Type I collagen is overexpressed in medulloblastoma

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common malignant brain tumor in children and account for. 25% of all pediatric brain tumors [1]. PNETs can arise in a supratentorial (cerebral) or infratentorial ...
J Neurooncol DOI 10.1007/s11060-007-9457-5

LAB INVESTIGATION-HUMAN/ANIMAL TISSUE

Type I collagen is overexpressed in medulloblastoma as a component of tumor microenvironment Yu Liang Æ Maximilian Diehn Æ Andrew W. Bollen Æ Mark A. Israel Æ Nalin Gupta

Received: 27 May 2007 / Accepted: 25 June 2007  Springer Science+Business Media B.V. 2007

Abstract Medulloblastoma is the most common malignant brain tumor of children, and more specific and effective therapeutic management needs to be developed to improve upon existing survival rates and to avoid sideeffects from current treatment. Gain of chromosome seven is the most frequent chromosome copy number aberration in medulloblastoma, suggesting that overexpression of genes on chromosome seven might be important for the pathogenesis of medulloblastoma. We used microarrays to identify chromosome seven genes overexpressed in medulloblastoma specimens, and validated using data from Electronic supplementary material The online version of this article (doi:10.1007/s11060-007-9457-5) contains supplementary material, which is available to authorized users. Y. Liang  N. Gupta Department of Neurological Surgery, Brain Tumor Research Center, University of California, San Francisco, CA 94143, USA Y. Liang (&) Division of Molecular Cell Biology, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA e-mail: [email protected] M. Diehn Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305, USA M. Diehn Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA A. W. Bollen Department of Pathology, University of California, San Francisco, CA 94143, USA M. A. Israel Departments of Pediatrics and Genetics, Norris Cotton Cancer Center, Dartmouth Medical School, Lebanon, NH 03756, USA

published gene expression datasets. The gene encoding the alpha 2 subunit of type I collagen, COL1A2, was overexpressed in all three datasets. Immunohistochemistry of tumor tissues revealed type I collagen in the leptomeninges, and in the extracellular matrix surrounding blood vessels and medulloblastoma cells. Expression of both type I collagen and the b1 subunit of integrin, a subunit of a known type I collagen receptor, localized to the same area of medulloblastoma. Adherence of D283 medulloblastoma cells to type I collagen matrix in vitro depends on the b1 subunit of integrin. Because medulloblastoma is characteristic of high vascularity, and because inhibition of type I collagen synthesis has been shown to suppress angiogenesis and tumor growth, our data suggest that type I collagen might be a potential therapeutic target for treating medulloblastoma. Keywords Medulloblastoma  Microarray  Extracellular matrix  Type I collagen  Adhesion

Introduction Brain tumors are the primary cause of non-traumatic death in children and young adults under the age of 20 years. Primitive neuroectodermal tumors (PNETs) are the most common malignant brain tumor in children and account for 25% of all pediatric brain tumors [1]. PNETs can arise in a supratentorial (cerebral) or infratentorial (cerebellar) location but most PNETs occurring in children arise within the cerebellum. These cerebellar tumors are also called medulloblastoma. The standard treatment for medulloblastoma is surgical resection followed by fractionated external beam radiation and/or chemotherapy [2]. Although medulloblastoma is sensitive to both therapeutic modalities,

