PROJECT 1 SUMMARY (CERVIX) The standard of care (SOC) for locally advanced cervical cancer, pelvic radiation (RT) with concurrent cisplatin, is associated with a 30-50% failure rate, and there is no cure for recurrent disease. Most cervical cancers are caused by infection with Human Papilloma Virus (HPV), and persistent expression of HPV oncogenes supports a state of chronic stromal inflammation mediated by macrophages. Preclinical studies suggest that tumor associated macrophages (TAMs) are the most abundant cell type in the tumor microenvironment (TME) after RT and exhibit “M2-like” phenotypes that suppress the development of anti-tumor immunity. In our previous work, we demonstrated that cervical tumor 18F-Fluoro-deoxy-glucose (FDG) uptake on pretreatment positron emission tomography (PET) is associated with increased TAMs with “M2-like” phenotypes in the cervix TME, and that co- culture of cervix tumor cells with TAMs increases tumor cell glucose uptake. We propose that TAMs and tumor cells in the TME co-evolve and mutually adapt their glucose and ROS metabolism to optimize tumor growth. In this proposal, we will test the hypothesis that SOC CRT induces ROS-mediated “metabolic switches” that alter gene transcription and metabolism in each cell type, providing a therapeutic opportunity to limit this metabolic co-dependency and improve outcomes after SOC CRT by promoting RT induced anti-tumor immunity. Here we will use a combination of state of the art single cell and spatially resolved profiling of human tumors together with state of the are imaging to determine the impact of SOC CRT induced changes in metabolism in tumor cells and TAMs on CRT sensitivity, and perform mechanistic preclinical studies to determine how this impacts the development of anti-tumor immunity. In Aim 1 we will determine the longitudinal impact of SOC CRT on metabolism and inflammatory signaling in cervix tumor cells. Our preliminary data supports targeting CRT induced upregulation of SOD2 and NRF2 in tumor cells. We will study CRT associated changes in ROS and mitochondrial metabolism using tumor organoids and novel mouse models. In Aim 2 we will determine the impact of SOC CRT on TAM and T cell phenotype and function in the cervix TME. We will study the effect of TAM targeting using novel imaging approaches, antibody depletion and CCR2 inhibitors in mouse models. In Aim 3 we will integrate longitudinal changes in PET imaging features with CRT associated changes in gene expression to improve patient classifiers and response prediction. In these studies, we will use FDG in addition to 64Cu- DOTA-ECLi, a novel PET tracer for CCR2+ TAMs. Finally, we will work with a trainee from our Training Core, METEORITE, to prioritize new leads from our human data. This trainee will explore the contribution of immune checkpoint blockade (ICB) when added to SOC CRT. ICB is well known to alter metabolism in immune cells, and we will synthesize our data and approaches to achieve our ulti...