Positron emission tomography imagingof drug-induced tumor apoptosis with a caspase-3/7specific [18F]-labeled isatin sulfonamideQuang-De Nguyena, Graham Smitha, Matthias Glaserb, Meg Perumala, Erik Årstadb, and Eric O. Aboagyea,1

aComprehensive Cancer Imaging Center, Department of Oncology, Imperial College London Faculty of Medicine, Hammersmith Hospital, Du Cane Road,London W12 0NN, United Kingdom; and bMDx Discovery (part of GE Healthcare) at Hammersmith Imanet Ltd., Hammersmith Hospital, Du Cane Road,London W12 0NN, United Kingdom

Edited by Robert Mach, Washington University, and accepted by the Editorial Board July 31, 2009 (received for review February 5, 2009)

Of the molecular biochemical alterations that occur during apo-ptosis, activation of caspases, notably caspase-3, is probably themost attractive for developing specific in vivo molecular imagingprobes. We recently designed a library of isatin-5 sulfonamides andselected [18F]ICMT-11 for further evaluation on the basis of sub-nanomolar affinity for activated capsase-3, high metabolic stabil-ity, and facile radiolabeling. In this present study, we have dem-onstrated that [18F]ICMT-11 binds to a range of drug-inducedapoptotic cancer cells in vitro and to 38C13 murine lymphomaxenografts in vivo by up to 2-fold at 24 h posttreatment comparedto vehicle treatment. We further demonstrated that the increasedsignal intensity in tumors after drug treatment, detected by wholebody in vivo microPET imaging, was associated with increasedapoptosis. In summary, we have characterized [18F]ICMT-11 as acaspase-3/7 specific PET imaging radiotracer for the assessment oftumor apoptosis that could find utility in anticancer drug devel-opment and the monitoring of early responses to therapy.

cell death � response to treatment � small animal imaging

Apoptosis is an essential process for eliminating unwantedcells during embryonic development, growth, differentia-

tion, and maintenance of tissue homeostasis. Deregulation ofapoptosis signaling pathways is, therefore associated with variouspathologies including autoimmunity, neurodegeneration, car-diac ischemia, and transplant rejection (1), and the capacity toevade apoptosis has been defined as one of the hallmarks ofcancer (2). Moreover, because effective anticancer therapy oftenrequires induction of tumor cell death through apoptosis, mon-itoring of this process could provide important predictive out-come information in the context of routine patient managementand early clinical trials (3, 4). A noninvasive apoptosis imagingtechnology will permit the detection of tumor biological changesthat evolve within hours of initiating treatment compared tochanges in tumor size, the basis for Response Evaluation Criteriain Solid Tumors (RECIST) guidelines (5) that evolves withinmonths.

In cancer, apoptosis is induced by a large variety of stimuliincluding cytotoxic and mechanism-based therapeutics, andradiotherapy. Although those stimuli trigger different apoptoticsignaling pathways, the molecular events in the execution phaseof apoptosis are largely shared and involve the caspases, whichconstitute a family of cysteine proteases that cleave their sub-strate after specific tetrapeptide motifs. Within the caspasefamily, the effector caspases (caspases-3, -6, and -7) orchestratethe demolition phase of apoptosis that results in the controlleddismantling of a range of key structures within the cell and itssubsequent disposal (6). Moreover, one of the most noticeableand specific features of apoptosis is the degradation of the DNAinto numerous fragments, often down to multiples of 200 basepairs, driven by the activation of caspase-3 (7), the centraleffector caspase, which makes it an attractive biomarker ofapoptosis.

Given its central role, various nonpeptide small moleculeinhibitors of the isatin sulfonamide class that bind selectively tothe active site of caspase-3 (and -7), have been described, initiallyas potential inhibitors of apoptosis (8, 9). Furthermore, recentreports described the utility of [18F]-radiolabeled isatins asprobes for PET imaging of apoptosis (10–14), one of them usingan experimental rat model of drug-induced liver apoptosis (15).To date, no study has however reported imaging of tumor modelsby this strategy. Given that patient tumors typically show lowlevels of apoptosis at baseline and a relatively small increase afterdrug treatment (2- to 6-fold increase corresponding to 5–15%apoptosis) (16, 17), it is important to demonstrate the potentialof new imaging agents in relevant tumor models. In the attemptto design a PET imaging tracer for the detection of anticancerdrug-induced tumor apoptosis, we previously screened a focusedlibrary of isatin-based caspase-3/7 inhibitors and identified aisatin 5-sulfonamide, ICMT-11 (isatin-15 in Ref. 18), with sub-nanomolar affinity for caspase-3 and moderate lipophilicity (18).In the same report, we synthesized the radiolabeled [18F]ICMT-11 (Fig. 1A) by a ‘click radiochemistry’ approach and demon-strated its high metabolic stability, suggesting that [18F]ICMT-11would make a suitable caspase-3/7 specific PET imaging tracer.

