Priority Report

Fluorophore-NanoLuc BRET Reporters EnableSensitive In Vivo Optical Imaging and FlowCytometry for Monitoring TumorigenesisFranz X. Schaub1, Md. Shamim Reza2, Colin A. Flaveny3,Weimin Li1, Adele M. Musicant4,Sany Hoxha5, Min Guo2, John L. Cleveland1, and Antonio L. Amelio6,7

Abstract

Fluorescent proteins are widely used to study molecular andcellular events, yet this traditionally relies on delivery of excitationlight, which can trigger autofluorescence, photoxicity, and photo-bleaching, impairing their use in vivo. Accordingly, chemilumi-nescent light sources such as those generated by luciferases haveemerged, as they do not require excitation light. However, currentluciferase reporters lack the brightness needed to visualize eventsin deep tissues. We report the creation of chimeric eGFP-NanoLuc(GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporterproteins coined LumiFluors, which combine the benefits of eGFPor LSSmOrange fluorescent proteins with the bright, glow-typebioluminescent light generated by an enhanced small luciferasesubunit (NanoLuc) of the deep-sea shrimp Oplophorus graciliros-

tris. The intramolecular bioluminescence resonance energy trans-fer that occurs between NanoLuc and the fused fluorophoregenerates the brightest bioluminescent signal known to date,including improved intensity, sensitivity, and durable spectralproperties, thereby dramatically reducing image acquisition timesand permitting highly sensitive in vivo imaging. Notably, the self-illuminating andbifunctional nature of these LumiFluor reportersenables greatly improved spatiotemporal monitoring of verysmall numbers of tumor cells via in vivo optical imaging and alsoallows the isolation and analyses of single cells by flow cytometry.Thus, LumiFluor reporters are inexpensive, robust, noninvasivetools that allow for markedly improved in vivo optical imaging oftumorigenic processes. Cancer Res; 75(23); 5023–33. �2015 AACR.

IntroductionA number of in vivo imaging technologies, for example, MRI,

PET, PET-MRI, PET-CT, and ultrasound have been developed andused in the clinic (1, 2). Theprohibitive costs and laborious natureof MRI and PET has limited their use for preclinical investigationsof developmental and pathologic processes, and for monitoringthe response of disease to therapeutic agents. To address this issue,a variety of bioluminescent imaging (BLI) and fluorescence imag-ing reporter systems have been developed for preclinical studies,

yet these reporters lack the in vivo penetration (sensitivity) orduration and strength (intensity) of signal that are needed toprovide quantitative, real-time, and inexpensive in vivo imaging(3–5). For example, the routinely used ATP-dependent firefly andclick beetle luciferases, as well as the ATP-independent Renilla andGaussia luciferases, are limited by light absorption and by theirreported physical instability to conditionsmanifest in vivo, includ-ing changes in temperature, pH, and urea concentration (6). As aconsequence, the utility of these luciferases reportedly benefitfrom imaging with long acquisition times, often in excess of5 minutes, and use within nude (nu/nu) or shaved mice becauseless fur or lighter fur allows more signal to reach the detector.Collectively, these features limit their utility, particularly in morehigh-throughput, preclinical drug screening efforts (7–9).

Somemultimodal imaging reporters have been developed thatpermit the analysis or isolation of single cells by methods such asflow cytometry and FACS, respectively. However, the signal inten-sities of these reporters have limited sensitivity in vivo, and thecassettes encoding these reporters are large, thus restricting theirapplication when using viral delivery methods that require spaceto encode transgenes or shRNAs (10–14). Accordingly, a compactmultimodal reporter having enhanced signal intensity is neededfor preclinical cancer studies. To meet this need, a variety ofreporters and knockin mouse models have been developed thatallow one to monitor, albeit at low resolution, the developmentand progression of neoplastic disease, and its response to ther-apeutics (2, 5, 9, 15–17).

The ideal in vivo reporter should combine the benefits of highfluorescent signal intensity with the low background associatedwith bioluminescent molecules, which would permit single-cellanalysis as well as spatial and temporal monitoring in live

1Department of Tumor Biology, Moffitt Cancer Center and ResearchInstitute,Tampa, Florida. 2Department of Cancer Biology,The ScrippsResearch Institute, Scripps Florida, Jupiter, Florida. 3Department ofPharmacological & Physiological Science, School of Medicine, SaintLouis University, St. Louis, Missouri. 4UNC Biological and BiomedicalSciences Graduate Program, University of North Carolina at ChapelHill, Chapel Hill, North Carolina. 5Scripps Graduate Program, TheScripps Research Institute, Scripps Florida, Jupiter, Florida. 6Lineber-ger Comprehensive Cancer Center, University of North Carolina atChapel Hill, Chapel Hill, North Carolina. 7Biomedical Research ImagingCenter, University of North Carolina at Chapel Hill, Chapel Hill, NorthCarolina.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

F.X. Schaub, Md. S. Reza, and C.A. Flaveny contributed equally to this article.

Corresponding Author: Antonio L. Amelio, Oral and Craniofacial HealthSciences, University of North Carolina at Chapel Hill, 385 South Columbia Street,Chapel Hill, NC 27599-7455. Phone: 919-537-3309; Fax: 919-966-3633; E-mail:antonio_amelio@unc.edu

doi: 10.1158/0008-5472.CAN-14-3538

�2015 American Association for Cancer Research.

