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Therapeutics, Targets, and Chemical Biology Molecular Imaging with Bioluminescence and PET Reveals Viral Oncolysis Kinetics and Tumor Viability Darshini Kuruppu 1 , Anna-Liisa Brownell 2 , Khalid Shah 2 , Umar Mahmood 2 , and Kenneth K. Tanabe 1 Abstract Viral oncolysis, the destruction of cancer cells by replicating virus, is an experimental cancer therapy that continues to be explored. The treatment paradigm for this therapy involves successive waves of lytic replication in cancer cells. At present, monitoring viral titer at sites of replication requires biopsy. However, repeat serial biopsies are not practically feasible for temporal monitoring of viral replication and tumor response in patients. Molecular imaging provides a noninvasive method to identify intracellular viral gene expression in real time. We imaged viral oncolysis and tumor response to oncolysis sequentially with bioluminescence and positron emission tomography (PET), revealing the kinetics of both processes in tumor xenografts. We demonstrate that virus replication cycles can be identied as successive waves of reporter expression that occur 2 days after the initial viral tumor infection peak. These waves correspond to virions that are released following a replication cycle. The viral and cellular kinetics were imaged with Fluc and Rluc bioluminescence reporters plus two 18F-labeled PET reporters FHBG [9-(4-18F-uoro-3-[hydroxymethyl] butyl) guanine] and FLT (18F-3 0 -deoxy-3- 0 uorothymidine), respectively. Correlative immunohistochemistry on tumor xenograft sections conrmed in vivo results. Our ndings show how PET can be used to identify virus replication cycles and for real-time measurements of intratumoral replicating virus levels. This noninvasive imaging approach has potential utility for monitoring viral oncolysis therapy in patients. Cancer Res; 74(15); 411121. Ó2014 AACR. Introduction Viral oncolysis by Herpes Simplex virus 1 (HSV-1) is cur- rently being investigated for cancers unresponsive to conven- tional treatment. Replication-conditional HSV-1 mutants are genetically engineered for effective oncolysis. These mutants replicate preferentially in cancer cells rather than in normal cells, which forms basis for safety and efcacy of this approach (1, 2). The process of oncolysis begins when the virus infects a tumor cell. Progeny virions that are liberated at the end of a replication cycle infect neighboring cancer cells to begin another wave of replication. The process continues until the cancer is eliminated or the virus is extinguished by the host defense mechanisms. Thus, the initially injected virus titer is amplied to a larger virus titer to destroy the cancer cells (3, 4). HSV-1 is among several oncolytic viruses that are studied, which include reovirus, vaccinia virus, poxvirus, adenovirus, and vesiculostomatitis virus (58). HSV-1 has an advantage over some of the other oncolytic viruses because of its large genome capacity, nonintegration of its DNA into the host genome, and high infectivity in cancer cells. The availability of antiherpetic drugs such as acyclovir and gancyclovir provide added safety as they can help terminate unwanted viral rep- lication if needed (911). Monitoring is needed during viral oncolysis therapy to identify virus titer at sites of virus replication. The current method of biopsy is not feasible for repetitive virus assessment. The absence of a reliable and clinically applicable method to measure viral activity in real-time is a major drawback for monitoring viral oncolysis therapy in clinical trials. The non- invasive and dynamic imaging capabilities of bioluminescence and positron emission tomography (PET) provide a means to image viral replication repetitively in vivo, preclinically for bioluminescence and both preclinically and for clinical trans- lation with PET. Using specic reporters for each imaging modality, the kinetics of virus replication can be studied by identifying sites and intensity of reporter expression. In addi- tion, the destruction of cancer cells undergoing viral oncolysis can be simultaneously studied. Bioluminescence imaging provides rapid, short interval, serial imaging for kinetic studies. Because of its high target to background ratio, bioluminescence offers a sensitive and robust tool for studying gene expression and function (1214). Through multireporter imaging of distinct sub- strates, temporal relationships between multiple biological processes and their pathological manifestations in cancer, artherosclerosis, and neurological disorders, and treatment response can be dened (1518). The kinetics of virus replication and the tumor response to lytic replication can be simultaneously studied in vivo, in preclinical models using this approach. Authors' Afliations: Departments of 1 Surgery and 2 Radiology, Massa- chusetts General Hospital, Boston, Massachusetts Corresponding Author: Kenneth K. Tanabe, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114. doi: 10.1158/0008-5472.CAN-13-3472 Ó2014 American Association for Cancer Research. Cancer Research www.aacrjournals.org 4111 on October 15, 2020. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst May 29, 2014; DOI: 10.1158/0008-5472.CAN-13-3472

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Page 1: Molecular Imaging with Bioluminescence and PET Reveals ......Molecular Imaging with Bioluminescence and PET Reveals Viral Oncolysis Kinetics and Tumor Viability Darshini Kuruppu 1

Therapeutics, Targets, and Chemical Biology

Molecular Imaging with Bioluminescence and PET RevealsViral Oncolysis Kinetics and Tumor Viability