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irradiation of the central nervous system (CNS) in young children often results in serious long-term side effects, such as deafness, cognitive decline, and neuroendocrine insufficiency. For this reason, a variety of chemotherapy regimens are used as initial treatment in the very young patients to delay the use of radiation therapy. Early diagnosis and improved treatment have increased the 5 year survival rate of patients with localized medulloblastoma to greater than 60% [3, 4]. Invasion of the leptomeninges by medulloblastoma cells and their dissemination via the subarachnoid spaces throughout the neuraxis to distant sites can occur either at presentation or at relapse and is associated with poor survival [5]. Genetic studies and the development of transgenic mouse models have provided important information about the cellular origin and oncogenic pathways underlying medulloblastoma [6]. The molecular mechanisms responsible for invasion into and dissemination within the neuraxis remain largely unknown. A recent study using DNA microarrays suggested that expression of plateletderived growth factor a and the RAS/mitogen-activated protein kinase signal transduction pathway might be up-regulated in disseminated medulloblastoma and could be targets for more effective treatments [7]. Both medulloblastoma and supratentorial PNET are malignant and invasive embryonal tumors that have similar histological features, but the survival for children with supratentorial PNET is much poorer, only 20–30% at 5 years [8–10]. The degree of surgical resection may play a role in the improved survival of medulloblastoma, since gross total resection of supratentorial PNET is usually more difficult to achieve. PNET arise from both locations exhibit high microvascular density and express wide range of angiogenic factors [11, 12]. Despite these similarities, recent evidence has shown that medulloblastoma and supratentorial PNET are two genetically distinct tumor types, primarily based upon gene expression profiling [13] and the patterns of chromosome copy number aberrations from comparative genomic hybridization [14–17]. One important observation is that majority of these studies also demonstrated that gain of chromosome seven is the most frequent copy number alteration shared by both medulloblastoma and supratentorial PNET [14–18]. For example, gain of chromosome seven was detected in 44–57% of medulloblastoma and in 66% of supratentorial PNET [17, 18]. For this reason, we sought to identify genes located on the chromosome seven which are consistently overexpressed in both tumor types might provide the genetic basis for shared features that characterize these tumors, such as tumoral vascularity. In the present study, we used cDNA microarrays to examine the expression profiles of chromosome seven genes in three medulloblastoma and three supratentorial

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PNET specimens, and in normal brain tissue. We validated the results with published datasets and confirmed overexpression of COL1A2 in both these tumor types. This gene and COL1A1, each encodes a distinctive subunit that together heterodimerizes forming type I collagen. We also detected increased accumulation of type I collagen in medulloblastoma by immunohistochemistry.

Materials and methods Cell culture D283 medulloblastoma cell line was obtained from the Brain Tumor Research Center Tissue Bank at UCSF. All cells were maintained in Eagle’s minimal essential medium with 15% fetal bovine serum and 5% CO2. Tissue specimens Medulloblastoma and normal brain specimens were obtained from the Brain Tumor Research Center Tissue Bank at UCSF after approval by the Committee on Human Research. Microarray and bioinformatic analyses The detailed microarray methods were published at the website http://microarray-pubs.stanford.edu/gbm/. Briefly, total RNA was extracted using Trizol followed by mRNA purification using FastTrack (Invitrogen). mRNA was reverse transcribed to cDNA and directly labeled with Cy dyes (Amersham Biosciences) before hybridization. The raw data of 22,636 features on the array were extracted from the Stanford Microarray Database using the ScanAlyze-featured extraction software with the following settings: regression correlation >0.6, channel 1 mean intensity/median background intensity ‡1.5, and channel two normalized mean intensity/median background intensity ‡1.5. The DNA sequences of individual clones from the extracted raw data were examined for chromosomal locations with the ‘‘Table Browser’’ function at the UCSC Genome Bioinformatics website (the Human May 2004 assembly at http://genome.ucsc.edu), and those localized on chromosome seven (1,116 features) were identified. Among these 1,116 features, only those with available data in three or more tumor specimens and in one or more normal brain specimens were selected for final analyses (757 features). Gene expression data of three independent published datasets (four groups of tumor samples in total) for verification of expression of the COL1A1 and COL1A2 genes were extracted from the Broad Institute Cancer Program

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Datasets (http://www.broad.mit.edu/cancer/datasets.html) and Serial Analysis of Gene Expression (SAGE) Anatomic Viewer (http://cgap.nci.nih.gov/SAGE/AnatomicViewer).

