Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals
Su, Y. et al. (2020) Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nature Methods. 17, 852–60. DOI: 10.1038/s41592-020-0889-6
Bioluminescence imaging with luciferases is a technique commonly used to track cells and report biological events in preclinical animal models. There is no excitation light required for bioluminescence and therefore, luciferase-based reporters can achieve high signal-to-background ratios due to a lack of autofluorescence. This sensitivity offers noninvasive imaging of deep tissue cells with lower detection limits. Current bioluminescent systems use NanoLuc®-based enzyme-substrate systems that include a blue-emitting NanoLuc® luciferase, furimazine (Fz) as a substrate, and reporters such as Antares, which consist of two cyan-excitable orange fluorescent protein copies fused to NanoLuc. Unlike firefly-derived luciferases, NanoLuc does not require ATP as a cofactor, making it uniquely suited for applications in extracellular spaces. A goal of bioluminescent imaging is the ability to sensitively detect two biological events in vivo, but current systems are limited by poor Fz solubility in aqueous solutions and the maximum substrate dose possible in vivo.
This study aimed at improving the NanoLuc® system by developing new substrates that are more water soluble than furimazine and can maximize the dose possible in vivo. Researchers were able to develop two substrates: hydrofurimazine (HFz) and fluorofurimazine (FFz). HFz has more intense and prolonged photon generation than Fz , allowing reporters to track in vivo events at higher resolutions and for extended periods of time. FFz, on the other hand, has even higher peaks and brightness than HFz. Additionally, with these new substrates researchers were able to demonstrate two-population imaging of tumor size and CAR-T cells in the same mice using Antares with FFz to track the size of tumors and a firefly luciferase reporter derivative AkaLuc with AkaLumine substrate to visualize the CAR-T cells.Results of this study have several implications for future research. Researchers suggest that there is interest in investigating how furimazine analogs for bioluminescence reporting perform in other cells and tumor types such as those in the brain. Particularly, they are interested in whether the newly developed substrates have higher sensitivity when imaging the brain. Additionally, more work could be done to improve the brightness of NanoLuc®-based systems in animals, similar to the new substrates developed in this research paper. As it stands, performing studies on the interactions between immune cells and cancer cells or pathogens using dual-bioluminescent imaging will be an important tool for furthering biomedical research.
Keywords: bioluminescent imaging, luciferase, NanoLuc, cancer cells, immune cells, preclinical animal models, two-population imaging, dual-luminescent imaging
