Development of a model of humanized mouse to study the cross-talk between tumor and immune system and of a therapeutic approach for pancreatic cancer with human dendritic cells loaded with post-apoptotic tumors

Pancreatic cancer has a poor prognosis and the 5-years survival rate is less than 1%, with a median survival of 4–6 months. For this reason, new therapies are needed and immunotherapy might be an option. The development of a preclinical model to study in vivo the cross-talk between tumor and immune system can be crucial. The initial aim of this study was the reconstitution of newborn immunodeficient mice with intrahepatically injection of bone marrow-derived leukocyte precursor CD34+ cells from pancreatic cancer patients, in order to obtain patient-specific humanized mice, to be implanted subcutaneously or orthotopically with pancreatic tumor cells from the same patient; such a system should allow to study in vivo the cross-talk between tumor and immune system and to evaluate immunotherapeutic approaches in a “patient-specific setting“. Unfortunately, burocratic problems hampered so far the acquisition of bone marrow-derived leukocyte precursor CD34+ cells from pancreatic cancer patients as well as from healthy donors. Therefore, we reprogrammed the the study and: 1- we established protocols of delivery of human PBMCs to C57BL/6-Rag2-/-  chain-/- immunodeficient mice, starting from either newborn or adult mice; 2- we developed an adoptive cell transfer (ACT) therapy based on human T lymphocytes activated in vitro by autologous DCs loaded with necrotic pancreatic tumor cells after hyperthermia or UVC treatment, to be delivered to C57BL/6-Rag2-/-  chain-/- mice bearing the same human pancreatic tumor from which the necrotic cells used for DC loading were derived. As expected, the reconstitution we obtained using human PBMCs regarded only the human T and B cells compartment, and not cells of the innate immunity. The differences observed in mice reconstituted by injection of human PBMCs at newborn versus mice injected at adult age concerned mortality and the presence of T cells in peripheral blood. Mice injected at newborn age showed very high mortality following injection (67%) and relatively low percentage of reconstituted animals (45% of alive animals). Nevertheless reconstituted mice presented human lymphoid cells either circulating in the blood or in lymphoid organs. On the contrary, mice injected with human PBMCs at adult age did not show any mortality effect, but the amounts of circulating human lymphoid cells in the blood was very low, even if engrafment of human cells in lymphoid organs was similar to that found in mice injected at newborn and adult age. The second part of work presented is focused on the development of an immunotherapeutic approach for pancreatic cancer. The immunogenic potential of dying tumor cells is receiving great attention on account of both its importance in enhancing T-cell directed immunotherapy and its indication of the best immunogenic source for ex vivo TAA DCs loading (Lake et al., 2005; Nowak et al., 2006). DCs are potent antigen-presenting cells and, as recently shown, can be activated by DAMPs, such as HMGB-1 and HSP-70, that are highly expressed after hyperthermia treatment of cancer cells and that induce anti-tumor immunity (Shi et al., 2006; Bianchi et al., 2007; Chen et al., 2009). Antigen presentation by DCs is essential to effective antitumor T cells responses in cancer patients, but it depends on DCs origin, maturation state and the environmental cytokine milieu. For this reason, we focused our attention on the activation of a tumor-protective immune response using human PaCa-44, PT-45, PANC-1 and PANC-2 pancreatic tumor cell lines. We developed an immunotherapeutic strategy based on human dendritic cells from healthy blood donors loaded with cancer cells in secondary necrosis due to incubation at 56°C or exposure to UVC. This immunotherapeutic approach appears promising, especially using UVC exposure as tumor death treatment. The treatments induced apoptosis followed by secondary necrosis and the release in the supernatant of levels of DAMPs much higher than without treatment. DAMPs are responsible for stimulation of DCs to antigen uptake and maturation, and consequently, for an increased immunogenity of tumor cells, as shown by the expression of higher levels of maturation markers such as CD80, CD83 and MHC-II. Secondary necrosis also resulted in an improvement of uptake efficiency by DCs for all the four different pancreatic cancer cell lines. The specific immune response obtained when T cells from the same donor as DCs were added to activated DCs according to standard protocols, was variable between different pancreatic cancer cell lines and it was also blood donor-dependent: a specific immune response was obtained in vitro only against PaCa-44 and PT-45 cancer cell lines, and not against PANC-1 and PANC-2 cell lines. These results were also confirmed in vivo in a protocol of curative immunotherapy using PaCa-44 and PT-45 cancer cells only, even if the low number of T-lymphocytes injected and of animals for each groups are a crucial points for the assessment of a functional preclinical model.