Induction of Apoptosis Improves Efficacy of Dendritic Cell-Based Immunotherapy in a Murine Model of Pancreatic Cancer

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS epithelial p ¼ 0.709); Table 1. Finally, over 90% of tumor tissue expressed HA, compared to 77% of normal tissue, and no normal tissue expressed high levels of the molecule (p < 0.01); Table 1. Also, in late stage tissue samples (Stage III and IV) there was significant difference in HA expression (p < 0.01). Conclusion: In this study we found that CD44 and HA expression is increased in human colorectal cancer tissue compared to normal colonic tissue. RHAMM expression was not significantly different between normal and tumor tissue, but there was a trend toward increased expression in tumor cells. Moreover, late stage cancer samples expressed higher levels of these molecules compared to early stage samples. These data suggest that CD44, RHAMM and HA may be involved in colorectal cancer progression, and therefore may offer new diagnostic and therapeutic targets.

43.8. Induction of Monocyte Chemoattractant Protein-1 by Nicotine in Pancreatic Ductal Adenocarcinoma Cells: Role of Osteopontin. M. Lazar, J. Sullivan, G. Chipitsyna, T. Aziz, A. Salem, Q. Gong, A. Witkiewicz, C. J. Yeo, H. A. Arafat; Thomas Jefferson University, Philadelphia, PA Introduction: Several epidemiological studies have identified cigarette smoke as the leading environmental risk factor for developing pancreatic ductal adenocarcinoma (PDA). Nicotine, the major component of cigarettes, exists at high concentrations in the bloodstream of smokers. However, the mechanisms by which nicotine contributes to the onset or development of PDA are not clear. We showed recently that in PDA cells, nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers. In this study, we explored the potential proinflammatory role of nicotine in PDA through studying its effect on the expression of monocyte chemoattractant protein- (MCP)-1 and evaluated the role of OPN in mediating these effects. We also analyzed the expression of MCP-1 and OPNc in invasive and premalignant PDA tissues from smokers and non smokers. Methods: MCP-1 mRNA and protein in MiaPaca, AsPC-1 and HS766 T PDA cell lines treated with or without nicotine (3-300 nM) were analyzed by real time PCR and ELISA, respectively. Transient transfection and luciferase-labeled promoter studies evaluated the effects of OPNc and OPN protein on the transcription and translation of MCP-1. Confocal microscopy examined the intracellular localization of OPN and MCP-1 in PDA cells. Real time PCR and immunohistochemistry analyzed the mRNA expression levels and localization of OPNc and MCP-1 protein in invasive PDA (n ¼ 73; 48 smokers and 25 non smokers) and intraductal papillary mucinous neoplasms, IPMN (n ¼ 6; 2 smokers, 4 non smokers). Serum levels of MCP-1 in the different patient groups were analyzed by ELISA. Results: PDA cells expressed variable basal levels of MCP-1. Nicotine treatment both dose-and time-dependently increased MCP-1 expression in all cell lines. Interestingly, blocking OPN with siRNA or OPN antibody prevented these effects. Transient transfection of the OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (P < 0.05) increased MCP-1 mRNA and protein and induced its promoter activity. Confocal microscopy analysis revealed intracytoplasmic colocalization of OPN and MCP-1 in PDA cells. MCP-1 was found in 60% of invasive PDA lesions, of which 66% were smokers. MCP-1 colocalized with OPN in the malignant ducts and correlated well with higher expression levels of OPNc in the tissue from patients with invasive PDA. Conclusions: Our data suggest that cigarette smoking and nicotine may contribute to PDA inflammation through inducing MCP-1 and provide a novel insight into a unique role for OPN in mediating these effects. Although the role of OPN-mediated induction of MCP-1 in pancreatic carcinogen-

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esis remains to be defined, the existence of OPN as a downstream effector of nicotine that is capable of mediating its proinflammatory effects in PDA cells is novel and could provide a unique potential target to control pancreatic cancer aggressiveness, especially in the cigarette smoking population.