SPSS for Windows (Release 11.5.0). A P value £ 0.05 was considered statistically significant. Immunoprecipitation and immunoblotting

Antibodies Anti-b1 subunit of integrin (ITGB1), anti-type I collagen, and anti-integrin linked kinase polyclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). AntiITGB1 monoclonal antibodies AIIB2 and P4G11 were from Developmental Studies Hybridoma Bank (Iowa City, IA). Anti-CD31 and anti-fibronectin antibodies were from Novocastro Laboratories (United Kingdom) and GIBCO-BRL (Gaithersburg MD), respectively. Peroxidase-conjugated and biotinylated secondary antibodies were from Vector Laboratories (Burlingame, CA). Fluorescine-conjugated and Rhodamine-conjugated secondary antibodies and normal serum were from Jackson ImmunoResearch Laboratories (West Grove, PA). Immunohistochemistry Frozen tissue sections used for immunohistochemistry were fixed in 4% formaldehyde, treated with H2O2, blocked with normal serum, incubated with primary antibodies at 4C overnight or room temperature (RT) 2 h, incubated with biotinylated secondary antibody and peroxidase-labeled streptavidin at RT for 30 min, and visualized using the DAB Reagent kit (KPL; Gaithersburg, MA). Some frozen sections were visualized using FITC- or Texas Red-conjugated secondary antibodies (Vector Laboratories). Staining of paraffin-embedded sections followed the same protocol, except for prior de-waxing and antigen retrieval by microwave heating. Adhesion assay Wells of 96-well tissue culture plates (Corning, Corning, NY) were coated with 100 lg/ml of rat-tail type I collagen (BD Biosciences, Bedford, MA) for 1 hour at room temperature, followed by two PBS washes. 1 · 105 D283 cells resuspended in serum-free medium were plated into either uncoated or type I collagen-coated wells for 1 hour. Nonadhered cells were removed by lightly tapping the plates and the number of cells remaining adherent was counted. Monoclonal antibodies AIIB2 and P4G11 were diluted to 1:20 into the serum-free medium during a 1-hour incubation to inhibit cell adhesion.

D283 cells were lysed in RIPA buffer (in 1% NP-40, 0.1% SDS, 0.5% Na deoxycholate, 50 mM Tris pH 8.0, and 150 mM NaCl) supplemented with 1 mM NaF, 1 mM Na3VO4, and Complete protease inhibitor cocktail tablets (Roche, Basel, Switzerland). After high-speed centrifugation, the lysate was pre-cleared twice with protein A Sepharose beads (Zymed Laboratories, South San Francisco) followed by mixing with 1:100 dilution of antibodies at 4C for 1 h. Immunoprecipitated proteins were captured by protein G Sepharose beads (Zymed Laboratories) and released by sample buffer. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes, followed by 10% skim milk blocking and antibody incubation, and visualized using the Super Signal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

Results Gene expression profiling identifies genes on chromosome seven overexpressed in primitive neuroectodermal tumors compared to normal brain We performed gene expression profiling of three medulloblastoma and three supratentorial PNETs using cDNA microarrays that consisted of ~23,000 features (representing ~18,000 unique UniGene clusters), and compared the results to the expression patterns we observed for two specimens derived from normal cerebrum and one from normal cerebellum. After data filtering, we selected approximately 750 genes located on chromosome seven (see Methods and Additional file 1 for data), and computed the difference of their expression between all six tumors and the three normal brain specimens. Six UniGene clones representing five different genes, ETV1, CDK6, COL1A2, TAC1, and CAV2, had average expression in tumors at least 4-fold higher when compared to normal tissues (Fig. 1). Differences in gene expression between tumor and normal brain specimens reached significance in three genes using the Student’s t test (ETV1, P = 0.006; CDK6, P = 0.0005; COL1A2, P = 0.009).

