gene signatures associated with mouse postnatal hindbrain ......may 24, 2012 · 2643-4626; fax:...

Gene Signatures Associated with MousePostnatal Hindbrain Neural Stem Cellsand Medulloblastoma Cancer Stem CellsIdentify Novel Molecular Mediators and Predict Human Medulloblastoma Molecular Classifi cationDaniela Corno1, Mauro Pala6, Manuela Cominelli7, Barbara Cipelletti1, Ketty Leto8, Laura Croci3, Valeria Barili3, Federico Brandalise9, Raffaella Melzi4, Alessandra Di Gregorio8, Lucia Sergi Sergi2, Letterio Salvatore Politi5, Lorenzo Piemonti4, Alessandro Bulfone6, Paola Rossi9, Ferdinando Rossi8, Gian Giacomo Consalez3, Pietro Luigi Poliani7, and Rossella Galli1

RESEARCH ARTICLE

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Published OnlineFirst May 3, 2012; DOI: 10.1158/2159-8290.CD-11-0199





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JUNE 2012�CANCER DISCOVERY | OF2

Authors’ Affi liations: 1Neural Stem Cell Biology Unit and 2Telethon Insti-tute for Gene Therapy, Division of Regenerative Medicine, Stem Cells & Gene Therapy; 3Division of Neuroscience; 4San Raffaele Diabetes Research Institute (HSR-DRI), Division of Immunology, Transplantation and Infec-tious Disease; 5Neuroradiology Unit and CERMAC, San Raffaele Scientifi c Institute, Milan; 6Biofl ag, Ltd, Scientifi c and Technologic Park of Sardinia Polaris, Pula (Cagliari); 7Department of Pathology, University of Brescia, Spedali Civili of Brescia, Brescia; 8Neuroscience Institute of Turin (NIT), Department of Neuroscience and Neuroscience Institute of the Cavalieri-Ottolenghi Foundation (NICO), University of Turin, Orbassano (Turin); and 9Department of Physiology, University of Pavia, Pavia, ItalyNote: Supplementary data for this article are available at Cancer Discov-ery Online (http://cancerdiscovery.aacrjournals.org/).M. Pala and M. Cominelli contributed equally to the work.Corresponding Author: Rossella Galli, Neural Stem Cell Biology Unit, Divi-sion of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Scientifi c Institute, Via Olgettina 58, 20132 Milan, Italy. Phone: 39-02-2643-4626; Fax: 39-02-2643-4621; E-mail: [email protected]: 10.1158/2159-8290.CD-11-0199©2012 American Association for Cancer Research.

INTRODUCTION

Medulloblastoma is the most common malignant brain tumor of childhood. Surgery alone is not suffi cient to eradi-cate this tumor, and both radiotherapy and chemotherapy are considered ineffi cient treatments, mostly due to the high vulnerability of young patients to the toxic effects of antimitotic treatments. The restricted incidence of medul-loblastoma during childhood relates to the peculiar ontog-eny of the cerebellum, which begins early during embryonic development, reaching its fi nal maturation only after birth

ABSTRACT Medulloblastoma arises from mutations occurring in stem/progenitor cells located in restricted hindbrain territories. Here we report that the mouse post-

natal ventricular zone lining the IV ventricle also harbors bona fi de stem cells that, remarkably, share the same molecular profi le with cerebellar white matter–derived neural stem cells (NSC). To identify novel molecular mediators involved in medulloblastomagenesis, we compared these distinct postnatal hindbrain-derived NSC populations, which are potentially tumor initiating, with murine compound Ptch/p53 mutant medulloblastoma cancer stem cells (CSC) that faithfully pheno-copy the different variants of human medulloblastoma in vivo. Transcriptome analysis of both hind-brain NSCs and medulloblastoma CSCs resulted in the generation of well-defi ned gene signatures, each reminiscent of a specifi c human medulloblastoma molecular subclass. Most interestingly, medulloblastoma CSCs upregulated developmentally related genes, such as Ebfs, that were shown to be highly expressed in human medulloblastomas and play a pivotal role in experimental medullo-blastomagenesis. These data indicate that gene expression analysis of medulloblastoma CSCs holds great promise not only for understanding functional differences between distinct CSC populations but also for identifying meaningful signatures that might stratify medulloblastoma patients beyond histopathologic staging.

SIGNIFICANCE: The functional and molecular comparison between the cell progenitor lineages from which medulloblastoma is thought to arise and medulloblastoma CSCs might lead to the identifi cation of novel, potentially relevant mediators of medulloblastomagenesis. Our fi ndings provide a rationale for the exploitation of mouse CSCs as a valuable preclinical model for human medulloblastoma, both for the defi nition of CSC-associated gene signatures with predictive mean and for the identifi cation of therapeutically targetable genes. Cancer Discov; 2(6); 1–15. ©2012 AACR.

(1). This delayed maturation results in a prolonged formative postnatal period, characterized by increased susceptibility to tumor formation. Whereas the majority of patients are diagnosed with medulloblastoma at a very early age, sug-gesting that medulloblastomas initiate from mutations occurring during embryonic development in hindbrain ger-minative regions (2, 3), 20% of medulloblastoma patients develop medulloblastomas in late adolescence or early adult-hood, indicating that medulloblastoma might also arise postnatally.

Different subsets of neural precursors persist postnatally within the hindbrain and, as such, are potentially involved in medulloblastomagenesis. In the mouse, the best character-ized progenitor population comprises the so-called granule cell progenitors (GCP) of the external granular layer (EGL), which are characterized by a peak of proliferation at postna-tal day (P) 7, followed by progressive decline and exhaustion within the third postnatal week. GCPs proliferate robustly in vivo and in vitro in response to Sonic Hedgehog (Shh), and their deregulated postnatal proliferation has been causatively implicated in the development of the desmoplastic medul-loblastoma variant, characterized by alterations in the Shh pathway (2). However, GCPs do not satisfy the requirements for stemness, as they are characterized by limited self-renewal and lack of multipotency. On the contrary, the other popula-tion of transiently neurogenic progenitors identifi ed within the cerebellum white matter during the same postnatal win-dow of EGL GCPs does contain bona fi de neural stem cells (NSC) that can be cultured in vitro in the presence of epi-dermal growth factor (EGF) and fi broblast growth factor-2




Corno et al.RESEARCH ARTICLE

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(FGF-2) and display typical features of NSCs, such as self-renewal and in vitro/in vivo multipotency (4–6).

Of note, a third subset of hindbrain neural progenitors has been retrieved in proximity of the IV ventricle (IVv), around which the cerebellum proper develops. IVv-derived progenitors are endowed with persistent neurogenic activity throughout adulthood (7, 8). However, the establishment of long-term self-renewing NSC lines from the IVv has never been reported. Interestingly, oncogenic transformation of progenitors located in the dorsal brainstem, as IVv NSCs are, has been associated with the development of a subtype of the “classic” medulloblastoma variant, whose main molecular feature is activation of the WNT pathway (3).

