Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals

Abstract

Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.

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Fig. 1: In vivo screening of new furimazine analogs.
Fig. 2: In vivo characterization and application of compound B (HFz).
Fig. 3: In vitro characterization and in vivo screening of new fluorinated furimazine analogs.
Fig. 4: In vivo characterization and application of FFz.
Fig. 5: Dual bioluminescence imaging of tumor xenografts and CAR-T cells in vivo.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

We thank H. Zeng and the Stanford Transgenic, Knockout, and Tumor Model Center for generating H11P-CAG-LSL-Antares and -FLuc transgenic mice, C. Manalac and L. Luo (Stanford University) for nestin-Cre transgenic mice, and G. Tao and K. Sylvester (Stanford University) for albumin-Cre transgenic mice. We thank J. Osterman and H. Lazaro in the analytical department at Promega Biosciences for their support in compound characterization. Cell sorting/flow cytometry analysis for this project was done on instruments in the Stanford Shared FACS Facility. We thank the Stanford Animal Histology Services for help with preparation of histologic specimens. This work was supported by American Heart Association Innovative Research grant no. 15IRG23290018 (M.Z.L.), NIH grant no. 1R21DA048252 (M.Z.L.), an American Heart Association Postdoctoral Fellowship (N.K.), NCI grant no. P5P30CA124435 (C.L.M.), a St Baldrick’s/Stand Up 2 Cancer Pediatric Dream Team Translational Cancer Research Grant (C.L.M.), Virginia and D.K. Ludwig Fund for Cancer Research (C.L.M.) and Stanford University School of Medicine Discovery Innovation Awards (J.R.C. and M.Z.L.). Stand Up 2 Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. C.L.M is a member of the Parker Institute for Cancer Immunotherapy, which supports the Stanford University Cancer Immunotherapy Program. L.L. received support from the National Science Foundation Graduate Research Fellowship, Stanford Graduate Fellowship and Stanford EDGE Fellowship. R.G.M. is the Taube Distinguished Scholar for Pediatric Immunotherapy at Stanford University School of Medicine.

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Authors

Contributions

Y.S. performed mouse experiments, analyzed data, prepared figures and wrote the manuscript. J.R.W. designed and synthesized new substrates. Y.P. performed solubility and mouse experiments, analyzed data and prepared figures. T.P.S. designed and prepared extended-release formulations. D.C.W. assisted with mouse experiments and created the FLuc integration plasmid. M.P.H. performed biochemical assays and prepared figures. L.X.L. performed mouse breeding and genotyping. R.H. assisted with protein purifications. L.P.E. assisted with cloning and protein expression. L.L. generated retroviral supernatants and prepared engineered T cells and tumor cells. N.K. created the Antares integration plasmid. K.M.C. performed mouse histopathology and prepared figures. F.Z. performed hydrodynamic transfection in mice. R.G.M., J.R.C., C.L.M. and M.A.K. provided supervision. T.A.K. designed experiments and provided supervision. M.Z.L. conceived the project, provided supervision, designed experiments, prepared figures and wrote the manuscript.

Corresponding authors

Correspondence to Thomas A. Kirkland or Michael Z. Lin.

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Competing interests

J.R.W., T.P.S., M.P.H., R.H., L.P.E. and T.A.K. are employees of Promega Corporation and inventors on a patent describing furimazine and furimazine derivatives. L.L. is a consultant for Lyell Immunopharma. R.G.M. is a consultant for Lyell Immunopharma, Xyphos Biosciences, Gamma Delta Therapeutics and Illumina Radiopharmaceuticals. Y.P., D.C.W., L.X.L., Y.S., N.K., K.M.C. and M.Z.L. declare no competing interests. C.L.M. is a founder, holds equity in and is a consultant for Lyell Immunopharma, consults for Neoimmune Tech, Apricity and Nektar, holds equity in Apricity and Allogene and has received royalties from NIH for a CD22-CAR licensed to Juno.

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Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 In vitro characterization of novel furimazine analogs.

a, Solubility of furimazine analogs at various concentrations in 35% PEG-300, 10% ethanol, 10% glycerol and 10% hydroxypropylcyclodextrin. Images were taken in ambient light, except for the right image of compound A at 28 mM, which was taken under backlit conditions to more clearly show an insoluble pellet. The experiment was repeated independently two times with similar results. b, Spectral profiles of Antares with furimazine and analogues. The experiment was repeated independently two times with similar results. c, Determination of kinetic parameters of relative kcat and absolute KM for Antares with each substrate. As the same concentration of purified Antares was used with each substrate, kcat relative to furimazine can be calculated from the relative asymptotic luminescence (Vmax) values. Error bars, standard error of the mean (s.e.m.). N = 3. d, Decay of signal over time of Antares signal with furimazine substrates. The experiment was repeated independently two times with similar results. e, Stability of furimazine substrates at 37 °C in the presence and absence of 10% FBS. Colored bars, mean of three biological replicates. Gray dots, individual values. Error bars, standard error of the mean (s.e.m.). P values, ordinary one-way ANOVA or two-tailed Student’s t test.