Data analysis

COL1A2 mRNA is overexpressed in independent groups of primitive neuroectodermal tumors

All statistical analyses used the Student’s t test (2-tailed and equal variance not assumed) and Pearson correlation in

We verified the increased expression of ETV1, CDK6, and COL1A2 using other gene expression datasets. In a recent

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Fig 1 Expression of genes located on chromosome seven in medulloblastoma and supratentorial PNET specimens compared to normal brain tissue. The average expression of each gene in three normal brain specimens was subtracted from its average expression in all six tumor specimens, and plotted against the base position of this gene on chromosome seven; the value of gene expression (Y-axis) is in log2 scale. Five genes with the values greater than two (4-fold higher expression in tumor than in normal) were selected for further consideration: from left to right ETV1, CDK6 (two clones), COL1A2, TAC1, and CAV2. The Student’s t test showed that expression of ETV1, CDK6, and COL1A2 was significantly greater in tumors than in normal brains, while expression of TAC1 and CAV2 was not

study, oligonucleotide microarrays were used to characterize gene expression from embryonal tumors of the CNS, and found a correlation between gene expression and the clinical outcome of patients with medulloblastoma [13]. Their study included two groups of medulloblastoma and supratentorial PNET specimens: the first group had 10 medulloblastoma and eight supratentorial PNET (Fig. 2a), and the second group consisted of 60 medulloblastoma and six supratentorial PNET (Fig. 2b). We reviewed the expression of AEBP1, CDK6, and COL1A2 in these two groups, and we found that COL1A2 but not the other two genes demonstrated higher expression in tumors than in normal cerebellum with a P value of less than 0.05 (see Additional file 2 for data). We also identified increased expression of COL1A2 in an additional published dataset [19] that included 10 medulloblastomas (Fig. 2c). Finally, we sought to validate this finding using gene expression data from the SAGE Anatomic Viewer [20] that displays gene expression in human normal and malignant tissues based on the number of SAGE tags corresponding to individual mRNAs [21]. In this database, expression of COL1A2 in 20 medulloblastoma specimens was significantly higher when compared to two normal cerebellum specimens (Fig. 2d, and see Additional file 3 for data). Type I collagen is overexpressed in medulloblastoma COL1A2 gene encodes the alpha 2 subunit of type I collagen. Unlike other types of collagen in which the subunits of the collagen fibrils heterodimerize in several possible

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combinations, type I collagen is always composed of one alpha 2 chain and two alpha 1 chains (encoded by the COL1A1 gene). Expression of type I collagen is regulated primarily at the transcriptional level, and common cisacting regulatory elements are present in the promoters of both COL1A1 and COL1A2 genes [22, 23] This is consistent with the coherent expression patterns between COL1A1 and COL1A2 in the two groups of normal, medulloblastoma, and supratentorial PNET specimens used by Pomeroy et al. [13] in which we verified COL1A2 expression in Figs. 2a,b (group 1, N = 20, P = 1 · 10–8; group 2, N = 68, P = 2.4 · 10–15; both by Pearson correlation and see Additional file 2 for data). As expected, COL1A1 mRNA was also significantly higher in these two sets of medulloblastoma and supratentorial PNET than in normal cerebellum (Figs. 2ab, and Additional file 2 for data), so was the number of SAGE tags corresponding to COL1A1 significantly higher in medulloblastoma than in normal cerebellum (Fig. 2d). Overexpression of both the COL1A1 and COL1A2 genes in both medulloblastoma and supratentorial PNET strongly suggests increased production of type I collagen in their tumor microenvironment. We examined the expression of type I collagen protein in a panel of 17 human medulloblastoma specimens using immunohistochemistry. In normal brain, type I collagen was detected in the leptomeninges and basement membrane associated with blood vessels but not in brain parenchyma (Fig. 3a). Two patterns of type I collagen expression were observed in the tumor specimens. The first pattern consisted of type I collagen immunoreactivity surrounding blood vessels, while the ECM between neoplastic cells was essential negative (Fig. 3b). The boundaries of the type I collagenpositive basement membrane were sharply demarcated. The second pattern of type I collagen immunostaining was not restricted in the ECM of blood vessels but was diffusely associated with thickened and distended basement membrane of the tumor vasculature and throughout the ECM within the tumor (Fig. 3c, e, g). We noticed that both immunostaining patterns occurred together in some tumors (data not shown). b1 integrin subunit is detected in type I collagensurrounded medulloblastoma cells and mediates their in vitro adhesion to type I collagen matrix Our immunohistochemical data indicate that type I collagen is present in the basement membrane associated with intra-tumoral vasculature as well as in the ECM surrounding neoplastic cells. This suggests that type I collagen-containing ECM interacts with at least a subset of neoplastic cells. The b1 subunit of integrin (ITGB1) is a known constituent of receptors for type I collagen, and