Cancer stem cells (CSC) are responsible for tumor growth and maintenance and, being resistant to standard therapies, are likely involved in tumor relapse. CSCs can be isolated from brain tumors and cultured in vitro long term in the presence of EGF and FGF-2. By this method, putative CSC lines have been established from desmoplastic medulloblas-tomas developing in Ptch+/− mice (9), alone or in combination with p53 loss (10). Medulloblastoma CSCs, characterized by expression of the CSC marker CD15 (9, 11), display the essen-tial requirement for a CSC, that is, the capability to generate new tumors, which closely mimic the human disease in terms of in vivo growth and phenotype.

To identify novel molecular determinants to be pursued as diagnostic markers and/or therapeutic targets in medullob-lastoma CSCs, we set out to establish long-term expanding postnatal NSC lines from the IVv as well as from the cerebel-lum white matter and to compare them with medulloblas-toma CSCs. IVv NSCs were capable of differentiating into the 3 main lineages of the central nervous system (CNS) both in vitro and in vivo, thus fulfi lling the requirements to qualify as bona fi de NSCs. By applying the same culture conditions to mouse medulloblastomas derived from Ptch+/− mutants with or without the concurrent loss of p53, we isolated sev-eral medulloblastoma CSC lines endowed with self-renewal, multipotency, and different tumorigenic potential. When subjected to microarray-based gene expression profi ling, nor-mal cerebellum white matter and IVv-derived SCs as well as medulloblastoma CSCs were characterized by distinctive gene signatures that hold predictive potential when tested against human data sets as previously shown for other stem cell types (12, 13). Most importantly, some of the genes upregu-lated in medulloblastoma CSCs were also expressed in human medulloblastoma and play a relevant role in the control of medulloblastomagenesis, thus validating medulloblastoma CSCs as a valuable preclinical model for medulloblastoma.

RESULTS

Long-term Expanding NSCs Can Be Isolated from the Postnatal Ventricular Zone of the IV Ventricle

The ventricular zone lining the IVv contains mitotically active cells (Fig. 1A) whose proliferation can be reactivated in vivo by infusion of mitogens (14). To assess whether IVv NSCs might give rise to long-term expanding NSC lines, we cultured the IVv region from the mouse hindbrain at P7, P14, P21, and P30 (Fig. 1B) according to the NeuroSphere

Assay. Cerebellum white matter and the subventricular zone lining the 2 lateral ventricles of the forebrain were used as controls.

At P14, P21, and P30, cell lines could be generated only from IVv and subventricular zone, confi rming that sus-tained proliferation of cerebellum white matter cells ceases by the fi rst postnatal week (5). However, at P7, long-term expanding cell lines could be established from all 3 regions; therefore, we analyzed NSCs at this time point. Cells were isolated from wild-type (wt), Ptch+/−p53+/+ and Ptch+/−p53+/− mice. Cerebellum white matter and IVv NSCs gave rise to a much lower frequency of primary neurospheres than cells from the subventricular zone, independently of genotype (Fig. 1B). Clonal NSC lines were established that self-renewed up to 40 subculturing passages in vitro, again independ-ently of genotype and, also, of the region of origin (Fig. 1B). The 3 regions and the clonal NSC lines derived from them comprised a higher frequency of CD15/SSEA1-IR cells than CD133/Prom1-IR cells (Fig. 1C). All NSC populations gave rise to neurons, astrocytes, and oligodendrocytes, thus show-ing full multipotency (Fig. 1D and Supplementary Table S1A). However, electrophysiologic analysis of terminally differenti-ated neurons obtained from region-specifi c NSCs indicated that they were intrinsically different in their electrorespon-siveness (Fig. 1E). In fact, cerebellum white matter and IVv NSC-derived neurons were characterized by inward fast Na+ and slow Ca2+ currents and outward inactivating K+ currents normally retrieved in functionally mature neurons. On the contrary, most neurons obtained from subventricular zone NSCs displayed K+ steady-state currents typical of immature neuronal cells (Fig. 1E and Supplementary Table S1B).

To formally prove the stem cell nature of IVv NSCs, GFP-labeled clonal IVv NSCs were transplanted into the cerebellum of neonatal mice (Fig. 1F). Similar to cerebellum white matter NSCs, IVv NSCs differentiated into astrocytes, neuron-like cells, and oligodendrocytes, which dispersed throughout the distinct layers of the cerebellum (Supplementary Table S1C). Thus, by showing extensive self-renewal ability and in vitro/in vivo multipotency, IVv-derived cells qualifi ed as bona fi de NSCs and, together with cerebellum white matter–derived NSCs, revealed intrinsic regional differences in terms of in vitro and in vivo differentiation potential as compared with subventricular zone NSCs.

Hindbrain- and Forebrain-Derived NSCs Are Molecularly Distinct

Wt and Ptch+/−p53+/+ NSCs isolated from the 3 different regions were subjected to whole-genome transcriptome anal-ysis at subculturing passages between 10 and 15. Notably, wt and Ptch+/−p53+/+ NSCs clustered together, with no genes resulting differentially expressed, suggesting that Ptch hetero-zygosity did not infl uence the global transcriptome of NSCs (not shown). Gene profi les of hindbrain NSCs were sharply distinguishable from that of subventricular zone NSCs, with 562 differentially expressed probe sets (Fig. 2A and Supple-mentary List S1). Forebrain-restricted transcription factors, such as Emx2 and FoxG1, were overexpressed in subventricu-lar zone NSCs; by contrast, transcription factors involved in hindbrain development, such as En2 and Pax3, were upregu-lated in cerebellum white matter and IVv NSCs (Fig. 2B).




Mouse Hindbrain NSCs and Medulloblastoma CSCs Predict Human Medulloblastoma Molecular Subclasses RESEARCH ARTICLE


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Figure 1. Long-term expanding NSCs can be isolated from the postnatal IVv region. A, expression of PH3, Ki67, and BrdUrd in cerebellum EGL and white matter (WM), IVv and subventricular zone at P7. Magnifi cation ×200. CB, cerebellum; SVZ, subventricular zone. B, Cerebellum white matter and the region lining the IVv were dissected as described in the drawing (top left). For IVv cultures, only the fl oor of the ventricle was included. Clonal effi ciency in hindbrain- and subventricular zone-derived primary cell cultures (top right; ***, P = 0.0001; ****, P < 0.0001). Long-term growth curves (bottom left) and clonogenic assay (bottom right) of NSC lines. C, stem cell marker expression in the different neurogenic regions (left), after exclusion of CD45+ stromal cells, and in their corresponding NSC lines (right). D, multipotency of cerebellum, IVv, and subventricular zone NSCs (Tuj1-IR neurons, green; GFAP-IR astrocytes, red; NG2-IR oligodendroglial progenitors, red; O4-IR oligodendrocytes, green; ×600). E, electrophysiologic profi le of cerebellum, IVv, and subventricular zone NSC-derived neurons. F, GFP-transduced, hindbrain NSCs after injection into the neonatal cerebellum (S100-IR astroglial cells and Olig2-IR oligodendrocytes. Neuronal-like cells were identifi ed based on cellular morphology). GL, granule layer; ML, molecular layer (×600).






