Deregulated TGF-b signaling in pancreatic cancer promotes tumor growth, invasion, metastasis, and a potent immunosuppressive network. A strategy for disrupting this tumor-promoting pathway is silencing TGF-b by siRNA. By introducing a triphosphate group at the 5 0 end of siRNA (ppp-siRNA), gene silencing can be combined with immune activation via the cytosolic helicase retinoic acid-inducible gene I (RIG-I), a ubiquitously expressed receptor recognizing viral RNA. We validated RIG-I as a therapeutic target by showing that activation of RIG-I in pancreatic carcinoma cells induced IRF-3 phosphorylation, production of type I IFN, the chemokine CXCL10, as well as caspase-9-mediated tumor cell apoptosis. Next, we generated a bifunctional ppp-siRNA that combines RIG-

Regulatory T cells (Treg) mediate tolerance towards self-antigens by suppression of innate and adaptive immunity. In cancer patients, tumor-infiltrating FoxP31 Treg suppress local anti-tumor immune responses and are often associated with poor prognosis. Markers that are selectively expressed on tumor-infiltrating Treg may serve as targets for immunotherapy of cancer. Here we show that CD103, an integrin mediating lymphocyte retention in epithelial tissues, is expressed at high levels on tumor-infiltrating FoxP31 Treg in several types of murine cancer. In the CT26 model of colon cancer up to 90% of the intratumoral FoxP31 cells expressed CD103 compared to less than 20% in lymphoid organs. CD1031 Treg suppressed T effector cell activation more strongly than CD103 neg Treg. Expression of CD103 on Treg closely correlated with intratumoral levels of transforming growth factor b (TGF-b) and could be induced in a TGF-b-dependent manner by tumor cell lines. In vivo, gene silencing of TGF-b reduced the frequency of CD1031 Treg, demonstrating that CD103 expression on tumor-infiltrating Treg is driven by intratumoral TGF-b. Functional blockade of CD103 using a monoclonal antibody did however not reduce the number of intratumoral Treg, indicating that CD103 is not involved in homing or retention of FoxP31 cells in the tumor tissue.In conclusion, expression of CD103 is a hallmark of Treg that infiltrate TGF-b-secreting tumors. CD103 thus represents an interesting target for selective depletion of tumor-infiltrating Treg, a strategy that may help to improve anti-cancer therapy.

Vaccines based on immune stimulatory complexes (ISCOM) induce T-cell responses against tumor antigen (Ag). However, immune responses are impaired in pancreatic cancer patients. We investigated the efficacy of an ISCOM vaccine in a murine pancreatic carcinoma model. Panc02 cells expressing OVA as a model Ag were induced subcutaneously or orthotopically in the pancreas of C57BL/6 mice. Treatment consisted of an OVA containing ISCOM vaccine, either used alone or in combination with the TLR9 agonist CpG Pancreatic cancer is one of the most fatal malignancies in the Western world. It is the fourth leading cause of cancer death in the United States. 1 The 5-year survival rate is less than 5% with a median overall survival time of 3-6 months. Despite advancements made over the past two decades in elucidating molecular pathways involved in pancreatic carcinogenesis and in the field of targeted therapy, the clinical outcome has not yet significantly improved. At the time of diagnosis the majority of patients present with locally advanced, unresectable tumors or metastatic disease. Even in the small number of patients who undergo surgery in a curative intention most patients succumb to recurrent and metastatic disease. Therefore, new treatments are urgently needed.Immunotherapy may offer a new treatment option. Pancreatic carcinoma cells can be recognized by T cells, which are found in the blood of pancreatic carcinoma patients.2 Tumor infiltration with T cells represents a positive prognostic factor.3 However, pancreatic carcinomas promote systemic and locally active immunosuppressive mechanisms.4 These include inhibition of T-cell activation, secretion of immunosuppressive cytokines, defects in Ag presentation and recruitment of regulatory T cells (Treg), a subgroup of CD4 þ T cells with suppressor function. 5,6 In patients with pancreatic carcinoma, increased numbers of Treg are found in the

<p>PDF file - 106K</p>

<p>PDF file - 702K, RIG-I expression in human pancreatic carcinoma specimens</p>

<p>PDF file - 60K, CXCL10 production and lymphocyte activation induced by ppp TGF-β treatment is independent of TRIF or TLR7 signaling</p>

<p>PDF file - 46K, Bi-functional ppp-siRNA directed against TGF-β1 combines gene silencing with RIG-I signaling in human pancreatic cancer cells</p>

A hallmark of pancreatic cancer is deregulated transforming growth factor-b (TGF-b) signaling which promotes tumor growth and entertains a potent immunosuppressive network correlating with poor prognosis. To brake immune suppression, we designed siRNA molecules silencing TGF-b1 and introduced a 5’ppp-modification (ppp-TGF-b) to additionally activate the cytosolic helicase retinoic acid-inducible gene I (RIG-I). RIG-I recognizes ppp-RNA motives in viral RNA and triggers type-I IFN production. We found that human pancreatic cancer cells express functional RIG-I. Treatment with ppp-RNA resulted in IRF-3 phosphorylation, type-I IFN and CXCL10 secretion, and tumor cell apoptosis. Treatment of mice with syngeneic orthotopic pancreatic tumors with ppp-TGF-b (i.v.) resulted in reduced tumor-associated TGF-b levels, systemic immune activation and high IFN levels in serum and tumor. Furthermore, ppp-TGF-b induced caspase-9-mediated tumor cell apoptosis in vivo and significantly prolonged survival of tumor-bearing mice. The development of bifunctional ppp-siRNAs that combine TGF-b silencing and RIG-I activation in one molecule represents an innovative strategy for the treatment of pancreatic carcinoma.