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Fig 2 Expression of COL1A2 (white boxes) and COL1A1 (grey boxes) genes is increased in medulloblastoma (Mb) and supratentorial PNET (PNET) compared to normal brain (Norm) in four independent groups of specimens (three groups in Pomeroy et al. [13] and Ramaswamy et al. [19] and one group in SAGE [20] depicted in these box plots. (a) Two normal brain specimens compared with 10 medulloblastoma (P = 0.007 for COL1A2, P = 0.009 for COL1A1) and eight supratentorial PNET specimens (P = 0.032 for COL1A2, P = 0.011 for COL1A1), (b) two normal brain specimens compared

with 60 medulloblastoma (P = 9.8 · 10–5 for COL1A2, P = 2 · 10– 10 for COL1A1) and six supratentorial PNET specimens (P = 0.034 for COL1A2, P = 0.04 for COL1A1), (c) three normal brain specimens compared with 10 medulloblastoma specimens (P = 0.001 for COL1A2), (d) SAGE libraries derived from two normal brain specimens compared with 20 medulloblastoma specimens (P = 0.001 for COL1A2, P = 0.01 for COL1A1). m, extremes; s, outliers

could be a candidate for mediating the cellular receptor to type I collagen. We investigated this possibility by first examining localization of the ITGB1 in medulloblastoma tumor tissues. We contrasted the CD31 immunoreactivity that stains endothelial cells (Fig. 3f) to the type I collagen immunoreactivity that stains basement membrane of the vessels and tumoral ECM (Fig. 3g). ITGB1 was detected in endothelial cells as well as neoplastic cells in regions where the ECM contained type I collagen (Fig. 3h). Dual immunofluorescence microscopy also showed that type I collagen-positive tumoral ECM overlapped with ITGB1 immunoreactivity (Figs. 3j–l). We then characterized whether type I collagen mediates medulloblastoma cell adhesion by examining the adhesion of D283 medulloblastoma cells to type I collagen matrix in vitro. One hour after plating, D283 cells started to adhere to a type I collagen-coated surface whereas they could be easily removed from an uncoated surface by

lightly tapping a culture dish (Fig. 4). Most D283 cells detached from either coated or uncoated surface after vigorous shaking (data not shown). AIIB2 and P4G11 are two monoclonal antibodies that have been previously characterized for their opposite effects on the ITGB1mediated functions, in that AIIB2 blocks binding of b1 integrin subunit to its ligand, [24] whereas P4G11 stimulates the binding [25]. Adherence of D283 cells to type I collagen is ITGB1-dependent, as AIIB2 abolished the attachment of cells to type I collagen, while P4G11 had no such effect (Fig. 4). Our preliminary experiments demonstrated that the integrin-linked kinase (ILK), a kinase downstream of ITGB1, was detectable only in neoplastic cells positive for ITGB1 (Fig. 3i). Anti-type I collagen polyclonal antibodies co-immunoprecipitated ILK from D283 cells (Additional file 4) suggesting that medulloblastoma cells interacting with type I collagen ECM via ITGB1 might trigger signaling pathways involving ILK.