Darshini Kuruppu1, Anna-Liisa Brownell2, Khalid Shah2, Umar Mahmood2, and Kenneth K. Tanabe1

AbstractViral oncolysis, the destruction of cancer cells by replicating virus, is an experimental cancer therapy that

continues to be explored. The treatment paradigm for this therapy involves successive waves of lytic replicationin cancer cells. At present, monitoring viral titer at sites of replication requires biopsy. However, repeat serialbiopsies are not practically feasible for temporal monitoring of viral replication and tumor response in patients.Molecular imaging provides a noninvasive method to identify intracellular viral gene expression in real time. Weimaged viral oncolysis and tumor response to oncolysis sequentially with bioluminescence and positron emissiontomography (PET), revealing the kinetics of both processes in tumor xenografts. We demonstrate that virusreplication cycles can be identified as successive waves of reporter expression that occur�2 days after the initialviral tumor infection peak. These waves correspond to virions that are released following a replication cycle. Theviral and cellular kinetics were imaged with Fluc and Rluc bioluminescence reporters plus two 18F-labeled PETreporters FHBG [9-(4-18F-fluoro-3-[hydroxymethyl] butyl) guanine] and FLT (18F-30-deoxy-3-0fluorothymidine),respectively. Correlative immunohistochemistry on tumor xenograft sections confirmed in vivo results. Ourfindings show how PET can be used to identify virus replication cycles and for real-time measurements ofintratumoral replicating virus levels. This noninvasive imaging approach has potential utility for monitoring viraloncolysis therapy in patients. Cancer Res; 74(15); 4111–21. �2014 AACR.

IntroductionViral oncolysis by Herpes Simplex virus 1 (HSV-1) is cur-

rently being investigated for cancers unresponsive to conven-tional treatment. Replication-conditional HSV-1 mutants aregenetically engineered for effective oncolysis. These mutantsreplicate preferentially in cancer cells rather than in normalcells, which forms basis for safety and efficacy of this approach(1, 2). The process of oncolysis begins when the virus infects atumor cell. Progeny virions that are liberated at the end of areplication cycle infect neighboring cancer cells to beginanother wave of replication. The process continues until thecancer is eliminated or the virus is extinguished by the hostdefense mechanisms. Thus, the initially injected virus titer isamplified to a larger virus titer to destroy the cancer cells (3, 4).HSV-1 is among several oncolytic viruses that are studied,which include reovirus, vaccinia virus, poxvirus, adenovirus,and vesiculostomatitis virus (5–8). HSV-1 has an advantageover some of the other oncolytic viruses because of its largegenome capacity, nonintegration of its DNA into the hostgenome, and high infectivity in cancer cells. The availabilityof antiherpetic drugs such as acyclovir and gancyclovir provide

added safety as they can help terminate unwanted viral rep-lication if needed (9–11).

Monitoring is needed during viral oncolysis therapy toidentify virus titer at sites of virus replication. The currentmethod of biopsy is not feasible for repetitive virus assessment.The absence of a reliable and clinically applicable method tomeasure viral activity in real-time is a major drawback formonitoring viral oncolysis therapy in clinical trials. The non-invasive and dynamic imaging capabilities of bioluminescenceand positron emission tomography (PET) provide a means toimage viral replication repetitively in vivo, preclinically forbioluminescence and both preclinically and for clinical trans-lation with PET. Using specific reporters for each imagingmodality, the kinetics of virus replication can be studied byidentifying sites and intensity of reporter expression. In addi-tion, the destruction of cancer cells undergoing viral oncolysiscan be simultaneously studied.

Bioluminescence imaging provides rapid, short interval,serial imaging for kinetic studies. Because of its high targetto background ratio, bioluminescence offers a sensitiveand robust tool for studying gene expression and function(12–14). Through multireporter imaging of distinct sub-strates, temporal relationships between multiple biologicalprocesses and their pathological manifestations in cancer,artherosclerosis, and neurological disorders, and treatmentresponse can be defined (15–18). The kinetics of virusreplication and the tumor response to lytic replication canbe simultaneously studied in vivo, in preclinical modelsusing this approach.

Authors' Affiliations: Departments of 1Surgery and 2Radiology, Massa-chusetts General Hospital, Boston, Massachusetts

Corresponding Author: Kenneth K. Tanabe, Massachusetts GeneralHospital, 55 Fruit Street, Boston, MA 02114.

doi: 10.1158/0008-5472.CAN-13-3472

�2014 American Association for Cancer Research.

