Monitoring macrophage trafficking in vivo with ultrasound

Introduction: Imaging immune cells trafficking has major implications for cancer diagnosis, prognosis, and therapy in addition to providing new insights in immune‐cell biology. While current imaging modalities offer increasingly detailed snapshots of immune cell distribution in tumors, real-time imaging methods with improved sensitivity and resolution deep in the tissues are needed to monitor and characterize this dynamic and spatially distributed in vivo process. Here we propose a new ultrasound-based imaging method to visualize immune cell trafficking in vivo that will not only allow to study the interaction of the immune cells with the host but also identify critical biomarkers (e.g., accumulation, residence time, etc.) to determine the state of cell-based treatments.

Materials and

Methods: We explore the potential of labelling macrophages with lipid-shelled microbubbles (MB) to enable ultrasound (US) imaging of macrophage accumulation in solid tumors. In our proof-of-concept investigations we employ i) rigorous characterization of the MB-labeled macrophages (optical and acoustical), ii) multiparametric analysis of macrophage properties and function (flow cytometry, immunofluorescence microscopy, and migration assay), iii) in vitro and in vivo experimentation (tumor-bearing rodents), and iv) linear and nonlinear US imaging methods for a range of cell concentrations, ultrasound exposure settings, and time points.

Results, Conclusions, and Discussions: Our study demonstrates that microbubble labeling and sonication at mechanical indexes below 0.35 do not affect macrophage viability, migration capabilities, and phenotype, providing evidence that ultrasound imaging can be used for nondestructive macrophage detection and tracking. Importantly, despite the significant damping exerted on the MB oscillations by the cellular compartments, the MB display highly nonlinear behavior upon sonication allowing to visualize MB-labeled macrophages with high specificity using nonlinear US imaging methods. Consistent with these findings, we observed that nonlinear ultrasound imaging can selectively monitor macrophage accumulation and extravasation in solid tumors in rodents for at least 8 hours after intravenous administration. Together, our investigations support the notion that ultrasound can provide a noninvasive and scalable platform to study the complex problem of immune cell trafficking in solid tumors while creating new possibilities for cancer diagnosis and therapy. These results could be extended to other diseases where macrophages play an important role such as atherosclerosis.

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