ONCOLOGY 6: APOPTOSIS 44.1. Induction of Apoptosis Improves Efficacy of Dendritic Cell-Based Immunotherapy in a Murine Model of Pancreatic Cancer. S. C. Agle, M. Carver, M. M. McNally, R. J. Tracy, E. E. Zervos; East Carolina University Brody School of Medicine, Greenville, NC Introduction: Manipulation of the tumor microenvironment aimed at enhancing shed tumor antigen through induction of apoptosis may be an effective adjunct to adoptive immunotherapy in solid tumors. The purpose of this study was to determine whether pretreatment with TNF prior to dendritic cell (DC) based immunotherapy improves survival in a murine model of pancreatic cancer (PCA). Methods: 6 treatment groups employing a syngeneic PCA cell line (Panc02) heterotopically implanted into C57BL6 mice were designed to measure the effect of TNFerade (a TNF expressing radiation inducible advector) administered prior to intratumoral DC injection. Treatments (control, DC alone, TNF þ XRT, XRT alone, XRT þ DC and TNF þ XRT þ DC) were administered over 4 day intervals for 3 consecutive weeks and consisted of TNFerade (4x1010 pu) followed by XRT (2 Gy on Monday, Tuesday and Wednesday) followed by intratumoral injection of 1 x 106 tumor lysate pulsed DC (Thursday). Intratumoral and serum TNF levels were determined by ELISA and apoptosis determined by cleaved caspase-3 targeted immunohistochemistry. Flow cytometry was used to characterize tumor infiltrating lymphocytes. Finally, tumors were measured twice weekly and survival analyzed using the Kaplan Meier method. Results: Animals receiving TNFerade with DC had significantly slower growing tumors resulting in a significant survival advantage (p < 0.001). This improved survival was associated with sustained and measurable intratumoral TNF levels compared to animals receiving radiation alone (4,1245.6 6 2,607.2 vs 4.7 6 7.4 pg/mg, p < 0.005), serum TNF levels (1205.7 6 895.4 vs 1.7 6 0.8 pg/ml, p ¼ 0.08) and intratumoral cleaved caspase-3 (33 vs 11 positively stained cells/100, p < 0.05). Flow cytometry confirmed enhanced DC infiltration of tumors in all animals receiving DC but failed to discriminate between specific T cell subsets.

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

Conclusion: Addition of temporally and spatially controlled TNF in a DC based adoptive immunotherapy model of PCA results in a significantly altered tumor microenvironment characterized by increased apoptosis resulting in slower tumor growth and improved survival.

44.2. Inhibition of Hyaluronan Synthase-3 Decreases Subcutaneous Tumor Growth by Increasing Apoptosis. B. Teng,1 E. Lai,1 Y. Zhao,2 C. LeVea,2 K. Bullard Dunn2; 1 University at Buffalo, Buffalo, NY; 2Roswell Park Cancer Institute, Buffalo, NY Introduction: Hyaluronan synthases (HAS) have been associated with cancer progression. HCT-116 is a human colon cancer cell line that is highly tumorigenic in mice and expresses high levels of HAS-3. We hypothesized that inhibition of HAS-3 would decrease subcutaneous tumor growth by altering apoptosis and/or proliferation. Methods: HAS-3 expression was inhibited by transfection with siRNA; a scrambled sequence served as a control. Decreased HAS-3 expression was confirmed by RT-PCR. Transfected cells were then injected into the flanks of nude mice. There were 10 mice injected with a scrambled RNA sequence and 18 mice injected with a silenced RNA sequence. Mice were euthanized after 30 days, and tumors measured and weighed. Apoptosis was assessed using Caspase-3 immunostaining; apoptotic index was calculated using the ratio of apoptotic cells to total cells per high powered field. Proliferation was assessed using Ki-67 immunostaining and graded from 1þ (low) to 4þ (high). Results were compared using the Student’s t-test. Results: siRNA transfection decreased HAS-3 expression by 70%. Injection with silenced cells resulted in tumors approximately 50% smaller than those from scrambled cells (Table 1). The silenced cells showed an increase in apoptosis compared to the scrambled cells (apoptotic index: silenced ¼ 3.9 6 2.9% vs. scrambled ¼ 2.6 6 1.4%, p ¼ 0.025). There was no difference in proliferation (silenced ¼ 2.5 6 0.7 vs. scrambled ¼ 2.6 6 0.6). Conclusions: Inhibition of HAS-3 decreased subcutaneous tumor growth by HCT116 colon cancer cells and increased apoptosis. These data suggest that inhibition of tumor growth by silencing HAS-3 is mediated by increased apoptosis. This observation suggests that HAS-3 may enhance colon cancer growth by inhibiting programmed cell death.

