Background Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase (MPO) is a central inflammatory enzyme secreted by activated macrophages, and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance (MR) agent can visualize MPO activity in atherosclerotic plaques in a rabbit model. Methods and Results We performed MR imaging of the thoracic aorta of New Zealand white (NZW) rabbits fed a cholesterol (n=11) or normal (n=4) diet up to 2 hours after injection of the MPO sensor bis-5HT-DTPA(Gd) (MPO(Gd)), the conventional agent, DTPA(Gd), or an MPO (Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hour post injection) showed focal areas of increased contrast (>2-fold) in diseased wall, but not in normal wall (p=0.84), compared to both DTPA(Gd) (n=11; p<0.001) and bis-tyr-DTPA(Gd) (n=3; p<0.05). Biochemical assays confirmed that diseased wall possessed three-fold elevated MPO activity compared to normal wall (p<0.01). Areas detected by MPO(Gd) imaging co-localized and correlated with MPO-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, p<0.0001). While macrophages were the main source of MPO, not all macrophages secreted MPO, suggesting that distinct subpopulations contribute differently to atherogenesis and supporting our functional approach. Conclusions Our study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme, to noninvasively provide both anatomic and functional information in vivo.

Metastasis is the main killer in cancer; consequently, there is great interest in novel approaches to prevent and treat metastatic disease. Brain metastases are particularly deadly, as the protection of the blood-brain barrier obstructs the passage of common anticancer drugs. This study used magnetic resonance imaging (MRI) to investigate the therapeutic effects of nanoparticles coated with a tumor-penetrating peptide (iRGD) against a preclinical model of breast cancer brain metastasis. Single doses of iRGD nanoparticle were administered intravenously, and the effect on tumor growth was observed over time. iRGD nanoparticles, when applied in the early stages of metastasis development, strongly inhibited tumor progression. Overall, this study demonstrated for the first time that a single dose of iRGD nanoparticle can have a significant effect on metastatic tumor progression and nonproliferative cancer cell retention when applied early in course of tumor development. These data suggest that iRGD nanoparticles may be useful in preventatively reducing metastasis after a cancer diagnosis has been established.

Invadopodia are cell protrusions that mediate cancer cell extravasation but the microenvironmental cues and signaling factors that induce invadopodia formation during extravasation remain unclear. Using intravital imaging and loss of function experiments, we determined invadopodia contain receptors involved in chemotaxis, namely GABA receptor and EGFR. These chemotaxis capabilities are mediated in part by PAK1 which controls invadopodia responsiveness to ligands such as GABA and EGF via assembly, stability, and turnover of invadopodia in vivo. PAK1 knockdown rendered cells unresponsive to chemotactic stimuli present in the stroma, resulting in dramatically lower rates of cancer cell extravasation and metastatic colony formation compared to stimulated cancer cells. In an experimental mouse model of brain metastasis, inhibition of PAK1 significantly reduced overall tumor burden and reduced the average size of brain metastases. In summary, invadopodia contain chemotaxis receptors that can respond to microenvironmental cues to guide cancer cell extravasation, and when PAK1 is depleted, brain tropism of metastatic breast cancer cells is significantly reduced, blocking secondary colony growth at sites otherwise permissive for metastatic outgrowth.

Cellular magnetic resonance imaging (MRI) is a means by which cells labeled ex vivo with a contrast agent can be detected and tracked over time in vivo. This technology provides a noninvasive method with which to assess cell-based therapies in vivo. Dendritic cell (DC)-based vaccines are a promising cancer immunotherapy, but its success is highly dependent on the injected DC migrating to a secondary lymphoid organ such as a nearby lymph node. There the DC can interact with T cells to elicit a tumor-specific immune response. It is important to verify DC migration in vivo using a noninvasive imaging modality, such as cellular MRI, so that important information regarding the anatomical location and persistence of the injected DC in a targeted lymph node can be provided. An understanding of DC biology is critical in ascertaining how to label DC with sufficient contrast agent to render them detectable by MRI. While iron oxide nanoparticles provide the best sensitivity for detection of DC in vivo, a clinical grade iron oxide agent is not currently available. A clinical grade (19) Fluorine-based perfluorcarbon nanoemulsion is available but is less sensitive, and its utility to detect DC migration in humans remains to be demonstrated using clinical scanners presently available. The ability to quantitatively track DC migration in vivo can provide important information as to whether different DC maturation and activation protocols result in improved DC migration efficiency which will determine the vaccine's immunogenicity and ultimately the tumor immunotherapy's outcome in humans.

Imaging reporter genes provides longitudinal information on the biodistribution, growth, and survival of engineered cells in vivo. A translational bottleneck to using reporter genes is the necessity to engineer cells with randomly integrating vectors. Here, we built homology-independent targeted integration (HITI) CRISPR-Cas9 minicircle donors for precise safe harbor-targeted knock-in of fluorescence, bioluminescence, and MRI (Oatp1a1) reporter genes. Our results showed greater knock-in efficiency using HITI vectors compared to homology-directed repair vectors. HITI clones demonstrated functional fluorescence and bioluminescence reporter activity as well as significant Oatp1a1-mediated uptake of the clinically approved MRI agent gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid. Contrast-enhanced MRI improved the conspicuity of both subcutaneous and metastatic Oatp1a1-expressing tumors before they became palpable or even readily visible on precontrast images. Our work demonstrates the first CRISPR-Cas9 HITI system for knock-in of large DNA donor constructs at a safe harbor locus, enabling multimodal longitudinal in vivo imaging of cells.