfere with growth of malignant GBM cell-derived xenograft tumors by activation of apoptosis and neuronal differentiation programs.
REST Controls SCP1 and PTPN12 Phosphatases Expression Levels in Human Tumorigenic-competent GBM Cells by Modulating miR-124 Levels
Taken together, our study suggests that self-renewal, growth and survival of GBM-derived GB cells are critically dependent on REST activity and that inhibition of REST-regulated pathways might disclose potentially exploitable strategies towards GBM treatment in human. Nevertheless, our study reports the presence of REST positive cells in diverse gliomas and defines a role for REST as a positive regulator of self-renewal in tumorigenic-competent GBM-derived cells. Noteworthy, analyses of REST transcript in relation to the TCGA subgroups does not provide any hint about REST mRNA levels in relation to the TCGA subgroups. This is in agreement with very recent findings reporting that analysis of REST mRNA levels cannot be used to strictly create such a correlation due to a not linear stoichiometric relation between REST mRNA and protein levels [33]. Analysis of protein levels and distribution is extremely important in the case of REST as its post-translational modifications have been shown to regulate cell maintenance [30], [31]. Interfering with mechanisms regulating REST post-translational modifications have been shown to affect NSCs differentiation and cellular transformation in non-neural epithelial cells [20], [31]. In particular, the net balance between the β-TrCP-mediated ubiquitylation and the HAUSP-mediated deubiquitylation has been shown to control REST protein levels and determine cellular fate in mouse and human NSCs [30]. Of note, it has been reported that the association of TRF-2 with REST also prevents the ubiquitin proteasome-mediated degradation of REST [34], [35]. Consistently, our interrogation of the Human Protein ATLAS database indicates strong expression of HAUSP and TRF2 and, conversely, reduced level of expression of β-TrCP in malignant glioma specimens with respect to non-pathologic brain tissue (not shown).
On the whole, our results support the hypothesis that in neural tissue, differently from non-neural compartments, REST favours the self-renewal and tumorigenic competence of tumor cells. The fact that REST can be either permissive or inhibitory for tumor formation, depending on tumor’s types, points to a context-specific modulation of REST-controlled pathways. miRNAs are crucial organizers of tissue-specificity [36] and REST has been shown to control the expression of different miRNAs [11], [37]. Among them, miR-124 represents the most differentially expressed miRNA between GBM and normal brain being poorly expressed in GBM [38], [39]. Of note, miR-124 levels directly correlated with patients’ survival [39]. miR-124 expression increases during NSC differentiation [40], and over-expression of miR-124 in putative GBM-derived stem cells has been shown to induce a dramatic increase in neuronal differentiation markers accompanied by reduced self-renewal and tumorigenicity [38]. We thus assayed miR-124 expression levels in normal and REST-depleted GB cells. Quantitative real-time PCR assay showed low miR-124 levels in CTRL and NT shRNA cultures, but a tenfold increase in shREST cultures (Figure 5A). We also found that the increase of miR-124 levels in REST-depleted GB cells resulted in a substantial reduction of SCP-1 and PTPN12 levels (Figure 5B), two phosphatases whose expression has been previously demonstrated to be controlled by a REST-miR-124 circuitry [41], [42]. Importantly, SCP-1 and PTPN12 play crucial roles in suppression of differentiative and oncogenic programs [42], respectively. These results indicated the conservation of REST inhibitory effect on miR-124 in GBM in human and we can speculate that one mechanism by which REST sustains self-renewal and tumorigenic competence of isolated GB cells might be through the repression of miR-124 expression (and deregulation of miR-124 targets) (Figure 6).
Our data define REST as an important mediator of GBM biology by demonstrating that REST is involved in GBM maintenance through regulation of the self-renewal, survival and differentiation of malignant GBM cells, and suggest that REST-regulated molecular circuitries may potentially turn into sensible targets for therapeutical intervention in GBM.
Materials and Methods
Ethics Statement
All the human tissues used in this study were collected only when necessary as part of treatment. Only samples in excess of what was needed for pathological assessment were used for research. Written informed consent to utilize excess tissue for research was obtained from each patient, and de-identified tissues, to protect anonymity, were used.
