Imaging agents that enable direct visualization and quantification of apoptosis in vivo have great potential value for monitoring chemotherapeutic response as well as for early diagnosis and disease monitoring. We describe here the development of fluorescently labeled activity based probes (ABPs) that covalently label active caspases in vivo. We used these probes to monitor apoptosis in the thymus of mice treated with dexamethasone (dex) as well as in tumor-bearing mice treated with the apoptosis inducing monoclonal antibody Apomab. Caspase ABPs provided direct readouts of the kinetics of apoptosis in live animals, whole organs and tissue extracts. The probes produced a maximum fluorescent signal that could be monitored non-invasively and that coincided with the peak in caspase activity as measured by gel analysis. Overall, these studies demonstrate that caspase-specific ABPs have the potential to be used for non-invasive imaging of apoptosis in both pre-clinical and clinical settings.

Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few druggable classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate- and activity- based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.

Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is co-expressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNAi-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a pro-metastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.