bioluminescent imaging: a critical tool in pre-clinical oncology research

Journal of PathologyJ Pathol 2010; 220: 317–327Published online 27 October 2009 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/path.2656

Review Article

Bioluminescent imaging: a critical tool in pre-clinicaloncology research

Karen O’Neill,1 Scott K Lyons,2 William M Gallagher,3 Kathleen M Curran1 and Annette T Byrne3,4*1UCD School of Medicine and Medical Science, Health Science Building, University College Dublin, Belfield, Dublin 4, Ireland2Cancer Research UK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK3UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland4Royal College of Surgeons in Ireland, Department of Physiology and Medical Physics, Reservoir House, Sandyford Industrial Estate, Ballymoss Road,Dublin 18, Ireland

*Correspondence to:Annette T Byrne, Royal College ofSurgeons in Ireland, Departmentof Physiology and MedicalPhysics, Reservoir House,Sandyford Industrial Estate,Ballymoss Road, Dublin18, Ireland.E-mail: [email protected]

No conflicts of interest weredeclared.

Received: 8 January 2009Revised: 6 October 2009Accepted: 17 October 2009

AbstractBioluminescent imaging (BLI) is a non-invasive imaging modality widely used in the field ofpre-clinical oncology research. Imaging of small animal tumour models using BLI involvesthe generation of light by luciferase-expressing cells in the animal following administrationof substrate. This light may be imaged using an external detector. The technique allowsa variety of tumour-associated properties to be visualized dynamically in living models.The increasing use of BLI as a small-animal imaging modality has led to advances in thedevelopment of xenogeneic, orthotopic, and genetically engineered animal models expressingluciferase genes. This review aims to provide insight into the principles of BLI and itsapplications in cancer research. Many studies to assess tumour growth and development, aswell as efficacy of candidate therapeutics, have been performed using BLI. More recently,advances have also been made using bioluminescent imaging in studies of protein-proteininteractions, genetic screening, cell-cycle regulators, and spontaneous cancer development.Such novel studies highlight the versatility and potential of bioluminescent imaging in futureoncological research.Copyright 2009 Pathological Society of Great Britain and Ireland. Published by JohnWiley & Sons, Ltd.

Keywords: bioluminescent imaging; animal models; pre-clinical; oncology; non-invasiveimaging

Overview

The in vitro study of cultured tumour cells fails toprovide insight into all processes involved in thedevelopment and progression of tumours within livingorganisms. To better recapitulate fundamental aspectsof human disease, such as angiogenesis and metas-tasis, it has been necessary to develop in vivo can-cer models. The use of such cancer models has ledto a need for sensitive, efficient, and non-invasivesmall-animal imaging modalities capable of follow-ing tumour development and progression in vivo, aswell as monitoring minimal residual disease [1]. Thesemodalities are directly related to their clinical counter-parts and facilitate true in vivo imaging of the patho-physiological changes that occur within organ sys-tems during the development of neoplastic disease [2].One important imaging modality (currently without aclinical counterpart), bioluminescence imaging (BLI),relies on the detection of light produced from cellstagged with luciferase and has proven to be a valu-able tool in furthering the utility of pre-clinical cancermodels.

Basic principles

Introduction

Bioluminescence refers to the enzymatic generationof visible light by living organisms. Many loweranimals are capable of undergoing a bioluminescentreaction (including species of bacteria, fungi, marinecreatures, and insects such as the firefly beetle). Innature, luciferase genes encode proteins that act onsubstrates including D-luciferin in the case of fireflyluciferase, and coelenterazine for Renilla luciferase.

The firefly luciferase gene is most commonly usedin animal tumour models. Luciferase oxidizes luciferinin the presence of ATP (adenosine tri-phosphate) andmolecular oxygen to form an electronically excitedoxy-luciferin species (see Figure 1). Visible yellow-green to yellow-orange light is emitted followingthe relaxation of excited oxy-luciferin to its groundstate [3].

