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Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about &minus, 5 &mdash, &minus, 10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

In designing new tracers consisting of a small peptide conjugated to a reporter of comparable size, particular attention needs to be paid to the selection of the reporter group, which can dictate both the in vitro and the in vivo performances of the whole conjugate. In the case of fluorescent tracers, this is particularly true given the large numbers of available dye moieties differing in their structures and properties. Here, we have investigated the in vitro and in vivo properties of a novel series of MMP-12 selective probes composed of cyanine dyes varying in their structure, net charge, and hydrophilic character, tethered through a linker to a potent and specific MMP-12 phosphinic pseudopeptide inhibitor. The impact of linker length has been also explored. The crystallographic structure of one tracer in complex with MMP-12 has been obtained, providing the first crystal structure of a Cy5.5-derived probe and confirming that the binding of the targeting moiety is unaffected. MMP-12 remains the tracers' privileged target, as attested by their affinity selectivity profile evaluated in solution toward a panel of 12 metalloproteases. In vivo assessment of four selected probes has highlighted not only the impact of the dye structure but also that of the linker length on the probes' blood clearance rates and their biodistributions. These experiments have also provided valuable data on the stability of the dye moieties in vivo. This has permitted the identification of one probe, which combines favorable binding to MMP-12 in solution and on cells with optimized in vivo performance including blood clearance rate suitable for short-time imaging. Through this series of tracers, we have identified various critical factors modulating the tracers' in vivo behavior, which is both useful for the development and optimization of MMP-12 selective radiolabeled tracers and informative for the design of fluorescent probes in general.

Background: The ability of tumor cells to spread and form metastases is one of the key parameters used to classify tumor aggressiveness. Metastasis detection at diagnosis or during followup post-surgery is associated with poor prognosis. The paucity of pertinent models to investigate the biology of metastasis renders problematic the set-up of anti-metastasis therapies. We took advantage of our human patient-derived xenograft (PDX) collection to develop models suitable to assess anti-metastatic therapy. To enable evaluation of metastasis response to treatment, we labeled tumors with lentivirus-mediated luciferase insertion, allowing longitudinal followup of metastasis formation by non-invasive imaging. A review of our metastatic bioluminescent PDX panel is described here with a particular focus on our most advanced bioluminescent breast model HBCx-14-Luc1. Methods: Tumor cells from freshly excised PDXs were dissociated and transduced with a defective lentivirus bearing the firefly luciferase gene. One million transduced cells were injected subcutaneously into immunocompromised mice (Nude or SCID), tumor growth and metastasis formation were monitored throughout passages by whole body visualization and ex vivo organ imaging at the end point using the Xenogen IVIS® Lumina. Bioluminescence was measured every two weeks, over 8 to 20 weeks, to assess metastasis appearance and progression. The presence of metastases was confirmed by histological analysis. Results: Lung metastases developed in a variety of PDX models (breast, prostate, pancreas, and melanoma) with different output according to the model and the mouse strain used. SCID mice were more permissive to metastasis development than nude mice. In our most advanced model, HBCx-14-Luc1, the influence of primary tumor resection was assessed on both lung metastasis apparition delay and frequency of metastasis positive mice. Regarding metastasis positive mice at the end point, no difference was observed between resected (7/8 positive at D88) and non-resected mice (11/13 at D93). However, while the first day of metastasis apparition was similar between non-resected (D48) and resected mice (D53), the number of metastasis positive mice at this early time-point was higher for the non-resected (7/8) compared to resected mice (3/18). Histological examination confirmed the adenocarcinomatous origin of metastases. Another breast PDX model, HBCx-5-Luc2, was metastatic for pancreas, brain, spleen, liver, lung, and bones as assessed by ex vivo screening. Additional PDX models were screened similarly to get a metastasis organ mapping. Conclusions and Perspectives: Bioluminescent metastatic PDX models of various tumor histotypes were successfully established by growing luciferase-engineered PDXs subcutaneously into appropriate mouse strains. These models will provide a useful tool to explore the biology of metastasis and evaluate anti-metastatic therapies in a selection of organs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B164. Citation Format: Ludovic Bourre, Mylène Bernes, Enora Leven, Vanessa Yvonnet, Olivier Déas, Myriam Lassalle, Jean-Gabriel Judde. A platform of metastatic bioluminescent PDX: Focus on HBCx14-Luc1 metastatic bioluminescent breast tumorgaft model for preclinical studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B164.