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Figure 2. Molecular comparison of hindbrain-derived NSCs with forebrain-derived NSCs indicates that regional identity is maintained. A, unsupervised whole-transcript expression analysis of region-specifi c NSC lines. CB, cerebellum; SVZ, subventricular zone. B, heatmap highlighting a selection of region-specifi c differentially expressed genes (DEG). Validation of the expression of DEGs by semiquantitative (right) and quantitative RT-PCR (bottom). Fold increase in Emx2 expression in subventricular zone NSCs was obtained with respect to cerebellum NSCs; fold increase in Pax3 expression in cer-ebellum and IVv NSCs was calculated with respect to subventricular zone NSCs. C, Pax3 protein nuclear expression in cerebellum and IVv NSCs (×200). D, ISH for En2 and Emx2 mRNA on P7 brain tissue (×200). E, immunohistochemistry for Pax3 and FoxG1 (×400). F, medulloblastoma/PNET-like lesions are generated by the coimplantation of GFP-labeled cerebellum/IV NSCs with pancreatic islets under the renal capsule. The tumor generated from cerebel-lum NSCs (asterisk, ×40, ×100; top) is GFP-IR (bottom left). High-magnifi cation fi elds depict typical rosette-like structures (bottom right panel, ×200 and ×400). Arrows point to grafted pancreatic islets. G, GSEA enrichment plots. Each gene signature was compared with molecular subgroups from a human medulloblastoma data set. The nominal P value and the FDR Q value are indicated below.






In silico data were validated in cultured NSCs (Fig. 2B and C) and, most relevantly, in tissue sections comprising the regions of origin (Fig. 2D and E). Thus, in vitro cultured region-spe-cifi c NSCs maintained their in vivo regionalization and their positional identity. Most remarkably, NSCs from the IVv, that is, an extracerebellar region, were molecularly indistinguish-able from cerebellum white matter-derived NSCs.

Given that many medulloblastomas arise postnatally, we challenged postnatal hindbrain NSCs with a pro-oncogenic stimulus, such as chronic exposure to insulin, to test whether they might act as potential medulloblastoma cells of origin (15). We transplanted C57BL/6 mice with syngeneic pan-creatic islets and GFP-labeled hindbrain NSCs under the kidney capsule. In line with previous studies in which tumor suppressor-deleted postnatal cerebellum white matter NSCs were tumorigenic (6), hindbrain NSCs developed GFP-IR tumors, which were characterized by the presence of rosette-like structures, typical of medulloblastoma/primitive neuro-ectodermal tumor (PNET) (Fig. 2F).

To investigate whether the gene signatures distinguishing hindbrain- versus forebrain-derived NSCs were enriched in distinct molecular subtypes of human medulloblastoma, we carried out Gene Set Enrichment Analysis (GSEA) on a pub-licly available data set (GSE21140), which contained expres-sion data from 103 human medulloblastoma samples (16). We selected 155 and 142 genes that were upregulated at least 2-fold in hindbrain and forebrain NSCs, respectively. Inter-estingly, the expression of genes upregulated in cerebellum/IVv NSCs was more strongly associated with the desmoplas-tic medulloblastoma subgroup than the subventricular zone gene signature (Fig. 2G, Supplementary Table S2 and Sup-plementary List S1). Accordingly, the same gene signature was exclusively upregulated in the Shh molecular subgroup, which comprises many desmoplastic medulloblastomas. When the global 297-gene signature was tested against all the medullo-blastoma subgroups simultaneously, it again sharply separated the Shh subgroup from the others (Supplementary Fig. S1).

Hindbrain- and Forebrain-Derived NSCs Are Shh Pathway Independent

To assess whether the Shh pathway, normally active in GCPs, might be operating also in hindbrain NSCs, we took advantage of the genetic confi guration of the Ptch targeting vector, which contains a sequence encoding a nuclear β-galactosidase (LacZ) and the neomycin resistance gene. If Ptch+/−p53+/+ cerebellum white matter and IVv NSCs express Ptch in vitro, they should be β-Gal positive and should survive neomycin selection.

The expression of Ptch-LacZ by β-Gal staining was retrieved in the subventricular zone, in the EGL, in the white matter, and in scattered cells around the IVv of P7 Ptch+/− mice in vivo (Supplementary Fig. S2A). Primary cells from subventricular zone, cerebellum white matter and IVv self-renewed long-term in the presence of sublethal concentration of G418, thus indicating that cerebellum white matter and IVv NSCs did express Ptch (not shown). However, when wt and Ptch+/− cer-ebellum white matter and IVv NSCs, cultured for more than 6 subculturing passages (SP), were exposed to 10 μmol/L cyclopamine, a Shh pathway inhibitor, no difference in terms of cell survival and proliferation were observed (Supplemen-tary Fig. S2B). Accordingly, Shh downstream targets were not

expressed in long-term cultured NSC lines, suggesting that they might grow independent of Shh signaling activation (Supplementary Fig. S2C and D). To formally prove this con-cept, we exposed to cyclopamine wt and Ptch+/− subventricular zone, cerebellum white matter and IVv NSC lines starting at SP0 (i.e., primary culture) up to SP14. A remarkable reduc-tion in survival was retrieved in all NSC lines at very early SPs, which was not observed in the same NSCs after additional SPs (Supplementary Fig. S2D). Accordingly, overexpression of the Shh pathway downstream targets Gli1 and Gli2 as well as downregulation of Gli3 were detected in short-termcultured NSCs, being absent in long-term cultured NSCs (Supplementary Fig. S2E).

CSC Lines Can Be Established In Vitro from Ptch�/� Mice, Independent of p53 Loss and Shh Pathway Activation

Cells isolated from adult Ptch+/−p53+/+, Ptch+/− p53+/–, and Ptch+/− p53−/– medulloblastomas were cultured under the NeuroSphere Assay conditions (9). Part of each cell suspen-sion was grown in the presence of serum and in the absence of mitogens, to generate serum-dependent cancer cell lines to be exploited as nonstem cell controls (17). After 30 to 60 days in vitro, stable clonal CSC lines could be obtained (n = 10 from Ptch+/−p53+/+ medulloblastomas, n = 4 from Ptch+/− p53+/– medulloblastomas, and n = 2 from Ptch+/− p53−/–