The current study aims to investigate and validate the abilityof [18F]ICMT-11 to noninvasively image the drug-induced tumorapoptotic process in vivo, as well as, its potential for the earlydetection and monitoring of response to anticancer therapy.

ResultsIn Vitro [18F]ICMT-11 Binding and Caspase-3/7 Activation in Drug-Treated Cancer Cells. We first investigated the binding potential of[18F]ICMT-11 in cells undergoing anticancer drug-induced celldeath in vitro and showed that the murine fibrosarcoma RIF-1and human pulmonary carcinoma LNM35 cells treated with thetopoisomerase inhibitor etoposide or the alkylating agent cis-platin showed higher [18F]ICMT-11 binding in drug-treated cellscompared to control by up to 2-fold (left panels Fig. 1 B and C,respectively). We demonstrated the relevance of this binding byconfirming that increased [18F]ICMT-11 binding in drug-treatedcells corresponded to higher cellular caspase-3/7 activity (rightpanels Fig. 1 B and C). Of note, the control baseline uptake for[18F]ICMT-11 was higher than that for the caspase-glo assay andthat may be inherent in the two detection methods, directbinding to activated caspase-3 and hence, cellular retentionversus caspase-3 enzymatic activity, respectively. Moreover,

Author contributions: Q.-D.N. and E.O.A. designed research; Q.-D.N. and M.P. performedresearch; G.S., M.G., and E.Å. contributed new reagents/analytic tools; Q.-D.N. and E.O.A.analyzed data; and Q.-D.N. and E.O.A. wrote the paper.

Conflict of interest statement: A patent on [18F]ICMT-11 has recently been filed.

This article is a PNAS Direct Submission. R.M. is a guest editor invited by the Editorial Board.

1To whom correspondence should be addressed. E-mail: eric.aboagye@imperial.ac.uk.

This article contains supporting information online at www.pnas.org/cgi/content/full/0901310106/DCSupplemental.

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there was no increased [18F]ICMT-11 binding in caspase-3-deficient MCF-7 human breast cancer cells (19) treated with thealkylating agent 4-hydroperoxycyclophosphamide (4-HC) com-pared to control (Fig. S1), further validating the specificity of[18F]ICMT-11 binding to activated caspase-3 in drug-treatedcancer cells.

In Vitro Detection of Apoptotic Cancer Cells with [18F]ICMT-11. Al-though caspase-3 activation occurs during apoptosis (20) thepresence of active caspase-3 and/or specific products of theirenzymatic activity can also be linked to nonlethal biologicalprocesses (21). Thus, as embodied in recent recommendations ofthe Nomenclature Committee on Cell Death (22), we charac-terized anticancer drug-induced apoptosis via caspase-3 activa-tion in conjunction with the loss of integrity of the plasmamembrane defined by cellular incorporation of vital dyes, suchas propidium iodide (PI) and compared this to [18F]ICMT-11cellular binding.

The treatment of 38C13 murine lymphoma cells with 4-HCinduced a 2-fold increase of [18F]ICMT-11 binding compared tocontrol (Fig. 2A). To further characterize the specificity of thisbinding, we introduced an additional 60 min ‘chase’ time inradioactivity-free medium after the initial 60 min radiotracerincubation and washing, and demonstrated a consistent 2-foldincreased binding of [18F]ICMT-11 in drug-treated cells, despitean overall lower [18F]ICMT-11 cellular binding, possibly due tothe washout of unbound tracer and leakage of caspase-3 boundtracer in late apoptotic cells. We then quantified the percentageof caspase-3 positive cells from the same samples used for thebinding assay (Fig. 2B, quadrants Q3�Q4 on the flow cytometrycharts); this variable reflects the percentage of overall apoptosis

(Q3 � early apoptosis and Q4 � late apoptosis). A 2-foldincrease in [18F]ICMT-11 binding following 4-HC treatment wasassociated with elevated caspase-3 positive apoptotic cell level ofmore than half of the total cell population (70.54% � 2.54). Wecurrently do not know the dynamic range (all-or-nothing, high orlow threshold) of [18F]ICMT-11 in relation to percentage apo-ptosis; this issue will be addressed in future studies.