All in vivo bioluminescent imaging approaches relyon the external detection of this internally generatedchemiluminescence and so permit the non-invasive

Copyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.www.pathsoc.org.uk

318 K O’Neill et al

Figure 1. Panel A depicts the typical architecture of a bioluminescent reporter transgene. (i) The promoter sequence used todrive the expression of the transgene. This is most commonly a constitutive promoter (eg CMV) capable of driving ubiquitousand high-level transgene expression in the majority of mammalian cell types. Alternatively, it could be a tissue-specific orcontext-specific (eg only at S-phase of the cell cycle) promoter. (ii) The bioluminescent reporter, typically encoding either aluciferin or a coelenterazine oxidizing enzyme (eg firefly or Renilla luciferase, respectively). (iii) A synthetic intron; although notrequisite, splicing of the transgenic transcript can enhance transgene expression. (iv) The poly-A signal sequence to enhance thestability of the transgenic mRNA, thus enhancing expression. Panel B illustrates the firefly luciferase–luciferin reaction that resultsin the production of light

study of biological processes within small animals [4].This principle has been extensively applied to pre-clinical cancer models. Light emission from consti-tutively expressed luciferase is proportional to tumourcell burden, and photon emission increases as the cellpopulation multiplies [5–7]. Cells may be transfectedor transduced in vitro to express luciferase prior toin vivo implantation or reporter gene expression maybe introduced through the germline to generate biolu-minescent transgenic tumour models.

Luciferase expression is normally imaged followingan intraperitoneal (IP) injection of substrate. Imagesare then acquired when signal intensity is maximal (tobe determined for each model, but typically between10 and 15 min). Substrate may also be delivered intra-venously (IV) and the subjects imaged immediately.Signal intensity is reportedly enhanced up to six times,but the clearance of substrate is also considerablyfaster [8]. Repeated IV administration of substratecan also result in tail vein damage, potentially lim-iting the number of images that can be acquired overtime. Additional methods of substrate administrationinclude direct intratumoural injection [7,9], subcu-taneous delivery [10,11], and oral delivery throughdrinking water [12]. Micro-osmotic pumps have alsobeen utilized. These result in sustained and contin-uous delivery of substrate for greater than 7 days,but signal intensity is lower due to reduced systemiclevels of luciferin [13]. These pumps eliminate sig-nal variability associated with substrate delivery andmay be best suited for models where luciferase expres-sion is induced (eg apoptosis or cell-cycle-dependentreporters).

Certain aspects of tumour-associated biology canalso affect bioluminescent read-out. For example,the expression of the ATP-binding cassette (ABC)family transporter, ABCG2/BCRP, has recently been

shown to pump D-luciferin out of cells [14]. Similarly,the substrate coelenterazine, used to image Renillaluciferase expression, has been shown to be pumpedout of MDR-1 P-glycoprotein-expressing cells [15].

A recently characterized and naturally secretedluciferase called GLuc (Gaussia luciferase, from themarine copepod Gaussia princeps) should not beprone to the effects of such small molecule transporteractivity. In addition to possessing a signal intensity upto 200 times greater than that of Renilla luciferasewhen expressed in mammalian cells under standardconditions [16], GLuc activity can be measured inthe extracellular environment. Indeed, in addition tolocally lighting up the primary site of implanted andlabelled tumours, it has been shown that biolumines-cence activity measured in peripheral blood correlatesstrongly with viable in vivo tumour burden [17].

Context-dependent or ‘pro-luciferin’ substrates havealso been developed for beta-galactosidase (beta-gal),activated caspases, and cytochrome 450 [18,19]. Thesesubstrates are inactive until metabolized and so pro-vide a specific bioluminescent read-out for the targetprotein in question.

Equipment

The bioluminescent reaction between luciferase andits substrate produces low-intensity light that can-not be seen with the human eye or by conventionalmicroscopy. Therefore a high sensitivity detector isrequired, particularly when imaging cells in vivo, asoverlying tissues cause absorption and signal scatter-ing. Several commercially available systems are capa-ble of detecting such low levels of light. In general,these systems comprise a light-tight imaging chamberinto which the subjects are placed. Above this chambersits a sensitive charged coupled device (CCD) camera

J Pathol 2010; 220: 317–327 DOI: 10.1002/pathCopyright 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bioluminescent imaging: a critical tool in pre-clinical oncology research 319

to detect the bioluminescence. The CCD is super-cooled to around −90 ◦C to reduce thermal noise,thereby increasing its ability to detect very low lev-els of light. Cameras broadly detect light within thevisible range of the spectrum, with some systems ableto detect light in the far-red and near-infra-red [20].This range is in fact well beyond that required forbioluminescence detection as the peak emission wave-lengths for firefly and Renilla luciferases are 560 and475 nm, respectively. Studies have shown that cooledintegrated CCD cameras provide consistent and repro-ducible results within ±8% standard deviation frommean values [21]. Image acquisition is also short, typ-ically ranging between 1 and 180 s, and is controlledby software that enables modification and analysis ofthe final image.

