Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

Abstract

Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep-sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity, and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, noninvasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.

Figure 1

Development and validation of an eGFP-NanoLuc (GpNLuc) bifunctional LumiFluor reporter. A, schematic of the GpNLuc reporter. The N-terminus of GpNLuc is derived from eGFP, which is followed by a flexible 5-residue linker (DISGG), and the C-terminus is derived from NanoLuc. Following hydrolysis of its substrate furimazine, the light emitted by the NanoLuc moiety activates the eGFP moiety via bioluminescence resonance energy transfer (BRET) in cis. B, structural model and functional evaluation of the GpNLuc LumiFluor. A model of GpNLuc was generated by combining the structure of eGFP (pdb4EUL) with a model of NanoLuc based on sequence homology to fatty acid binding protein (pdb1B56). The in-frame 5-residue DISGG linker was added between the C-terminus of eGFP and the N-terminus of NanoLuc. The distance between the NanoLuc active site and the eGFP fluorophore ranges between 30 to 70 Å based on this model, with a mean of 52 Å. C, normalized spectral emission scans of native proteins. Equimolar amounts of expressed and purified recombinant NanoLuc, Renilla, GpNLuc, and Nano-lantern BRET fusion proteins were aliquoted and emission intensities measured in triplicate in the presence of either furimazine (FZ; 50 μM) or coelenterazine (CLZ, 100 μM). D, expression and functional comparison of the GpNLuc fusion reporter to eGFP and NanoLuc alone. Left, HEK293T cells were transfected with equal concentrations of each respective retroviral construct and luciferase assays were performed 24 hr post-transfection (n = 4; mean ± s.e.m.). Right, western blot analyses of whole cell lysates from HEK293T cells transfected with NanoLuc (lane 1), eGFP (lane 2), or GpNLuc (lane 3). E, approach used to validate the functional utility of the bifunctional GpNLuc reporter for in vivo bioluminescent imaging and ex vivo flow cytometry analyses.

Figure 2

GpNLuc signal strength and stability in A549 NSCLC xenograft and orthotopic transplants. A, A549-GpNLuc cells or A549 cells engineered to also express the LKB1 tumor suppressor (A549-LKB1-GpNLuc) were injected subcutaneously into the front and rear flanks of recipient NOD/SCID mice with the indicated numbers of tumor cells to gauge limits of signal detection (minimum number of detectable cells) 1 day following injection. Furimazine was injected intraperitoneally (i.p.) and bioluminescence images were captured for the two cohorts, which were monitored longitudinally from day 1 to day 28 post-transplantation (lens aperture = f/1; image exposure time = 60 seconds on Day 1 or 7 seconds on Day 28; binning = 8; field of view = 13.3 cm; and emission set to open filter). B, in vivo dose-response kinetics of GpNLuc signal strength. Mouse subcutaneous xenografts were established with A549-GpNLuc cells (5 × 10⁵) and signal strength was monitored temporally in response to i.p. furimazine administration at the indicated doses when tumor volume reached 1500 mm³ (n = 3). C, direct comparison of subcutaneous tumor growth monitored temporally by bioluminescent imaging (BLI; top) and caliper measurements (bottom) for mouse xenografts (5 × 10⁵ cells) from A549-GpNLuc or A549-LKB1-GpNLuc cohorts (n = 3). A significant difference was detectable between the A549-GpNLuc and A549-LKB1-GpNLuc cohorts on day 11 by BLI but not until day 21 by caliper measurements (*P < 0.05; **P < 0.01; ***P < 0.001). D, tissue penetrating ability of GpNLuc signal was evaluated by orthotopic transplantation of A549-GpNLuc cells (1 × 10⁶) injected intravenously (via tail vein) into NOD/SCID mice. Furimazine was injected intravenously (i.v.) and 2D (left) and 3D (right) bioluminescence images were captured. Images are representative of mice monitored longitudinally from day 1 to day 49 post-transplantation (lens aperture = f/1; image exposure time = 60 seconds; binning = 8; field of view = 6.6 cm; and emission set to open filter).

Figure 3

Longitudinal bioluminescence imaging quantification and flow cytometry analyses of GpNLuc-expressing Eμ-Myc lymphoma transplants. A, allografts of Eμ-Myc mouse lymphoma cells stably expressing GpNLuc (1 × 10⁶) that were injected i.v. into syngeneic Albino C57Bl/6 recipient mice (n = 10). Left, furimazine was injected intravenously (i.v.) and ventral and dorsal bioluminescence images were captured from day 1 to day 14 post-transplantation (lens aperture = f/1; image exposure time = 6 seconds; binning = 8; field of view = 22.6 cm; and emission set to open filter). Right, quantification of bioluminescent signal intensities in vivo from indicated lymph nodes and tissues colonized by B cell lymphoma. B, direct comparison of tumor burden on day 2 versus 14 post-transplantation by 2D (left) or 3D (right) bioluminescence imaging. Representative images are shown. C, ex vivo confirmation of tumor burden by flow cytometry analyses of surgically resected lymph nodes and tissues identified by BLI. Graphs are representative of mice analyzed on day 14 post-transplantation (n = 3).

Figure 4

Intramolecular BRET in LumiFluors drives fluorophore excitation/emission and is essential for sensitive in vivo imaging. A, comparison of catalytically inactive GpNLuc-Y67A and GpNLuc-Y67C mutants, as well as the red-shifted LSSmOrange-NLuc (OgNLuc) fusion, to either GpNLuc or NanoLuc alone. HEK293T cells were transfected with equal concentrations of each respective retroviral construct and luciferase assays were performed 24 hr post-transfection (n = 3; mean ± s.e.m.). B, flow cytometric analysis of Eμ-Myc mouse lymphoma cells, along with serially passaged Eμ-Myc lymphoma cells engineered to express NanoLuc, GpNLuc, GpNLuc-Y67A, GpNLuc-Y67C, GpNLuc-T66G or OgNLuc, confirmed effects of mutagenesis on the fluorescence excitation capacity of NanoLuc on eGFP and on LSSmOrange. C, allografts of Eμ-Myc mouse lymphoma cells expressing either NanoLuc, GpNLuc, GpNLuc-Y67C, or OgNLuc (1 × 10⁶) were injected i.v. into syngeneic Albino C57Bl/6 recipient mice (n = 3). Furimazine was injected i.v. and ventral bioluminescence images (BLI) were captured on day 7 post-transplantation (lens aperture = f/1; image exposure time: NanoLuc = 25 seconds, GpNLuc = 6 seconds, GpNLucY67C = 40 seconds, or OgNLuc = 3 seconds; binning = 8; field of view = 22.6 cm; and emission set to open filter). D, model comparing and contrasting conventional in vivo fluorescent imaging to new methods offered by GpNLuc and OgNLuc LumiFluor reporters. Ectopic excitation of fluorescent reporters in vivo results in significant autofluorescence whereas local excitation of fluorophores by intramolecular energy transfer from a fused NanoLuc partner prevents global autofluorescence and augments overall signal output and detection.