Caspases are well known for their role as executioners of apoptosis. However, recent studies have suggested that these lethal enzymes also have important non-canonical roles in the activation and proliferation of T cells. Effective antitumor immune response is based on the ability of T cells to recognize and destroy cancer cells, for which activation of caspase-3 (c-3) is key. Therefore, the assessment of tumor response based upon the activation of c-3 following immunotherapy, may represent a promising strategy for early prediction of therapy outcome. The current set of c-3-targeted positron emission tomography (PET) radiotracers does not provide adequate resolution and signal-to-noise ratio to precisely visualize c-3 activity during the course of immunotherapy. In addition, monitoring of CAR T-cell trafficking to the tumor site is still not possible in cancer patients. Therefore, the goal of this application is to develop PET radiotracers for selectively imaging c-3 and to investigate their value for prediction and evaluation of responses to immunotherapy. We propose to use novel c-3 specific cell-permeable activity-based probes to visualize dying tumors following immunotherapy, and c-3 specific cleavable metabolic probes for bioorthogonal monitoring of T cell activation and trafficking to tumor cells. Probes will be evaluated in vitro to assess c-3 affinity and selectivity, and in vivo using cancer xenograft models treated with immunotherapy for response evaluation.