In the attempt to enhance the characterization of [18F]ICMT-11 binding in apoptotic cells, we have also subjected the same

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Fig. 1. [18F]ICMT-11 binding and active caspase-3/7 content analysis in cancercells undergoing treatment-induced apoptosis. (A) Chemical structure of[18F]ICMT-11: the caspase-3/7 selective isatin sulfonamide labeled with2-[18F]fluoroethylazide. (B,C) The effect of cisplatin (100 �M; 24 h) or etopo-side (100 �M; 24 h) treatment on [18F]ICMT-11 binding (left panels) andcorresponding caspase-3/7 activity (right panels) in RIF-1 fibrosarcoma (B) andLNM35 lung carcinoma (C) cells. For each set of treated and control samples,radioactivity data were expressed as decay-corrected counts per milligram oftotal cellular protein; caspase-3/7 activity data were expressed as relative lightunit (RLU) per milligram of total cellular protein. Data are mean � SEM.

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Fig. 2. [18F]ICMT-11 binding and apoptosis flow cytometry analysis in cancercells undergoing treatment-induced apoptosis. (A). [18F]ICMT-11 binding in38C13 lymphoma cells treated with 4-hydroperoxycyclophosphamide 4-HC(4-HC;1 �g/mL; 24 h) to induce apoptosis. The two panels represent data(decay corrected to the same time) from cells that were not further processed(left panel) and cells that were subjected to additional 60 min ‘chase’ incuba-tion in normal growth medium (right panel). For all treated and controlsamples, radioactivity data were expressed as decay-corrected counts permilligram of total cellular protein. (B) Flow cytometry analysis of control and4-HC-treated cells stained with fluorescent inhibitor of caspases-3/7 (FLICA)and propidium iodide (PI). Flow cytometry charts of representative controland treated cells are shown. The quadrants Q were defined as Q1 � viable(FLICA negative/PI negative), Q2 � necrosis (FLICA negative/PI positive), Q3 �early apoptosis (FLICA positive/PI negative) and Q4 � late apoptosis (FLICApositive/PI positive). (C) Flow cytometry analysis of control and 4-HC-treatedcells stained with Annexin V-FITC and PI. Flow cytometry charts of represen-tative control and treated cells are shown. The regions R were defined as R1 �viable (Annexin V negative/PI negative), R2 � apoptosis (Annexin V positive/PInegative) and R3 � necrosis (Annexin V positive/PI positive). (D) Effect of 4-HCon the binding of the low affinity caspase-3/7 inhibitor, [18F]ICMT-18, in 38C13lymphoma cells. Data are mean � SEM.

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cells used for the binding assay to an Annexin V/PI flowcytometry analysis, as it is commonly used for in vitro cell deathassessment. This assay is based on the Annexin V detection ofactively externalized phosphatidylserine on the plasma mem-brane outer surface of apoptotic cells and the PI staining ofnecrotic cells with compromised plasma membrane. Three cellpopulations were defined on the flow cytometry chart: viablecells (R1), apoptotic cells (R2), and necrotic cells (R3) (Fig. 2C).According to this definition, the 2-fold increase [18F]ICMT-11binding in 4-HC-treated cells was associated with 17.93% apo-ptotic cells. However, the R3 region cannot discriminate be-tween the late apoptotic cell population, containing activatedcaspase-3, and necrotic cells as both of them are characterizedby a double Annexin V/PI positive staining (membrane phos-phatidylserines external exposure and loss of their membraneintegrity) (23). As the R3 region should mirror the Q2�Q4(necrosis and late apoptosis, respectively) quadrants in Fig. 2B,the FLICA/PI flow cytometry analysis might be more appropri-ate than the Annexin V/PI analysis for the characterization ofactivated caspase-3 positive apoptotic cells.

