Chimeric antigen receptor (CAR)-T cell therapy has resulted in dramatic, durable responses, especially for patients with B cell malignancies that are refractory to standard treatment. Numerous clinical trials are focused on improvements of the approach and applications for a much broader range of hematologic malignancies and solid tumors. However, this targeted therapy is also associated with high relapse rates and the development of severe toxicities that are challenging to predict. Thus, improved tools to optimize and monitor response are critically needed. Our ultimate goal is to develop a simple, quantitative, and non-invasive point-of-care device to monitor CAR-T cell interactions in vivo dynamically with the needed frequency. The objective of this proposal is to demonstrate proof-of-principle of the approach and its translational potential for human studies. Our approach relies on in vivo flow cytometry (IVFC) assessments using endogenous and exogenous sources of optical contrast and its optimization and use to detect CAR-T/B cell interactions in vitro and in vivo. The proposed method relies on the confocal detection of endogenous light scattering and fluorescence (label-free or LF) signals of flowing CAR-T/B cells that are interacting. Previous studies support the hypothesis that interacting CAR-T/B cells will likely yield unique optical signatures. We anticipate that utilization of advanced signal processing approaches will enable us to optimize detection of these signals in the challenging milieu of flowing blood in vitro and in vivo. Initial measurements with fluorescent protein (FP)-expressing human CAR-T and B cells spiked in whole blood will allow us to develop, implement, and assess enhancements in hardware and software to realize accurate LF-IVFC assessments of interacting CAR-T and B cells. (Aim 1.1). The translational potential will be tested on blood samples isolated from patients undergoing CAR-T cell therapy at different time points over the first month of therapy. We expect to detect variations in the numbers of interacting CAR-T and B cells using LF- IVFC and this expectation will be corroborated with antibody-based flow cytometry measurements (Aim 1.2). Finally, we will perform in vivo LF and FP-based IVFC measurements using mice infused with human B lymphoblasts and CAR-T cells at concentrations known to induce variations in the relative circulating levels of these cells. Our goal will be to demonstrate that LF-IVFC measurements can be performed with high enough accuracy to detect dynamic changes in the numbers of interacting CAR-T and B cells over a month. We expect to generate a robust set of data that will motivate development and application of LF-IVFC monitoring of interacting CAR-T/B cells for dynamic, non-invasive monitoring of patients receiving CAR-T cell therapy. Such a device may enable acquisition of information regarding individual patient responses with a combined time resolution and sensitivity/specificity t...