CancerResearch

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animals. However, the utility of fluorescentmolecules is hinderedin vivo by the requirement for externally provided excitation lightthat generates autofluorescence and has limited penetration dueto absorption by tissues. Conversely, bioluminescent enzymes arelimited by wide variations in signal intensity and duration. Toresolve these problems, bioluminescence resonance energy trans-fer (BRET)-based reporters employing direct fusion of a donorluciferasemoiety and a fluorescent acceptormoiety have emergedas promising tools for monitoring complex biologic processes,including tumor development and progression (18, 19). Themajority of BRET reporters are designed with Renilla luciferase(RLuc) and variants thereof, which serve as the donor molecule toa yellow fluorescent acceptor molecule, although firefly luciferase(FLuc) BRET fusions have also beenmade. Although several BRETreporter fusions have been described, these reporters suffer fromsuboptimal acceptor activation, due to the poor overall levels andkinetics of light production generated by most luciferases, whichis in part due to auto-inactivation by enzymatic by-products(4, 20–27). To overcome these challenges, we utilized theenhanced small luciferase subunit (NanoLuc) of the deep-seashrimp Oplophorus gracilirostris, which displays extremely bright,stable, glow-type luminescent properties and physical stability,with >150-fold brighter luminescence compared with firefly andRenilla luciferases and >2 hours signal half-life (6, 28).

Here we report the creation and markedly improved imagingproperties of novel BRET reporters we coin LumiFluors, which arefusions of enhanced GFP [eGFP, quantum yield (QY) ¼ 0.6] orlong stokes shiftmOrange [LSSmOrange,QY¼ 0.45; a red-shiftedGFP variant (29)] to NanoLuc (GpNLuc and OgNLuc, respective-ly). Specifically, we document that these LumiFluors are highlysensitive optical reporters for monitoring tumorigenesis, andmechanistically show that the much brighter in vivo signals ofthese BRET reporters is due to intramolecular energy transfer fromthe intense luminescent signal of NanoLuc to the fused fluoro-phore. This creates an optical reporter that is activatedwithout theneed for UV excitation, has little autofluorescence, and that can beused to FACS sort cells that stably or inducibly express thesereporters. Further, the small size of LumiFluor reporter cassettesallows their incorporation into several viral vector delivery sys-tems. Finally, assessments of theGpNLuc andOgNLuc LumiFluorreporters in both solid and soft tumor models demonstratedexquisitely sensitive monitoring of tumor development at bothshallow and deep tissue levels and facile analyses of tumor cells exvivo by flow cytometry. Thus, LumiFluor reporters are broadlyapplicable and highly sensitive optical reporter tools that can beused for real-time, noninvasive in vivo spatiotemporal monitoringof molecular and cellular events.

Materials and MethodsCell lines and luciferase assays

HEK293T cells (ATCC;CRL-11268)weremaintained inDMEMmedium (Invitrogen) supplemented with 10% FBS (PremiumSelect; Atlanta Biologicals), GlutaMAX (Invitrogen), and penicil-lin, streptomycin, and L-glutamine (PSG; Invitrogen). HumanRaji Burkitt lymphoma cells (ATCC; CCL-86) were transducedwith concentrated RIEP retroviral particles (plasmid was kindlyprovided by C. Miething, Uniklinikum Freiburg, Freiburg inBreisgau, Germany) in the presence of Ecotropic Receptor Booster(Clontech). Cells were then selected with puromycin (1 mg/mL)and maintained in RPMI 1640 media supplemented with 10%

FBS and PSG. All cell lines procured from the ATCC were char-acterized by short tandem repeat profiling. NSCLC A549-pBABEand A549-LKB1 cells were previously characterized and kindlyprovided by Dr. Frederic J. Kaye (Department of Medicine, Divi-sion of Hematology and Oncology, College of Medicine, Univer-sity of Florida, Gainesville, FL; ref. 30) and maintained in RPMI1640 media supplemented with 10% FBS and PSG antibiotics.Mouse lymphoma cell lines were generated by crossing the Eu-Myc transgenic mouse with the Rosa 25rtTA transgenic mouse(JAX#006965). At the age of 8 weeks, offspring carrying Eu-Mycand Rosa 25rtTA alleles were closely monitored for tumor devel-opment. Lymphomas were then harvested and homogenized inPBS with 10% FBS and the erythrocytes were lysed. The cellswere then filtered through a 40-mm nylon filter and plated in45% Iscove's Modified Dulbecco's Medium (with 25 mmol/LHEPES; GIBCO), 45% DMEM (high glucose, GIBCO), 10%FBS with 1% penicillin/streptomycin, 4 mmol/L L-glutamine,25 mmol/L b-mercaptoethanol, 1� sodium pyruvate, and 10ng/mLmouse IL7 (R&D Systems). The cells were passaged severaltimes to create a stably growing cell line. All cells were cultured instandard, humidified conditions (37�C, 5% CO2).

Transfection of HEK293T cells for luciferase assays was carriedout using Lipofectamine 2000 (Life Technologies). Luminescencewas measured 24 hours after transfection on an Envision platereader (Perkin Elmer) following addition of Nano-Glo LuciferaseAssay Substrate (Promega).

Construction and expression of the GpNLuc and OgNLucLumiFluor reporters

The eGFP or LSSmOrange cDNAs lacking a stop codon werePCR amplified and cloned with 50 BspMI and 30 EcoRV restrictionenzyme sites into the pRetroX-Tight-Puro vector (Clontech) inplace of the PuromycinR cassette. Orientation of the eGFP andLSSmOrange inserts and the lack of a stop codon were confirmedby sequencing. In-frame fusion of a DISGG peptide linker andNanoLuc to eGFP or LSSmOrange was achieved by a restrictionenzyme-free, two-step PCR cloning protocol. Briefly, two separatesets of PCR primers were designed with overlapping regionsof homology to the new pRetroX-eGFP or pRetroX-LSSmOrangevectors and NanoLuc. These were then used to amplify eachrespective region, and then transformed into competentEscherichia coli. Recombination of the DISGG peptide linker-NanoLuc fragment into the pRetroX-eGFP and pRetroX-LSSmOr-ange vectors was confirmed by sequencing.