It is possible that [18F]ICMT-11 simply enters cells and isretained in dead cells irrespective of its affinity for caspase-3. Weruled out this possibility in a control experiment with a lowaffinity caspase-3/7 inhibitor, [18F]ICMT-18, which lacks the5-sulfonamide group conferring the isatins high affinity forcaspase-3/7 (24). Unlike [18F]ICMT-11, no increased[18F]ICMT-18 binding in 4-HC-treated cells was observed com-pared to control (Fig. 2D). This finding suggests that [18F]ICMT-11 cell uptake is not related to increased cell permeability inapoptotic cells. In contrast, the apparent lower [18F]ICMT-18uptake in 4-HC-treated cells compared to control is probablydue to a more active washout of the unbound tracer in membranecompromised apoptotic cells. We further examined the speci-ficity of [18F]ICMT-11 in a model of necrosis. The data wereinconclusive and a more appropriate in vivo experimental modelof tumor necrosis is desirable to elucidate specificity of bindingto apoptotic versus necrotic cells (Fig. S2).

[18F]ICMT-11 PET Imaging of Tumor Bearing Mice Following AnticancerDrug-Induced Apoptosis Treatment. We then investigated the invivo potency of [18F]ICMT-11 as an apoptosis PET imagingtracer using an experimental model of tumor apoptosis.[18F]ICMT-11 PET imaging was performed 24 h after thetreatment of 38C13 xenograft-bearing mice with 100 mg/kgcyclophosphamide (CPA) or vehicle, and tumor was subse-quently excised for tracer biodistribution assessment and histol-ogy. In our experimental model of apoptosis, no overall toxicitywith respect to body weight loss was observed at the dose ofcyclophosphamide used; the drug is tolerated at a dose 170mg/kg cyclophosphamide every 6 days in mice (25). PET imagesshowed low uptake of the radiotracer in tumors from vehicle-treated mice (Fig. 3A). Overall, high background uptake wasseen in liver, small intestine and urine (data not shown). This wasconsistent with our previously reported normal tissue biodistri-bution of [18F]ICMT-11 that indicated rapid distribution of theradiotracer to tissues together with rapid elimination mostly viahepatic and renal routes, as suggested by the high localization of[18F]ICMT-11 in the liver, small intestine, kidney and urine (18).The high uptake in small intestine could also be due in part tohigh physiological apoptosis of enterocytes at the villus tips andof macrophages within the intestinal lamina propria (26); thisaspect requires further investigation. In contrast to the lowtumor radiotracer uptake in vehicle-treated mice, PET images ofCPA-treated mice showed a clear increased tumor uptake of[18F]ICMT-11 (Fig. 3A). This was confirmed by gamma countingof tumor radioactivity after imaging; there was a significant1.5-fold increased tumor uptake of [18F]ICMT-11 in CPA-treated mice compared to vehicle (Fig. 3B). The tumor time

versus radioactivity curves (TACs) determined from region ofinterest analysis of the imaging data (Fig. 3C) were shifted upfollowing treatment with CPA suggesting radiotracer retention.Furthermore, PET imaging variables (NUV60, AUC and FRT)extracted from TACs were higher with CPA compared to vehicletreatment (2.1-, 1.6-, and 1.6-fold higher, respectively, Fig. 3D).Notably, voxel-wise analysis of the PET data, depicting hetero-geneity, showed an increase in the number of voxels with highintensity in tumor ROIs of CPA injected mice compared tovehicle (a 1.5-fold group average induction in the 75%–90%percentiles and maximum intensity values, Fig. 4 A and B). Wefurther established a parallel between tumor [18F]ICMT-11uptake and apoptosis. Histological analysis of formalin fixedtumor tissues showed that CPA treatment significantly increasedapoptosis defined by increased apoptotic bodies (visualized withH&E staining), increased active caspase-3 and fragmented DNAstaining (6.5- and 9.8-fold induction, respectively, 8.2-fold in-duction for combination), together with a significant decrease ofDNA content (Fig. 5). Of note, there was also a markedreduction in tumor sizes at 24 h in the CPA-treated mice(approximatively 83% that of vehicle-treated mice tumors; notshown). These results establish [18F]ICMT-11 as a good phar-macodynamic marker of apoptosis.

DiscussionWe have demonstrated from in vitro binding and PET imaginganalysis that [18F]ICMT-11 has utility for detection and quan-

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Fig. 3. [18F]ICMT-11 PET imaging analysis. 38C13 xenografts bearing micewere treated with 100 mg/kg cyclophosphamide CPA (n � 3) or vehicle (n � 3)for 24 h and subsequently subjected to 60 min dynamic [18F]ICMT-11 PETimaging. (A) [18F]ICMT-11 PET images of two representative 38C13 xenograft-bearing mice treated with CPA or vehicle. White arrowheads indicate thetumor. (B) Tumor and blood were removed after the scan and analyzed for[18F]ICMT-11 tissue uptake. (C) The tumor time versus radioactivity curve(TAC). (D) Semiquantitative imaging variables extracted from the TAC. Dataare mean � SEM.