medulloblastomas). Some CSC lines grew as small clusters similar to neurospheres, whereas other as adherent cells. Medulloblastoma cells were capable to expand in culture not only in the presence of both EGF and FGF2 (Fig. 3A) but also in the presence of either growth factor alone (data not shown). Importantly, all cell lines expanded for more than 100 passages, with a doubling time between 2 and 5 days. Medullo-blastoma CSC lines were heterogeneous in terms of growth rate and clonal effi ciency, which was independent from the genotype (Fig. 3A and B). Similar to normal NSCs, all clonally derived medulloblastoma cell lines differentiated into Tuj1-IR neuron-like cells, GFAP-IR astrocyte-like cells, and NG2/O4-IR oligodendrocyte-like cells, with few cells aberrantly colabeled with neuronal and glial markers (ref. 18; Fig. 3C and Supplementary Table S3). When exposed to cyclopamine, only a few medulloblastoma CSC lines showed a very modest decrease in survival, which was in line with the low expression of Shh downstream targets in the same cells (Supplementary Fig. S3A–C). To test the presumptive independence of medulloblastoma CSCs from Shh signal-ing for in vitro growth, we generated stable medulloblastoma CSC lines in which Smo was silenced by lentiviral-based short hairpin RNA (shRNA; Supplementary Fig. S3D). Notably, Smo inhibition, and, as such, Shh pathway inactivation, did not signifi cantly affect medulloblastoma CSC proliferation and survival (Supplementary Fig. S3D), thus suggesting that medulloblastoma CSCs might rely on different growth path-ways for effi cient growth.

Loss of p53 Is Required for the Tumorigenicity of CSCs Isolated from Ptch�/� Mouse Medulloblastomas

Because the majority of Ptch+/− tumors lose expression of the Ptch wt allele (9), we tested whether our medulloblastoma cells






Figure 3. CSCs can be isolated from mouse medulloblastomas and give rise to phenotypically distinct experimental tumors. (A) growth curves and (B) clonogenic assays of medulloblastoma CSC lines. (C) medulloblastoma CSC differentiation into Tuj1-IR neuron-like cells (green), GFAP-IR astrocyte-like cells (red), NG2-IR oligodendrocyte precursors (red) and O4-IR (green) oligodendrocyte-like cells (×400). (D) LOH of the Ptch and p53 alleles by genomic DNA PCR. (E) immunohistochemical characterization of CSC-derived experimental tumors. Hematoxylin and eosin (H&E) stain, ×200; Tuj1, NeuN (GC, tumor-entrapped granule cells), synaptophysin, GFAP, and Sox2 staining, ×200.






were also characterized by LOH for Ptch (Fig. 3D). By DNA genotyping, LOH for Ptch was detected in all Ptch+/− p53+/+

medulloblastoma CSCs due to the deletion of a large fragment of the genomic locus. Conversely, Ptch LOH was absent in their tumor of origin and in the tail tissue. Intriguingly, Ptch LOH was never observed in Ptch+/−p53−/− and Ptch+/−p53+/− medul-loblastoma CSCs, which, conversely, showed LOH for p53 (Fig. 3D). Both Ptch and p53 LOH resulted in the absence of the corresponding transcripts (data not shown).

When injected into immunosuppressed animals, both intracranially (striatum and/or cerebellum) and subcutane-ously, either in adult or early postnatal animals, Ptch+/− p53+/+

medulloblastoma CSCs gave rise to slowly growing grafts that never evolved into full-blown lesions (data not shown). Con-versely, Ptch+/−p53+/− and Ptch+/−p53−/− medulloblastoma CSC lines reproducibly established tumors, with a take effi ciency of 70% and 100% (intracranially and subcutaneously, respec-tively). Of note, Ptch LOH did not infl uence the tumorigenic potential of medulloblastoma CSC lines, as all CSC lines that were tumorigenic retained the expression of the wt Ptch allele. Rather, loss of p53, occurring by genomic deletion or by somatic LOH (Fig. 3D), was associated with medulloblastom-agenesis. Intracranial tumors were detected by MRI after 10 to 12 weeks after transplantation and fully developed within30 weeks (data not shown). Conversely, subcutaneous tumors were observed as early as at 2 weeks after transplantation and progressed very rapidly. Only NeuroSphere Assay–culturedmedulloblastoma CSC lines generated tumors, whereas matched serum-dependent, nonstem cancer cell lines did not.

CSC-derived intracranial tumors closely resembled sponta-neous Ptch+/− tumors, although their morphology was slightly more malignant and pleomorphic, with marked atypia and the presence of infi ltrating cellular nests; they also expressed mark-ers typical of human medulloblastoma, such as Tuj1, NeuN, synaptophysin, and GFAP (Fig. 3E). In a few circumstances, CSC-derived intracranial tumors escaped the site of implanta-tion, through either leptomeningeal or extracranial dissemina-tion. Tumors infi ltrating the meninges expressed lower levels of neuronal and glial markers, whereas extracranial tumors were highly undifferentiated, with signs of pleomorphism and increased malignancy. CSC-derived subcutaneous tumors were the most highly undifferentiated, showing a myxoid and het-erogeneous morphology, and containing high numbers of cells expressing the putative CSC marker Sox2. Tuj1-IR cells were also detected, while NeuN- and synaptophysin-IR cells were absent. Thus, the site of tumor formation affects the histologic phenotype of experimental CSC-derived medulloblastomas, with a progressive malignant evolution from lowly malignant intracranial tumors to highly malignant extra-CNS neoplasias.

Molecular Analysis of Medulloblastoma CSCs and Normal NSCs Identifi ed Novel Potential Medulloblastoma Targets

To identify dysregulated genes that may play a role in the pathogenesis of medulloblastoma, we fi rst compared the global gene expression profi les of medulloblastoma CSCs with that of normal NSCs. Unsupervised clustering analy-sis showed that medulloblastoma CSCs shared many more molecular determinants with hindbrain NSCs than with subventricular zone NSCs. Notably, many more genes were

differentially expressed between hindbrain NSCs and Ptch+/−

p53−/− medulloblastoma CSCs than between hindbrain NSCs and Ptch+/−p53+/+ medulloblastoma CSCs (Fig. 4A).

These gene expression data were then exploited to gener-ate gene signatures that refl ected the behavior of the dis-tinct populations of medulloblastoma CSCs (Fig. 4B). The fi rst signature was upregulated exclusively in tumorigenic Ptch+/− p53−/− medulloblastoma CSCs versus hindbrain NSCs (MB1_EXCLUSIVE, 980 genes, Supplementary List S2), the second exclusively in nontumorigenic Ptch+/− p53+/+ medul-loblastoma CSCs versus hindbrain NSCs (MB2_EXCLUSIVE, 105 genes, Supplementary List S2), and the third comprised the probe sets whose expression was shared by both tum-origenic and nontumorigenic CSCs versus hindbrain NSCs (MB1_MB2 COMMON, 81 genes, Supplementary List S2). The MB1_EXCLUSIVE and MB1_MB2 COMMON gene signatures were specifi cally enriched in the desmoplastic subgroup of human medulloblastomas. However, although MB1_MB2 COMMON gene signature was associated with the Shh molecular subgroup, MB1_EXCLUSIVE gene signa-ture was enriched in the WNT molecular subgroup (Fig. 4B and Supplementary Fig. S4). Accordingly, the WNT pathway mediator β-catenin was expressed in Ptch+/− p53+/− sponta-neous tumors, in Ptch+/−p53−/− CSC-derived intracranial and subcutaneous tumors in vivo, and in Ptch+/− p53−/− medul-loblastoma CSCs in vitro (Supplementary Fig. S5A and B). Transcripts for several WNT pathway mediators included in the MB1_EXCLUSIVE gene signature were also signifi cantly upregulated in Ptch+/− p53−/− medulloblastoma CSC lines as compared with Ptch+/− p53+/+ medulloblastoma CSCs (Sup-plementary Fig. S5C). Thus, human medulloblastomas can be distinguished based on their degree of resemblance to distinct mouse medulloblastoma CSC molecular phenotypes.

