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Adunka, Tina (2014): Characterization of murine pancreatic carcinoma models regarding immunosuppressive mechanisms and therapy with bifunctional siRNA targeting galectin-1. Dissertation, LMU München: Medizinische Fakultät



Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor that is characterized by abundant tumor stroma and a potent immunosuppressive microenvironment. Further studies to clarify why T cells infiltrate the tumor but are not able to perform effector functions as well as to find new effective therapies to overcome immunosuppression are urgently needed. The aims of the present study were (1) to characterize different murine PDAC tumor models in regard to their utility for studying novel immunotherapeutic approaches, (2) to assess the therapeutic efficacy of a novel bifunctional ppp-siRNA that combines silencing of the immunosuppressive molecule galectin-1 and RIG-I-mediated immune activation in murine PDAC and (3) to characterize the immunosuppressive mechanisms leading to T cell inhibition in the tumor microenvironment. (1) This study revealed that the pancreatic cancer cell line T110299, which was developed from a primary tumor of the KPC mouse model, is a new valuable tool for studying novel treatment strategies for PDAC. The histological appearance of T110299 tumors reflects in many aspects the primary tumors in KPC mice, which harbor mutations in the Kras oncogene and p53, and the human disease with regard to tumor differentiation, extensive tumor stroma development, poor vascularization and expression of immunosuppressive molecules, like indoleamine 2,3-dioxygenase (IDO) and galectin-1 (Gal-1). In contrast, Panc02 tumors were found to have a sarcomatoid architecture with very little tumor stroma. In Panc02 cells, galectin-1 was strongly expressed by the tumor cells, which differs from the situation found in humans, KPC mice and T110299 tumors, where galectin-1 is preferentially expressed in tumor-associated pancreatic stellate cells (PSC). However, expression of the cytosolic helicase RIG-I was functional in Panc02 cells, but defective in T110299 cells. As RIG-I is expressed in all human PDAC cell lines tested, the Panc02 model appears to be better suited to study RIG-I-based immunotherapies (Ellermeier et al., 2013). Thus, the histological and functional characterization of the tumor models in this thesis will allow selecting the best-suited tumor model for addressing specific aspects of immunotherapy. (2) Treatment studies of PDAC were performed with the 5’ppp-modified siRNA molecule ppp-Gal-1 in the orthotopic Panc02 tumor model. The dual activities of this molecule were confirmed in vitro, leading to (i) reduced galectin-1 expression via RNAi; and (ii) production of CXCL10 and IFN-, MHC-I up-regulation and apoptosis of tumor cells via RIG-I activation. Treatment of mice with orthotopic pancreatic tumors with ppp-Gal-1 significantly prolonged survival, as compared to unmodified OH-Gal-1 or control RNA. In addition, 20% of the mice completely rejected their tumors leading to long-term tumor control. Thus, bifunctional 5’ppp-modified siRNA is a promising treatment strategy for PDAC deserving further pre-clinical evaluation. (3) Pancreatic tumor cells employ multiple mechanisms for suppression of T cell responses. This study identified TGF-β and IDO as two potent mechanisms leading to inhibition of T cell proliferation. Minute amounts of PDAC supernatants effectively blocked T cell proliferation induced by CD3 and CD28 triggering. This could be partially prevented by SD-208, a small molecule inhibitor of TGF-β receptor signaling, or by blocking IDO activity with D-1-MT. Interestingly, tumor supernatants induced up-regulation of IDO mRNA expression in T cells. Furthermore, blocking IDO activity in T cells appeared to be more effective than blocking IDO in tumor cells. This leads to a new hypothesis that factors secreted by the tumor cells induce IDO expression in T cells, which in turn leads to auto-intoxication of the T cells via kynurenine production and eventually T cell apoptosis. Further studies confirming this hypothesis are warranted.