GB cell lines described in this manuscript were generated from tumor samples obtained during planned neurosurgical procedures for resection of newly diagnosed glioma at the Neurosurgical unit of the IRCCS Policlinico S. Matteo, Pavia, Italy. Surgery was performed according to the recommendation of the institutional review boards and in full agreement with the principles of the Declaration of Helsinki. For the use of the tumor samples for the generation of GB cell lines a written informed consent was obtained from each patient and the study approved by The Ethics Committees of the IRCCS Policlinico S. Matteo, Pavia, Italy. Cancer cell lines G144, G166 and G179, as well as the human NS (hNS; CB 541 and CB 192 cell lines) cells, were donated by Prof. Austin Smith (University of Cambridge, UK) who reported their generation and characterization elsewhere [29], [43]. GBM neurosphere line (NSGBnR1) were donated to our lab by Dr. Gaetano Finocchiaro (Istituto Neurologico C. Besta, Italy); these cells have been previously generated and described by the lab of Dr. Gaetano Finocchiaro [44]. Tumor specimens used for the immunohistochemistry analyses were collected in Italy at the Center of Policlinico di Monza Foundation and approval was obtained from the Institutional Ethics Board and in in full agreement with the principles of the Declaration of Helsinki.
All animal procedures were carried out in strict accordance with the legislation and guidelines for animal care and handling in Italy (DL116 92) approved by the Italian Ministry of Health, and those from the European Union (2003/65/CE from the European Parliament and Council July 2003). The protocols were approved by the Bioethical Committee of the University of Milan and by the Committee on the Ethics of Animal Experiments of the Fondazione IRCCS Istituto Nazionale dei Tumori. All experiments were designed to minimize the numbers of animals used and their discomfort.
Tumor Samples and Cell Culture
The GB cell lines were established from fresh GBM surgical specimens collected directly from the operating theatre in PBS plus 0,6% glucose and immediately transported on ice to the cell culture room and processed and cultured as monolayer cultures as previously described [29]. Briefly, fresh tumor samples or xenografts were manually dissociated into single cells by enzymatic digestion with Accutase (Sigma) for 15–20 min at 37°C. Tumor cells suspension was then seeded into a fresh laminin-coated plastic plate. GB cells and control foetal human Neural Stem cells (hNS cells) were derived and grown in self renewal serum-free medium (SFM) containing EGF and FGF-2 (20 ng/ml; Peprotech), as previously described [29], [43]. Culture vessels were coated with 5 µg/ml Laminin (Invitrogen) for 3 hr prior to use. The phenotype of these cultures was confirmed by analysis of stem cell markers expression, in vitro assays of self-renewal, differentiation potential and in vivo tumor formation and propagation. In our analysis were included also other GB lines (G166 and G179 cells) and GBM neurospheres (NSGBnR1) previously described in other studies [29], [44]. For differentiation, single-cell suspensions of GB cells and normal hNS cells were seeded on Laminin-coated 48-well plates at 1.2×104 cells/well density in proliferation medium and subjected to lentiviral transduction [29], [43]. 24 hours after infection, the cells were shifted in the same basal media deprived of both EGF and FGF-2 (growth factor withdrawal condition) and supplemented with a double amount of B27 supplement (4%) for 15 days. Half of the medium was replaced every 2–3 days along the differentiation period. GB7 and GB8 cell lines have been deposited in the cell repository at Biorep (Milan, Italy).
REST Targeting in vitro
Knockdown of REST was achieved by means of lentiviral vector–mediated short hairpin RNA (shRNA) interference, using validated commercial lentiviral particles for the NRSF sequence (Santa Cruz Biotechnology), or through small interference RNA (siRNA), by employing oligonucleotides targeting human REST transcript sequence (Invitrogen). For the infection, lentiviral particles were gently diluted (1∶70) in proliferation medium supplemented with 5 µg/ml polybrene. Sub-confluent cultures were exposed to viral particles for 18–24 hours and then restored to classic compete medium. Knockdown efficiency was determined by immunocytochemistry, real time PCR and Western Blot analysis on GB infected cells. Control nontargeting shRNA (NT shRNA) Lentiviral Particles and cop GFP Control Lentiviral Particles (Santa Cruz Biotechnology) were used as controls. The latters were also used to monitor the transduction efficiency in vitro and in vivo. For REST- siRNA knockdown three different siRNAs, directed against different portion of human REST transcript, were characterized for their knockdown efficiency by real time PCR analysis on GB cells transfected with each REST targeting siRNAs or with universal negative scrambled controls (Invitrogen). The most efficient siRNA was used for all further experiments. Nucleofection technology (Amaxa, Lonza) was used to transfect the cells. For each nucleofection 4–5×106 cells and 100pmole of siRNA were used following the protocol according to the manufacturer’s instruction. 50pmole of pMAX plasmid were used as control.
