Noninvasive Imaging of Apoptosis and Its Application in Cancer Therapeutics

Abstract

Purpose: Activation of the apoptotic cascade plays an important role in the response of tumors to therapy. Noninvasive imaging of apoptosis facilitates optimization of therapeutic protocols regarding dosing and schedule and enables identification of efficacious combination therapies. Experimental Design: We describe a hybrid polypeptide that reports on caspase-3 activity in living cells and animals in a noninvasive manner. This reporter, ANLucBCLuc, constitutes a fusion of small interacting peptides, peptide A and peptide B, with the NLuc and CLuc fragments of luciferase with a caspase-3 cleavage site (DEVD) between pepANLuc (ANLuc) and pepBCLuc (BCLuc). During apoptosis, caspase-3 cleaves the reporter, enabling separation of ANLuc from BCLuc. A high-affinity interaction between peptide A and peptide B restores luciferase activity by NLuc and CLuc complementation. Using a D54 glioma model, we show the utility of the reporter in imaging of apoptosis in living subjects in response to various chemotherapy and radiotherapy regimens. Results: Treatment of live cells and mice carrying D54 tumor xenografts with chemotherapeutic agents such as temozolomide and perifosine resulted in induction of bioluminescence activity, which correlated with activation of caspase-3. Treatment of mice with combination therapy of temozolomide and radiation resulted in increased bioluminescence activity over individual treatments and increased therapeutic response due to enhanced apoptosis. Conclusion: The data provided show the utility of the ANLucBCLuc reporter in dynamic, noninvasive imaging of apoptosis and provides a rationale for use of this technology to optimize dose and schedule of novel therapies or to develop novel combination therapies using existing drugs.

Noninvasive molecular imaging of apoptosis in vivo using a modified firefly luciferase substrate, Z-DEVD-aminoluciferin

“…[27][28][29][30][31][32][33][34] Cleavage of the linker permits the interacting peptides to associate, allowing reconstitution of luciferase activity. Coppola et al 15 described development of a split firefly luciferase reporter strategy in which DEVD is used as the intervening cleavage sequence and also reported the ability to detect activation of apoptosis in vivo after treatment with chemotherapeutic agents. However, a limitation to these strategies is the relatively large molecular size of the reporters that need to be transfected or transduced into the host cell.…”

“…11,12 Several recent reports have described the development of fluorescent and/or bioluminescent reporter constructs that specifically allow monitoring of caspase activation in vivo. These molecular imaging studies have mainly focused on generating cells that express a modified reporter protein, commonly a fusion protein or a split luciferase protein complementation strategy [13][14][15] that takes advantage of the specificity of caspases to cleave exclusively after aspartic acid residues and, in particular, the DEVD (aspartic acid-glutamic acid-valine-aspartic acid) tetrapeptide sequence, which is optimal for apoptotic effector caspases-3 and -7. 16,17 In these models, light production is silenced unless the cell is undergoing apoptosis, at which time activated caspase liberates the reporter.…”

Apoptosis Imaging: Beyond Annexin V

“…Bioluminescence imaging has been applied to image apoptosis with several seminal apoptosis-responsive reporter gene constructs (22)(23)(24)(25). One reporter gene contains firefly luciferase gene flanked by ER (residues 281-599 of the modified mouse estrogen receptor sequence) with DEVD linker.…”

Beyond annexin V: fluorescence response of cellular membranes to apoptosis

“…Detection of apoptosis is of great importance for many areas of biological research, for understanding the pathological conditions and for the development of new drugs. In cancer, the balance between cell proliferation and apoptosis shifts toward the cell proliferation, and this stimulates an active search for the most efficient and safe anticancer therapeutic regimens that induce apoptosis (Coppola et al 2008). The ability to monitor apoptosis using noninvasive sensing and imaging techniques would markedly enhance early assessment and continuous evaluation of the efficacy of anticancer drugs (Kim et al 2002).…”