Transmembrane Protein 18 Enhances the Tropism of Neural Stem Cells for Glioma Cells Jaana Jurvansuu, 1 Ying Zhao, 1,2 Doreen S.Y. Leung, 1 Jerome Boulaire, 1 Yuan Hong Yu, 3,4 Sohail Ahmed, 3,4 and Shu Wang 1,2 1 Institute of Bioengineering and Nanotechnology; Departments of 2 Biological Sciences and 3 Physiology, National University of Singapore; 4 Institute of Medical Biology, Singapore, Singapore Abstract The failure of current glioma therapies is mainly due to the abilityofthetumorcellstoinvadeextensivelythesurrounding healthy brain tissue, hence escaping localized treatments. Neural stem cells (NSC) are able to home in on tumor foci at sites distant from the main tumor mass, possibly enabling treatment of scattered glioma clusters. To make the strategy more effective, we performed a cDNA expression library screening to identify the candidate genes that once overex- pressedwouldenhancethetropismofNSCsforgliomas.Here, weshowthatapreviouslyunannotatedgene,theoneencoding transmembrane protein 18 (TMEM18), is one such gene. Overexpression of TMEM18 was seen in the current study to provide NSCs and neural precursors an increased migration capacity toward glioblastoma cells in vitro and in the rat brain. Functional inactivation of the TMEM18 gene resulted in almost complete loss of the migration activity of these cells. Thus, TMEM18 is a novel cell migration modulator. OverexpressionofthisproteincouldbefavorablyusedinNSC- based glioma therapy. [Cancer Res 2008;68(12):4614–22] Introduction Glioma cells misregulate the expression of growth factors, proteases, and extracellular matrix and cell surface proteins to gain their devastating invasion capacity (1, 2). Localized treatments are thus inefficient and comprehensive treatments are too damaging to the delicate brain. A solution is to find a treatment that can specifically locate the tumor cells. Neural stem cells (NSC) and neural precursor cells (NPC) have an intrinsic tropism for sites of brain injuries, including gliomas, and as shown first by Benedetti and colleagues (3) and Aboody and colleagues (4), engrafted primary and immortalized NSCs can be used in gene therapy of gliomas in animal models. These engrafted stem cells have been shown to spread through the existing migratory pathways in healthy brain as well as nontypical routes when gliomas are present (4, 5). Besides primary and immortalized NSCs/NPCs, embryonic stem cell–derived NPCs seem to have the same aptitude for glioma cell tracking (6). Moreover, NSCs are able to locate not only gliomas but also tumors of a nonneural origin, suggesting that there exist common regulators of cell trafficking probably composed of secreted factors from a tumor site and receptors present on NSCs (7, 8). Candidate signals to attract NSCs to the sites of brain injuries and tumors have been studied. Among them are cytokines released from the immunoreactive microglial cells of the brain during inflammation (9), which also provide cues for NSC migration in brain development (10). Stromal cell–derived factor-1 (SDF-1) chemokine can attract NSCs too. When its receptor CX chemokine receptor 4 (CXCR4) is blocked, SDF-1 hinders the NSC migration to the site of injury (9, 11, 12). In addition, chemokine monocyte chemoattractant protein-1, the expression of which can be induced by tumor necrosis factor-a, can activate migration of NSCs (13). Cytokine stem cell factor, expressed by glioma cell lines and overexpressed in neurons at the sites of brain injury, is another possible contributing attractant for NSCs (14–16). Similar to cytokines and chemokines, growth factor–mediated signaling [e.g., vascular endothelial growth factor and epidermal growth factor (EGF) receptor] has been shown to regulate NSC and NPC migration (17, 18). Glioma invasion depends largely on the ability of the cell to modify the extracellular matrix, and interestingly, the extracellular matrix secreted from glioma cell lines is able to promote NSC motility (19). The picture emerging from the above studies seems to support a model of the complex interaction of several factors in regulating NSC migration toward tumors. We hypothesized that other regulators are likely to exist and their genes can be identified through expression cloning based on gene function in influencing the tropism of NSCs toward glioma cells. We were particularly interested in the molecules that once overexpressed in NSCs and NPCs are able to enhance cell migration toward gliomas, as the manipulation of the expression of these molecules could then facilitate the use of NSCs/NPCs as gene therapy vectors to reach scattered glioma cells. We used Boyden chambers in the current study to select the cells that were primed by gene transfer of a tumor cDNA expression library. A novel gene encoding transmem- brane protein 18 (TMEM18) emerged from the screen and was selected for extensive characterizations. Materials and Methods Cells. NT2, U87MG, H4, and NIH3T3 cell lines were purchased from the American Type Culture Collection and HEK293FT cells were purchased from Invitrogen. All the cell lines were maintained in DMEM supplemented with 10% FCS (Life Technologies), penicillin-streptomycin (Life Technolo- gies), normoxin (Invivogen), and nonessential amino acids (Life Techno- logies). C17.2 cells were kindly provided by Prof. E. Arenas (Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden) and maintained in DMEM supplemented with 10% FCS, 5% horse serum (Life Technologies), penicillin-streptomycin, normoxin, and nones- sential amino acids. The NIH Human Embryonic Stem Cell Registry listed human embryonic stem (hES) cell line, HES-1, and its feeder cell K 4 mouse embryonic fibroblasts were obtained from ES Cell International (ESI), Singapore. The hES cells were amplified and maintained according to the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). J. Jurvansuu and Y. Zhao contributed equally to this work. Requestsforreprints: Shu Wang, Institute of Bioengineering and Nanotechnology, Singapore 138669, Singapore. Phone: 65-6824-7105; Fax: 65-6478-9083; E-mail: swang@ ibn.a-star.edu.sg. I2008 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-07-5291 Cancer Res 2008; 68: (12). June 15, 2008 4614 www.aacrjournals.org Research Article Research. on February 12, 2016. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from Research. on February 12, 2016. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from Research. on February 12, 2016. © 2008 American Association for Cancer cancerres.aacrjournals.org Downloaded from