Project Summary A critical step in the success of adoptive cell transfer (ACT) T cell immunotherapy in solid cancers is achieving trafficking and persistence of T cells at tumor sites, while avoiding toxicities due to T cell attack of off-target tissues and organs. Non-invasive quantitative imaging would be a powerful tool to understand mechanisms of action and failure of T cell immunotherapies, evaluate the impact of T cell modifications and delivery routes, monitor off-target T cell accumulation, and stratify response to therapy on the basis of measures of T cell tumor accumulation. This Bioengineering Research Grant project will pioneer non-invasive and quantitative tracking of adoptive T cell cancer immunotherapy using magnetic particle imaging (MPI), a new molecular imaging modality that enables non-invasive, unambiguous, and tomographic analysis of the whole-body distribution of superparamagnetic iron oxide nanoparticles (SPIONs). Preliminary results demonstrate non-invasive quantitative tracking of ACT T cells in solid intracranial tumors, synthesis of tracers with enhanced MPI sensitivity, and current sensitivity of 5x103 T cells. The proposed work aims to improve sensitivity to 5x102 T cells and demonstrate the accuracy of MPI in quantifying T cell biodistribution in mouse models of cancer. Modeling of MPI physics by the PI demonstrates that tracers optimal for MPI must have uniform physical and magnetic properties and low magnetocrystalline anisotropy, to enable fast dipole switching at large SPION diameters. The PI has developed a new synthesis that yields defect-free SPIONs with uniform magnetic properties and low magnetocrystalline anisotropy. The proposed work (Aim 1) will couple this new synthesis with modeling of MPI physics and comprehensive physical and magnetic characterization to gain fundamental understanding of the relation between SPION properties and MPI performance and to obtain SPIONs with superior sensitivity. Imaging approaches to track T cells must not compromise their viability or function and T cells pose unique challenges for nanoparticle labeling. The proposed work (Aim 2) will define an upper limit for labeling primary T cells with MPI tracers without compromising viability or function using tracers that associate with T cells through charge interactions. Preliminary studies demonstrate non-invasive tracking of T cell biodistribution in mice using MPI, and that SPION-labeled T cells reach solid tumors after systemic administration in murine models. The proposed work (Aim 3) will validate in vivo tracking of ACT T cell therapy using MPI against T cell counting using flow cytometry and will evaluate dynamics of T cell accumulation in tumors longitudinally using MPI. The proposed biomaterials-development research plan is enabled by the complementary expertise of the PI (SPIONs and MPI physics) and Co-I (ACT T cell therapies) and access to state-of-the-art instrumentation to characterize SPION MPI performance ex vivo...