In vitro of characterization of recombinant luciferasesFor in vitro characterization of recombinant luciferases, lumi-

nescence intensitywasmeasured inwhite opaque 384-wellmicro-plates (OptiPlate-384 HS, PerkinElmer Inc.) using the 2104EnVision Multilabel Plate Reader (PerkinElmer Inc.). The assayreagent contained 50 mmol/L Tris-HCl, pH 8.0, 150 mmol/LNaCl, and 50 mmol/L Nano-Glo substrate (furimazine; PromegaCorporation) or 100 mmol/L coelenterazine (Biosynth Interna-tional, Inc.). Luminescence intensity was recorded 3.0 minutesafter adding the assay reagent to the respective luciferase dilutions(5 � 10�2

–1.56 � 10�3 mmol/L). Emission spectral scans of allrecombinant luciferases (50 nmol/L each) were performed inwhite opaque 96-well microplates (OptiPlate-96, PerkinElmerInc.) using SpectraMaxM5fluorescencemicroplate reader (Molec-ular Devices, LLC). Emission spectra were recorded from 390to 600 nm using the integration time of 1,000 milliseconds with

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5-nm step increments. The assay reagent for the emission spectralscans contained 50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl,and50mmol/L furimazine (PromegaCorporation) or 100mmol/Lcoelenterazine (Biosynth International, Inc.).

Subcutaneous tumor xenograftsA549-pBABE-GpNLuc and A549-LKB1-GpNLuc cells were cul-

tured, dissociated via trypsin digestion, and suspended in a 1:1mixture of PBS andMatrigel (BDBiosciences). Cell suspensions ofeither A549-pBABE or A549-LKB1 (200 mL) containing 5 � 102,5� 103, 1� 105, or 5� 105 cells were subcutaneously implantedas shown (Fig. 3) in 4- to 6-week-old NOD/SCID mice. In vivoluminescence of transplanted cells was measured on days 1 and 2after implantation and once every 7 days using an in vivo imager(IVIS Spectrum; Xenogen).

Xenograft tumor volume measurementsTo determine tumor volume by external caliper, the greatest

longitudinal diameter (length) and the greatest transversediameter (width) were determined. Tumor volume based oncaliper measurements were calculated using the modified ellip-soidal formula, Tumor volume ¼ 1/2(length � width2), asdescribed (31).

Orthotopic NSCLC xenograftsA549-pBABE-GpNLuc and A549-LKB1-GpNLuc cells were

dissociated via trypsin digestion, suspended in PBS, and 5 �105 cells were injected into the tail vein ofNOD/SCIDmice. Theluminescence signal from implanted tumors cells were mea-sured once every 7 days using an in vivo imager (IVIS Spectrum;Xenogen).

Orthotopic Em-Myc lymphoma allografts or Burkitt lymphomaxenografts

Em-Myc or Burkitt lymphoma cells stably expressing GpNLucwere resuspended in PBS and 1 � 106 sorted GFPþ cells wereinjected via tail vein into syngeneic AlbinoC57Bl/6 orNOD/SCIDrecipients, respectively. The luminescence signal from implantedtumors cells were measured once every 7 days unless otherwisespecified using an in vivo imager (IVIS Spectrum or Bruker XtremeOptical and X-ray small animal imaging system). Tissues infil-trated with tumor cells that were identified by BLI were collectedfor flow cytometry analysis.

BLIIn vitro BLI was performed using an IVIS Spectrum 1 minute

after addition of Nano-Glo Luciferase Assay Substrate (furima-zine; 2-furanylmethyl-deoxy-coelenterazine) following the man-ufacturer's specifications (Promega). In vivo BLI was performed onisoflurane-anesthetized animals 5 minutes after injection of theindicated doses of furimazine either i.p. or i.v. tail vein. Imageswere capturedwithopenfilter and acquisition times of 60 secondsor less at the indicated settings. Data were analyzed using theLiving Image software.

ResultsDevelopment and analyses of bifunctional LumiFluor BRETreporters

Brighter reporter proteins that exhibit durable signal emissionare needed for spatial and temporal imaging of molecular andcellular processes in vivo. The advent of intramolecular BRETfusion reporters has made significant strides in this effort, where

the ability to autoilluminate fused fluorescent probes generateshigher QY than that offered by luciferase molecules alone (4).However, previous attempts at creating chimeric fluorescent–bioluminescent fusions have primarily employed the flash-typelight-emitting properties of Renilla luciferase (RLuc), and variantsthereof, but these fail to generate the intense, stable, and durablesignals required for truly sensitive in vivo imaging applications(Table 1).

Brightness of luciferase proteins is a function of QY, catalyticrate, and sensitivity to product inhibition (4). Thus, we reasonedthat one could engineer a more robust, autoregulatory BRETfusion reporter using the enhanced luciferase variant of Oluc-19 (NanoLuc; NLuc), previously generated by directed evolution.Notably, NanoLuc produces three orders of magnitude moreluminescence than RLuc when provided with an optimized sub-strate (furimazine), due to combined improvements in all threeparameters governing brightness (6). We predicted that theintense and stable glow-type light emitted by NanoLuc, whichranges from 440 to 480 nm, could be successfully employed forBRET to excitefluorescent proteins having ahighQY, such as eGFPin cis, by substrate-dependent chemical energy transfer (Fig. 1A).The crystal structure of eGFP (32) andmolecular modeling of thestructure ofNanoLucwere used to guide the designof the chimericreporter (Fig. 1B). Using this model, a short flexible (5-residue)amino acid linker between the N-terminal eGFP and C-terminalNanoLuc (GpNLuc LumiFluor) moieties was optimized to allowindependent folding of the twoproteins and tomaintain the closephysical proximity (range of 30 to 70 Å) required for efficientintramolecular energy transfer (Fig. 1B and Table 1).