CancerResearch

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In contrast, PET is readily translatable to human studies. It isroutinely used with [18F]FDG as a sensitive noninvasive imag-ing method to identify and stage tumors, determine treatmentsuccess and disease recurrence at the deep tissue level withoutbackground tissue interference. The prototype enzyme report-er, HSV-thymidine kinase (TK) is ideal for imaging virusreplication in tumors with PET. Based on the promiscuity ofHSV-TK, the guanine derivative 9-[3-fluoro-1-hydroxy-2-propoxymethyl] guanine (FHBG; refs. 19 and 20) serves as asubstrate for viral TK. The phosphorylated [F18-labeled]FHBG, which is trapped inside cells is detected with PET. Wehave previously reported that HSV-1 oncolysis can be imagedwith PET using FHBG as the substrate for HSV-TK (21).However, because of the large viral titer used in the study,PET imaging was restricted to the early stages of virus repli-cation. Imaging beyond this stage was not possible becausemost of the cancer cells were destroyed. The kinetics of virusreplication can be better understood by sequential study ofoncolysis. To address this in amore clinically relevant scenario,we assessed lower titers and used the replication-conditionaloncolytic HSV-1 mutants, hrR3 and HSV-Luc. Because of LacZgene insertion into the gene locus for viral ribonucleotidereductase, these mutants replicate preferentially in cancercells where the abundant nucleotides substitute for the absentribonucleotide reductase gene product (22, 23). HSV-Luc inaddition expresses the Fluc gene.

Here we present our investigations on the kinetics of HSV-1 replication and the accompanying tumor response tooncolysis with dual bioluminescence and PET imaging.Through optimization of dose, dosing intervals, and sub-strate for reporters, we identify, for the first time, virusreplication cycles as waves in bioluminescence and PETreporter expression. In addition, by studying the kineticsof tumor cells subjected to oncolysis we show a reduction intumor burden subsequent to lytic replication. These resultssupport the translatability of monitoring virus replicationwith serial PET scans using [18F]FHBG.

Materials and MethodsDevelopment of stable cell lines for bioluminescenceimaging

Human cancer cell lines [MDA-MB-231 breast cancer (ATCCHTB-26), A2058 amelanoticmelanoma (ATCCCRL-11147), andHT29 colon cancer (ATCC HTB-38)] and mouse cancer celllines [4T1 breast cancer (ATCC CRL-2539) and MC26 coloncancer] were engineered to express Rluc. The human cell lineswere maintained in RPMI-1640 medium supplemented with10% FBS, whereas the murine cell lines were maintained inDMEM supplemented with 10% FBS. The cell lines wereobtained from ATCC and were used in this study for less than6 months after resuscitation. They are authenticated by ATCCafter a comprehensive quality control before shipment. MC26was kindly provided by Dr. R.L. Martuza. The cell lines weretested for mycoplasma, Hoechst DNA staining, PCR, andculture testing for contaminant bacteria, yeast, and fungi.Authentication procedures used include species verificationby DNA bar coding and identity verification by DNA profiling.

The cell lines were infected with lentivirus-containing cDNAfor Rluc and mCherry as well as puromycin resistance. Within18 to 24 hours after infection, approximately 90% of cellsexpressed mCherry fluorescence. These cells were detachedwith trypsin and plated in 6-well dishes in media containingpuromycin (10 mg/mL). This allowed growth of cells with thenewly incorporated lentivirus DNA containing the mCherry,Rluc, and the puromycin resistance gene. Over the followingweek, colonies that developed in media containing puromycinwere examined for mCherry expression under an invertedphase contrast fluorescence microscope. The colony thatexpresses the strongest mCherry signal for each of the celllines was expanded for our experiment. The Rluc expressionwas validated with coelenterazine.

Replication-conditional HSV-1 mutantsThe replication-conditional HSV-1 mutant that expresses

Fluc (HSV-Luc) was generated by a bacterial artificial chro-mosome (BAC)-based HSV cloning system (kindly provided byDr. R.L. Martuza) utilizing Flip-Flop HSV-BAC technology (23).The HSV-Luc contains the luciferase expression cassette driv-en by the cytomegalovirus (CMV) promoter and the LacZ geneinserted in themiddle of the UL39 (ICP6) gene. The replication-conditional HSV-1 mutant hrR3 has the ICP6 inactivated byinsertion of the LacZ gene (22). The viruses were propagated inVero cells and replication assays were performed to determinethe titer as previously described (24).

In vitromeasurement of viruswith Fluc bioluminescenceCancer cells (MDA-MB-231 and MC26) were plated in 24-

well plates at a concentration of 6� 104 cells/well. A day later,the cells were exposed to different titers of HSV-Luc [1� 103 to1� 108 plaque forming units (pfu)]. The virus was imaged withbioluminescence using luciferin. To determine the kinetics ofvirus replication, Fluc expression was imaged over sequentialtime points at 6, 12, 18, 24, 30, and 36 hours after infection. Thisbioluminescence signal was expressed as the net intensity perarea of triplicate wells. A viable cell count was performed usingtrypan blue exclusion for each well. This was done at each ofthe time points that were imaged.

In vitro measurement of virus with TK PETCancer cells (MC26) and Vero cells were plated in 6-well

plates at a concentration of 4 � 105 cells/well. Once the cellswere 80% confluent they were infected with 1 � 108 pfu hrR3.Virus expression in the cells was determined by incubating thecells with [18F]FHBG for 2 hours, followed by 2 washes. The[18F]FHBG uptake by the cells was measured by a g-counter(Packard Cobra 5005), and was expressed per cell. The con-centration of cells in each well was determined by a viable cellcount.