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tification of tumor apoptosis as early as 24 h after treatment, anddescribe for the first time a caspase-3/7 specific PET tracer fortumor apoptosis imaging. A number of noninvasive apoptosisimaging approaches have been developed based on the recog-nition of selective apoptotic biochemical and morphologicalfeatures coupled to dedicated imaging modalities. These includemagnetic resonance spectroscopy (MRS) measurements of cy-toplasmic lipid droplet accumulation (27), magnetic resonanceimaging (22) measurements of the apparent diffusion coefficientof water (28) and the C2A domain of synaptotagmin (29), highfrequency ultrasound detection of cell morphology (30), and thewell known annexin V and membrane active nuclear imagingmethods (31–33). Of these, the annexin V method, based on thedetection of phosphatidylserine asymmetric distribution pertur-bation in apoptotic cells, is arguably the most widely used,preclinically and in humans. This approach has some majordrawbacks limiting its routine clinical application including poorbiodistribution and clearance of the radiotracer, and nonspeci-ficity to apoptosis. More recently attention has turned to theisatin sulfonamide class of compounds as potential PET radio-tracers (8, 10, 12, 13, 15, 18).

In the present study, we have evaluated a [18F]-labeled isatinsulfonamide, [18F]ICMT-11, with desirable attributes for PET

imaging of apoptosis: high affinity for its target (activecaspase-3), high metabolic stability, reduced lipophilicity, andease of radiosynthesis. A feature of apoptosis is the cleavageof pro-caspase-3 to active caspase-3 (6). The prevailing theoryis that isatin-sulfonamides bind to nucleophilic cysteine andhistidine targets within the catalytic site of the active caspaseto form a thiohemiketal (9). Thus, [18F]ICMT-11 is expectedto distribute reversibly into cells but only trapped specificallywithin cells with active caspase-3 (and -7). The cellular uptakeof [18F]ICMT-11 in four different cell lines and with threedifferent anticancer agents showed that radiotracer uptake wasselectively increased with drug treatment by up to 2-fold.Increased cellular uptake of [18F]ICMT-11 was associatedbiochemically with the activity of caspase-3/7 and the foldincrease in cellular uptake compared favorably with the per-centage of activated caspase-3 positive apoptotic cells in vitro.A number of control studies, including use of a low affinityisatin ([18F]ICMT-18), chase experiments with [18F]ICMT-11,and binding studies of [18F]ICMT-11 in caspase-3 deficientcells, demonstrated that the binding of [18F]ICMT-11 in cellswas specific to altered caspase-3 activity. A further study in anin vitro necrosis model was not conclusive due to the existenceof activated caspase-3 in the cells. A particular difficulty indefining necrosis is that in the absence of phagocytosis,apoptotic cells become secondary necrotic cells with manymorphological features of primary necrosis (23).

In vivo PET imaging studies showed a measurable increase in[18F]ICMT-11 signal of up to 2-fold at 24 h after drug treatment.Other imaging time points will be explored in future studies. Thislevel of change is comparable to that seen preclinically and inpatients with other radiotracers used for therapy monitoringsuch as [18F]fluorothymidine ([18F]FLT), albeit in the oppositedirection; decreased [18F]FLT signal and increased [18F]ICMT-11 signal posttreatment (34–36). Notably, however, baselineuptake in tumors was low, representing nonspecific reversibledistribution. This means that use of anatomical imaging, such ascomputed tomography for localization of the tumor may bemandatory in the translation of this technology into clinicalpractice. Furthermore, the PET images showed a high retentionof the tracer in the liver and intestines, which might limitinterpretation of [18F]ICMT-11 PET images of the abdominalregion. The high tissue uptake is unlikely to be due to nonspecificrecognition of other caspases as [18F]ICMT-11 shows �10,000-fold selectivity for caspase-3 over other caspases (except caspase-7), activated or otherwise. It is likely that this high backgroundlocalization is due to metabolic turnover in liver and eliminationvia the gut (18).

