Project Summary Despite decades of research, the prognosis for patients with malignant brain tumors remains poor and novel treatment strategies are urgently needed. Malignant gliomas are particularly refractory to treatment, uniformly fatal, and have not seen improvement in outcomes for over three decades. Immunotherapy has tremendous promise for eradicating cancers with exquisite precision by leveraging the cytotoxic capabilities of tumor antigen- reactive lymphocytes. Immune checkpoint blockade therapy blocking the programmed death 1 (PD-1) pathway is used clinically against melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, renal cell carcinoma, and Hodgkin's lymphoma. Although anti-PD-1 therapy alone is ineffective against malignant gliomas, recent clinical trials with recurrent glioblastoma multiforme (GBM) suggest benefit of neoadjuvant anti- PD-1 therapy prior to tumor resection. Preliminary results with an immunocompetent mouse model of recurrent GBM suggests a role for CD8+ T cell recruitment during neoadjuvant anti-PD-1 therapy prior to tumor resection. This Exploratory/Developmental Bioengineering Research Grant will develop technology to enable non-invasive quantitative imaging of CD8+ tracer recruitment to the site of tumor resection using magnetic particle imaging (MPI), a new imaging modality with potential advantages over immunoPET imaging. MPI tracers will be formulated consisting of superparamagnetic iron oxide nanoparticles (SPION) with targeting motifs to selectively label CD8+ T cells, enabling non-invasive, unambiguous, and quantitative imaging of their biodistribution. Preliminary results show the PI has already developed MPI tracers with high sensitivity and long blood circulation time. Selective in vivo labeling of T cells will be achieved by functionalizing these optimized MPI tracers with antibody fragments that bind to T cell surface markers, resulting in internalization or persistent binding to the T cell membrane. An important consideration in labeling T cells in vivo and in situ in solid tumors is penetration of the imaging label. Preliminary results demonstrate accelerated and enhanced tumor accumulation of systemically administered SPIONs through magnetic targeting. The PI hypothesizes that magnetic targeting of T cell targeting SPIONs at the site of tumor resection will lead to increased SPION/T cell interactions and increased sensitivity to the presence of T cells at the tumor resection site. In Aim 1 T cell labeling with the MPI tracers will be optimized ex vivo and the effect of labeling on T cell viability and cytotoxic phenotype will be evaluated. Then, in Aim 2 in vivo T cell labeling using systemically administered tracers will be evaluated in the context of neoadjuvant anti-PD-1 therapy prior to tumor resection in a mouse model of recurrent GBM. The proposed work will capitalize on magneto responsiveness of the SPION tracers to accelerate and enhance tumor penetration using magne...