Flank tumor modelBALB/c nude mice were obtained from the COX institu-

tional animal breeding services (Steele Laboratories). Allexperiments were performed according to the institutionalsubcommittee on research animal care. Themice were housedin an authorized animal facility with free access to food and

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water. Flank tumors were created in mice using each of thestable cell lines that were developed and grown in culture. Thecells were trypsinized and washed in medium containingserum to inactivate trypsin. After a PBS wash, the cells wereprepared for injections. Each cell line was injected into groupsof tenmice. 1� 105 cells from each cell line in 100 mL PBS wereinjected bilaterally or to one side of the flank. Experimentswere commenced when the tumors reached 5mm in diameter.

In vivo virus and tumor imaging with dual Fluc and RlucbioluminescenceThe animals were randomized into control and virus treat-

ment groups. Tumors in both groups were imaged for Rlucbioluminescence every other day for 8 days. Images wereacquired for 10 minutes after coelenterazine injection (7.5 mgi.v./mouse) at 8 � 8 binning (Carestream Molecular Imaging).HSV-Luc (1� 108 pfu/50mL in PBS) was injected into the tumorcenter and imaged daily for 8 days. The virus-treated groupunderwent dual imaging for Fluc and Rluc. Fluc biolumines-

cencewasacquired for10minutesat8�8binningafter luciferininjection (3.5 mg i.p./mouse). Fluc images were acquired aminimum of 6 hours after Rluc images were captured to allowfor complete clearance of the Rluc signal. Each of the Rluc andFluc images was overlaid onto an X-ray image captured for 20seconds. Tumor volume was calculated in the control andtreatment groups with caliper-measured tumor diameter. Bio-luminescence signal was quantified using the imaging software(Carestream). Regions of interest were normalized to back-ground intensity and expressed as net intensity per unit area.

In vivo virus and tumor imaging with microPETWhen the bilateral flank tumors reached 5 mm in diameter,

viruswas injected directly into the left tumor, whereas the righttumor served as the control. Themice were positioned into thescanner under isoflurane anesthesia (1%–1.5% isoflurane with2 L/min oxygen). We used a microPET scanner with a reso-lution of 4.1 mm3 (microPET-P4; Concorde Microsystems,Inc.). Transmission images were gathered with the aid of 57Co

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Figure 1. Schematic characterization ofmolecular reporters, cells, and virus. A, the dual bioluminescence detection system is based on the enzymatic reactionof Fluc and Rluc, where luciferin and coelenterazine serves as substrates. The PET ligand [18F]FHBG, serves as the substrate for HSV-TK, whichphosphorylates and traps it intracellularly. B, stable cancer cell lines that express Rluc andmCherry can be identifiedwith Rluc bioluminescence andmCherryfluorescence. TransformedMC26cells imaged for Rluc in vitro are shown. C,HSV-Luc expresses the Fluc and TKgenes. The replicating viruswas identified inMC26 cells with Fluc bioluminescence in vitro (MC26þHSV-Luc), and the signal was comparable to MC26 cells that transiently express Fluc (MC26-Fluc).The virus replicating in MC26 cells was detected with the PET tracer [18F]FHBG in vitro (MC26þhrR3). The [18F]FHBG signal was comparable to thatof MC26 cells, which express the TK gene transiently (MC26sr39TK).

Imaging of Virus Replication and Oncolysis Kinetics

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to correct for attenuation. Dynamic volumetric data wereobtained for 120 minutes after [18F]FHBG (100 mCi t.v./mouse)injection. Volumetric images were reconstructed with filtered-back projection after the data were corrected for uniformity,scatter, attenuation, decay, and injected activity using Asi-Pro4.1 software. Time-activity curves were generated from theselected regions of interest from the control tumor, virusinjected tumor, the liver, and the heart. PET signal wasexpressed as the % radiolabel accumulation/injected dose/cc.Imaging studies with [18F]FHBG were conducted at 2, 6, 24, 48,and 72 hours for different virus titers (hrR3 at 1� 109, 1� 107,1 � 105, and 1 � 103 pfu). The background activity wasmeasured before injecting the [18F]FHBG at each time pointto correct for any residual signal from the dose that preceded it.Although negligible, this measurement was corrected with theinjected dose. The tumor response to lytic replication wasimaged with [18F]FLT-PET.

Identificationof viruswith LacZ in frozen tumor sectionsFrozen tumors were sectioned at 5 mm and mounted on

superfrost-coated microscope slides (Fisher Scientific). The

sections were fixed in 3% glutaraldehyde before LacZ stainingforb-galactosidase. The sectionswere incubated in 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) solution at37�C overnight. The X-gal solution was prepared as follows:X-gal at 20 mg/mL in dimethyl formamide, 0.1 M potassiumferricyanide, 0.1 M potassium ferrocyanide, and 0.1 M MgCl2(Sigma). Areas of LacZ expression appeared blue. Sectionswere coverslipped and studied under a light microscope.