Advantages and limitations

Some of the advantages of BLI include high sensi-tivity, comparatively reduced equipment costs, highthroughput, short image acquisition times, relative easeof use, and minimal image post-processing require-ments [5]. BLI is also a non-radioactive imagingmodality, in contrast to other modalities such aspositron emission tomography (PET), single photonemission computed tomography (SPECT), and com-puted tomography (CT).

BLI allows longitudinal monitoring of tumourgrowth, spread, and response to treatment in pre-clinical cancer models within the same individual, withno requirement to euthanize [22,23]. This is of signifi-cant benefit as each animal comprises its own control;therefore fewer animals are needed per study, whichreduces overall costs and labour.

Although BLI is not an absolutely quantitativeimaging approach like PET or SPECT, it does allowtumour growth dynamics to be accurately determinedby quantifying relative changes in light emission overtime [24]. As a result, BLI images provide measuresof tumour biology that are neither subjective norqualitative [25].

Firefly luciferase bioluminescent activity is depen-dent on the presence of oxygen and ATP, and con-sequently, photons are only emitted from metaboli-cally active cells. This facilitates ready assessment ofnew therapeutic effects, as light emission is propor-tional to the number of viable labelled cells [6,26].In vivo studies assessing tumour burden relative tobioluminescent signal have shown strong correlationsbetween viable cells and bioluminescence [27]. Deador necrotic regions within a tumour, potentially indica-tive of a positive drug response, contribute to volume;therefore the ability to solely measure viable cells soonafter treatment is advantageous for rapidly assessingdrug efficacy over traditional volumetric-based mea-surements [28]. The high sensitivity of BLI also allowsfor the early detection of tumours. Malignant cellsmay be visualized using BLI at least 13 days before

Table 1. Advantages and limitations of BLI

Advantages Limitations

• Easy to use • Dependent on tissueproperties

• High sensitivity • Light absorption byhaemoglobin

• Non-invasive • Signal attenuation bymelanin and fur

• Short acquisition times • Limited 3Dreconstruction

• High-throughput capability • Currently not applicableto human studies

• Animals may act as own control• Images metabolically active cells only

they form palpable tumours in certain xenograft mod-els [29]. BLI may therefore be useful in ‘preventiontrials’ for drugs specifically targeting the early stagesof tumour development [29]. As the whole animal isimaged, unpredictable sites of metastasis are evidentand can be rapidly identified [30].

Although it is clear that BLI has many advan-tages, like all imaging modalities, there are limita-tions (Table 1) [21]. Firstly, the efficiency of biolu-minescent light transmission through an animal fromits origin depends largely on the type and depth ofoverlying tissue as well as its scattering properties.Haemoglobin absorbs light; therefore highly vascular-ized organs tend to have lower levels of light trans-mission compared with skin or muscle. Secondly, BLItraditionally has provided two-dimensional (2D) pla-nar images with limited spatial and depth resolution[31]. Recent advances have enabled the translationof BLI data into 3D tomographic imaging, therebytheoretically providing better quantification and signallocalization [32–34]. Signal depth can be estimatedby measuring the ratio of red to green emitted light onthe surface of the animal from multiple aspects andcomputing its likely origin.

Hypoxic regions within tumours may also affectsignal intensity. Bioluminescence is dependent onoxygen and a number of studies have found thatthe amount of light emitted from luciferase-labelledcells is reduced as the oxygen concentration decreases[9,35,36]. Oxygen tension limits for BLI are stillnot fully defined, however, and the HIF-1 (hypoxia-inducible factor 1) promoter has been successfullyused to express luciferase and to report hypoxiain vivo [37,38].

Similar to PET, the spatial resolution of BLI isrelatively low (1–2 mm), due to the scattering anddiffraction of light through tissue. This can make theprecise localization of the signal source difficult toascertain [39] and a detailed assessment of primarytumour anatomy is often limited [5]. Comparisonsbetween different animal subjects may also be compli-cated by the variable depth of tumours in each, as thelight signal will be variably attenuated [40]. Althoughthis problem may be overcome by using each animalas its own control, it is important to ensure that the



animal is accurately and reproducibly positioned eachtime an image is acquired. Post-processing techniquessuch as landmark-based image registration can be per-formed to align a time series of 2D data but consistentanatomical positioning is still important for accurateregistration [41].