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Fig 3 Type I collagen is overexpressed in medulloblastoma and codistributed with ITGB1. In normal brain, expression of type I collagen is associated with the basement membrane of blood vessels (arrow in a). Two patterns of type I collagen expression were observed in the medulloblastoma specimens. The first pattern consisted of type I collagen immunoreactivity in the basement membrane surrounding blood vessels (arrow in b), but not in the ECM between neoplastic cells (* in b). The second pattern of type I collagen immunostaining was diffusely associated with the ECM of the tumor (* in c, e, g) as

well as the vasculature indicated by arrows in from d to i (endothelial cells were highlighted by CD31 staining in d and f). Tumor cells surrounded by type I collagen-positive ECM also expressed ITGB1 (h) and ILK (i). Panels d/e and f–i were consecutive sections from the same specimen. Dual immunofluorescence microscopy showed that ITGB1 (j) and type I collagen (k) were co-distributed (yellow color in l) in the ECM surrounding tumor cells counterstained by DAPI (blue color in l). Bar in a represents 45 lm for from a to i. Bar in j represents 15 lm for from j to l

Discussion

Fig 4 D283 medulloblastoma cells adhered to type I collagen matrix via the ITGB1 in vitro. The inhibitory monoclonal antibody, AIIB2, blocked this interaction, whereas the activating monoclonal antibody, P4G11, did not. The data were plotted from a representative experiment with quadruplicates, and * indicates a P value of 0.0017

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Microarrays provide a high throughput platform to characterize gene expression profiles of tumor specimens and to identify clusters of differentially expressed genes that may mediate specific biological functions or clinical phenotypes or mark specific cell lineages from which tumors are derived [26]. For example, we have previously used cDNA microarrays and sequential supervised and unsupervised algorithmic analyses to identify a group of genes highly expressed in rapidly progressing glioblastoma tumors. We validated the prognostic value of the expression of one of the genes, FABP7, in two independent sets of specimens [27]. FABP7 was found to be preferentially expressed in

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glioma cells with astrocytic features, [28] and its prognostic value for glioblastoma was associated with EGFR overexpression of the tumor cells [29]. Because the availability of only a limited number of medulloblastoma specimens might easily confound the reliability of clustering analysis, we utilized a different strategy in this investigation to identify candidate genes of pathological importance. We built upon recognition of a frequently occurring chromosomal aberration in medulloblastoma, gain of chromosome seven, that is shared by supratentorial PNET, and sought to identify chromosome seven genes that were overexpressed in both tumor types using cDNA microarrays. We validated our finding in published databases that used distinctive assay platforms, and repeatedly found increased expression of the genes encoding the two subunits of type I collagen. Immunohistochemistry further confirmed increased deposition of type I collagen protein in the ECM of medulloblastoma. These findings provide evidence for roles of type I collagen in the pathogenesis of medulloblastoma. ECM components and the cellular machinery that mediates the interaction of tumor cells with the ECM are of great importance in efforts to understand the molecular mechanisms regulating angiogenesis of malignant cells. We found high levels of expression of type I collagen in medulloblastoma specimens associated with tumor vasculature. In normal tissues, type I collagen is a major component of basement membranes associated with blood vessels, and it is required for maintaining the integrity of the vasculature and for regulating angiogenesis, [30, 31] but type I collagen is not expressed in normal brain parenchyma. Type I collagen is believed to directly modulate the behavior of tumor cells mainly based on data from studies using halofuginone, a low molecular weight quinazolinone alkaloid that inhibits synthesis of type I collagen. While halofuginone inhibits neovascularization by inhibiting vascular sprouting, tubular formation, and ECM deposition by endothelial cells both in vitro and in vivo, [32] it also inhibits tumor invasion and tumor growth in an animal model for glioma and chemically induced mouse bladder carcinoma [33–35]. The best-characterized cellular receptors for type I collagen are a family of integrin membrane proteins. Integrins are heterodimeric glycoproteins composed of one a chain and one b chain. All type I collagen-binding integrins share a common b1 integrin subunit [36]. Anti-a2b1 integrin antibodies inhibit the attachment of endothelial cells to type I collagen matrix but not to fibronectin or laminin that are also the components of vasculature ECM [37]. ITGB1 plays central roles in tumor progression, ECM remodeling, and angiogenesis by transmitting mechanical signals as it mediates cell adhesion and migration by interacting with the cytoskeleton proteins, [38] and it also conveys chemical

