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Combination therapy with TRA-8 anti-death receptor-5 antibody significantly reduces pancreatic adenocarcinoma cell viability in vitro and growth in vivo
Leo C. DeRosier1, Zhi Huang1, Jeffrey Sellers2, Kurt Zinn3, Donald Buchsbaum2, Selwyn Vickers1; 1Department of Surgery, University of Alabama, Birmingham, AL; 2Department of Radiation Oncology, Univeristy of Alabama School of Medicine, Birmingham, AL; 3Department of Medicine, Univeristy of Alabama School of Medicine, Birmingham, AL

Gemcitabine (Gem) is a first line agent for pancreatic cancer, but yields minimal survival benefit. This study evaluated in vitro and in vivo effects of a monoclonal antibody (TRA-8) to human death receptor 5 (TRAIL R2), combined with gem, using two human pancreatic cancer cell lines, S2VP10 (S2) and MIA PaCa-2 (M2). A subcutaneous and intrapancreatic model of pancreatic cancer with ultrasound monitoring was developed to test in vivo efficacy. S2 and M2 cells were treated with varying doses of gem and TRA-8. Cell viability was determined using an ATP assay and apoptosis was verified with Annexin V staining. Mitochondrial membrane destabilization was evaluated with FACS analysis of JC-1 stained cells. Signal transduction was evaluated by Western blot analysis. M2 subcutaneous xenografts in athymic nude mice were evaluated for response to treatment: 200µg of TRA-8 (days 9, 13, 16, 20, 23, 27 post-implant) and 120mg/kg gem (days 10, 17, 24). Tumors were measured with calipers. Intrapancreatic tumors were established by injecting 2.5x106 M2 cells into the pancreas of SCID mice. Tumor growth was measured by ultrasound imaging on post-implant days 21 and 41. M2 cells demonstrated sensitivity to TRA-8 (IC50=15.9ng/ml) and additive reduction in cell viability in combination with gem. Combination treatment produced synergistic reduction in cell viability in S2 cells (TRA-8 resistant, IC50>1000ng/ml). M2 and S2 cells receiving combination treatment demonstrated enhanced Annexin V staining and mitochondrial destabilization compared to either agent alone. X-linked inhibitor of apoptosis (XIAP) decreased more in S2VP10 cells receiving combination treatment, than with either agent alone. Enhanced caspase-3 activation was observed in both cell lines receiving combination treatment. In vivo studies demonstrated mean subcutaneous tumor doubling times of 38d-untreated, 32d-gem, 49d-TRA-8, and 64d-combination. Using ultrasound measurements, the mean intrapancreatic tumor size (n=10) at 21d post implant was 42±9mm2 and 106±15mm2 at 41d. TRA-8 is an apoptosis inducing monoclonal antibody with synergistic effects with gem, possibly through enhanced caspase activation and downregulation of XIAP. These findings, with substantial inhibition of tumor growth in a mouse xenograft model receiving combination therapy, are encouraging for anti-death receptor therapy in the treatment of pancreatic cancer. An intrapancreatic model of pancreatic cancer can be followed using ultrasound measurement without sacrifice of study animals, providing a relevant model for evaluating novel therapies for pancreatic cancer.

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