To rigorously evaluate the optical properties of the optimizedGpNLuc BRET reporter fusion, we compared GpNLuc directlywith another recently described BRET fusion reporter based onenhanced RLuc (RLuc8.6) and a YFP variant (Venus), Nano-lantern-YNL, whose spatial arrangement was optimized by circu-lar permutations of Venus rather thanmolecular modeling (Table1; ref. 21). As expected given their optimized donor/acceptorconfigurations, analysis of purified GpNLuc compared withNano-lantern protein revealed similar BRET ratio and efficiencyprofiles. However, the GpNLuc BRET ratio was 3- to 4.5-foldgreater compared with that reported for BRET3 and BRET6 fusionreporters (Fig. 1C and Table 1). Further, although Nano-lanternexhibits improved brightness over other reported BRET fusions(21), the GpNLuc LumiFluor displays 70-fold higher peak emis-sion intensity, with 85-fold more luminescence than Nano-lan-tern when provided with furimazine, or 8-fold higher peakemission intensity, with 45-fold more luminescence thanNano-lantern when provided with each luciferases-preferred sub-strate, despite similar energy transfer characteristics (Fig. 1C andSupplementary Fig. S1A).

Given its compact size, the GpNLuc fusion was cloned into aretrovirus (pRetroX-Tight-MCS-GpNLuc) and used to generatecell lines stably expressing the reporter (SupplementaryFig. S1B). To verify the spectral characteristics and proper expres-sion of the GpNLuc fusion protein (46 kDa), HEK293T cells weretransiently transfected with increasing concentrations of thisretroviral construct constitutively expressing GpNLuc or withequivalent concentrations of eGFP alone or NanoLuc alone(Fig. 1D and Supplementary Fig. S1C). GpNLuc has a 10-foldincrease in total light output over NanoLuc alone, which wasslightly higher but similar to that observed by analysis of purifiedprotein, and, not surprisingly, several orders of magnitude more

Chimeric Fluorescent–Luminescent Fusions for In Vivo Imaging

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Table

1.BRETreporter

fusionpairs

Nam

eFluoropho

re(exm

ax/em

max)

Pep

tidelin

ker

Molecu

lar

modeling

Luciferase

(em

max)

Substrate

Spec

tral

separation

BRETratioa

BRETeffcienc

ybReferen

ce

LumiFluor-GpNLu

cEGFP(488/509)

5aa

-DISGG

Yes

Nan

oLu

c(460)

FZ

49nm

2.60�

0.02

3.02�

0.10

LumiFluor-OgNLu

cLS

SmOrang

e(437

/572

)5aa

-DISGG

No

Nan

oLu

c(460)

FZ

112nm

ND

ND

Nan

o-lan

tern

Ven

usDC

10(515/528

)2aa

-GT

No

Ren

illa_

Rluc8

DN3_

S25

7G(480)

CLZ

-h48nm

2.26

�0.03c

2.82�

0.00c

21NR

EYFP(513/527

)12

aa-SGLR

SRAQALA

TNo

Ren

illa_

Rluc(480)

CLZ

47nm

NR

NR

25BAF-Y

EYFP(513/527

)12

aa-SGLR

SAAQALA

TNo

Ren

illa_

Rluc(480)

CLZ

47nm

NR

NR

25eB

AF-Y

EYFP(513/527

)12

aa-SGLR

SAAQALA

TNo

Ren

illa_

Rluc8

(485)

CLZ

42nm

NR

NR

25BRET1

EYFP(513/527

)11aa

-RARDPRVPVAT

No

Ren

illa_

Rluc(480)

CLZ

47nm

NR

NR

26BRET2

GFP2(400/511)

NR

No

Ren

illa_

Rluc(400)

CLZ

-400a

111nm

NR

NR

27NR

GFP2(400/511)

18aa

-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc-C(480or400)

CLZ

orCLZ

-400a

31or111nm

NR

NR

24NR

GFP2(400/511)

18aa

-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc-M

(485or400)

CLZ

orCLZ

-400a

26or111nm

NR

NR

24NR

GFP2(400/511)

18aa

-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc8

(485or400)

CLZ

orCLZ

-400a

26or111nm

NR

NR

24BRET3

mOrang

e(548/564)

18aa

-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc8

(480)

CLZ

84nm

0.79�

0.01

NR

23BRET3.1

mOrang

e(548/564)

18aa

-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc8

(515)

CLZ

-v49nm

0.74�

0.02

NR

22,2

3BRET4.1

Tag

RFP(555

/584)

18aa

-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc8

(515)

CLZ

-v69nm

NR

NR

23BRET5

Tag

RFP(555

/584)

18aa

-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc8

.6(535

)CLZ

49nm

NR

NR

23BRET6

TurboFP(588/635

)18

aa-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc8

.6(535

)CLZ

100nm

0.58�

0.02

NR

23BRET6.1

TurboFP(588/635

)18

aa-SGSSLT

GTRSDIGPSRAT

No

Ren

illa_

Rluc8

.6(570

)CLZ

-v65nm

0.78�

0.04

NR

23TurboLu

cTurboFP635

14aa

-QSTVPRARDPPVAT

No

Firefl

y_Fluc2

(560)

D-Luciferin

75nm

NR

NR

20

Abbreviations:a

a,am

inoacid;F

Z,furim

azine,

2-furany

lmethy

l-deo

xy-coelen

terazine

;CLZ

-h,coelen

terazine

-h,2-D

eoxyco

elen

terazine

;CLZ

,coelen

terazine

;CLZ

-400a,co

elen

terazine

-400a,

1-bisdeo

xyco

elen

terazine

(Dee

pBlueC

);CLZ

-v,coelen

terazine

-v;ND,n

otdetermined

;NR,n

otreported

.aBRETratio¼[FusionRep

orter

Bioluminescent

emission(long

wavelen

gth)/Bioluminescent

emission(sho

rtwavelen

gth)]�[D

ono

rOnlyBioluminescent

emission(long

wavelen

gth)/Dono

rOnlyBioluminescent

emission

(sho

rtwavelen

gth)],ref.2

2.bBRETefficien

cy¼

(FusionRep

orter

Accep

torPea

kem

ission/FusionRep

orter

Dono

rPea

kem

ission).

c Calculatedin

thisreport.