signals to control cell survival and proliferation by activating a spectrum of kinases and downstream adaptor proteins [38]. ILK is a serine-threonine protein kinase that binds to the cytoplasmic domain of ITGB1 and regulates ITGB1-mediated signaling following growth factor stimulation [39]. Identification of ILK-binding proteins has recently demonstrated that the functions of ILK in ITGB1 signaling pathways include anchoring actin filaments and activating signaling molecules downstream of growth factor receptors [40]. Type 1 collagen may be of importance in the pathobiology of medulloblastoma because of its role in blood vessel formation. Medulloblastoma is a highly vascular tumor and several studies have suggested that anti-angiogenesis-directed therapy may be a potential strategy for future approaches to the treatment of medulloblastoma. For example, an integrin av antagonist peptide successfully inhibited angiogenesis induced by human DAOY medulloblastoma cells growing in chicken chorioallantoic membrane [41] and also reduced growth of DAOY cells implanted into nude mice [42]. Whether inhibitors of type I collagen, such as halofuginone, can suppress the spread and growth of medulloblastoma cells via inhibition of angiogenesis or by a direct effect on neoplastic cells deserves further investigation. Type I collagen is also a major constituent of leptomeninges, [43] and invasion of the leptomeninges is a frequent route over which medulloblastoma cells spread throughout the neuraxis and establish systemic metastatic disease [44]. However, expression of type I collagen might not be involved in medulloblastoma invasion into adjacent brain structures or metastasis through cerebrospinal fluid, as analyses using the published dataset did not show correlation between type I collagen expression and tumor/metastasis stages (data not shown). This is consistent with the findings that the vascularity of medulloblastoma is not associated with either metastasis or patient survival [12, 45]. We demonstrated that type I collagen is also present in the ECM associated with medulloblastoma cells in vivo. The type I collagen-positive interstitial matrix may be essential for the angiogenic behavior of medulloblastoma cells. Co-distribution of ITGB1 with type I collagen also suggests the possibility that by binding to its ligand, ITGB1 triggers intracellular signaling pathways that might regulate the pathophysiology of medulloblastoma cells, probably through ILK. In one study, ILK expression was not seen in normal brain but was detected in all three medulloblastoma and four supratentorial PNET specimens examined [46]. We had preliminary data suggesting that ILK might be recruited to the type I collagen/ITGB1 complex in D283 cells. These findings support the notion that the type I collagen/ITGB1 interaction in medulloblastoma may activate a signaling cascade via ILK. It

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would be interesting to investigate whether interaction of type I collagen and ITGB1 relates to angiogenic activities of medulloblastoma cells. Our findings suggest the possibility of a role for the inhibition of type I collagen and its signaling pathways in the treatment of medulloblastoma. The therapeutic potential of inhibiting type I collagen synthesis or downstream signaling events in medulloblastoma might have a twoprong effect: inhibition of angiogenesis and the tumoral vasculature in which type I collagen is actively synthesized, and blocking type I collagen-mediated tumor cell interaction by reducing the activated integrin signaling. Such therapeutic activities seem very likely to affect the pathobiology of this tumor in a manner that would have a significant impact on the clinical behavior of the tumor and thereby contribute to an improved outcome for patients. Acknowledgements We thank the Brain Tumor Research Center Tissue Bank of UCSF for contributing tissue specimens in this study. This work was supported by funding from the Department of Neurological Surgery at UCSF, and by National Institute of General Medical Sciences training grant GM07365 (M.D.), and the Theodora B. Betz Foundation and Kyra Memorial Fund (M.A.I.). UCSF is an NCI-designated Specialized Program of Research Excellence for Brain Tumors.

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