Reactivation of Developmentally Regulated Molecular Programs Promotes Medulloblastomagenesis

Early B-cell factors (Ebfs), such as Ebf2 and Ebf3, are expressed in the cerebellum during embryonic development and regulate progenitor cell proliferation and differentiation. Interestingly, they were highly expressed in medulloblastoma CSCs and in their corresponding tumor tissues (Fig. 4C). Ebf3 expression, in particular, was observed in cycling progenitors located in the mouse EGL as early as P3, peaked at P7, and decreased around P15. Accordingly, Ebf protein expression was retrieved in EGL progenitors and also in cells in the prospective white matter and around the IVv (Fig. 4E). In line with gene expression data, Ebf protein expression was high in both spontaneous and CSC-derived intracranial tumors, being very low in intrameningeal and subcutaneous CSC-derived tumors (Fig. 4E).

EBF3 expression was detected in silico in human medullo-blastomas and in a small fraction of neuroblastomas, whereas it was not retrieved in glial tumors, suggesting a possible associ-ation between the expression of the gene and a neuronal tumor phenotype (Supplementary Fig. S5A and B). Accordingly, EBF3 transcript and EBF proteins were expressed in human medulloblastomas and not in glioblastoma multiforme (GBM, Fig. 4F and G and Supplementary Table S5). The pattern of expression of the EBF proteins varied among histologic






Figure 4. Molecular comparison of medulloblastoma CSCs with normal NSCs pinpoints potential medulloblastoma targets. A, unsupervised analysis of global gene expression profi les of medulloblastoma CSCs and NSCs (left). Scatter plot of probe sets differentially expressed between medulloblastoma CSCs and cerebellum/IVv NSCs, between highly malignant Ptch+/−p53−/− CSCs and cerebellum/IVv NSCs and between low-malignant Ptch+/−p53+/+ CSCs and cerebellum/IVv NSCs (right). DEGs with |log2FC| > 1 are plotted outside the region between the 2 oblique lines. DEGs with a P value <0.05 are red or green. CB, cerebellum. B, GSEA enrichment plots. Each gene signature was compared with molecular subgroups from a human medulloblastoma data set. The nominal P value and the FDR Q value are indicated below. MB, medulloblastoma. C, semiquantitative and quantitative RT-PCR for Ebf2 and Ebf3 in NSCs, medulloblastoma CSCs and tissues. WB for Ebf3 in NSCs, medulloblastoma CSCs, and medulloblastoma tissues. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. D, ISH for Ebf3 in combination with BrdUrd incorporation analysis. DAPI, 4′,6-diamidino-2-phenylindole; PCL, Purkinje cell layer. E, Ebf protein expression in P7 hindbrain (top, ×100 and ×200). Ebf-IR cells in Ptc spontaneous tumors, and in intracranial, intrameningeal, and subcutaneous CSC-derived tumors (×400). F, quantitative EBF3 mRNA expression in human medulloblastoma, GBM, and healthy brain tissue. G, EBF protein expression in human medulloblastoma specimens (×100 and ×400).






variants of medulloblastoma. In particular, classic medullo-blastomas displayed intense EBF staining in the majority of tumor cells, whereas desmoplastic medulloblastomas showed EBF expression being restricted to desmoplastic portion of the tumor and excluded from the nodular part. Of note, EBF proteins were also strongly expressed in the pleomorphic giant cells found in large-cell anaplastic medulloblastomas.

To address the putative role of Ebf3 in medulloblastom-agenesis, we transduced both NSCs and medulloblastoma CSCs with an Ebf3-FLAG lentiviral construct, obtaining effi -cient expression of Ebf3 transcript and protein (Fig. 5A and B). Mock- and Ebf3-tranduced NSC and CSC lines did not show any difference in proliferation, long-term self-renewal, migration, and apoptosis. However, under proliferative cul-ture conditions (i.e., in the presence of EGF and FGF2), Ebf3-transduced NSC and CSC lines acquired the appearance

of cells undergoing differentiation. In fact, whereas normal mock-transduced IVv, cerebellum, and subventricular zone NSCs were almost negative for lineage differentiation mark-ers, Ebf3 overexpressing NSCs showed a consistent increase in the frequency of FLAG-positive cells expressing the neuronal marker Tuj1 and displaying a mature cellular morphology (Fig. 5C and Supplementary Fig. S7). Likewise, Ebf3 overex-pressing FLAG-positive CSCs gave rise to Tuj1-IR cells display-ing a more mature morphology than Tuj1-IR cells retrieved in mock CSC cultures (Fig. 5C). Upon differentiation, Ebf3 overexpressing NSC/CSC cultures mostly generated FLAG-positive neuronal-like cells featuring extended processes and well-developed neurites (Fig. 5D). The frequency of astrocytes and oligodendrocytes was similar between mock- and Ebf3-transduced NSC/CSCs under both proliferative and differen-tiative conditions (not shown).

Figure 5. Enforced expression of Ebf3 in NSCs and medulloblastoma CSCs results in premature neuronal differentiation. A, Ebf3 mRNA expression in mock- and Ebf3-transduced NSCs and CSCs. Fold increase of Ebf3 mRNA was calculated with respect to matched mock-transduced samples. CB, cerebellum. B, Ebf3 protein expression upon lentiviral mediated gene transfer in NSCs and medulloblastoma CSCs. C, neuronal differentiation of Ebf3-transduced NSCs and CSCs under proliferative conditions (FLAG, green; Tuj1, red; ×400, inset ×800). D, neuronal differentiation of Ebf3-transduced NSCs and CSCs under differentiative conditions (FLAG, green; Tuj1, red; ×400, inset ×800). *, P < 0.01; ***, P = 0.0001.