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intense than eGFP in the absence of excitation light. Importantly,fluorescence microscopy confirmed functional eGFP activity ofGpNLuc, and luciferase assays confirmed concentration-depen-dent luciferase activity of this chimeric reporter (SupplementaryFig. S1C). Finally,Western blot analysis confirmed the presence ofthe predicted 46kDaGpNLuc fusion protein (Fig. 1D). Thus, boththe eGFP and NanoLuc moieties of the GpNLuc chimera arefunctional and their fusion creates a markedly improved BRETreporter that can be used to transduce, image, and FACS sort targetcells to allow, for example, the evaluation of tumor cell growth invitro and in vivo (Fig. 1E).

Fluorescent–bioluminescent properties of GpNLuc in reportertumor cells ex vivo

To characterize the in vitroproperties of GpNLuc reporter, mouseEm-Myc lymphoma and human A549 non–small cell lung cancer(NSCLC) tumor cell lines were transducedwith retroviruses expres-singGpNLuc and stable clones selected by FACS sortingGFPþ cells.TheseGpNLuc-expressing tumor cellswere then seriallydilutedandthe total light emitted was imaged using a cooled CCD camerafollowing treatment with furimazine (Supplementary Fig. S2A andS2B, top). Quantifying bioluminescence as a function of cellnumber revealed that the minimum number of detectable cells

was about 8 to 16 cells/well for both cell lines, which is 40- to 60-fold better than the numbers of NSCLC cells that are required fordetection using conventional firefly luciferase (33).

The imaging data were also evaluated using a photo multi-plier tube-based plate reader equipped with an enhancedluminometer capable of ultrasensitive luminescence measuresless than 5 amol/well. The minimum number of detectable cellsby fluorescence was approximately 31,000 lymphoma and125,000 NSCLC cells/well. Strikingly, however, this numberwas as low as 4 to 8 cells/well for both cell lines when assessingluminescence intensity (Supplementary Fig. S2A and S2B, bot-tom). The highly sensitive detection of GpNLuc versus eGFPfluorescence alone corresponds to >3 orders of magnitude morelight signal and to detecting 8,000- to 30,000-fold fewer cells,respectively. Finally, stable GpNLuc expression and single-cellanalysis of serially passaged cells showed that GpNLuc-expres-sing lymphoma and NSCLC cell lines were �95% GFP-positiveafter 2 weeks of culture, validating their use for noninvasive invivo imaging (Supplementary Fig. S2C and S2D).

GpNLuc signal intensity and sensitivity in vivoBlue-shifted light emissions are scattered by tissues and are

absorbed by hemoglobin in vivo. Thus, the narrow, blue-shifted

A BFlexiblelinker

Excitation light460 nm

eGFP NLuc

Peak light emission range460–508 nm

C

350 400 450 500 550 600 650

0

2 105

4 105

6 105

8 105

Wave Length (nm)

Emis

sion

inte

nsity

(a.u

.)

1 107

Rel

ativ

e lig

ht u

nits

(RLU

)6 106

8 106

4 106

2 106

0

eGFP

NanoL

uc

GpNLu

c

Renilla

LumiFluorNanoLucNanolantern + FZ

Renilla

LumiFluorNanoLucNanolantern + CLZ

NanoLuceGFP

GpNLuc

NanoLuc

GFP

Actin

D

EPackage retrovirus

Transduce target cells

Fluorescence-activated cell sorting (FACS )

Animal transplantation in vivo bioluminescent imaging (BLI)

Tumor explant Ex vivo flow cytometry/FACS

1 106

52 A

eGFP

NanoLuc

GpNLuc LumiFluor

°

5 aaLinker

Figure 1.Development and validation of an eGFP-NanoLuc (GpNLuc) bifunctional LumiFluor reporter. A, schematic of the GpNLuc reporter. The N-terminus of GpNLuc isderived from eGFP, which is followed by a flexible 5-residue linker (DISGG), and the C-terminus is derived from NanoLuc. Following hydrolysis of its substratefurimazine, the light emittedby theNanoLucmoiety activates the eGFPmoiety viaBRET in cis. B, structuralmodel and functional evaluationof theGpNLucLumiFluor.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-bindingprotein (pdb1B56). The in-frame 5-residue DISGG linker was added between the C-terminus of eGFP and the N-terminus of NanoLuc. The distance betweenthe NanoLuc active site and the eGFP fluorophore ranges between 30 and 70 Å based on this model, with a mean of 52 Å. C, normalized spectral emission scans ofnative proteins. Equimolar amounts of expressed and purified recombinant NanoLuc, Renilla, GpNLuc, and Nano-lantern BRET fusion proteins were aliquoted andemission intensities measured in triplicate in the presence of either furimazine (FZ; 50 mmol/L) or coelenterazine (CLZ; 100 mmol/L). D, expression and functionalcomparison of the GpNLuc fusion reporter to eGFP and NanoLuc alone. Left, HEK293T cells were transfected with equal concentrations of each respectiveretroviral construct and luciferase assays were performed 24 hours after transfection (n¼ 4; mean� SEM). Right, Western blot analyses of whole cell lysates fromHEK293T cells transfected with NanoLuc (lane 1), eGFP (lane 2), or GpNLuc (lane 3). E, approach used to validate the functional utility of the bifunctional GpNLucreporter for in vivo bioluminescent imaging and ex vivo flow cytometry analyses.

