The broad goal of this proposal is to develop new imaging approaches to understand the complex biology of drug activity in vivo. Specifically, I will develop and optimize a fluorescence polarization microscopy approach to image target engagement of cancer drugs at cellular resolution in mouse tumor models. This technique, entitled competition multiphoton fluorescence anisotropy microscopy (compMFAM), enables imaging of unlabeled drug target engagement through competition with fluorescently labeled drug of the same target. Measuring target engagement provides insight into the mechanism of treatment failure when a drug proves ineffective. These data can be used to determine if failure arises from inefficient drug distribution or inability of the target to effect the cell. The candidte, J. Matt Dubach, PhD, has extensive experience in novel sensor development for fluorescent imaging of difficult to measure targets. The long term goal of the candidate is to develop novel imaging approaches to better understand cancer at the cellular level in biological systems. The proposed work will be conducted at the Center for Systems Biology at Massachusetts General Hospital and Harvard Medical School under the mentorship of Ralph Weissleder, MD, PhD and Tim Mitchison, PhD. The mentorship team will assist in the candidate's development during the mentored phase of the pathway to independence and help the candidate transition into an independent researcher. The specific aims of this proposal focus on two areas: technological development of the imaging system; and biological applications to measure the role of PARP target engagement in treatment of breast cancer through mouse models. There are several promising PARP inhibitors currently in clinical trials for a variety of cancers. Largely due to the high prevalence of BRCA mutations in breast cancer, PARP inhibitors are currently be explored as single agent treatment in triple negative breast cancer. Preliminary feasibility experiments demonstrate that this is a valid approach to quantitatively measure target engagement of drug in vivo. There are two specific aims in this proposal: 1) Validate compMFAM technique to measure cellular level target engagement; and 2) Determine how target engagement governs successful PARP inhibitor treatment. Aim 1 will be accomplished under the mentorship team, while Aim 2 will be performed as an independent investigator. Successful completion of this proposal would provide insight into how PARP target engagement governs cellular response in successful and unsuccessful treatment while providing a generalizable approach to study target engagement of nearly any inhibitor drug class.