Tenascin-C Stimulates Glioma Cell Invasion through Matrix Metalloproteinase-12 Susobhan Sarkar, 1 Robert K. Nuttall, 3 Shuhong Liu, 1 Dylan R. Edwards, 3 and V. Wee Yong 1,2 Departments of 1 Oncology and 2 Clinical Neurosciences, University of Calgary, Calgary, Alberta, Canada and 3 School of Biological Sciences, University of East Anglia, Norwich, Norfolk, United Kingdom Abstract The capacity of glioma cells to invade extensively within the central nervous system is a major cause of the high morbidity rate of primary malignant brain tumors. Glioma cell invasion involves the attachment of tumor cells to extracellular matrix (ECM), degradation of ECM components, and subsequent penetration into adjacent brain structures. These processes are accomplished in part by matrix metalloproteinases (MMP) within a three-dimensional milieu of the brain parenchyma. As the majority of studies have used a two-dimensional mono- layer culture system, we have used a three-dimensional matrix of collagen type I gel to address glioma-secreted proteases, ECM, and invasiveness of glioma cells. We show that in a three- dimensional collagen type I matrix, the presence of tenascin-C, commonly elevated in high-grade gliomas, increased the invasiveness of glioma cells. The tenascin-C–mediated inva- siveness was blocked by metalloproteinase inhibitors, but this did not involve the gelatinases (MMP-2 and MMP-9) commonly implicated in two-dimensional glioma growth. A thorough analysis of 21 MMPs and six members of a disintegrin and metalloproteinase domain showed that MMP-12 was increased in gliomas by tenascin-C in three-dimensional matrix. Fur- thermore, examinations of resected specimens revealed high MMP-12 levels in the high-grade glioblastoma multiforme tumors. Finally, a function-blocking antibody as well as small interfering RNA to MMP-12 attenuated the tenascin- C–stimulated glioma invasion. These results identify a new factor, MMP-12, in regulating glioma invasiveness through interaction with tenascin-C. (Cancer Res 2006; 66(24): 11771-80) Introduction Gliomas are the most common primary tumors that arise within the central nervous system in adults. The most malignant form, glioblastoma multiforme, is resistant to current modalities of treatment. The hallmark of gliomas is local invasion of single tumor cells to adjacent and distant brain structures. Such inva- siveness is mediated in part by the interaction of glioma cells with the extracellular matrix (ECM), followed by degradation of matrix by tumor cell–derived proteases, particularly the matrix metal- loproteinases (MMP; refs. 1–7). The overexpression of the gelatinase subfamily of MMPs, MMP-2 and MMP-9, is well documented in malignant gliomas (2–4, 6, 7). More recently, the membrane-type MMPs have also been found to be overrepresented in glioma specimens (4, 8, 9). There are 24 human MMP members, however, and the possible roles of nongelatinase or membrane-type MMPs in glioma biology remain uncertain. Moreover, the specific interaction between glioma cells and particular ECM molecules and how ECM may alter MMP expression are, for the most part, unclear. The roles played by the ECM in tissue repair and neoplastic transformation are complex. In malignant gliomas, many ECM components are overexpressed both in the tumor stroma and at the advancing edge of the tumor within brain parenchyma. These ECM molecules include vitronectin, collagen I, collagen IV, osteo- pontin, tenascin-C, secreted protein acidic and rich in cysteine, and brain enriched hyaluronan binding (10–21). Glioma invasion is thought to occur along ECM protein–containing structures, such as along tracts of myelinated fibers (22–24). Besides creating a more permissive substrate for invasiveness, ECM proteins can also affect other tumorigenic properties, such as survival, cell cycle progres- sion, and angiogenesis. Here, we have evaluated whether particular ECM proteins may modulate the invasiveness of glioma cells and whether this involves specific MMPs. We focused on a three-dimensional model of invasiveness, in which glioma cells are encased within a collagen I gel supplemented with test ECM proteins. Previous studies of glioma invasiveness in vitro have largely used a two-dimensional monolayer growth but it has become apparent that a three- dimensional matrix allows a more physiologic representation of tumor behavior (25–27). In addition, rather than focusing on a few MMPs, we have extended our analyses to include all MMP members and also a related family of metalloproteinases, a dis- integrin and metalloproteinases (ADAM). Our results show that tenascin-C is a permissive substrate for glioma invasiveness and that it does so by up-regulating MMP-12. These results provide new insights and targets to inhibit glioma invasiveness. Materials and Methods Cell culture. U178 and U251 cell lines (28) were maintained in MEM supplemented with 10% fetal bovine serum (FBS). For two-dimensional cell culture, a six-well plate was first coated with polyornithine (10 Ag/mL) for 30 minutes at room temperature. Wells were then further coated with specific ECM protein (10 Ag/mL) at 37jC for 24 hours. Glioma cells were trypsinized with 0.25% trypsin-EDTA and seeded onto the ECM substrate. For three-dimensional cell culture, collagen I gel was prepared on ice according to the manufacturer’s instruction (Chemicon, Temecula, CA). In brief, for a 500-AL volume, 400 AL of cold collagen I solution were mixed with 100 AL of cold 5Â DMEM and the pH was neutralized. One million glioma cells were added to the cold 500-AL collagen I solution, supplemented with or without 5-AL test molecules (e.g., tenascin-C). Following pipetting of mix cells into the collagen I solution, 70 AL (140,000 cells) were distributed onto the center of the top compartment of trans- well inserts (see below). The plate was then placed immediately at 37jC for 1 hour to allow collagen I to polymerize, trapping cells with it. The collagen I gel was then covered with culture medium. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Requests for reprints: V. Wee Yong, Departments of Oncology and Clinical Neurosciences, University of Calgary, 3330 Hospital Drive, Calgary, Alberta, Canada T2N 4N1. Phone: 403-220-3544; Fax: 403-283-8731; E-mail: vyong@ucalgary.ca. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-0470 www.aacrjournals.org 11771 Cancer Res 2006; 66: (24). 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