Caspase 8-activated bioluminescence probe for in vivo imaging of programmable cell death

Abstract

Programmable cell death, including apoptosis and pyroptosis, is central to physiological homeostasis, with Caspase-8 serving as a pivotal molecular switch. However, no Caspase-8-specific self-illuminating bioluminescence probe has been reported for in vivo imaging of these pathways. Here, we report a Caspase-8-activated bioluminescence probe Ac-Ile-Glu-Thr-Asp-_D-Aminoluciferin (Ac-IETD-Amluc). In vitro experiments confirmed that Ac-IETD-Amluc was efficiently and specifically cleaved by Caspase-8 to release bioluminescent Amluc, with a linear relationship of bioluminescence versus Caspase-8 concentration (limit of detection: 0.082 g/L). Upon cisplatin-induced apoptosis and H₂TCPP-sensitized laser irradiation-induced pyroptosis in firefly luciferase-transfected 4T1 (fLuc-4T1) cells, Ac-IETD-Amluc treatment led to cell bioluminescence signals peaking at 40 min (3.3-fold higher than the inhibitor control group) and 10 min (3.7-fold higher than the inhibitor control group), respectively. In fLuc-4T1 tumor-bearing mice, bioluminescence intensities within tumors peaked at 10 min post-injection of Ac-IETD-Amluc, with 4.2-fold (apoptosis group) and 6.8-fold (pyroptosis group) increases compared to inhibitor control groups. Given its superior capacity for real-time monitoring of cell death pathways in vivo, this probe is anticipated to be applied for diagnosing diseases involving dysregulated cell death, such as cancers, neurodegenerative disorders, and inflammatory syndromes.