Identifying luciferase-expressing cells at the end ofan experiment can also be a challenge. Poor anti-bodies for immunohistochemistry (IHC) may requiretechnically difficult in situ hybridization techniques[42]. Several groups have also developed multi-modalreporters (ie reporters that confer fluorescence aswell as bioluminescence) [43–45] to enable lightmicroscopy or FACS (fluorescence-activated cell sort-ing) of reporter-expressing cells. Most critically, asthere is no equivalent imaging modality for humansubjects, this method is not directly applicable forclinical use [21]. As the stable integration of biolumi-nescent reporter genes into the human genome wouldbe a random mutagenic event, it is unlikely that BLIwill be used as an optical imaging technique in humanstudies in the near future [46]. Sensitivity would alsolikely be reduced significantly by the increased tissuedepths associated with human imaging.

Sensitivity

Non-labelled cells do not emit light; therefore BLIhas an excellent signal-to-noise ratio and represents anextremely sensitive means of detecting labelled cells.Many factors affect the overall sensitivity of BLI,including signal depth, transgene expression level, andthe extent of background bioluminescence. It is easierto detect labelled cells located at the surface of thebody than those at deeper tissue locations. As tissuesover-lying the target cells substantially attenuate light,a population of labelled cells located deep withinthe body will appear less bright than an equivalentnumber of labelled cells located near the surface ofthe skin [47]. Emitted light from cells residing belowhighly pigmented organs is also prone to quenching.Another consideration is signal quenching due toanimal fur pigmentation. Dark fur quenches signalsignificantly more than white fur. Therefore, if albinostrains of mice cannot be employed, local depilationcan dramatically improve image sensitivity [48].

As mentioned, the high sensitivity of BLI is inpart due to minimal background noise [49]. As non-labelled tissues do not emit significant levels oflight, any light detected may be accounted for bycells expressing the bioluminescent reporter. How-ever, some exceptions exist. These include transgenicmodels in which there may be background expres-sion of luciferase from non-transformed but labelledcells within the same organ or from non-specificectopic transgene expression of luciferase in differentorgans. Also, the sensitivity of detection of metastaticlesions residing close to a primary tumour is reducedwhen the luciferase signal from the primary tumourdrowns out the light emitted from the small metastases.

Differences in BLI sensitivity at various locations mayalso arise from altered metabolic activity between sitesdue to spatial confinements [50].

When all the factors mentioned are favourable, smallnumbers of cells may be detected in animal tumourmodels. Early studies involving luciferase-labelled cer-vical carcinoma cells found that 1 × 103 cells couldbe detected in the peritoneal cavity, 1 × 104 at subcu-taneous sites, and 1 × 106 circulating cells followinginjection. Furthermore, signals were apparent immedi-ately following injection, even though tumour growthwas not visible until after 40–50 days [50]. Morerecent studies have reportedly detected cells in theperitoneal cavity ranging in number from 2500 cellsto as few as 100 cells [6,50]. Such high sensitivityindicates that BLI is an ideal approach for detectingminimal residual disease states and micro-metastaseslocated at a site removed from the primary light source.

BLI in oncology research

Pre-clinical models

To date, the majority of published BLI studies usingcancer models have been xenograft-based, as estab-lished tumour cell lines can be rapidly rendered bio-luminescent in vitro [51]. However, more sophisti-cated genetically engineered luciferase ‘reporter’ mod-els (GEMs) of human cancer have also been devel-oped which enable the study of in situ spontaneoustumour development and response to treatment inimmunocompetent animals [40,42,52]. This is highlyadvantageous as such GEMs are currently the mostrepresentative models of human cancer available andmany develop tumours with variable latency and innon-visible locations. Thus, real-time imaging of drugeffects in individual animals using BLI can facilitatestringent assessment of drug efficacy and help to eluci-date the mechanism of action. The ability to monitortumours over time within the same animal, possiblybefore and after treatment, greatly improves the over-all quality of data as well as reducing the number ofanimals needed to generate it.

An example of a bioluminescent GEM is a luciferasereporter mouse that enables the imaging of bothnormal and spontaneously transformed prostate [42].This particular model facilitated the non-invasiveidentification of androgen-dependent versus androgen-independent disease rapidly following androgen abla-tion. The latter is currently untreatable in the clinic, sothis model may facilitate the development of new treat-ments against this tumour type. Several other prostatereporter mice have now also been described [53,54].

