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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The data that support the findings of this study are available from the corresponding authors upon request.
Prescher, J. A. & Contag, C. H. Guided by the light: visualizing biomolecular processes in living animals with bioluminescence. Curr. Opin. Chem. Biol. 14, 80–89 (2010).
Genevois, C., Loiseau, H. & Couillaud, F. In vivo follow-up of brain tumor growth via bioluminescence imaging and fluorescence tomography. Int. J. Mol. Sci. 17, 1815 (2016).
Levin, R. A. et al. An optimized triple modality reporter for quantitative in vivo tumor imaging and therapy evaluation. PLoS ONE 9, e97415 (2014).
Contag, C. H. et al. Visualizing gene expression in living mammals using a bioluminescent reporter. Photochemistry Photobiol. 66, 523–531 (1997).
Iwano, S. et al. Single-cell bioluminescence imaging of deep tissue in freely moving animals. Science 359, 935–939 (2018).
Hall, M. P. et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7, 1848–1857 (2012).
Chu, J. et al. A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo. Nat. Biotechnol. 34, 760 (2016).
Suzuki, K. et al. Five colour variants of bright luminescent protein for real-time multicolour bioimaging. Nat. Commun. 7, 13718 (2016).
Zhao, H. et al. Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J. Biomed. Opt. 10, 41210 (2005).
Yeh, H.-W. et al. Red-shifted luciferase–luciferin pairs for enhanced bioluminescence imaging. Nat. Methods 14, 971 (2017).
Stacer, A. C. et al. NanoLuc reporter for dual luciferase imaging in living animals. Mol. Imaging 12, 1–13 (2013).
Germain-Genevois, C., Garandeau, O. & Couillaud, F. Detection of brain tumors and systemic metastases using NanoLuc and Fluc for dual reporter imaging. Mol. Imaging Biol. 18, 62–69 (2016).
Taylor, A., Sharkey, J., Plagge, A., Wilm, B. & Murray, P. Multicolour in vivo bioluminescence imaging using a NanoLuc-based BRET reporter in combination with firefly luciferase. Contrast Media Mol. Imag. 2018, 2514796 (2018).
Mezzanotte, L., van ‘t Root, M., Karatas, H., Goun, E. A. & Löwik, C. W. G. M. In vivo molecular bioluminescence imaging: new tools and applications. Trends Biotechnol. 35, 640–652 (2017).
England, C. G., Ehlerding, E. B. & Cai, W. NanoLuc: a small luciferase is brightening up the field of bioluminescence. Bioconjug Chem. 27, 1175–1187 (2016).
Gopalakrishnan, R. et al. A novel luciferase-based assay for the detection of chimeric antigen receptors. Sci. Rep. 9, 1957 (2019).
Nath, N., Flemming, R., Godat, B. & Urh, M. Development of NanoLuc bridging immunoassay for detection of anti-drug antibodies. J. Immunol. Meth. 450, 17–26 (2017).
Edinger, M. et al. Noninvasive assessment of tumor cell proliferation in animal models. Neoplasia 1, 303–310 (1999).
Inouye, S. & Shimomura, O. The use of renilla luciferase, oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate. Biochem Biophys. Res. Commun. 233, 349–353 (1997).
Tasic, B. et al. Site-specific integrase-mediated transgenesis in mice via pronuclear injection. Proc. Natl Acad. Sci. USA 108, 7902–7907 (2011).
Yeh, H. W. et al. ATP-independent bioluminescent reporter variants to improve in vivo imaging. ACS Chem. Biol. 14, 959–965 (2019).
Bodratti, A. M. & Bodratti, P. Formulation of poloxamers for drug delivery. J. Funct. Biomater. 9, 11 (2018).
Johnston, T. P. et al. Potential downregulation of HMG-CoA reductase after prolonged administration of P-407 in C57BL/6 mice. J. Cardiovasc. Pharm. 34, 831–842 (1999).
Oh, Y. et al. An orange calcium-modulated bioluminescent indicator for non-invasive activity imaging. Nat. Chem. Biol. 15, 433–436 (2019).
Gillis, E. P., Eastman, K. J., Hill, M. D., Donnelly, D. J. & Meanwell, N. A. Applications of fluorine in medicinal chemistry. J. Med. Chem. 58, 8315–8359 (2015).
Suda, T. & Liu, D. Hydrodynamic gene delivery: its principles and applications. Mol. Ther. 15, 2063–2069 (2007).
Mašek, T., Vopalenský, V. & Pospíšek, M. The Luc2 gene enhances reliability of bicistronic assays. Open Life Sci. 8, 423–431 (2013).
Yeh, H. W., Wu, T., Chen, M. & Ai, H. W. Identification of factors complicating bioluminescence imaging. Biochemistry 58, 1689–1697 (2019).
Shakhmin, A. et al. Three efficient methods for preparation of coelenterazine analogues. Chemistry 22, 10369–10375 (2016).
Shrestha, T., Troyer, D. & Bossmann, S. Strategies for large-scale synthesis of coelenterazine for in vivo applications. Synthesis 46, 646–652 (2014).
Walker, A. J. et al. Tumor antigen and receptor densities regulate efficacy of a chimeric antigen receptor targeting anaplastic lymphoma kinase. Mol. Ther. 25, 2189–2201 (2017).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
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.
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.
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.
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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.
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.
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.
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.
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.
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.
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 Figs. 1–7 and Tables 1–3.
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