44.3. In Vitro and in Vivo Radiosensitization of Colorectal Cancer HT-29 Cells by the Smac-Mimetic JP-1201. S. Huerta, X. Gao, T. Anthony, E. H. Livingston; University of Texas Southwestern, Dallas, TX Background: Ionizing radiation (IR) is currently used neoadvantly in stage II/III rectal cancer. IR, however, results in a wide range of a clinical response. The mechanisms of radioresistance in rectal cancer remain unclear. However, the inhibitors of apoptosis (IAPs) have been shown to be involved in radioresistance. This study was undertaken to assess the radiosensitizing properties of the Smac-mimetic JP-1201 in radioresistant HT-29 colorectal cancer cells in vitro and its established xenografts in SCID mice. Methods: survival was determined by clonogenic assays, apoptosis by PARP-1 cleavage assessed by Western blot analysis. SCID mice bearing HT-29 xenografts were treated with ionizing radiation [2.0 Gy X 5; n ¼ 8], JP1201 [6 mg/Kg i.p. n ¼ 5] or combination treatment (n ¼ 7) and com-

pared to control (n ¼ 8). Results: colorectal cancer HT-29-p53-null cells were resistant IR. Pre-treatment of HT-29 cells with JP-1201 [5.0 mM] 16 h prior to IR significantly radiosensitized these cells at 2.0, 4.0, and 6.0 Gy. Radiosensitization was achieved with a substantial elevation of cleaved PARP-1 in JP-1201-HT-29-treated vs. control cells. Following 40 d of treatment, SCID mice bearing HT-29 xenografts demonstrated no difference in tumor load in the group receiving exclusively JP-1201 vs. control (2660.7 6 680.3 vs. 2715.9 6 281.9 mm3, respectively; Figure 1). There was a 64.0% reduction in tumor load in the IR-treated group vs. control (969.6 6 281.9 vs. 2715.9 6 281.9 mm3; p < 0.001). An 80.4% decrease in tumor load in the JP-1201 þ IR-treated group vs. control was observed (525.3 6 110.5 vs. 2715.9 6 281.9 mm3; p < 0.001). Conclusions: The smac-mimetic JP-1201 radiosensitized HT-29 cells in vitro and in vivo by an additive effect in apoptosis.

44.4. Bioengineered Human L-Arginase Is Cytotoxic to Hepatocellular and Pancreatic Carcinoma Cells through Induction of Proapoptotic Pathways. E. S. Glazer,1 E. M. Stone,2 W. D. Kaluarachchi,1 L. Chantranupong,2 K. L. Massey,1 G. Georgiou,2 S. A. Curley1; 1The University of Texas M.D. Anderson Cancer Center, Houston, TX; 2The University of Texas-Austin, Austin, TX Introduction: Hepatocellular and pancreatic carcinomas are two human malignancies that have some degree of auxotrophy for Larginine, an otherwise non-essential amino acid. L-arginine is brought into the cell via non-specific transporters or produced from citrulline via the urea cycle. Hepatocellular and pancreatic cancers are not able to produce arginine from citrulline and are thus dependent on circulating arginine. The purpose of this study was 1) to demonstrate anti-cancer activity for a cobalt-substituted bioengineered human L-arginase and 2) to define the mechanism of L-arginase-induced anti-cancer activity. Methods: A pancreatic carcinoma cell line, Panc-1, and a hepatocellular carcinoma cell line, Hep3B, were exposed to bioengineered (increased activity at physiologic pH) L-arginase at concentrations of 100 nM, 1 nM, or 0 nM (PBS controls) in replicates of n ¼ 4. Nuclear Ki-67 expression, activation of the apoptotic enzyme caspase-3, and expression of the urea cycle enzyme argininosuccinate synthetase-1 (ASS-1) was determined by multi-parameter flow cytometry after treatment. In addition, MTT assays determined proliferation at multiple time points after treatment. Results: Viability decreased by 31% 6 2% for Panc-1 cells (p < 0.0001) and 34% 6 1% (p < 0.0001) for Hep 3B cells after high-dose (100 nM) L-arginase treatment compared to controls. In addition, there was an over four-fold increase in cells with activated caspase-3 expression in both cell lines (p < 0.01) after high-dose treatment (See figure for Hep3B cells). At the same time,