Extended Data Fig. 2 Comparing in vivo brightness of Antares with furimazine to new substrates.

Bioluminescence imaging was performed in mice doubly hemizygous for Albumine-cre a-b, or nestin-Cre c-f, and CAG-loxP-stop-loxP-Antares (CAG-LSL-Antares) genes, which express Antares protein in the liver or kidney. For liver imaging (a-b), 4.2 μmol or 13.3 μmol of each substrate was injected intraperitoneally (a), with quantitation of the mean bioluminescence intensity over time shown (b). Similarly, for kidney imaging (c-f), 1.3 μmol (c-d) or 13.3 μmol (e-f) of each substrate was injected intraperitoneally. Exposure = 1 s, Binning = 1, Fstop = 8. Error bars, standard error of the mean (s.e.m.). Numbers of mice are indicated in parentheses.

Extended Data Fig. 3 Individual bioluminescence traces of luciferase–luciferin pairs in deep tissues of live mice.

Traces of total bioluminescence intensity in the liver for each hydrodynamically transfected mouse in the groups of Antares + compound B (n = 10), Antares2 + DTZ (n = 6), and AkaLuc + AkaLumine (n = 11) were displayed in gray. The bioluminescence trace of selected mouse shown in Fig. 2a was plotted in black curve. Arrows indicate the time points of displayed images in Fig. 2a.

Extended Data Fig. 4 Characterization of compound B in a P-407-based formulation.

a, 4.2 μmol (1.7 mg) of compound B and 11.1 mg P-407 can be dissolved in ethanol, evaporated, redissolved in water, and lyophilized to create a lyophilized cake (top). 480 μL of water can then be added to resolubilize the compound B and P-407 (bottom). b, Sections of lung, liver, and kidney show no signs of toxicity following administration of P-407 or compound B with P-407. Mice received three daily intraperitoneal injections of 12 mg P-407 in 480 μL of water or of 1.7 mg compound B and 12 mg P-407 in 480 μL of water, then were sacrificed on the fourth day. Organs were fixed in formalin, embedded in paraffin, sectioned, de-paraffinized, and stained with hematoxylin and eosin. c, Comparison of bioluminescence intensity and persistence in vivo between the published PEG-300-based formulation and a poloxamer-407-based extended-release formulation of HFz (compound B). d, Mean bioluminescence intensity over time for the injected formulations. Error bars, standard error of the mean (s.e.m.). Numbers of mice are indicated in parentheses.

Extended Data Fig. 5 In vitro characterization of novel fluorinated furimazine analogs.

a, Spectral profiles of Antares with fluorinated furimazine analogues. The experiment was repeated independently two times with similar results. b, Decay of signal over time of Antares signal with furimazine substrates. The experiment was repeated independently two times with similar results. c, Stability of furimazine substrates at 37 °C in the presence and absence of 10% FBS. Colored bars, mean of three biological replicates. Gray dots, individual values. Error bars, standard error of the mean (s.e.m.). P values, ordinary one-way ANOVA or two-tailed Student’s t test. d, Solubility of fluorinated furimazine analogs at various concentrations in an aqueous formulation containing 35% PEG-300, 10% ethanol, 10% glycerol and 10% hydroxypropylcyclodextrin, or a formulation containing 12 mg P-407 in 0.5 mL water. The experiment was repeated independently two times with similar results.

Extended Data Fig. 6 Establishing optimal dosage and vehicle for FFz administration in mice.

a, Mean bioluminescence intensity over time for bioluminescence imaging results in mice doubly hemizygous for albumin-Cre and CAG-loxP-stop-loxP-Antares or luc2 (CAG-LSL-Antares or FLuc) genes, which express Antares or FLuc protein in the liver, and injected with indicated amount of luciferins to establish saturating dosage for each luciferin b, Mean bioluminescence intensity over time for comparing the effects of formulation on fluorofurimazine (FFz). Error bars, standard error of the mean (s.e.m.). Numbers of mice are indicated in parentheses. c, Renal and hepatic histologic lesions are most severe in mice receiving FFz (4.2 µmol) following 3 days of intraperitoneal administration. Mice receiving FFz (4.2 µmol) exhibited renal tubular degeneration (white arrow), renal tubular dilation (asterisks), and hepatic capsular degeneration with neutrophilic infiltrates (black arrows). Renal and hepatic lesions were minimal to absent in mice receiving compound FFz (1.3 µmol) or vehicle (P407) alone. No lesions were noted in the lungs across any groups. Hematoxylin and eosin, scale bar = 20 µm. The experiment was repeated independently three times with similar results.

Extended Data Fig. 7 Details of background and signal comparisons.