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emission range of NanoLuc is not optimal for penetratingmammalian tissues and sensitive in vivo optical imaging(34). To initially assess the utility of the enhanced spectralprofile and intense signal properties of the GpNLuc LumiFluorin vivo, subcutaneous xenografts were performed with varyingnumbers of (LKB1-null) A549-GpNLuc NSCLC cells (A549-GpNLuc), and these were compared with A549-GpNLuc cellsthat were also engineered to express the tumor suppressor LKB1(A549-LKB1-GpNLuc; Fig. 2A). Previous reports have claimedthe ability to detect fewer than 10 cells in vivo using conven-tional luciferases (7, 8). However, in these studies images werecaptured with an open filter and acquisition times of 5 minutesor more, and in some cases several days after transplant. To testthe in vivo sensitivity of the GpNLuc LumiFluor reporter, imageswere captured as indicated with an open filter and acquisitiontimes of 60 seconds or less. Longitudinal monitoring with BLIrevealed that �500 GpNLuc-expressing cells are easily detectedusing brief image acquisition times on the first day aftertransplant and that GpNLuc effectively tracks the inhibitoryaffects of LKB1 on NSCLC tumor growth (Fig. 2A and Supple-mentary Fig. S3A–S3C). Quantitation of signal-to-noise ratiosfor 500 cells revealed that the GpNLuc signal is 2 to 3 orders ofmagnitude above background signal generated in control micesimilarly injected with substrate; thus, far fewer cells can besuccessfully imaged. To define the optimal dose of furimazinesubstrate and the stability of the resulting GpNLuc signal,subcutaneous xenografts were established with 500,000GpNLuc-expressing tumor cells, and tumors were allowed todevelop to 1,500 mm3. Recipient mice were then administeredwith specific doses of furimazine and followed by periodic BLI(Fig. 2B). Analysis of signal intensity and stability revealed that250 and 500 mg/kg furimazine produced 12- to 16-fold highersignal than a 50 mg/kg dose, although all three doses displayeda remarkably stable signal output, with a t1/2 of 40 minutes(Fig. 2B). These remarkable in vivo properties for the GpNLucLumiFluor are in stark contrast with the apparent rapid in vivosignal decay rate of secreted NanoLuc, which has a t1/2 of 5 to 10minutes (35).

The sensitivity of the GpNLuc reporter was also assessed by adirect comparison of BLI and external caliper measurements insubcutaneous xenografts (Fig. 2C). Temporal analysis identifieda significant difference between the A549-GpNLuc and A549-LKB1-GpNLuc NSCLC cohorts as early as day 11 after transplantusing BLI, which was not evident until day 21 using calipermeasurements. We next tested the ability of the GpNLuc signalto penetrate through deep tissue using an orthotopic lung tumormodel. As few as 500,000GpNLuc-expressing A549NSCLC cellswere injected via tail vein into recipient mice, allowed to col-onize the lungs, and recipients were followed by longitudinalBLI monitoring (Fig. 2D). Three-dimensional (3D) reconstruc-tion performed using the Living Image DLIT algorithm con-firmed that GpNLuc signal was easily detected from deep withinthe lungs and ex vivo BLI of these surgically resected lungsvalidated the DLIT reconstruction (Fig. 2D, SupplementaryFig. S4A–S4C and Supplementary Video S1). Notably, the inten-sity of the GpNLuc signal allowed the detection of microme-tastases at regional lymph nodes (Supplementary Fig. S4D).

GpNLuc monitoring of soft tumors by BLI and flow cytometryIn vivo monitoring of models of hematologic malignancies is

challenging, as experimental parameters often rely on endpoint

analysis such as overall survival, or periodic blood sampling,white blood cell counts, and flow cytometry analyses. To testthe utility of GpNLuc LumiFluor reporter in detecting suchmalignancies, we established orthotopic allografts followingintravenous (tail vein) transplantation of two independentlyderived Em-Myc B cell lymphomas expressing the GpNLucreporter (Fig. 3 and Supplementary Fig. S5). Longitudinalmonitoring revealed a progressive increase in signal intensity,which increased by more than two orders of magnitude on day14 after transplant versus that manifest on days 1 and 2 (Fig. 3Aand Supplementary Figs. S6 and S8A). Temporal analysis con-firmed that monitoring tumor development in deep tissueswith this LumiFluor reporter is technically feasible given thedetection of lymphoma cells in the lungs of recipient mice(Supplementary Fig. S5C and S5D). Homing and colonizationof lymphoma cells into the spleen, inguinal lymph nodes, andspinal bone marrow was easily and strongly detected as early as2 days after transplant, followed shortly thereafter by detectionwithin the axial, cervical, and lumbar/sacral lymph nodes (Fig.3B and Supplementary Figs. S5E and S6). This represents asignificantly reduced time frame for detection compared withthe 2 weeks required for most leukemia models using conven-tional luciferase reporters having inferior light emission (15).Differences in observed signal intensity and half-life could beattributed to the route of substrate administration. Although ani.p. administration route was used for monitoring the NSCLCtumor models (Fig. 2), substrate was administered via anintravenous route for the Em-Myc B cell lymphoma orthotopicallografts or human Burkitt lymphoma xenografts, and analysisof signal intensity and stability revealed that a 250 mg/kg doseadministered intravenously also displays stable signal output(Supplementary Fig. S7A). Both DLIT 3D reconstruction withthe IVIS Spectrum and optical imaging coupled with X-rayperformed with a Bruker In Vivo Xtreme optical/X-ray imagerconfirmed the anatomic origins of observed signals (Fig. 3B,Supplementary Figs. S6B and S7B and Supplementary VideoS2). Finally, ex vivo BLI of surgically resected tissues or flowcytometry analyses of surgically resected tissues on day 14following lymphoma transplant validated these findings(Fig. 3C and Supplementary Fig. S5D and S5E, and Fig. S8).

Enhanced output of LumiFluor reporters requiresintramolecular energy transfer

To test if increased light output and broader optical profilegenerated via the intramolecular BRET within GpNLuc trulyenabled more sensitive in vivo imaging, Y67A and Y67CGpNLuc substitution mutants within the chromophore of theeGFP (35) moiety were generated (Fig. 4A). As a control,mutation of the adjacent threonine residue not predicted todisrupt the eGFP chromophore (T66G) was also generated inGpNLuc. Finally, a second LumiFluor reporter was generatedthat has an even broader optical profile, by fusing LSSmOrange(a red-shifted GFP variant) to NanoLuc (OgNLuc). HEK293Tcells were transfected with equal concentrations of retroviralconstructs constitutively expressing NanoLuc, the GpNLuc orOgNLuc LumiFluors, or the GpNLuc point mutants (Fig. 4A).As predicted, like GpNLuc, there were 10-fold increases in totallight output of OgNLuc or GpNLuc-T66G over that of NanoLucalone, and there was a marked attenuation in light output bythe GpNLuc-Y67A and GpNLuc-Y67C mutants. Further, FACSanalyses of retrovirus-transduced Em-Myc B cell lymphomas