Cell Assays
For colorimetric MTT-based cell viability assay cells were plated in triplicate into 24 well plate at a density of 2×104 cells/well in self-renewal medium. At each time point considered, cells were exposed to 3-[4.5-dimethylthiazol-2-phenyl]-2.5-diphenyl-tertrazolium bromide (1 mg/ml) for 1 hour at 37°C and formazan release was quantified at 560 nm using a Microplate Reader (Biotek). For Active Caspase 3 analysis, 4×106 cells were transfected with control and specific siRNA as indicated above and after 24 hours 1.5×104 of nucleofected cells were plated in triplicate into 96 well plate in proliferation medium. Degree of apoptosis was assayed using Caspase-Glo® 3 Assay (Promega) on a Veritas™ Microplate Luminometer (Promega) according to the manufacturer’s protocol. For Annexin V staining, we used the Annexin V Apoptosis Detection Kit (BD Pharmingen™). Cells were nucleofected with NT siRNA and specific anti REST siRNA as indicated in the previous section and stained with Annexin V at different time points post nucleofection. The cells stained with Annexin V and relative controls were analyzed by cytofluorimetry with at least 10,000 events/determination, using FACSCantoII and DiVA software for acquisition and analysis (BD Pharmingen™).
Mouse Xenograft Assay
Xenotransplantation experiments were performed on SCID mice (6–8 weeks old; Charles River Laboratories) housed in pathogen-free conditions. For s.c. inoculation SCID mice were anesthetized with i.m. ketamine and xylazine and a suspension 1∶1 of 0.5×106 cells and Matrigel was injected bilaterally (200 µl/injection). Mice were monitored until the appearance of a mass and were sacrificed by CO2 asphyxiation when the tumors were in the exponential phase of growth. The explanted tumors were mechanically dissected into 2 mm fragments used for in vitro serial cell lines derivation and immunohistochemistry analysis. For in vivo REST knockdown analysis 107 cells/mouse of viable GB cells non transfected and transfected with NT siRNA or the selected REST targeting siRNA were transplanted (48 hours after transfection procedure) into mice through s.c. injection following the same protocol described above. At least three SCID mice, receiving a bilateral s.c. cell inoculation for each experimental condition were used. For lentiviral-mediated REST shRNA treatment, GB cells were first s.c. injected to establish glioma xenografts as described above and the same quantity of NT lentiviral particles or REST targeting lentiviral particles were delivered to tumor sites through intratumoral injection. A second virus injection was performed one week after the first treatment. The lentiviral injection dose was defined by delivery of cop GFP Control Lentiviral Particles; 80 µl lentiviral particles in 300 µl PBS (i.e. 1.2–1.5×106 viral infectious units was chosen as injection dose. For intracranial graft, SCID mice were anesthetized with i.m. ketamine and xylazine. Thereafter, animals received a stereotaxic injection of 1.5 µl (105 cells/µl) of GB cells suspension in the left corpus striatum. Animals were monitored for 6 months for disease symptoms and were sacrificed when they showed weight loss or any other sign of disease.
Statistical Analysis
Statistical significance was assayed using a Student’s unpaired t test or one-way ANOVA analysis of variance using the GraphPad Prism software. P<0.05 was considered statistically significant.
Supporting Information
Acknowledgments
We thank Giovanna Altobelli for technical assistance with tissue immunohistochemical procedures and Sara Brignani and Manuela Marullo for assistance with quantitative Real-Time experiments. We thank Prof. Austin Smith (University of Cambridge, UK) and Dr. Steve Pollard (UCL Cancer Institute, UK) for providing G144, G166 and G179 cell lines and Dr. Gaetano Finocchiaro (Istituto Neurologico C. Besta, Italy) for providing the NSCGBnR1 neurosphere line.
Footnotes
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by NeuroScreen (FP6, European Union Health-2007-B-222943) and Progetto Giovani Ricercatori (Italian Ministry of Health) to Luciano Conti and by Progetto Oncologico “Characterization of the molecular mechanisms regulating brain tumors growth and chemioresistance” (Italian Ministry of Health, 2008-2011) and partially by NeuroStemcell (European Community’s Seventh Framework Programme grant agreement nr.222943) to EC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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