To assess background luminescence for furimazine, HFz, FFz, AkaLumine, or D-luciferin mice lacking any luciferase genes were injected with the indicated amounts of substrates. a, No luminescence was observed for all the luciferase substrates at the low sensitivity settings (Exposure = 1 s, Binning = 1, Fstop = 8). The experiment was repeated independently two times with similar results. b, Under the high sensitivity imaging settings (Exposure = 60 s, Binning = 2, Fstop = 1.2), no significant luminescence except for the occasional signal at the injection site was observed for all the mice injected with furimazine analogues, while liver signal were observed with AkaLumine injected. The experiment was repeated independently two times with similar results. c, Traces of total bioluminescence intensity in the liver for each hydrodynamically transfected mouse in the groups of AkaLuc + AkaLumine (n = 8) and Antares + FFz (n = 7) were displayed in gray. The bioluminescence trace of selected mouse shown in Fig. 4a was plotted in black curve. Arrows indicate the time points of displayed images in Fig. 4a. d, Mean signal intensity over time from 103 HeLa[Antares-P2A-AkaLuc] cells implanted subcutaneously after injection of FFz or AkaLumine. Note the slower kinetics compared to bioluminescence time courses in the liver. This can be explained by lack of vascularization of the implanted HeLa cells, which were imaged within 24 h of implantation. Note AkaLumine signals have reached a plateau whereas FFz signals have not at 20 min, so the peak and integrated signals of FFz relative to AkaLumine are likely underestimated. Error bars, standard error of the mean (s.e.m.). Numbers of mice are indicated in parentheses.

Extended Data Fig. 8 Bioluminescence imaging engrafted luciferase-expressing EW8 tumors in nod scid mice.

a, Flow cytometry characterization of EW8 cells stably expressing indicated luciferase. Wild-type EW8 cells were lentiviral transduced with indicated constructs, expanded, and then sorted based on CyOFP reporter fluorescence. The experiment was repeated independently two times with similar results. b, Bioluminescence imaging in nod scid mice engrafted with luciferase-expressing EW8 tumors in one leg (at day 0). 1.3 µmol FFz (0.15 mL in P-407) or 1.5 µmol AkaLumine (0.10 mL in 0.9% saline) were injected IP on the indicated days. c, Raw grayscale images. Imager settings: Exposure time = 2 s (FFz) or 60 s (AkaLumine), Binning = 2, Fstop = 1.2.

Extended Data Fig. 9 Bioluminescence imaging engrafted AkaLuc-expressing MG63.3 tumors in NSG mice.

a, Flow cytometry characterization of MG63.3 cells stably expressing AkaLuc-p2A-mNeonGreen. Wild-type MG63.3 cells were retroviral transduced with indicated constructs, expanded, and then sorted based on mNeonGreen reporter fluorescence. The experiment was repeated independently two times with similar results. b, Representative in vivo bioluminescence time course from grafted AkaLuc-expressing MG63.3 tumor after AkaLumine injection. Arrow indicates time point used for quantitation. The experiment was repeated independently ten times with similar results. c, Bioluminescence imaging in NSG mice engrafted with AkaLuc-expressing MG63.3 tumors in one leg (at day 0). 3.0 µmol AkaLumine (0.10 mL in 0.9% saline) were injected IP on the indicated days. d, Raw grayscale images. Imager settings: Exposure time = 1 s, Binning = 1, Fstop = 1.2.

Extended Data Fig. 10 Dual bioluminescence imaging MG63.3 tumors and non-immune T cells in NSG mice.

a, Flow cytometry characterization of MG63.3 cells stably expressing Anatares-p2A-mNeonGreen. Wild-type MG63.3 cells were retroviral transduced with indicated constructs, expanded, and then sorted based on mNeonGreen reporter fluorescence. The experiment was repeated independently two times with similar results. b, Representative in vivo bioluminescence time course from grafted Antares-expressing MG63.3 tumors after FFz injection. Arrow indicates timepoint used for quantitation. The experiment was repeated independently ten times with similar results. c-d, Raw grayscale images of NSG mice engrafted with Antares-expressing MG63.3 tumors in one leg (at day 0) and intravenously injected with AkaLuc-expressing cells (d) B7-H3 CAR-T (Fig. 5a) or (e) mock CAR-T (native T) cells (Fig. 5b).

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and Tables 1–3.

Reporting Summary

Supplementary Video 1

A mouse was injected with 4.2 μmol hydrofurimazine in P-407-based formulation. Vasopressin was added before the 30-min frame, with its presence indicated by the white circle. The localized nature of calcium oscillations can be more easily seen after time 45 min.

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Su, Y., Walker, J.R., Park, Y. et al. Novel NanoLuc substrates enable bright two-population bioluminescence imaging in animals. Nat Methods 17, 852–860 (2020). https://doi.org/10.1038/s41592-020-0889-6

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