[CANCER RESEARCH 64, 6858 – 6862, October 1, 2004] Advances in Brief Convection-Enhanced Delivery of Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand with Systemic Administration of Temozolomide Prolongs Survival in an Intracranial Glioblastoma Xenograft Model Ryuta Saito, John R. Bringas, Amith Panner, Matyas Tamas, Russell O. Pieper, Mitchel S. Berger, and Krystof S. Bankiewicz Brain Tumor Research Center, Department of Neurological Surgery, University of California at San Francisco, San Francisco, California Abstract Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of cell death, preferentially killing neoplastic cells over normal cells, the efficacy of TRAIL for the treatment of glioma might be limited due to cellular resistance and, importantly, poor distri- bution after systemic administration. TRAIL and temozolomide (TMZ) were recently shown to have a synergistic antitumor effect against U87MG glioma cells in vitro. Convection-enhanced delivery (CED) can effectively distribute TRAIL protein throughout a brain tumor mass. In this study, we evaluated CED of TRAIL, alone and in conjunction with systemic TMZ administration, for antitumor efficacy. CED of TRAIL demon- strated safe and effective distribution in both normal brain and a U87MG intracranial xenograft model. Individually, both CED of TRAIL and systemic TMZ administration prolonged survival in tumor-bearing rats. However, the combination of these two treatments was significantly more effective than either treatment alone. CED of TRAIL in conjunction with systemic TMZ treatment is a promising strategy for the treatment of malignant gliomas. Introduction Treatment of malignant gliomas remains a challenge. The combi- nation of surgery, radiation therapy, and chemotherapy yields a me- dian survival of only 9 months (1). Therefore, there is a strong need for new treatment strategies. Convection-enhanced delivery (CED) is a promising local drug delivery technique. By using bulk flow, it allows the direct delivery of small and large molecules to targeted sites in clinically significant volumes of tissue, offering an improved volume of distribution over simple diffusion techniques (2). CED of therapeutic agents bypasses the blood– brain barrier, delivers a high concentration of therapeutic agents to the injection site, provides wider distribution of therapeutic agents within the target site, and minimizes systemic exposure, resulting in fewer side effects. How- ever, because CED distributes therapeutic agents not only to the tumor mass but also beyond the tumor margin into normal surrounding brain tissue, the need for selective tumor cytotoxic activity remains. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; also called Apo2L) is a promising candidate for CED infusion because it is a potent endogenous activator of the cell death pathway and preferen- tially kills neoplastically transformed cells. Because glioma cells have greater expression of the transcripts (DR4 and DR5) encoding the TRAIL death receptor than normal astrocytes (3), they may represent a potential target for selective killing by TRAIL. Cellular resistance to TRAIL, however, has also been reported (4). To improve the efficacy of TRAIL administration and overcome TRAIL resistance, potentially synergistic TRAIL-based combination chemotherapeutic regimens are being studied extensively in many cancers in vitro and in vivo.A recent in vitro study using U87MG human glioma cells showed the synergistic effect of TRAIL and temozolomide (TMZ; ref. 5) against tumor cells. Because TMZ is generally well tolerated, can easily be administrated orally, and has been shown to cause objective response or stabilization of disease in 50% to 60% of patients with glioblastoma (6), we were encouraged to further explore the TRAIL-TMZ drug combination in vivo. In this study, we evaluated the combination therapy of CED infusion of TRAIL and systemic administration of TMZ for efficacy against a U87MG intracranial xenograft model. Materials and Methods Recombinant TRAIL and Temozolomide. Two recombinant human TRAIL proteins were used in this study. One was soluble recombinant human TRAIL composed of residues 114 to 281 (Calbiochem, San Diego, CA), and the other was the NH 2 -terminal His 6 -tagged recombinant human TRAIL com- posed of residues 95 to 281 (R&D Systems, Minneapolis, MN). All studies were done with the recombinant protein purchased from Calbiochem, except for the distribution study, which used the protein purchased from R&D Systems; to detect only the distribution of injected TRAIL protein without detecting TRAIL protein endogenously expressed by gliomas (7), His-tagged protein and anti-His tag antibody were used for evaluation of distribution. TMZ was provided by the Drug Synthesis and Chemistry Branch, Develop- mental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (Bethesda, MD) and was dissolved in dimethyl sulfoxide (DMSO; Sigma Chemical Co., St. Louis, MO). Tumor Cell Line. An established human glioblastoma multiforme cell line, U87MG, was obtained from the Brain Tumor Research Center Tissue Bank at the University of California, San Francisco. Cells were maintained as monolayers in a complete medium consisting of Eagle’s minimal essential medium supplemented with 10% fetal calf serum and nonessential amino acids. Cells were cultured at 37°C in a humidified atmosphere consisting of 95% air and 5% CO 2 . In vitro Exposure to TRAIL and Temozolomide. On the day before treatment, 5  10 4 cells per well were seeded into a 24-well plate (Corning Inc., Corning, NY). After 24 hours of incubation, cells were exposed to TMZ (0 –50 mol/L), TRAIL (0 –350 ng/mL), or both agents in serum-free medium. DMSO, which was used to dissolve TMZ, had no effect on cell growth at the concentration used [0.1% (v/v)] in this study. After another 24 hours of incubation, cell survival was estimated using a trypan blue exclusion assay. Western Blotting for Cleaved Caspase-8, Cleaved Caspase-3, and Actin. On the day before treatment, 80  10 4 cells per well were seeded into a 6-well plate (Corning Inc.). After 24 hours of incubation, cells were exposed to TMZ (15 mol/L), TRAIL (200 ng/mL), or both agents in serum-free medium. After 6 hours of incubation, cells were collected, and protein was extracted using cell lysis buffer (Cell Signaling Technology, Beverly, MA). Equal amounts of protein were separated by 15% SDS-PAGE and blotted onto PDGF membrane (Bio-Rad, Hercules, CA). The PDGF membrane was incubated in blocking Received 5/12/04; revised 7/28/04; accepted 8/11/04. Grant support: Accelerate Brain Cancer Cure (K. Bankiewicz). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Krystof S. Bankiewicz, Department of Neurological Surgery, University of California at San Francisco, 1855 Folsom Street, Mission Center Building Room 230, San Francisco, CA 94103. Phone: 415-502-3132; Fax: 415-514-2177; E-mail: kbank@itsa.ucsf.edu. ©2004 American Association for Cancer Research. 6858 Research. on November 7, 2015. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from