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

Abstract

Apoptosis is a highly regulated process of programmed cell death essential for normal physiology. Dysregulation of apoptosis contributes to the development and progression of various diseases, including cancer, neurodegenerative disorders, and chronic heart failure. Quantitative noninvasive imaging of apoptosis in preclinical models would allow for dynamic longitudinal screening of compounds and facilitates a more rapid determination of therapeutic efficacy. In this study, we report the in vivo characterization of Z-DEVD-aminoluciferin, a modified firefly luciferase substrate that in apoptotic cells is cleaved by caspase-3 to liberate aminoluciferin, which can be consumed by luciferase to generate a luminescent signal. In two oncology models, namely SKOV3-luc and MDA-MB-231-luc-LN, at 24, 48, and 72 h after treatment with docetaxel, animals were injected with Z-DEVD-aminoluciferin and bioluminescent images were acquired. Significantly more light was detected at 24 (Po0.05), 48 (Po0.01), and 72 h (Po0.01) in the docetaxel-treated group compared with the vehicle-treated group, with caspase-3 activation at these time points confirmed using immunohistochemistry. Importantly, whereas significant differences between groups were detected as early as 24 h after treatment by molecular imaging, caliper measurements were unable to detect a difference for 4-5 additional days. Taken together, these data show that in vivo imaging of apoptosis using Z-DEVD-aminoluciferin could provide a sensitive and rapid method for early detection of drug efficacy, which could potentially be used by numerous therapeutic programs.

In vivo imaging of early stage apoptosis by measuring real-time caspase-3/7 activation

“…Our results support previous data from similar studies, which used different therapeutics and different tumor models as the ones evaluated here, and have demonstrated once more that monitoring the early apoptosis induction might serve as a suitable biomarker for the prediction of anti-cancer drug efficacy with respect to tumor mass reduction [18,19].…”

“…However, the first preparation of Z-DEVDaminoluciferin that was used in vivo appeared to be toxic and did not allow a second imaging session of the animal [17], and the new formulated Z-DEVD-aminoluciferin substrate that was utilized in most recent studies was not toxic but had solubility issues [18,19]. In our study, we have used a new preparation of Z-DEVD-aminoluciferin that is not toxic and has an improved solubility.…”

“…Previous reports have already demonstrated that measuring caspase-3/7 activation using Z-DEVD-aminoluciferin in a bioluminescent assay is an extremely sensitive approach to measure apoptosis in vitro and also in vivo [17][18][19]24]. However, the first preparation of Z-DEVDaminoluciferin that was used in vivo appeared to be toxic and did not allow a second imaging session of the animal [17], and the new formulated Z-DEVD-aminoluciferin substrate that was utilized in most recent studies was not toxic but had solubility issues [18,19].…”

Apoptosis Imaging: Beyond Annexin V

“…Instead of modifying the reporter gene to respond to caspases, luciferase reporter gene substrate can also be modified to reflect caspase activity. One such example is a proluminescent, caspaseactivated DEVD-aminoluciferin reagent (Caspase-Glo 3/7; Promega) (27,28).…”

Bioluminescence Applications in Preclinical Oncology Research

“…Animals were injected with the Z-DEVD-aminoluciferin before BL images were acquired. This group shows that more light was detected in the docetaxel-treated group compared with the untreated group (Hickson, Ackler et al 2010). …”