DENDRITIC CELLS IN A MURINE MODEL OF BREAST CANCER: FUNCTIONAL AND PHENOTYPIC EVALUATION USING NANOPARTICLES

Dendritic cells play a pivotal role in antigen presentation and adaptive immune response activation. Studies on mice models reported that tumoral antigens-loaded DCs can induce therapeutic and protective anti-tumour immune responses, both in vitro and in vivo. These results provide an evidence of the possible use of DCs as vaccines. However, the efficacy of such therapeutic vaccination was recently questioned due to the limited degree of actual tumour regression in some clinical trials. Thus, it is necessary to better characterize immunological and clinical parameters of DCs immunization to increase the therapeutic efficay of the vaccines. In vivo imaging tecniques, as magnetic resonance imaging (MRI) and single photon emission tomography (SPET), are used to study cell trafficking mechanisms in vivo and state anti-tumoral treatments efficacy, allowing an accurate assessment. In this work we initially set the optimal culture conditions to obtain functional immature DCs. Evaluations based on DCs number, cell viability and immature DCs percentage led to establish day 6 as the one in which the highest number of functionally active immature DCs can be achieved. We then set a DCs labelling protocol with commercial SPIO (Endorem®) evaluating Iron content using Perl’s staining and relassometry. Time and dose-response kinetic evaluations allowed to define as optimal labelling conditions the use of 200 μg Iron of Endorem®/ ml of culture for 16 hours. Cell viability and phenotypic markers analysis proved that SPIO labelling had no influence on immature DCs viability or activation status. Images acquired by transmission electron microscopy showed that Endorem® were inside cells, in particular inside lysosomes, and were taken up by endocytosis mechanisms. We then focused on DCs antigens loading. Tumoral antigens were obtained lysing neoplastic masses extracted from a mammary tumour mouse model (MMTV-Ha-Ras). DCs pulsed with different antigen concentrations resulted mature by means of high levels of activation and maturation markers. Moreover, such cells were able to stimulate T lymphocytes extracted from MMTV-Ha-Ras spleens. 3H-thymidine and CFSE proliferation assays, together with IFN-γ production measured by ELISA, showed an increase in T lymphocytes activation when co-cultured with antigen-pulsed DCs. Cytokines production assessed by flow cytometry and ELISA allowed us to verify that tumoral antigen-pulsed DCs were able to elicit a specific T-lymphocyte response toward a Th1 type. Indeed, high amounts of IFN-γ were produced by T lymphocytes, high levels of IL-12 were produced by DCs and low levels of IL-10 were detectable. In vitro migration showed that tumoral antigen-pulsed DCs displayed migratory abilities toward MIP3β and 6Ckine chemockines, responsible in vivo of mature DCs attraction. Flow cytometric analysis confirmed that migrated cells displayed a mature phenotype and expressed high levels of CCR7. We then performed in vivo experiments in MMTV-Ha-Ras mice bearing neoplastic lesions and in control mice without tumours. MRI showed that Endorem®-labelled DCs pulsed with tumour antigens are able to migrate to the draining lymph site in vivo. Histological analysis on ex vivo lymph nodes showed the presence of Iron-labelled cells in the cortical area. Immunohistochemistry reveals co-localization of Iron-containing cells with CD208-labelled DCs, but not with macrophages. In vivo SPET migration of antigen-pulsed DCs labelled with 111In-oxine showed specific radiotracer presence in the lymph nodes omolateral to injection site; γ-counter analysis of ex-vivo organs confirmed such results. Taken together, these results suggest that this protocol could candidate for experimental use on animal models for in vivo tumour immunotherapy efficacy imaging.