Figure 6. Enforced expression of Ebf3 in medulloblastoma CSCs results in enhanced tumorigenesis. A, expression of GFP, FLAG, and Ebf in intracranial tumors generated by mock- and Ebf3-transduced CSCs, respectively (×200, ×400). B, expression of GFP, FLAG, and Ebf in subcutaneous tumors generated by mock- and Ebf3-transduced CSCs, respectively (×200, ×400). C, quantitative assessment of tumor development (L51, P < 0.0005; LB, P < 0.005). D, H&E, Tuj1, and GFAP staining in mock- and Ebf3-transduced CSC-derived experimental tumors (×200) Inset in H&E: perivascular rosette. E, the frequency of Ki67 and Sox2-IR cells in Ebf3 overexpressing and mock CSC-derived tumors (P < 0.001). **, P < 0.001; ***, P = 0.0001. F, Ebf2/Ebf3 transcripts and Ebf3 protein were reduced in Ebf-silenced CSCs. G, quantitative assessment of tumor development (L21, P < 0.01). H&E and GFP staining in mock- and Ebf-silenced CSC-derived experimental tumors (×200). H&E, Hematoxylin and eosin.






Mock- and Ebf3-transduced CSC lines were then trans-planted intracranially and subcutaneously into adult immune-defi cient mice. In spite of very high effi ciency of Ebf3-FLAG overexpression (Fig. 6A and B), signifi cant dif-ferences in the tumorigenic ability of Ebf3 overexpressing CSCs with respect to mock CSCs were observed only upon subcutaneous transplantation. Indeed, the frequency of cells endogenously expressing Ebf in mock CSC-derived intracra-nial tumors was very high and comparable with that in Ebf3 expressing tumors. As such, Ebf3 overexpression in CSCs under intracranial transplantation settings was not function-ally relevant. On the contrary, given that endogenous Ebf expression in mock CSC-derived subcutaneous tumors was low (Fig. 4E, Fig. 6B), enforced expression of Ebf3 in medul-loblastoma CSCs accelerated the formation of highly malig-nant subcutaneous tumors (Fig. 6C and D). Whereas mock CSC-derived tumors were characterized by sarcomatoid/myxoid morphology and by the presence of Tuj1- and GFAP-IR cells, Ebf3-transduced CSC-derived tumors were more highly undifferentiated, showed a pleomorphic and malig-nant appearance, contained perivascular cellular rosettes, and comprised only Tuj1-IR cells. In line with these fi ndings, both the mitotic index as measured by Ki67 staining and the frequency of Sox2-IR cells were signifi cantly higher in Ebf3-transduced CSC-derived tumors than in mock CSC-derived tumors (Fig. 6E). Overall, subcutaneous mock CSC-derived tumors were reminiscent of classic medulloblastomas, and Ebf3 overexpressing tumors resembled highly malignant PNET-like anaplastic medulloblastomas.

To inhibit Ebf3 endogenous expression in medullob-lastoma CSCs by RNA interference (RNAi), given that Ebf family members function as active heterodimers, we took advantage of a GFP-coding retroviral construct that allows the concomitant expression of siRNAs against Ebf1-, Ebf2-, and Ebf3 (19). Signifi cant downregulation of Ebf2 and Ebf3 transcript and protein was observed in Ebf-silenced medulloblastoma CSCs (Fig. 6F). Mock- and Ebf-silenced medulloblastoma CSCs were then injected subcutane-ously. Ebf-silenced tumors were signifi cantly smaller than those generated by mock-transduced cells and, again, this defect was refl ected in the histologic appearance of tumors (Fig. 6G). In fact, whereas mock CSC-derived tumors showed the typical sarcomatoid/myxoid morphology, tumors in which Ebfs were silenced were characterized by a homoge-neous, well-differentiated morphology and reduced prolif-eration, with cells displaying large and eosinophil cytoplasm, suggestive of a less malignant phenotype.

DISCUSSION

To identify novel molecular targets involved in medul-loblastomagenesis to be exploited for selective and effective therapies, we took advantage of the biologic relationship existing between medulloblastoma and the cell progenitor lineages from which it is thought to arise. In fact, different clinical/molecular medulloblastoma subtypes are believed to originate from mutations occurring in distinct stem/precur-sor cells located in cerebellar germinative regions, such as the rhombic lip derivative and the ventricular zone matrix (20), as

well as in extracerebellar domains, as the dorsal brainstem (3), some of them persisting postnatally (2).

NSCs Can Be Isolated from Extracerebellar Anatomic Sites as the IV Ventricle and Share Functional and Molecular Features with Cerebellar NSCs

During embryonic development, 2 primary cerebellar neu-rogenic epithelia, that is, the rhombic lip and the IVv ven-tricular zone, segregate as early as embryonic day 9 (E9) and represent the birthplace of all GABAergic and glutamatergic lineages, respectively (1, 21). Early postnatally, the rhombic lip derivative located at the cerebellar surface, the EGL, undergoes a massive expansion due to GCP proliferation that peaks at P7 and gradually ceases, being completely exhausted within the third postnatal week in rodents and the fi rst year in humans (22). Very recently, a second postna-tal cerebellar neurogenic compartment has been identifi ed within the cerebellar white matter (4, 5). Cerebellum white matter NSCs were shown to self-renew shortly in culture and to be multipotent in vitro and in vivo (4). We also generated P7 cerebellum white matter neurospheres, which were subcul-tured for many passages, giving rise to long-term expanding NSCs (6). Most interestingly, by applying the same culture conditions to cells obtained from the P7 brain parenchyma surrounding the IVv, from which short-term proliferating precursors were previously isolated (7, 8), we established long-term self-renewing multipotent bona fi de IVv NSCs.

As previously shown for embryonic NSCs (23), the distinct postnatal NSC populations did maintain their original posi-tional identity. In fact, both cerebellum and IVv NSCs over-expressed transcription factors, such as Pax3 and En2, which are well-known regulators of hindbrain development. Likewise, subventricular zone NSCs displayed enhanced expression of genes involved in forebrain development, for example, Emx2, Foxg1, and Lhx2. Most interestingly, IVv NSCs, which were iso-lated from an extracerebellar region, were molecularly indistin-guishable from cerebellum white matter NSCs, thus suggesting that progenitors residing in different anatomic sites might have a common origin. Relevantly, when challenged oncogenically, hindbrain-derived postnatal NSCs generate medulloblastoma/PNET-like lesions, suggesting that multiple populations of hindbrain NSCs may be at the origin of medulloblastoma (3). Accordingly, IVv NSCs could be isolated not only from early postnatal mice but also from adults, in line with the observa-tion that medulloblastomas deriving from brainstem progeni-tors show a distributed age of onset, ranging from late infancy to adulthood, with a peak in older children (24).