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Figure 2.GpNLuc signal strength and stability in A549 NSCLC xenograft and orthotopic transplants. A, A549-GpNLuc cells or A549 cells engineered to also expressthe LKB1 tumor suppressor (A549-LKB1-GpNLuc) were injected subcutaneously into the front and rear flanks of recipient NOD/SCID mice, with theindicated numbers of tumor cells to gauge limits of signal detection (minimum number of detectable cells) 1 day following injection. Furimazine was injectedi.p. and bioluminescence images were captured for the two cohorts, which were monitored longitudinally from day 1 to day 28 after transplantation(lens aperture ¼ f/1; image exposure time ¼ 60 seconds on day 1 or 7 seconds on day 28; binning ¼ 8; field of view ¼ 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) andsignal strength was monitored temporally in response to i.p. furimazine administration at the indicated doses when tumor volume reached 1,500 mm3 (n ¼ 3).C, direct comparison of subcutaneous tumor growth monitored temporally by BLI (top) and caliper measurements (bottom) for mouse xenografts (5 � 105

cells) from A549-GpNLuc or A549-LKB1-GpNLuc cohorts (n ¼ 3). A significant difference was detectable between the A549-GpNLuc and A549-LKB1-GpNLuccohorts 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 signalwas evaluated by orthotopic transplantation of A549-GpNLuc cells (1 � 106) injected intravenously (via tail vein) into NOD/SCID mice. Furimazine wasinjected intravenously and 2D (left) and 3D (right) bioluminescence images were captured. Images are representative of mice monitored longitudinally fromday 1 to day 49 after transplantation (lens aperture ¼ f/1; image exposure time ¼ 60 seconds; binning ¼ 8; field of view ¼ 6.6 cm; and emission setto open filter).

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confirmed that OgNLuc displays a red-shifted fluorescentsignal and that the GpNLuc-Y67C mutant cannot generate aGFP signal (Fig. 4B and Supplementary Fig. S9).

To compare their in vivo activity, BLI analyses of orthotopicEm-Myc lymphomas allografts expressing these reporters wereperformed. These analyses confirmed that the BLI potential ofthe GpNLuc-Y67C mutant was comparable to NanoLuc alone,with much inferior in vivo optical properties requiring 4- to 6-fold longer exposure times (25 and 40 seconds, respectively)versus the GpNLuc LumiFluor (6 seconds; Fig. 4C). Notably,

despite having an apparent equivalent total light output in vitroand half the exposure time in vivo (3 seconds), OgNLuc dis-played an even greater (2- to 4-fold) increase for in vivo signaloutput relative to GpNLuc, likely owing to its red-shiftedemission that is capable of enhanced tissue penetration. Basedon these image acquisitions, signal-to-noise ratios were quan-tified and revealed that GpNLuc and OgNLuc have a signalworking range between 3 and 4 orders of magnitude abovebackground signal generated in control mice similarly injectedwith substrate. Thus, compared with conventional in vivo

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Figure 4.Intramolecular BRET in LumiFluors drives fluorophore excitation/emission and is essential for sensitive in vivo imaging. A, comparison of chromophore mutantsGpNLuc-Y67A and GpNLuc-Y67C, as well as the red-shifted LSSmOrange-NLuc (OgNLuc) fusion, to either GpNLuc or NanoLuc alone. HEK293Tcells were transfected with equal concentrations of each respective retroviral construct and luciferase assays were performed 24 hours after transfection(n ¼ 3; mean � SEM). B, flow cytometric analysis of Em-Myc mouse lymphoma cells, along with serially passaged Em-Myc lymphoma cells engineered to expressNanoLuc, GpNLuc, GpNLuc-Y67A, GpNLuc-Y67C, GpNLuc-T66G, or OgNLuc, confirmed effects of mutagenesis on the fluorescence excitation capacity ofNanoLuc on eGFP and on LSSmOrange. C, allografts of Em-Mycmouse lymphoma cells expressing either NanoLuc, GpNLuc, GpNLuc-Y67C, or OgNLuc (1� 106) wereinjected intravenously into syngeneic Albino C57Bl/6 recipient mice (n ¼ 3). Furimazine was injected intravenously and ventral BLI were captured on day 7 aftertransplantation (lens aperture ¼ f/1; image exposure time: NanoLuc ¼ 25 seconds, GpNLuc ¼ 6 seconds, GpNLucY67C ¼ 40 seconds, or OgNLuc ¼ 3 seconds;binning¼ 8; field of view¼ 22.6 cm; and emission set to open filter). D, model comparing and contrasting conventional in vivo fluorescent imaging to newmethodsoffered by GpNLuc and OgNLuc LumiFluor reporters. Ectopic excitation of fluorescent reporters in vivo results in significant autofluorescence, whereas localexcitation of fluorophores by intramolecular energy transfer from a fused NanoLuc partner prevents global autofluorescence and augments overall signal outputand detection.

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fluorescent or BLI, the GpNLuc and OgNLuc LumiFluor BRETreporters generate a robust, high-intensity signal that is per-fectly suited for sensitive, noninvasive in vivo optical imaging(Fig. 4D).

DiscussionThe development of luciferase molecules having enhanced

light-emitting properties such as NanoLuc is as active arena ofstudy (4, 5, 36–39). However, due to absorption and scatteringof the blue-shifted, short wavelength light emitted by NanoLuc,this reporter cannot penetrate tissues and the signal generatedby NanoLuc has a short half-life in vivo (6, 34). Here we describethe generation and characterization of a novel class of in vivoBRET imaging reporters coined LumiFluors that overcome thesedeficiencies. Specifically, LumiFluors have the desired fluores-cent–bioluminescent spectral and optical properties thatallow sensitive imaging both ex vivo and in vivo, and they alsoallow one to isolate and fully characterize target cells using flowcytometry. Indeed, the enhanced strength, stability, and dura-tion of signal, and the deep tissue penetration capabilities ofthe GpNLuc and OgNLuc LumiFluors dramatically reduceimage acquisition times making them more desirable thanconventional reporters for in vivo imaging. The increased sen-sitivity and imaging speed offered by LumiFluors providesimproved monitoring of tumor development and the detectionof small metastatic lesions (40).