Some GEMs rely on site-specific recombinases toirreversibly switch on or off gene expression in acell-type-specific manner. Tumour suppressors can beswitched off and oncogenes switched on, thus result-ing in tumourigenesis [55,56]. The development ofconditional reporters has made it possible to visualize



spontaneously arising tumours in such models usingBLI. One example of this is the use of the Cre/loxPsystem to conditionally ‘switch on’ bioluminescence tomeasure conditional Kras2v12-induced lung tumoursin mice [51]. Theoretically, this reporter facilitatesthe imaging of any Cre-dependent tumour irrespectiveof tissue and has also shown demonstrable utility inthe imaging of a conditional PTEN (tumour suppres-sor gene)-dependent prostate adenocarcinoma model[52]. This prostate model is also unique, as luciferaseexpression is prostate-specific but completely indepen-dent of androgens.

The ability to measure two parameters non-invasively in vivo can be advantageous and some mod-els have now been dual-labelled with both Fluc (fireflyluciferase) and Rluc (Renilla luciferase) [57]. As theseluciferases use different substrates, each can be imagedseparately in the same subject by varying the time ofsubstrate administration [58]. For example, one studyinvolving a human glioma model quantified tumourcell burden using firefly luciferase, while simultane-ously measuring the efficiency of tumour cell trans-duction by a therapeutic virus using Renilla luciferase.This allowed gene delivery efficiency to be correlatedwith therapeutic efficacy over time, all within the sameanimal [58,59].

Two-step transcriptional amplification (TSTA) isan approach used to increase the level of tissue-specific transgene expression, which can often beweak. TSTA has been used to augment the transcrip-tional activity of the relatively weak VEGF (vascularendothelial growth factor) promoter (pVEGF) usedto drive luciferase reporter expression. In the resul-tant transgenic mouse (pVEGF-TSTA-fl), the induc-tion of VEGF gene expression could be imaged non-invasively using a wound-healing model and a sub-cutaneous mammary tumour model [60]. The TSTAsystem has been further used to amplify the expres-sion of firefly luciferase and an enhanced thymidinekinase transgene in a prostate cancer model [58].

Replication competent viruses (armed therapeuticviruses — ATVs) represent a novel anti-cancer strat-egy in which the action of therapeutic proteins is com-bined with the anti-tumour properties of viral infec-tion. BLI has been used to visualize the levels andduration of transgene expression delivered by theseviruses. A study using an intracranial glioma mousemodel found that cellular replication decreased afterintratumoural injection of oncolytic adenovirus andthat treatment with cyclophosphamide increased viral-mediated gene expression [61] (Figure 2).

In summary, BLI of reporter GEMs has now beenapplied in a wide variety of pre-clinical oncologystudies. Even though GEMs lack the complexityof environmental influences associated with humancancer development [47], these models represent themost accurate models of tumourigenesis currentlyavailable. Further discussion on the applications ofspecific models is provided in subsequent sections(Table 2).

Assessing therapeutic efficacy and mechanismof action

When bioluminescence is constitutively expressed incells, light output is proportional to tumour burden[5,7]. Subsequent application of this uncomplicatedstrategy has facilitated the monitoring of cancer ther-apeutic effect on tumour growth in vivo in a largenumber of models [7,10,27,62–64]. Advances usingsuch simple models have also been made. For exam-ple, an orthotopic multiple myeloma model (KMS-11-luc) has been developed to express a mutant versionof a fibroblast growth factor receptor (FGFR3) knownto contribute to tumour progression. In one study,CHIR-258, an anti-myeloma drug, was administeredto inhibit FGFR3 signalling. This agent was found tostrongly inhibit tumour growth and dissemination inthis model [26].

Luciferase expression can also be regulated in differ-ent ways by different classes of promoter to providedifferent tumour-related information. For example, aGEM has been developed whereby luciferase expres-sion is controlled by the human E2F1 promoter. Asthis promoter is only active during S-phase, this mousewas used to directly assess effects on the proliferativeactivity of glioma cells [40]. It was shown that dis-ruption of the RB (retinoblastoma tumour suppressorgene) pathway in endogenously induced brain tumourcells could be measured (due to a resultant increase incell cycling), followed over time, and used to evaluatethe efficacy of drug treatment in vivo [40].