Hematoxylin and eosin and IHC staining for HSV-TK,proliferating cell nuclear antigen, and terminaldeoxynucleotidyl transferase dUTP nick end labelingstaining of tumors

Formalin-fixed tumors were processed, paraffin embedded,and serial sections were mounted for hematoxylin and eosin(H&E), HSV-TK, proliferating cell nuclear antigen (PCNA), andterminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) staining. HSV-TK IHC was performed after antigenretrieval in citrate buffer. Endogenous peroxidase and serumwere blocked before HSV-TK antiserum was applied at 1:5,000dilution overnight at 4�C. The sections were incubated in

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Figure 2. Kinetics of virus and cells in vitro determined with bioluminescence and PET substrates. A, the replication kinetics of HSV-Luc inMC26 cells studiedwith Fluc bioluminescence. Two peaks (arrows) were observed in the Fluc signal in the high virus titers (1� 108, 1� 107, and 1� 106 pfu), whereas the signalwas undetectable in the lower virus titers. Fluc signal is expressed as the mean net intensity/area � SD. B, viability of MC26 cells infected with virus.The cell counts decreased when infected with high viral titers (1 � 108, 1 � 107, and 1 � 106 pfu). Cell count is expressed as the mean � SD. C, [18F]FHBGuptake in MC26 and Vero cells 24 hours after HSV-Luc (1 � 108 pfu) infection. Data are the mean% of ID of [18F]FHBG uptake/cell � SD. D, changesinMC26 andVero cell concentration once infectedwith virus. TheMC26 cell concentrationwasmarkedly reduced comparedwith cells not infectedwith virus.Cell concentration is expressed as the mean � SD.

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biotinylated goat anti-rabbit secondary antibody, followed byhorseradish peroxidase, before chromogen development withperoxidase substrate 3,30-diaminobenzidine (DAB). HSV-TK–positive cells appeared brown. PCNA IHCwas performed usingthe PCNA antibody (raised in mice; Millipore) overnightat 1:1,000 at 4�C. The antibody was detected with biotinylatedantimouse secondary antibody as described above. PCNA-positive cells appeared brown. Apoptosis staining was per-formed using the ApopTag Fluorescein In-Situ ApoptosisDetection Kit (Millipore), which detects DNA strand breaks bythe TUNELmethod. After proteinase K treatment, the sectionswere incubated with terminal deoxynucleotidyl transferaseenzyme, followed by incubation with antidigoxigenin-peroxi-dase conjugate and counterstained with propidium iodide.ApopTag-positive cells were detected under fluorescence.

Statistical analysisDatawere expressed as themean� standard deviation (SD),

unless otherwise specified as the mean� standard error of themean (SEM)] and were compared using an unpaired 2-tailed ttest. The P values were compared with the controls dataset.

ResultsStable cell lines and virus can be detected throughbioluminescence and PET imagingThe cell lines (human MDA-MB-231, A2058, HT29, and

mouse 4T1 and MC26) stably transformed to express mCherryand Rluc were detected through red fluorescence and biolu-minescence, respectively (Fig. 1B). The replication-conditionalHSV-1 mutant (HSV-Luc), which contains the Fluc and TKgenes, can be imaged with bioluminescence and PET (Fig. 1C).Virus replication in MC26 cancer cells was imaged with Flucbioluminescence. The Fluc signal was comparable to that ofMC26 cells, which stably express Fluc (Fig. 1C, top graph). Virusreplication was identified with [18F]FHBG-PET. Radiolabel

uptake in MC26 cells infected with virus was comparable toMC26 cells that stably express the mutant viral TK (MC26-sr39TK; Fig. 1C, bottom graph).

Kinetics of virus replication measured in vitro withbioluminescence and PET tracers

The kinetics of virus replication was studied in vitro withFluc bioluminescence. The Fluc signal intensity correlatedwith the virus titer while it increased over time. In the highestvirus titer (1� 108 pfu HSV-Luc), two peaks were identified at18 and 30 hours after infection, which corresponded to virusreplication cycles (Fig. 2A). The cell counts decreased over timesuggestive of lytic replication. Low titers (1� 103, 1� 104, and 1� 105 pfu) were undetectable with bioluminescence, whereasthe cell counts increased over time, similar to the uninfectedcontrols (Fig. 2B). The kinetics of virus replication (hrR3 at 1�108 pfu) studied in vitro with the PET tracer [18F]FHBGdemonstrated a marked radiolabel uptake in virus-infectedMC26 and Vero cells (Fig. 2C). This was accompanied by areduction in cell concentration (Fig. 2D), indicative of lyticreplication.

