A. Specific Aims Identification of high-risk populations suitable for screening and chemoprevention of hepatocellular carcinoma (HCC) has been proposed as an effective and efficient strategy to impact HCC-related mortality. Screening and earlier detection of HCC has had some impact on HCC mortality. However, the absence of tools to accurately predict which patients will progress to HCC remains a significant barrier to development of effective chemoprevention agents. Progress in prediction of who will progress to HCC and whether chemoprevention is likely to be effective would have a major clinical impact. Liver fibrosis is a common pathway shared by all progressive chronic liver diseases that raise risk for HCC, regardless of the underlying etiologies. Many lines of evidence suggest that increased fibrosis and fibrogenesis are independent predictors of HCC transformation, and that reducing liver fibrosis reduces HCC risk. Our lab has developed molecular probes for noninvasive, quantitative imaging of liver fibrosis and fibrogenesis throughout the entire liver. With a type I collagen targeted peptide-based MR probe we showed that we could accurately stage liver fibrosis in 6 different rodent models. We developed a second type of probe in prior work funded by this grant: molecular MR probes that target the aldehyde-containing allysine residue on oxidized collagen and showed that we could specifically quantify fibrogenesis (fibrotic disease activity). In this Renewal, we propose to expand this toolbox further and develop a third novel probe that targets inflammation associated with fibrogenesis (IAF). This probe is based on the low relaxivity Fe2+-PyC3A chelate which is activated by rapid oxidation to high relaxivity Fe3+-PyC3A in the presence of peroxidases released during inflammation and which then binds to allysine residues that are present during fibrogenesis, retaining the probe and its high relaxivity signal at the site of IAF. We will deploy these three probes (fibrosis, fibrogenesis, IAF) to characterize the combinations of these biochemical features that best predict transformation to HCC in rat and mouse models. Next, we will use molecular MR for real-time monitoring of response to chemoprevention agents that have been proven effective in preclinical models, and to guide effective dosing. The overarching goal of this project is to leverage molecular imaging of fibrosis to accurately predict risk of progression to HCC, and real-time monitoring of efficacy of interventions that reduce the risk. Aim 1. Develop a turn-on molecular probe to detect fibrogenesis when it is associated with inflammation. 1.1. Synthesize a small focused library of allysine-targeted, Fe2+-PyC3A probes 1.2. Screen the probes for relaxivity in the presence/absence of peroxidase, target affinity, non-specific binding, and allysine binding kinetics to a model proteiny 1.3. Confirm the ability of the IAF probe to detect inflammation associated fibrogenesis in a mous...