MRI evaluation of dendritic cell vaccination protocol in a murine model of breast cancer (MMTV-hRas)

Introduction: Dendritic cells (DCs) play a pivotal role in the regulation of immune responses and in tumor immunosurveillance. The aim of this study was the set up and the evaluation using MRI of a tumor-specific DC-based vaccine . Methods: Total bone marrow cells were extracted from wt mice; DC differentiation, culture, labelling and antigen loading was performed as previously published (Martelli et al, Mol Imaging Biol 2011). Cell activation was analysed by RealTime PCR. For the vaccination protocol, MNP labelled and antigen loaded DCs were injected weekly into the footpad of transgenic mice (MMTV-hRas) for three weeks (n=4, starting at 7 weeks of age, n=4 control mice) in presence of TNFα stimulation. MRI was performed -4, 24 and 48h after cells injection- with a 7T Bruker Pharmascan instrument. Weekly MRI screening were done to identify the presence of pre-neoplastic lesions and the early tumor onset. Notably, MRI analysis also permitted to evaluate the composition of the tumor mass (haemorrhagic, necrotic or vital areas). Plasma was collected at multiple time points. Mice were sacrificed 2 weeks after the tumor onset; and spleen, lymph nodes and tumors were collected for ex vivo analysis. Splenocytes were re-exposed to antigen loaded DCs, to evaluate the presence of a specific immune response against tumor antigens. Paraffin embedded tumors were used for immunohistochemical analysis. Results: Gene expression profile showed the up-regulation of multiple genes involved in the processing (e.g. B2m) and presentation (e.g. CD36 and CD40) of antigens, and in the production of cytokines (e.g. IL-12, CXCL10 and CXCL12). MRI showed lymph nodal homing of the iron labelled activated DCs cells injected into the footpad of MMTV-hRas mice; these data were confirmed by immunohistochemistry, showing co-localization of CD208 positive cells (but not macrophages) and iron. MRI screening of mammary glands showed a delay in tumor onset in vaccinated mice compared to control ones (4 weeks). Cytokine quantification in plasma samples showed a statistically significant increase of IL-12 production in vaccinated mice, compared with controls, at tumor onset. Cytofluorimetric analysis of immune cells derived from spleen demonstrated a systemic activation, characterized by an augmented production of perforin by CD8 and NK cells and of activatory cytokines (e.g. IL-2, TNFα e IFNγ) by CD4 and CD8 cells after re-exposition to antigen. Immunohistochemical analysis suggested a shift in the M1/M2 tumor associated macrophages ratio towards the anti-tumoral ones (M1>M2) in vaccinated mice. Conclusions: Dynamic in vivo MRI monitoring during the vaccine protocol provides insight for the early evaluation of a vaccine potential (DC migration to LNs) and for the assessment of tumor onset time. This non-invasive approach offers a powerful and highly translational tool for the set up and optimization of cell mediated treatment in clinical trials.