Identification of virus replication cycles in vivo withbioluminescence and PET imaging

The kinetics of HSV-1 replication was studied sequentiallywith bioluminescence and PET in mice bearing tumor xeno-grafts (MDA-MB-231 and MC26). Virus (HSV-Luc at 1 � 109, 1� 108, and 1 � 107 pfu) replication in MDA-MB-231 tumorsimaged with bioluminescence showed peaks in Fluc reporterexpression over time. The intense peak at day 1was followed bytwo smaller peaks at day 3 and day 5 as the signal decreased(Fig. 3A). The peaks correlated with virus released followinga replication cycle, confirmed by plaque assay. Althoughthe "wave" pattern was consistent, the signal intensity corre-latedwith the virus titers. Virus (hrR3 at 1� 109, 1� 107 1� 105,

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Figure 3. Dynamics of virus replication in vivo imaged with bioluminescence and PET. A, kinetics of virus replication in flank tumors studied with Flucbioluminescence. The Fluc signal expressed three peaks (arrows) that decreased in intensity over time. Fluc signal is presented as themean net intensity/area� SD. B, kinetics of virus replication imaged with [18F]FHBG- PET. The [18F]FHBG uptake in tumors peaked (arrows) at 6 hours (0.3 days) in the highvirus titers. It decreased in highest virus titer (1� 109 pfu), whereas it peaked again at 3 days in the lower titer (1� 105 pfu). The lowest titer (1� 103 pfu) had nosignal. The radiolabel uptake is presented as the mean � SD of the % injected dose/cc.

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and 1 � 103 pfu) replication in MC26 flank tumors imagedwith PET showed a profile similar to that observed withbioluminescence. The [18F]FHBG reporter accumulation incancer cells infected with 1 � 105 pfu hrR3 expressed a"wave"-like pattern over 3 days. The peaks were identified at6 hours (0.3 days) and 72 hours (3 days) and correlated withvirus titers confirmed by plaque assays on tumors after imag-ing. The [18F]FHBG signal in the highest titer (1 � 109 pfu)decreased after the initial peak at 6 hours (Fig. 3B), whereasinfection with 1 � 103 pfu was undetectable.

Tumor growth kinetics identified with bioluminescencecorrelates with in vitro volumetric data

Growth kinetics of human (MDA-MB-231-Rluc, A2058-Rluc,and HT29-Rluc) and mouse (4T1-Rluc and MC26-Rluc) cancercells growing in the flanks were studied with sequential Rlucbioluminescence imaging over 8 days. Tumor volume wascalculated from caliper measurements at each time point forcomparison. The Rluc expression in the bioluminescence scans

that defines tumor borders increased over time in all threetumor xenografts (Fig. 4A, C, and E). The calculated Rluc signalintensities for each tumor xenograft correlated with externaltumor volume measured from caliper readings (Fig. 4B, D, andF). These parameters define tumor growth. We observedsimilar growth dynamics with bioluminescence and correla-tion with volumetric measurements from caliper readings inflank tumors from mouse breast cancer cells (4T1-Rluc; Sup-plementary Fig. S1).

Dynamics of virus replication and tumors undergoingoncolysis imaged with dual bioluminescence

The dynamics of virus replication and tumor response wasstudied with dual Fluc and Rluc bioluminescence followingHSV-Luc infection of MDA-MB-231-Rluc tumors. Mice wererandomized into treatment and control groups when thetumors reached 5 mm in diameter. Virus (1 � 108 pfu) wasinjected to the treatment group once baseline Rluc readingswere obtained. Both groups were imaged over 8 days. The area

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Figure 4. Tumor growth imaged with Rluc bioluminescence correlates with volumetric measurements obtained with caliper readings. The growth of humantumor xenografts imagedwith Rluc bioluminescence. The Rluc signal areas in scans of bilateral MDA-MB-231-Rluc flank tumors increased over time (A). TheRluc signal intensity increased and corresponded with caliper-measured tumor volume (B). A similar expression was observed where the Rluc signal increasecorrelated with caliper-measured tumor volume in A2058-Rluc (C, D) and HT29-Rluc (E, F) flank tumors. Data are expressed as the mean � SEM.

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of Rluc expression that defines tumor borders in the controlgroup increased (Fig. 5A) whereas that in the virus-treatedgroup decreased over time (Fig. 5B). The corresponding cal-culated Rluc signal intensities rose exponentially in the controlgroup whereas it was markedly inhibited in the virus treatedgroup (P< 0.004; Fig. 5C). The decrease in reporter expression issuggestive of lytic replication. The caliper-measured tumorvolume showed a profile similar to Rluc measurements for thecontrol group, but diverged for the treatment groups (Fig. 5D).This difference is attributable to the process that is measuredin each case: Rluc defines a functional measurement thatidentifies viable cells, whereas caliper measurements are fromexternal tumor diameters that also incorporate the necrotictumor core (viable and nonviable tumor). The Fluc signalpeaked 6 hours after virus injection and decreased over time(Fig. 5E). Quantified signal intensities revealed peaks every 2days after the initial peak expression (Fig. 5F), which areindicative of newly released virions following a replicationcycle as measured in plaque assays. Lytic replication reducesthe number of viable cells for virus replication, which accountsfor the decreasing Fluc signal. A similar response in virus andtumor dynamics was observed in the mouse colon cancer cellline (MC26-Rluc; Supplementary Fig. S2).

