Project Summary Earlier gains in reducing prostate cancer (PCa) mortality, partially due to advances in screening and the use of androgen deprivation therapy to treat metastatic PCa (mPCa), have not been sustained in recent years. Current clinical and research challenges include overtreatment of patients with low probability of progression, no effective therapy for metastatic castration-resistant PCa (mCRPC), and the persistent health disparities in PCa for African American men. A better understanding of the molecular dynamics and interactions among cancer and non-cancer cells within the tumor microenvironment CTME) at high resolution and in a spatial context across time will be crucial for addressing these challenges. Against the backdrop of recent advances in multi-omics and spatial technologies, we propose to use our expertise in the latest technological platforms, experience in atlas building, and strengths in PCa research and treatment at Washington University School of Medicine (WUSM) to extend our prior HTAN work to PCa. The proposed Human Prostate Tumor Atlas Center (HPTAC) will elucidate PCa architecture, cellular dynamics, TME interactions, and temporal evolution via the building of a molecularly resolved spatial atlas that integrates multi-omics, imaging, and clinical data, with a focus on spatial characterization with maximum optical display of molecular and cellular features. We will procure both retrospective and prospective PCa specimens with detailed clinical data at WUSM, which maintains a comprehensive repository of PCa specimens and has a high PCa patient volume (Aim 1). These specimens will span two critical transitions: from indolent to aggressive PCa and from hormone-sensitive to castration-resistant metastatic PCa. The high percentage of African American patients in our PCa patient population permits a 50% composition of African American specimens to investigate the biological basis for disparities associated with African ancestry. Aim 2 concentrates on molecular and spatial characterization of PCa specimens. At the core of the spatial characterization are spatial-omics, including sequencing-based (Visium/Curio Seeker) and imaging-based (Xenium/CosMx) platforms, 2D/3D imaging modalities, including CODEX, 3D in vitro Light Sheet Fluorescence Microscopy, and in vivo MRI/DBSI imaging. These technologies will be augmented by bulk and single cell omics for mutations/ancestry detection and overall molecular profiling. In Aim 3, data will be analyzed and integrated into a molecularly resolved spatial atlas of PCa. Co-registration, reconstruction, and integration will be achieved by employing computational tools, such as PASTE2, Morph, Mushroom. The resulting atlas will describe molecular, cellular, and spatiotemporal relationships of cellular and non-cellular components of the PCa ecosystem across critical transitions. In Aim 4, we will establish a collaborative infrastructure within our HPTAC and foster collaborations across HTAN...