Split luciferase reporters have also been developedthat only emit light following the onset of a biolog-ical process (eg caspase-3 activity during apoptosis).Luciferase has been split into two complementary butnon-functional N-terminal and C-terminal fragments.These can then be fused to pepA and pepB domains(peptide partners with strong binding affinity). Whena caspase cleavage motif (DEVD) is cleaved at theearly stages of apoptosis, the pepA and pepB domainscan bind one another, resulting in reconstitution ofluciferase function and light emission. The utilityof this reporter was demonstrated with a gliomamodel and imaging apoptosis in vivo in responseto various chemotherapy and radiotherapy regimens[65]. The ability to detect apoptosis non-invasivelyin vivo is important for anti-cancer drug develop-ment and several additional caspase-dependent biolu-minescent reporter strategies have been additionallydescribed [65–67].

BLI has also been used to monitor tumour regressionand apoptosis following the induction of S-TRAIL(secretable tumour necrosis factor-related apoptosis-inducing ligand) activity via a caspase-3 activatableaminoluciferin in a murine glioma model [59].

One important factor contributing to a cancer cell’sability to avoid cell death is loss of p53 function,which occurs in almost half of all human tumours. BLIhas been used to perform a high-throughput cell-basedmolecular screen to identify small molecules capable



Figure 2. In vivo imaging of mice bearing U87� EGFR intracranial tumours treated with an intratumoural injection of a range oftherapeutic virus doses (Ad� 24.CMV-Luc; 106, 107, 108 pfu) or of a control virus (Ad.CMV-Luc; 107, 108 pfu). Individual mice areshown with a colour-coded overlay on days 4, 9, and 12 after virus injection [61] [Reprinted with kind permission from NaturePublishing, Licence No 1872030160888]

Table 2. BLI in pre-clinical oncology

Application Example

• Animal models Xenograft, orthotopic, and GEM models of humancancer have been developed which expressluciferase

• Drugdevelopment

BLI allows therapeutic efficacy of cancer drugs to beestablished

• Monitoring ofgenes

Luciferase-labelled cells may be used to monitorgene delivery and gene expression in vivoGenetic screening has also been performed usingBLI, allowing identification of specific oncogenes

• Tumourdevelopment

BLI may be used to study processes such asangiogenesis, apoptosis, and adhesion in cancer cells

• Metastasis High sensitivity of BLI allows the imaging ofmetastasis and minimal residual disease states incancer models

• Proteininteractions

BLI has been used to image protein–proteininteractions in vivo

of restoring wild-type p53 activity. This work wasfollowed up by in vivo studies using a human colon

cancer xenograft model to demonstrate the anti-tumoureffects of such compounds [68]. BLI reporters havealso enabled in vivo measurements of p53 expressionin response to DNA damage at both transcriptional andtranslational levels [69,70].

Assessing cell signalling pathways and proteininteractions

BLI has also been used to monitor the cell cycle[32]. A p27-luciferase fusion molecule was devel-oped and shown to be regulated by Skp2 in acell-cycle-dependent manner. In vivo BLI visualizedthe accumulation of p27Luc in human tumour cellsafter the administration of Cdk2 inhibitory drugs[71] (Figure 3). The glioma model (E2f-luc) previ-ously discussed also highlights cells in S-phase andhas facilitated the investigation of downstream sig-nalling pathways involving mTOR (mammalian tar-get of rapamycin) and PDGF (platelet-derived growth



factor) receptor activation, both critical for gliomamaintenance [40].

A reporter molecule has been developed to measureAkt activity in animals via BLI [72]. The reportercomprises a uniquely engineered luciferase moleculethat undergoes a conformational change and gainsfunctionality in response to phosphorylation by Akt.This reporter should result in a better understandingof the role of Akt in tumour progression, as well asproviding a means to evaluate the efficacy of agentsdesigned to modulate this pathway.

Protein regulators of cellular function have alsobeen studied using BLI. A split synthetic Renillaluciferase (hRLUC) complementation-based biolumi-nescence assay was successfully applied to studyhomodimeric protein–protein interactions in mice[73]. Protein–protein interactions in vivo have alsobeen examined using an inducible yeast two-hybridsystem adapted for mammalian cells and having bio-luminescent reporter function. Such approaches willlikely facilitate the evaluation of drugs modulatingprotein–protein interactions in the future [74].