Dynamics of virus replication and tumor response tolytic replication imaged with PET

Virus replication in MC26 flank tumors was imaged withPET using [18F]FHBG over 3 days after injecting 1 � 105 pfuhrR3 into the right flank tumor. Sites of virus replication wereidentified by intracellular phosphorylated [18F]FHBG accumu-lation. The intensity of the signal correlated with the magni-tude of replicating virus (Fig. 6A). The control tumor had no[18F]FHBG accumulation on PET. The time activity curvesshowed [18F]FHBG accumulation in the virus-infected tumorwhereas the radiolabel was washed out of the control tumor,heart, and the liver (Fig. 6B). The fate of the cancer cellsundergoing oncolysis was studied with [18F]FLT-PET imaging,which identified a highly proliferative tumor before virusinjection. After virus injection, the signal decreased and thesite of lytic replication was identified as central photopenia,which correlated with the area of tumor cell lysis (Fig. 6C).

Molecular PET and bioluminescence imaging datacorrelate with in vitro findings

Tumors that were imaged in vivo with bioluminescence andPET were excised to study growth characteristics before andafter viral replication by immunohistochemical staining.

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Tumor cell proliferation imaged with [18F]FLT-PET, mCherryfluorescence, and Rluc bioluminescence correlated with PCNAimmune-staining of ex vivo tumor sections (Fig. 7A). Tumordestruction following virus replication was identified as acentral loss of signal when imaged with Rluc bioluminescence,mCherry fluorescence, and [18F]FLT-PET. On ex vivo H&Estaining, this central region was necrotic with a markedreduction in proliferating tumor cells (PCNA; Fig. 7B). Viralreplication was identified with [18F]FHBG-PET and Fluc bio-luminescence. Virus was detected with LacZ and HSV-TK IHCon ex vivo tumor sections (Fig. 7B). The central core consistedof apoptotic cells.

DiscussionSuccessful treatment outcomes in preclinical animalmodels

have set the stage for clinical trials of viral oncolysis as a novelcancer therapy (25). In these studies, the virus must be closelymonitored to determine the accuracy of virus delivery to thetumor, as well as assessing viral titer locally and following viralreplication at nontarget sites. Viral replication kinetics andbiodistribution can be studied noninvasively by imaging virusgene expression that occurs intracellularly. Such information

can be used to modulate virus dose, delivery routes, and theviral construct. In this study, we identified virus replicationcycles as waves in the reporter gene expression in real-timewith bioluminescence and PET imaging. The peaks in thesewaves correspond to virions that are released at the end of areplication cycle. To our knowledge, this is the first report ofsequential measurement of virus replication with biolumi-nescence and PET, and the first to report on in vivo assess-ment of intratumoral viral waves that provide continuedtumor lysis after the initial wave of infection and oncolysishas occurred.

Bioluminescence is widely used in tumor biology to studygrowth characteristics and treatment response in cancers (26–29). In thesemodels, bioluminescence reporters identify tumorgrowth, angiogenesis, and apoptosis in response to differenttreatments. And, bioluminescence studies have been con-ducted for oncolytic vesicular stomatitis virus on prostate(26) and bladder cancers (30), oncolytic vaccinia virus onpancreatic (18), and oncolytic adenovirus therapy of gliomas(31) and ovarian cancer (32), as well as HSV-1 strains onhepatocellular carcinoma (HCC) (33). These studies limit theirinvestigations to evaluating tumor response to lytic replica-tion, without the study of viral replication itself. In addition, the

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Figure 6. Dynamics of viral replication and cell proliferation imaged with microPET. A, localization of sites of virus replication in tumors with [18F]FHBG-PET.Virus was injected into the left of bilateral flank tumors. [18F]FHBG uptake was seen in the virus infected tumor but not in the control tumor. B, time activitycurves obtained from dynamic PET scans after [18F]FHBG injection. [18F]FHBG accumulated in the virus infected tumor, whereas it was washed off from theheart, liver, and control tumor. C, identification of proliferating tumor with [18F]FLT-PET. Photopenia identifies the site of virus replication in the tumor core.