Molecular Analysis of Medulloblastoma-Derived CSCs and Hindbrain-Derived NSCs Identifi es Novel Potential Molecular Mediators of Medulloblastomagenesis

In agreement with previous observations in medullo-blastoma cell cultures and serum-cultured colon cancer cells (25, 26), the expression of downstream mediators of Shh pathway in NSCs and medulloblastoma CSCs was modest, and, accordingly, Shh pathway activation was marginally relevant for proliferation and self-renewal of NSC/CSCs






in vitro. Indeed, we showed that addiction to Shh pathway occurs in NSCs only at early subculturing passages, when Shh pathway is still active. Accordingly, Shh inhibition in long-term cultured medulloblastoma CSCs by Smo RNAi does not infl uence CSC proliferation. These results are in contrast with studies reporting full activation of Shh path-way in normal NSCs (27) as well as in CSCs isolated from the same Ptch mouse model (28) or other brain tumors (29). Indeed, both NSC/CSC-derived neurospheres employed in those studies were maintained in vitro for very few subcul-turing passages. Short-term NSC/CSC cultures are mostly composed by committed progenitors (30). As such, lineage-restricted precursors, rather than NSC/CSCs might have been addicted to Shh signaling and, therefore, responded to Shh inhibition by reduced survival and proliferation. On the contrary, long-term propagated cultures, which are highly enriched in the NSC/CSC component and devoid of com-mitted precursors (30), might not be Shh dependent and may self-maintain through different signaling pathways. Alternatively, chronic exposure of NSC/CSCs to mitogenic stimulation, in particular to FGF2 (31), might repress Shh pathway activation and promote the activity of different compensatory effectors.

Notably, although Shh pathway activation was not retrieved in postnatal NSCs, the cerebellum/IVv NSC gene signature is signifi cantly associated with the Shh human molecular subgroup. This observation might imply cerebel-lum white matter and IVv NSCs as additional cells of origin for medulloblastomas, as they share the expression of the Shh pathway mediators with EGL GCPs, which are well known to be causatively involved in medulloblastoma tumorigenesis (32, 33).

Most relevantly, the genes characterizing Ptch+/−p53+/+ medulloblastoma CSCs were associated with the Shh molecular subgroup of human medulloblastomas, whereas those overexpressed in Ptch+/−p53−/− medulloblastoma CSCs were enriched in the WNT subgroup, thus emphasizing the exploitability of these medulloblastoma CSCs as in vitro pre-clinical model of Shh- and Wnt-dependent medulloblas-tomas, respectively. Moreover, the observation that the site of transplantation of medulloblastoma CSCs under experi-mental in vivo settings results in tumors endowed with vary-ing degree of malignancy, ranging from well-differentiated desmoplastic intracranial tumors to highly undifferenti-ated anaplastic subcutaneous tumors, further underline the relevance of medulloblastoma CSCs as a versatile model, highly representative of the different variants of human medulloblastomas.

In line with a recent report (9), we established long-term self-renewing CSC lines from medulloblastomas, which spon-taneously develop in Ptch heterozygous mice (34) and in compound Ptch/p53 mutants (10). However, only medullo-blastoma CSCs, characterized by genetic or somatic p53 loss, were able to generate fully developed tumors closely mimick-ing the different histologic variants of the human disease. Interestingly, the gene signature distinguishing Ptch+/−p53−/− medulloblastoma CSCs is enriched in the WNT subgroup, in agreement with the recent fi nding that, in medulloblastoma patients, p53 mutations are mostly found in favorable-risk WNT-subtype medulloblastomas (35).

Reactivation of Developmentally Regulated Proneural Molecular Programs Favors Medulloblastomagenesis

During development, Ebf transcription factors are selec-tively expressed in early postmitotic neurons (36). However, we observed that Ebf3 expression was retrieved not only in postmitotic GCPs of the inner EGL but also in bromodeox-yuridine (BrdUrd)-incorporating GCPs undergoing neuronal fate choice in the outer EGL. Accordingly, enforced expres-sion of Ebf3 in NSCs and medulloblastoma CSCs did not induce cell-cycle arrest or apoptosis, but, rather, premature and enhanced neuronal commitment, which was already evident under proliferative conditions and was maintained upon differentiation.

Most interestingly, enforced expression of Ebf3 in medul-loblastoma CSCs greatly increased their tumorigenic ability, indicating that the Ebf3-mediated induction of a neuronal phenotype in medulloblastoma cells might be required to promote medulloblastoma initiation and pro-gression, in agreement with recent reports proposing that the acquisition of granule cell neuronal commitment is essential for medulloblastomagenesis (32, 33). Of note, the molecular subgroup of human medulloblastomas showing a highly malignant behavior is associated with a neuronal signature (24).

Ebf3 expression is known to be undetectable in many human cancers, including leukemias, pancreatic cancers, head and neck squamous cell carcinomas and, most interest-ingly, GBMs. In many of these cancers, EBF3 inactivation is due to epigenetic silencing by promoter methylation, genomic deletion, or point mutations. As expected, Ebf 3 expression in these tumor cells induces cell-cycle arrest and apoptosis, indicating that Ebf3 could act as tumor suppres-sor (37). By contrast, our fi ndings suggest that Ebf3 expres-sion in malignancies belonging to the neuronal lineage, such as medulloblastomas and, possibly, neuroblastomas, might play a pro-oncogenic role, in contrast with glial tumors and other cancers.

Transcriptional data derived from molecular comparisons between normal NSCs and medulloblastoma CSCs thus holds great potential as preclinical molecular tools for the identifi cation of novel molecular determinants and meaning-ful gene signatures in medulloblastoma.

METHODS

Isolation of NSCs and CSCsNSC cultures were established from the subventricular zone, the

cerebellum, and the region surrounding the IVv of P7 mice, whereas CSCs were obtained from tumors developed in 3- to 6-months-old animals. NSC lines were established from tissues pooled from 4 to 6 mice, whereas CSC lines were from single tumors. NSC and CSC lines were cultured and propagated in standard medium as described by Galli and colleagues (38).

Differentiation of NSCs and CSCsNSCs and CSCs were differentiated by withdrawal of mitogens

from the culture medium and addition of 2% FBS for 7 days (38) and processed for immunocytochemistry. The lists of the antibodies used are available in the Supplementary Methods section.






Whole-Cell Patch Clamp RecordingSee Supplementary Methods.

In Situ HybridizationDigoxygenin-labeled riboprobes were transcribed from plasmids

coding for Emx2, En2, and Ebf3 cDNAs. Sixteen micrometer cryostat sections of P3, P7, and P15 brains were processed as described in Sup-plementary Methods.

Flow Cytometric AnalysisTissue samples and NSC/CSC lines were enzymatically and/or

mechanically dissociated to obtain a single cell suspension. Single cells were suspended in PBS/BSA 5 mg/mL 2 mmol/L EDTA pH 8 and incubated on ice for 20 minutes. Specifi c antibodies were diluted in the same solution and then incubated together with the cells for 30 minutes on ice. The acquisition was carried out on BD FACS CANTO II (Becton and Dickinson) and the analysis by FCS express 3.0 soft-ware. Antibodies used were fl uorescein isothiocyanate (FITC)-conju-gated CD15 (Becton and Dickinson) 1:20; PE-conjugated Prominin-1 (eBioscience) 1:50, APC-conjugated CD45 (Becton and Dickinson), 1:100, PE-conjugated Ter119 (Becton and Dickinson), 1:100.