Assessing key processes in tumour developmentand progression

BLI can functionally assess critical processes intumour development and progression, such as angio-genesis and metastasic dissemination [47]. Angiogen-esis is an essential process required to sustain tumourgrowth. VEGF (vascular endothelial growth factor)signalling via the VEGFR2 receptor is believed toplay an important role in the growth of new bloodvessels and the establishment of a functional vascu-lar network during tumourigenesis. Several biolumi-nescent transgenic animal models have been devel-oped to better understand the role of VEGF duringin vivo tumour angiogenesis. In addition to the TSTAVEGF-luc [75], a VEGFR2-luc transgenic mousemodel has been developed and has shown utility formonitoring angiogenesis during wound healing [76].Similar approaches may be successful in the futurefor imaging tumour angiogenesis (see Figure 4). Amulti-modality approach using BLI, MRI, and PEThas also been used to successfully assess the anti-angiogenic effects of a vasculature-targeting fusiontoxin (VEGF121/rGEL) in an orthotopic glioblastomamodel [77].

The high sensitivity of BLI makes it an idealapproach to monitor minimal residual disease, and hasbeen extensively used to study metastatic spread [78].Bone metastases derived from a prostate carcinomamodel could be detected by BLI up to 2 weeks earlierthan by radiography [79]. BLI has also been foundto be more accurate in the measurement of tumourviability and growth than radiography measurements,which only take into account the osteolytic area [80].Intra-cardiac injection of a bioluminescent breast can-cer cell line, MDA-MB-231-luc, demonstrated thatbone marrow metastases could be localized and

monitored via BLI before tumour-induced osteolysisoccurred [81]. Thus, further studies have used BLIto measure the therapeutic efficacy of bisphospho-nate on bone metastases [80]. Other studies involvingthe anti-osteolytic drug SU11 248 and a 435/HAL-luc tumour model helped to establish the effectivedose of drug against the growth of established bonemetastases [82].

The role of bone morphogenetic protein 7 (BMP7)in inhibiting the development of osteolytic bone metas-tases has also been examined with BLI [83]. Over-expression of this protein in a bioluminescent breastcancer model showed a decrease in metastatic poten-tial. In addition, intravenous administration of BMP7(100 µg/kg per day) to the model also inhibited cancercell growth.

In vivo BLI has also been used to help identifyand rapidly validate the results of large-scale geneticscreens aimed at identifying candidate metastasis-associated oncogenes. These include validating sup-pressors of anoikis (apoptosis induced by loss ofcell–matrix interactions) [84] and assessing genes thatpromote breast cancer metastasis to the lung [85–87].

Recent advances in BLI

Advances in the field of BLI continue to be maderapidly. One activity has focused on improving theability of BLI-generated light to transmit through tis-sues by red-shifting the predominantly yellow/greenemission of luciferase. The colour of emitted lighthas recently been related to the structure of luciferase.Studies have found that residue Ile288 affects molec-ular rigidity, which in turn influences the wave-length of the light emitted. Mutants have beencreated (LcrLuc 1288 V and 1288A) which werefound to shift the emission spectrum from yel-low/green to orange/red, enhancing tissue penetra-tion [3] (Figure 5). Similarly, light emission from amodified version of luciferase of a different speciesof firefly (Lampyris turkestanicus) has been shiftedfrom green to red [88]. As red light has enhancedtissue penetration properties, these mutant versionsof luciferase may be more suited to future in vivostudies.

Advances have also been made in the combina-tion of BLI with various other imaging modalities.Tri-fusion reporter genes have been designed whichfacilitate the multi-modality imaging of gene expres-sion. These triple reporters, typically comprising aluciferase, a fluorescent protein, and a thymidinekinase gene, allow the simultaneous imaging of targetcells using bioluminescence, fluorescence, and PET[45,89,90]. Such fusion reporters have been used tocharacterize the metastatic properties of a breast can-cer cell line [91], as well as to verify the presence ofpulmonary metastases within a CT-defined anatomicalframework [92].

As non-invasive BLI does not provide anatomi-cal information, software has been developed that can



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(c) (d)