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properties of HSV-1, such as the infectivity of wild-type virus inhealthy mice (34), host immune responses to infection (35),transcriptional activity of early and late promoters (36), and theinfectivity of various strains for cancer therapy (33) have beenstudied with bioluminescence. In these studies, virus wasmeasured at a specific endpoint of the experiment. In contrast,we have dynamically studied HSV-1 replication and oncolysiswith sequential imaging.Inmost imaging studies involving HSV-1, the virus is used as

an immunotherapy (e.g., T lymphocytes) for cancers (37) or asan amplicon to deliver cell-based therapies (e.g., TNF-relatedapoptosis-inducing ligand; ref. 38). The TK gene of the virus isoften used for prodrug activation with gancyclovir, or as theprototype PET reporter to image tumor growth and responseto treatment. Most often the HSV-TK gene is inserted into thegenome of other oncolytic viruses to image those viruses withPET (17). However, the use of HSV-TK to track the replicationkinetics of its own virus, that ofHSV-1, is limited.Wepreviouslydemonstrated that HSV-1 oncolysis can be imaged with PETusing the HSV-TK as the reporter (21). In this study, we showthat HSV-1 oncolysis and virus replication cycles can be

studied with dual reporter imaging with bioluminescence andPET. We did not focus on imaging HSV-1 replication inmetastases. However, in other studies, we have identifiedmetastases at distant sites in the gut with bioluminescenceand PET while imaging viral oncolysis treatment of livermetastases. With regard to immunity, we have previouslyreported no difference in antitumor activity of hrR3 in immu-nocompetent and immunocompromised mice (22). The activeimmune system in immunocompetent mice neither enhancednor attenuated antitumor effect of the virus.

We imaged two replication-conditional HSV-1 viruses, HSV-Luc and hrR3, with bioluminescence and PET. These mutantsselectively replicate in proliferating tumors (10, 22, 23). Wehave analyzed tumor and normal liver parenchyma after hrR3was injected into the spleen to deliver virus to treat livermetastases. No virus was detected in the normal liver. Thisobservation has also been confirmed with in vivo imaging withbioluminescence and PET. Virus (hrR3 or HSV-Luc) injectedinto a normalmouse (via the tail vein) was observed in the liverwithin 6 to 24 hours with a signal in the entire liver. This signalhad completely resolved by 48 hours. However, in the case of a

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Figure 7. Correlation ofbioluminescence, fluorescence,and microPET imaging datain vitro. A, tumor characteristicsbefore virus administration. Thetumor is identified with Rlucbioluminescence, mCherryfluorescence, and [18F]FLT-PET(small arrows). H&E and PCNAshow highly proliferative tumorsin vitro. B, changes in tumorcharacteristics following lytic virusreplication. A destructed tumorcore is identified with Rluc,mCherry, and [18F]FLT-PET(arrows), 48 hours after intratumorvirus (1 � 108 pfu) injection. Thechanges are seen on H&E andPCNAwhere the destructed tumorcore (arrow) is surrounded by a rimof viable tumor (asterisk). C,replicating virus is identified withFluc bioluminescence and[18F]FHBG-PET. IHC staining forLacZ and HSV-TK (arrow heads) inexcised tumors confirm thepresence of virus. Apoptosis isseen in the destructed tumor core.

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mouse bearing liver metastases, virus was observed in thetumors beyond 48 hours whereas the signal had resolved innormal liver.

Out of the 2 replication-conditional HSV-1 viruses used inthis study, hrR3 is more robust and has a greater replicationrate than HSV-Luc, explaining the difference between the twoinitial imaging peaks for the viruses. The more robust hrR3replication generated a replication peak at 6 hours instead of 24hours as seen with HSV-Luc. An additional contribution islikely related to different parent tumor lines undergoing ther-apy in these studies. What is striking is that replication cyclescan be monitored noninvasively with bioluminescence andPET, irrespective of the type of virus mutants or the type ofcancer, suggesting the broad applicability of this approach tofollow viral oncolysis in vivo, both preclinically and for clinicaltranslation. The ability to assess oncolytic viral replicationkinetics and spatial distribution in vivohas the potential to helpoptimize this therapeutic paradigm and accelerate clinicaltrials using targeted viral therapy.

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

Authors' ContributionsConception and design: D. Kuruppu, U. Mahmood, K.K. TanabeDevelopment of methodology: D. Kuruppu, A.-L. Brownell, U. Mahmood,K.K. TanabeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Kuruppu, A.-L. BrownellAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Kuruppu, A.-L. Brownell, U. Mahmood,K.K. TanabeWriting, review, and/or revision of the manuscript: D. Kuruppu,A.-L. Brownell, K. Shah, K.K. Tanabe, U. MahmoodAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): D. Kuruppu, K. Shah, K.K. TanabeStudy supervision: A.-L. Brownell, U. Mahmood, K.K. Tanabe

Grant SupportThis work was supported by NIH grants 5R01CA076183 and 5R21CA119600

(K.K. Tanabe), U01CA084301 and P50CA127003 (U. Mahmood), 5R01 EB001850,1R01EB012864, and 1S10RR023452 (A.-L. Brownell), and Department of Defense(DOD) grant W81XWH-11-1-0388 (D. Kuruppu).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received December 11, 2013; revised May 3, 2014; accepted May 20, 2014;published OnlineFirst May 29, 2014.

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2014;74:4111-4121. Published OnlineFirst May 29, 2014.Cancer Res   Darshini Kuruppu, Anna-Liisa Brownell, Khalid Shah, et al.   Oncolysis Kinetics and Tumor ViabilityMolecular Imaging with Bioluminescence and PET Reveals Viral

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