ImmunoblottingEach sample was homogenized in 10× volume of radioimmunopre-

cipitation assay lysis buffer. Samples were then diluted in Laemmli’s SDS sample buffer. Proteins were separated by electrophoresis on 8% SDS-PAGE and transferred onto trans-blot nitrocellulose mem-branes (Amersham). Ponceau staining (Sigma) was carried out to con-fi rm that the samples were loaded equally. The primary antibodies/antisera used were mouse anti-Ebf3 (1:1,000; Abnova), mouse anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase, 1:10,000; Sigma), and goat anti-Smo (1:300; Santa Cruz).

Gene Overexpression and SilencingMouse Ebf3 cDNA was cloned into the monocistronic transfer len-

tiviral vector (LV) pCCL.sin.cPPT.PGK.GFP.WPRE11. GFP was excided and substituted with the FLAG-Ebf3 cassette. Sister cultures were infected with the same vector coding for GFP, as mock control. NSCs and CSCs were transduced with 1 × 107 TU/mL of each LV for 16 hours. Ebf3 silencing was achieved by using retroviral constructs coding for short-hairpin RNA directed against the 3 Ebf genes (19). CSCs were transduced through 3 rounds of infection. Smoothened silencing was done by exploiting commercially available LVs coding for gene-specifi c shRNA clones (Mission RNAi; Sigma-Aldrich). Infection of CSCs with shRNAs was carried out according to the manufacturer’s instructions.

Molecular AnalysisTotal RNA from all samples was extracted using the RNeasy Mini kit

(Qiagen). One microgram of total RNA was reverse transcribed by using fi rst strand synthesis kit Superscript III RNaseH Reverse Transcriptase (Invitrogen) and OligodT primers. All the semiquantitative reverse tran-scriptase PCRs (RT-PCR) were carried out using home-designed specifi c primers and cDNAs were normalized on �-actin housekeeping gene. Quantitative real-time PCR was carried out by IQ SybrGreen technol-ogy (BioRad), following manufacturer’s instructions. β-Actin was used as housekeeping gene. Mouse- and human-specifi c Ebf2 and Ebf3 prim-ers were employed for qPCR (Qiagen, Sa Bioscience).

Microarray-Based Gene Expression Profi lingThe subculturing passages at which medulloblastoma CSCs and

NSCs were collected for molecular analysis were comprised between the 8th and 20th. Quality control of hybridization was done by Image Quality, MAplots, Boxplot and Density Plot, Array normali-

zation was executed by the RMA and GCRMA algorithms. Divisive clustering algorithms were used to obtain dendrograms, in which the biologic samples were clustered on the basis of the differentially expressed genes. The hierarchical clustering algorithms employed were (i) distances (Euclidian, correlation), (ii) linkage (complete, sin-gle, mcquitty, ward, and centroid). The differentially expressed genes (DEG) were obtained based on (i) t test moderated empirical Bayes, (ii) P value [false discovery rate (FDR) adjusted 0.05], (iii) cut-off (1 log2 Fold Change, FC). Microarray data are available at NCBI GEO (GEO accession number GSE37316).

NSCs Injection into Neonatal MiceGFP-labeled clonal cerebellum white matter and IVv NSC lines

were injected into neonatal cerebella as described in Leto and col-leagues (21).

Evaluation of TumorigenicityFor subcutaneous injection, 1 × 106 to 2 × 106 CSCs were resus-

pendend in 100 μL PBS and injected into the right fl ank of 45 to 60 days old nu/nu female mice. Mice were sacrifi ced at different time points comprised between 4 to 12 weeks postinjection, according to the cell line originally injected. For intracranial transplantation, 2 × 105 CSCs were resuspended in Dulbecco’s modified Eagle’s medium and DNase (Sigma) and delivered into the right striatum or the cerebellum by stereotactic injection through a 5 μL Hamilton microsyringe. The following coordinates were used: AV = 0; ML = +2.5 mm; DV = −3.5 mm from bregma for intrastriatal injections and AV = −3.5; ML = 0; DV = 2 from interaural line for intracerebellar injections. Animals were sacrifi ced 2 to 8 months after transplanta-tion. Transplantation of hindbrain NSCs under the renal capsule was done as described in Melzi and colleagues (15).

Disclosure of Potential Confl icts of InterestNo potential confl icts of interest were disclosed.

Authors’ ContributionsConception and design: D. Corno, R. GalliDevelopment of methodology: D. Corno, B. Cipelletti, V. Barili, R. Melzi, L. Sergi Sergi, L. Piemonti, R. Galli, G.G. Consalez, L. Croci, P.L. PolianiAcquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Corno, M. Cominelli, K. Leto, L.S. Politi, L. Piemonti, A. Bulfone, P. Rossi, F. Rossi, P.L. Poliani, R. Galli, G.G. Consalez, L. CrociAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Corno, M. Pala, K. Leto, F. Brandalise, L.S. Politi, P. Rossi, F. Rossi, P.L. Poliani, R. GalliAdministrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Corno, L. Croci, A. Di Gregorio, R. GalliWriting, review, and/or revision of the manuscript: P. Rossi, F. Rossi, R. Galli, G.G. ConsalezStudy supervision: R. GalliDesign and analysis of Ebf3 gene expression in cerebellar granules: L. Croci, G.G. ConsalezDesign of Ebf3 silencing: G.G. Consalez, D. Corno, R. Galli

AcknowledgmentsThe authors thank Laura Magri and Matteo Zanella for help and

assistance with molecular analysis.

Grant SupportThis work was supported by Fondazione Pierfranco and Luisa

Mariani for Child Neurology and by Fondazione Guido Berlucchi for Cancer Research to R. Galli.






Received August 15, 2011; revised April 12, 2012; accepted April 12, 2012; published OnlineFirst May 3, 2012.

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856 | CANCER DISCOVERY�SEPTEMBER 2012 www.aacrjournals.org

Correction: Gene Signatures Associated with Mouse Postnatal Hindbrain Neural Stem Cells and Medulloblastoma Cancer Stem Cells Identify Novel Molecular Mediators and Predict Human Medulloblastoma Molecular Classifi cation

In this article (Cancer Discovery 2012;2:554–68), which was published in the June 2012 issue of Cancer Discovery (1), the name of the fi rst author is incorrect. The correct name is Daniela Corno. The publisher regrets this error.

REFERENCE 1. Corno D, Pala M, Cominelli M, Cipelletti B, Leto K, Croci L, et al. Gene signatures associated with mouse post-

natal hindbrain neural stem cells and medulloblastoma cancer stem cells identify novel molecular mediators and predict human medulloblastoma molecular classifi cation. Cancer Discovery 2012;2:554–68.

Published OnlineFirst August 1, 2012.doi: 10.1158/2159-8290.CD-12-0284©2012 American Association for Cancer Research.

CORRECTION

Published OnlineFirst May 3, 2012.Cancer Discovery Daniela Corno, Mauro Pala, Manuela Cominelli, et al. Molecular ClassificationNovel Molecular Mediators and Predict Human MedulloblastomaNeural Stem Cells and Medulloblastoma Cancer Stem Cells Identify Gene Signatures Associated with Mouse Postnatal Hindbrain

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