Figure 3. Monitoring Cdk2 inhibition in vivo using bioluminescent imaging. (a) Hollow fibres filled with polyclonal U2OS cellsproducing luciferase (Luc; left flank) or p27Luc (right flank) were implanted subcutaneously into nude mice. Left: baselinebioluminescent images taken 7 days later. Right: repeat images obtained after two doses of flavopiridol (5 mg/kg, once a day byintraperitoneal injection); fold induction is indicated. (b) Normalized fold induction (p27Luc/Lucpost−treatment ÷ p27Luc/Lucpretreatment)of p27Luc in seven mice after treatment with flavopiridol. (c) Normalized fold induction of p27Luc in mice treated with theindicated doses of flavopiridol (eight mice per treatment group) and imaged as in a. (d) Polyclonal H1299 cells producing luciferase(left flank) or p27Luc (right flank) were injected subcutaneously into nude mice. Left: bioluminescent images were obtained6 weeks later, when tumours of comparable size (∼5 mm) had formed bilaterally. Right: repeat images were obtained aftertwo doses of flavopiridol (5 mg/kg, once a day by intraperitoneal injection). Fold induction is indicated and was calculated asp27Luc/Lucpost−treatment ÷ p27Luc/Lucpretreatment. Error bars indicate standard error (SEM) [71] [Reprinted with kind permissionfrom Nature Publishing, Licence No 1872030927154]

Figure 4. Non-invasive in vivo bioluminescent imaging of tumour angiogenesis in mice. BLI of a representative VEGFR2-luc-KImouse following subcutaneous implantation of LL2 cells on the dorsal side. Characteristically, a robust induction of bioluminescenceis observed, predominantly from within the developing tumour. All VEGFR2-luc-Kl mice were imaged with an IVIS 100 seriesimaging system and associated Living Image software (Caliper LS) for 20 s with medium binning, 14 min after intraperitonealinjection of luciferin (150 mg/kg). Note that this mouse possesses bioluminescent vasculature and emits light to a varying degreefrom all regions of the body. The non-specific glowing snout and hindquarters apparent in these images merely reflect this point.[Imaged provided by Caliper LS. All work performed at Caliper is strictly regulated under IACUC guidelines. The InstitutionalAnimal Care and Use Committee (IACUC) is a self-regulating entity that, according to US federal law, must be established byinstitutions that use laboratory animals for research or instructional purposes to oversee and evaluate all aspects of the institution’sanimal care and use programme]



Figure 5. Comparison of the luciferin binding site in theLcr luciferase structures. (a) Superposition of the LcrLuc(WT) DLSA (green) and LcrLuc(WT).AMP/oxy-luciferin (white)complexes. (b) Superposition of the structures of LcrLuc(6286N).DLSA (pink) and LcrLuc(WT). AMP/oxy-luciferin(white) complexes. Polar interactions are shown as dashed lines.(c) Comparison of van der Waals interactions in the structuresof wild-type (left) and S285N (right) luciferase complexed withDLSA. The van der Waals radii of DLSA (blue), Ile 288 ofwild-type (green and light green) luciferase, and Ile288 of S286N(red and pink) luciferase are drawn in colour. The atoms in vander Waals contact with DLSA are highlighted in green and red[3]. [Reprinted with kind permission from Nature Publishing,Licence No 1872020241748]

superimpose virtual murine organs within a 3D bio-luminescent image. This ‘mouse atlas’ can help topredict the internal origin of bioluminescent sig-nal when used in combination with a 3D BLItechnique [93].

The recent development of dynamic BLI hasenabled the monitoring of acute vascular changes andrelative tumour perfusion in response to therapeutics.In this way, the effects of a vascular disrupting agent(combretastatin A4 phosphatase) have been success-fully imaged in xenograft models of breast cancer [94].This should have wide implications in the study ofnovel anti-vascular therapeutic agents.

Conclusion

BLI is now well established as an important tool forthe non-invasive imaging of pre-clinical tumour mod-els. Its high sensitivity and high-throughput capabili-ties allow the rapid measurement of tumour biology-associated parameters and, as a result, its applica-tions in pre-clinical oncology are wide and varied.These include the development of sophisticated cancermodels, assessment of therapeutic efficacy, monitor-ing of cell signalling and protein–protein interactions,visualization of critical processes in tumour

development and progression, such as apoptosis andmetastasis, and for genetic screening.

As with all imaging modalities, there are certainlimitations associated with BLI, such as minimalspatial resolution and depth sensitivity. However,continued refinement of this technique is steadilyovercoming each of these disadvantages, thus ensuringthat BLI will continue to remain a critical modality forimaging the tumour models that are fundamental to theadvancement of the field of pre-clinical oncology.

Acknowledgements

Funding is acknowledged from Science Foundation Ireland,the Health Research Board of Ireland, and University CollegeDublin. UCD Conway Institute is funded by the Programmefor Third Level Institutions (PRTLI), as administered by theHigher Education Authority (HEA) of Ireland. Unpublisheddata provided in Figure 4 were kindly provided by Caliper LS.

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