Experimental strategies for the in vivo imaging of cell-mediated therapies in cancer and myopathies

Introduction. Cell-mediated therapies have acquired great importance in the last years and numerous protocols based on the use of dendritic cells in anti-neoplastic immunotherapies and of stem cells in regenerative medicine were described. However, important parameters such as the distribution and survival of the injected cells, the target organ localisation and the cell proliferation and differentiation deserve being deeply evaluated in order to improve the efficacy of cell-mediated treatments. Here we propose some experimental strategies for in vivo imaging by MRI and SPET of dendritic cell-mediated immunotherapy treatments, in a transgenic murine model of breast cancer (MMTV-v-Ha-Ras) and labelling protocols for in vivo visualisation by MRI of human mesoangioblasts to be used in the treatment of inflammatory myopathies. Materials and Methods. Total bone marrow cells were extracted from wt mice. DC differentiation was studied by flow cytometry; on the 6 day of culture DCs were labelled with commercial MNPs. Dose-response and kinetic of labelling studies were performed (100-200-400 μg of iron/ml, for 6-16-24-48 hours). Labelling efficiency was evaluated by optical microscopy after Perl’s staining and relaxometric analysis. Tumour lysates from breast cancer lesions of transgenic mice were used to load immature DCs (iDCs) 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; their migratory ability was evaluated in the presence of 6Ckine, MIP-3, MIP-1 and MIP-1. Two millions of MNP-labelled DCs loaded with tumour antigens were then injected into the footpad of a transgenic tumour bearing mouse. Then, imaging was performed by use of a 7T MR scanner (Bruker) after 4, 24 and 48 hours from cell administration by use of MSME and FLASH sequences. After 48 hours, Perl’s staining was carried out on the section of the collected draining lymph node to evaluate DC migration. DCs were also labelled with different amounts of 111In-oxine, and labelling efficiency and viability analyses were performed. Two millions of labelled and antigen-loaded DCs were injected in the murine model, and SPET imaging with a new prototype of γ-camera (DRAGO, in collaboration with the Politecnico of Milan) was performed after 4, 24 and 48 hours from cell injection. Ex vivo analysis of the collected lymph nodes was then carried out to evaluate the presence of radioactivity due to migrating DCs. Human mesoangioblasts were cultured in collaboration with a team from the Catholic University of Rome, and labelled for 24 and 48h with different amounts of MNPs (Endorem®, 0-100-200-400 μg of iron/ml) in the presence or absence of transfection agents (Poly-L-Lysine or Polybrene). Cells were washed with PBS and analysed for viability, iron content (Perl’s staining) and morphology or plated for further analysis of multipotency or differentiation capability. One million of mesoangioblasts labelled with 200 μg of iron/mL in presence of PLL for 48h were washed twice and injected intramuscularly or in the femoral artery into SCID mice after N2 damage of the tibial posterior muscle. MRI imaging was performed 1h and 3 days after injection using a 7T MR scanner (Bruker). Mice were acquired prone: multislice Spin-Echo (MSME) and 3D gradient Echo (FLASH 3D) sequences were performed. Results. The cytofluorimetric analyses identified the best time point to perform labelling of DCs with MNPs (maximum iDCs:mDCs ratio) on day 6 of culture. Results showed that the labelling efficiency was proportional to the iron content in the medium and to the incubation time. The ideal labelling condition (85% efficiency) was identified as being 200ug Fe/ml for 16h. Viability was not deeply affected by the MNP internalisation. Relassometric assay showed that the T2 reduction is proportional to the iron content. Labelling with MNPs did affect neither DC immune-phenotype nor functionality as demonstrated by the expression levels of CD86 and CD83 and by the observation that LPS stimulation induced maturation of the labelled DCs. Antigen-loaded DCs induced T-cell proliferation and INF-γ production; the 1:5 and 1:10 (DCs-tumour cells) ratio resulted in the highest efficiency of DC maturation as demonstrated by the MHCII expression levels. Migration assays showed that antigen-loaded DCs, also in presence of MNPs, were able to migrate in the presence of stimulatory chemokines (6Ckine and MIP3). Imaging through Magnetic Resonance and SPET evidenced the presence of MNPs- or 111In-labelled and antigen-loaded DCs in the draining lymph nodes. Perl’s staining of lymph node sections confirmed these data, indicating that mature and labelled DCs migrate in vivo from the site of injection to the draining lymph node; moreover ex vivo analysis of colleted lymph nodes showed the presence of radioactivity in the nodes omolateral to the injection site. After thawing, human mesoangioblasts were cultured and incubated for 24 or 48 hours with 0-50-100-200 μg of iron/ml in presence of transfection agents (PLL and PB), and no difference in terms of viability was observed between labelled and non-labelled cells in the presence or absence of PLL or PB, except for the case of 200 μg of iron/ml for 24 hours in the presence of PLL. On the contrary, the percentage of Iron-positive cells increased in proportion to the iron content in the medium and in the presence of PLL. In particular we obtained 99,6%+1 iron-positive cells in the samples incubated with 200 μg of iron/ml for 48 hours with PLL present. The labelling procedures determined a reduction in T2 time in all the conditions, revealing that the signal was stronger at 24 hours than at 48 hours: the signal was proportional to the iron content. Iron-labelled cells, cultured for a further 5 passages, maintained the phenotypic characteristics of non labelled human mesoangioblasts, as revealed by cytofluorimetric analysis. Growth of labelled cells was comparable to control cells and they maintained their ability to differentiate and form myotubes. As no differences between controls and cells incubated with 200 μg of iron/ml for 48 hours in presence of PLL were present, this condition was deemed ideal for cell labelling and in vivo visualisation by MRI. Imaging evidenced the presence of MNPs-labelled mesoangioblasts in the site of intramuscular injections and in the damaged muscles. Perl’s staining of muscle sections confirmed these data, indicating that labelled MBs migrate in vivo from the site of injection to the muscle injury. Conclusions. The isolation of the murine dendritic cell population proved efficient while the labelling protocols with MNPs did not affect DC physiology and functionality. Cultured DCs maintained their ability to migrate in response to specific chemokines and to activate T cells. Moreover, the protocol for 111In-oxine permitted labelling cells with high efficiency and without influencing the cell viability. Dynamic in vivo MRI and SPET monitoring of these cells permitted to follow the migration and localization of DCs in the animal model. The labelling of human mesoangioblasts with MNPs for visualisation by MRI was as efficient as DCs protocols and no alterations in cell phenotype and morphology was observed. MRI allowed to study the fate of these cells once injected into the recipient SCID mice after muscle injury. The possibility of tracking injected cells will shed light on the fundamental parameters responsible for the cell-mediated therapy efficacy (such as the administration route, the amount of cells, the adjuvant treatment, etc.), while the use of clinically approved MNPs will speed up the transfer from pre-clinical investigations to clinical applications.