PROJECT SUMMARY Adoptive cellular immunotherapies are an established therapeutic weapon in the fight against cancer. However, there is a large variability in anti-tumor responses and toxicity between patients, due primarily to differences in immune cell trafficking to tumor and lymph tissues. This has urged the development of surveillance tools that can monitor adoptive immune cell tissue migration in vivo to inform treatment regimens, empower interpretation of therapeutic outcomes, and aid in the early detection of adverse events. To address this technologic need, our goal is to develop nano-scale contrast agents that enable continuous and high-fidelity ultrasound (US) imaging of immune cells in deep tissues, employing tumoricidal chimeric antigen receptor macrophages (CAR-M) as an exemplary cellular model. Fundamental to this strategy is our development of phase-changing peptide nanoemulsions (NPep) that are rapidly internalized and persist within macrophages for multiple days and can generate echogenic bubble imaging nuclei on-demand without compromising cell viability. Our recent studies show these capabilities allow NPeps to provide real-time and long-term monitoring of macrophages in tissues using diagnostic B-mode and Doppler US imaging. Additional studies demonstrate NPeps provide a distinctive Doppler ‘twinkling’ feature that permits unparalleled spatiotemporal resolution of contrast-enhanced features from the tissue background to improve imaging resolution and identification of cellular locale. Bringing together a multi-disciplinary team of materials scientists, ultrasound imaging experts, and immunologists, our objective is to rationally tune NPep design to improve Doppler twinkling in cells (Aim 1), controllably modulate macrophage behavior in situ via US release of immunostimulants loaded into the NPep carrier (Aim 2), and demonstrate non- invasive, real-time, high contrast, and continuous imaging of NPep-labeled CAR-M intratumoral migration and persistence in vivo (Aim 3). To achieve this, in aim 1 we perform multiplexed imaging of NPep formulations with varying emulsion size and surface tension, two parameters linked to particle US response, to establish design rules that can be used to improve the consistency, power, and duration of NPep Doppler twinkling. Aim 2 will evaluate changes in macrophage viability, phenotype, and phagocytic function after uptake of NPep contrast agents, as well as demonstrate our ability to productively modulate anti-tumor behavior via US-actuated intracellular release of a loaded PPARγ agonist demonstrated to stimulate tumoricidal responses. In aim 3, we evaluate the potential of this tool to provide real-time and long-term monitoring of adoptive CAR-M infiltration into xenograft neuroblastoma tumors, as an exemplary in vivo model, using standard diagnostic US imaging modalities. Collectively, this work will provide the foundation for a clinically relevant tool that can be broadly adopted by the oncology community for real-time deep tissue imaging of adoptive cellular immunotherapies.

PROJECT NARRATIVE Technologies that allow real-time monitoring of engineered immune cell distribution within tissues would transform patient treatment and management in oncology. This project will develop stimuli-responsive contrast agents that provide on-demand and long-term deep tissue imaging of cellular immunotherapies via diagnostic ultrasound, using anti-tumor macrophages as an exemplary model. Through this work we will establish a platform technology that can be broadly adopted for in vivo monitoring and modulation of cellular immunotherapies.