Dendritic Cell Labelling with Paramagnetic Nanoparticles, 111In-Oxine and PKH26 for In Vivo Magnetic Resonance, SPECT and Optical Imaging of their Migration

Aim: The aim of this study is the development and the evaluation of a tumour-specific DC vaccine in a transgenic murine model of breast cancer (MMTV-v-Ha-Ras). DC labelling with commercial paramagnetic nanoparticles (MNPs, Endorem®), 111In-Oxine or PKH26 will permit to study their distribution and migration to local lymph-nodes by MRI, SPECT or optical imaging, respectively. Methods: Total bone marrow cells were extracted from wt mice. DC differentiation was studied by flow cytometry; at the 6th day of culture DCs were labelled with commercial MNPs, 111In-Oxine or PKH26. Dose response and kinetic of labelling studies were performed. Labelling efficiency was evaluated. iDCs were loaded with tumour antigens and maturation was monitored by flow cytometry. Stimulatory activity of antigen-loaded DCs was evaluated by T-cell proliferation and IFN-γ production by T cells; DC migration was evaluated in the presence of 6Ckine, MIP-3β, MIP-1α and MIP-1β. MNP-labelled DCs, loaded with tumour antigens were injected into the footpad of transgenic tumour bearing mice. Perl’s staining of the draining lymph nodes was carried out to evaluate DC migration. Results: FACS analyses identified the best time point to perform DC labelling. Results showed that labelling efficiency with MNPs was proportional to the medium iron content and to the incubation time (ideal conditions 200 μg Fe/ml, for 16h with 85% efficiency) as confirmed by relaxometric assay. Labelling with MNPs did not affect DC immune-phenotype or functionality as demonstrated by CD86 and CD83 expression levels, and by LPS induction of labelled DC maturation. Labelling with 30 and 60 μCi of 111In-Oxine resulted in a 75% and 72% efficiency, respectively. Concurrent DC labelling with PKH26 and 30 μCi of 111In-Oxine did not impair labelling efficiency. DC vitality was not highly affected by all the labelling procedures. Antigen-loaded DCs induced T-cell proliferation and INF-γ production. Migration assays showed that antigen-loaded DCs were able to migrate in the presence of stimulatory chemokines (6Ckine and MIP3β). Perl’s staining of lymph node sections after in vivo injection of labelled DCs loaded with the antigens, showed the presence of iron within the node, indicating that mature and labelled DCs migrate in vivo from the site of injection to the draining lymph node. Conclusions: Cell labelling with different probes for multimodal imaging of DCs will permit to overcome the limitation of the single imaging techniques and to in vivo visualise their migration and limph-node localisation, shedding light on the fundamental parameters responsible for the anti-neoplasic efficacy