Title: Two-way Magnetic Resonance Tuning Nanoprobe Enhanced Subtraction Imaging for Precision Diagnosis of Brain Metastasis Project Summary/Abstract Metastasis from systemic cancers to the brain is a leading cause of cancer mortality. The current diagnostic method is sensitive only to larger tumors when therapeutic options are limited. Visualizing early brain metastases by non-invasive imaging approaches with high sensitivity and spatial resolution followed by timely treatment is crucially important to reduce their high mortality rate. While effective interventions (e.g., surgery, radiation, targeted therapy, and immunotherapy) strongly depend on our ability to detect brain metastases at an early stage, imaging small brain metastases hidden in a large population of normal cells presents a unique challenge. It is essential to design novel imaging approaches to detect small brain metastases with the highest possible tumor-to-normal tissue ratios (TNRs). The goal of this application is to develop a new molecular nanoprobe with activatable magnetic resonance contrast integrated with a new computational subtraction approach to improve the TNR of imaging for small brain metastases. We recently developed a new two-way magnetic resonance tuning (TMRET) nanoprobe with dually activatable T1&T2 magnetic resonance signals coupled with dual-contrast enhanced subtraction imaging (DESI) to dramatically enhance contrast in targeted tissues and suppress the background signal from normal tissue. This integrated platform could sensitively detect very small tumors in the brain by magnetic resonance imaging (MRI) in patient-derived xenograft (PDX) models with a TNR >10. We also developed a Sequential Targeting In CrosslinKing (STICK) nano-delivery strategy to “stick in” central nervous system (CNS) tumors and metastases, which will be applied to our TMRET nanoplatform to enhance its specific delivery to brain metastases. In this project, we will develop novel blood-brain barrier (BBB)-traversing and deep tumor-penetrating TMRET (bt-TMRET) nanoprobes with superior TNR for sensitive and specific detection of brain metastases. The STICK strategy will be used to improve the CNS pharmacokinetics (PK) of TMRET nanoprobes by pHe-cleavable crosslinkers to maximize the time window for transcytosis through the BBB. Our STICK strategy will further enhance the efficiency of BBB traversal by manipulation of glucose transporter 1 (GLUT1) on the BBB by optimization of the polyvalent interaction of nanoprobes with GLUT1 via fine-tuning the surface targeting moieties. The STICK strategy optimizes the pH-responsive size transformation for improved tumor tissue penetration and sialic acid-targeting selectivity for enhanced tumor cell specificity. The MRI signal can be turned ON specifically at the brain metastases after BBB traversal and tumor penetration via size transformation in acidic tumor microenvironment. Our hypothesis is that the proposed nanoprobes can improve the TNR for MRI...