The development and characterization of the GpNLuc andOgNLuc BRET reporters was achieved by a molecular model-ing-guided approach that resulted in a short, flexible peptidelinker that enables highly efficient donor energy transfer to thepaired acceptor to generate intense bioluminescent signals. Oplo-phorus gracilirostris luciferase (OLuc) naturally has a high QY (28),but even brighter signals were achieved by pairing the enhancedversion ofOLuc (NanoLuc) to high QY fluorophores (eGFP QY¼0.6 and LSSmOrange QY ¼ 0.45). Compared with NanoLucalone, LumiFluor BRET reporters are more than 10-fold brighterand thus display increased tissue penetration thereby overcomingcurrent limitations associated with reporters used for in vivoimaging. A key advantage of LumiFluor reporters is that theyprovide the user with the ability to noninvasivelymonitor specificcell populations in vivo and to then isolate these cells by FACS. Thisis, for example, particularly useful for characterization of sub-populations and heterogeneity in primary andmetastatic tumors,and in circulating tumor cells, in orthotopic or even geneticallyengineered mouse models (GEMM). Moreover, their strength ofsignal suggests that LumiFluors may allow one to locate, isolate,and characterize rare cancer stem/initiating cells and dormant/resistant tumor cells.

A potential limitation to the use of LumiFluor reporters istheir dependence on the substrate furimazine. Although fur-imazine was previously shown to be stable in media in thepresence of serum (6), it is a coelenterazine analogue andcoelenterazine is known to be a substrate for multidrug resis-tance (MDR1) P-glycoprotein, which can lead to its rapidexport from cells that express MDR1, thereby impacting signalintensities (41). Future experiments will evaluate the transportproperties of furmazine by MDR1 as well as its ability to crossthe blood-brain barrier.

Many uses of LumiFluors are feasible, for example, as reportersin GEMM, as fusions with proteins to monitor real-time biologic

processes in cells, and as biosensor tags for antibodies or smallmolecule probes that home to select target cells, which could beused to visualize responses in vivo and to determine themargins ofselect tissues and/or tumors, to aid in surgical procedures(5, 42, 43). Finally, LumiFluors can also be used in traditionalBRET assays to study protein:protein or ligand:protein interac-tions by developing a split LumiFluor reporter for complemen-tation assays (44, 45).

Thefluorescent component of LumiFluor reporters alsopermitsmultiplexing. For example, the distinct spectral characteristics ofGpNLuc and OgNLuc allow one to simultaneously monitorsignals coupled tomultiplemolecular and cellular events in eitherin vitro or in vivo formats. Importantly, the sustained intensesignals produced by these two LumiFluors also increases theconfidence for monitoring rare coincident events in preclinicalmodels, for example, the interplay of immune cells with tumors,tumor–stromal interactions and how these, and the fate of pri-mary tumors andmicrometastases, are affected by treatment withtherapeutics. Finally, engineering the NanoLuc moiety of Lumi-Fluors, so that it emits light at different wavelengths, should allowfor the intramolecular activation of a broad spectrum of fluores-cent proteins that will expand the repertoire and imaging capa-bilities of these novel reporters.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A.L. AmelioDevelopment of methodology: F.X. Schaub, Md. S. Reza, C.A. Flaveny, W. Li,S. Hoxha, M. Guo, A.L. AmelioAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F.X. Schaub, Md. S. Reza, C.A. Flaveny, W. Li,J.L. Cleveland, A.L. AmelioAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F.X. Schaub, Md. S. Reza, C.A. Flaveny, W. Li,J.L. Cleveland, A.L. AmelioWriting, review, and/or revision of the manuscript: F.X. Schaub, Md. S. Reza,C.A. Flaveny, W. Li, A.M. Musicant, M. Guo, J.L. Cleveland, A.L. AmelioStudy supervision: M. Guo, J.L. Cleveland, A.L. Amelio

AcknowledgmentsThe authors thank Promega Corporation for their generous gifts of

plasmids for NanoLuc, the NanoLuc substrate furimazine, and the poly-clonal antibody to NanoLuc and Dr. Frederic J. Kaye for the NSCLC A549-pBABE and A549-LKB1 cell lines. The authors also thank SollepuraD. Yogesha, Krishna Chinthalapudi, Rangarajan Erumbi, and Pengfei Fangfor helpful technical assistance and Drs. Shawn Hingten, Chad Pecot, andKlaus Hahn and members of the Amelio Lab for helpful comments, sugges-tions, and scientific review of this manuscript.

Grant SupportThis work was supported by a Howard Temin Pathway to Independence

Award in Cancer Research from the NCI R00-CA157954 and UNC UniversityCancer Research Funds (A.L. Amelio), NIH/NCI R01 grant CA167093(J.L. Cleveland), by a Swiss National Science Foundation PostdoctoralFellowship (F.X. Schaub), by a NIH/NIGMS T32 training grant GM007092(A.M. Musicant), and by monies from the State of North Carolina to UNC-Chapel Hill. This work was also supported in part by NCI ComprehensiveCancer Center Grants P30-CA076292 awarded to the H. Lee Moffitt CancerCenter & Research Institute and P30-CA016806 awarded to the LinebergerComprehensive Cancer Center.

Received December 5, 2014; revised August 18, 2015; accepted August 28,2015; published OnlineFirst September 30, 2015.

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Chimeric Fluorescent–Luminescent Fusions for In Vivo Imaging

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In VivoFluorophore-NanoLuc BRET Reporters Enable Sensitive

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