Project Summary Advances in molecular profiling technologies have enabled the identification of ever-increasing numbers of disease biomarkers, each raising the potential for more precise, and possibly even patient-specific, diagnoses. Current methods of quantifying protein biomarkers, however, are slow and laborious, often require large quantities of biopsied tissues, and ultimately fail to provide a complete, intact quantification of functional protein activity. To address these limitations, we have developed an innovative activity dependent proximity ligation platform (ADPL), with the ultimate goal of obtaining multiplexed, ultrasensitive quantification of protein activity directly in complex biological samples. In the context of cancer specimens, we hypothesize that defining a molecular profile based on protein activity – the end result of biologic signaling – can more precisely and accurately inform diagnoses and treatment regimes, allowing early intervention and improving clinical outcomes. This proposal outlines the advanced development and validation of two reliable, high-resolution ADPL-based platforms, with the power to alternately visualize and quantify dozens of active enzymes simultaneously from tumor tissue, cells and biofluids. Our current knowledge and expertise in the use of this integrated platform will be leveraged in the service of the following aims: 1) development of novel chemoproteomic probes for multiplexed, family-wide activity mapping of cancer-relevant enzymes, including kinases; 2) advanced development of on-tissue Exchange-ADPL and on-chip single-cell ADPL (scADPL) platforms for simultaneous, high resolution visualization and quantification of a panel of active enzymes in situ; and 3) applying these technologies to breast cancer tissues and cells to create accurate and precise activity signatures and identify potential correlations between these signatures and clinical outcomes. To accomplish these aims we will develop new chemical proteomic molecular probes, validate a panel of ADPL reagents for cancer-associated enzyme biomarkers, and perform rigorous, metric-driven benchmarking analyses in cell lines and tumor tissues. Completion of this project will validate ADPL platforms for use in diverse cancers, provide the first single-cell activity profiles of breast tumors, and provide novel correlations between breast cancer tumor activity signatures, disease severity and response to therapy.