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This study suggests that [18F]ICMT-11 PET imaging will beuseful for the monitoring of response to therapy rather thandiagnosis and localization of tumors per se assessed throughconventional clinical imaging. The dynamic range of the imagingapproach was found to be lower than that of histology. The8.2-fold increase in histological tumor apoptosis relative to theup to 2-fold increase in [18F]ICMT-11 NUV60 is likely due todifferences in the experimental assessment of apoptosis by thetwo methods (immunohistochemistry versus radiotracer phar-macokinetics and selective binding). Moreover, because of tu-mor heterogeneity, the strength of [18F]ICMT-11 imaging inter-pretations could benefit from a systematic voxel-wise dataanalysis allowing the selection of specific regions of interestwithin the tumor tissue. By analogy to flow cytometry analysisof cells, we speculate that these parameters will be more sensitivewhen subjects are used as their own controls as in clinical PETimaging protocols. Our current protocol did not permit thisstrategy to be assessed since we planned to compare the histol-ogy of individual mice to radiotracer uptake. Future studies willexplore this possibility.

In summary, we have demonstrated that [18F]ICMT-11 is asensitive and selective pharmacodynamic marker of caspase-3/7activation and apoptosis in vivo with potential utility in nonab-dominal malignancies. [18F]ICMT-11 detects caspase-3/7 cleav-age—an early event in apoptosis—and the paradigm has thecapacity to be improved in the future for measuring moreprecisely the amount of cell death in a reproducible manner.

Materials and MethodsRadiopharmaceuticals and Drugs. The radiosynthesis of the caspase-3/7 highaffinity inhibitor [18F]ICMT-11 has been described previously (18). Briefly,(S)-1-(2-propynyl)-5-(2-(2,4-difluorophenoxymethyl)-pyrrolidine-1-sulfonyl-)isatin was reacted with 2-[18F]fluoroethyl azide via copper catalyzed Huisgen[3 � 2] cycloaddition or ‘click chemistry’. The specific activity was 37–148GBq/�mol. The caspase-3/7 low affinity inhibitor [18F]ICMT-18 was radiosyn-thesized according to the same methodology as for [18F]ICMT-11. The anti-cancer drugs cisplatin, etoposide, and cyclophosphamide were obtained fromSigma-Aldrich. Cyclophosphamide is considered inactive in vitro in most cells,because it requires liver hepatocyte P450 system to generate active cytotoxicmetabolites (37). Therefore, 4-hydroperoxycyclophosphamide (4-HC), whichspontaneously converts to 4-hydroxycyclophosphamide in aqueous solution(the primary active metabolite of cyclophosphamide) was purchased fromNiomech (IIT GmbH) and used in the cell-based assays.

In Vitro [18F]ICMT-11 Binding and Caspase-3/7 Activation Assays. Cells wereplated in triplicate in 12-well plates 2 or 3 days before the experiments andtreated with indicated drug or corresponding vehicle (DMSO and water forcisplatin/etoposide and 4-HC respectively) for 24 h to induce apoptosis, orsubmitted to a 56 °C heat shock for 30 min to induce necrosis. On the day ofthe experiment, �0.37 MBq/well of [18F]ICMT-11 (or [18F]ICMT-18) was addedand allowed to accumulate into cells for 60 min at 37 °C. Cells were collected,washed, and resuspended in PBS. For the chase time experiment, the cells arewashed once and subjected to additional 60 min incubation in normal growthmedium. Samples were transferred into counting tubes and fluorine-18 ra-dioactivity was immediately determined using a Packard Cobra II gammacounter (Perkin–Elmer). Luminescent caspase-3/7 activation assay was per-formed on each sample according to the manufacturer’s instructions (Caspase-Glo 3/7 assay, Promega). Briefly, cells were transferred in a white opaque96-well plate, incubated for 1 h with Caspase-Glo reagent and the enzymaticactivity of caspase-3/7 was measured using a Multiskan Luminometer (ThermoElectron). To enable normalization of data to total cellular protein content,the Bicinchoninic acid (BCA) protein assay (Pierce) was performed for allsamples according to the manufacturer’s instructions.

In Vitro Apoptosis Flow Cytometry Assay. Aliquots of a 38C13 cell suspensionused for the binding assay were withdrawn before addition of [18F]ICMT-11and the cells were doubly stained with either fluorescent inhibitor of caspases-3/7 (FLICA) and propidium iodide (PI) or Annexin V-FITC and PI, according tothe instructions of the manufacturer (Vybrant FAM Caspase-3 and -7 andVybrant Apoptosis assays respectively, Invitrogen). A flow cytometry analysiswas then performed using the CyAn™ ADP cytometer and Summit v4.3software (Dako). Data were analyzed by setting quadrants Q and regions Rbased on the single staining controls.

[18F]ICMT-11 Tumor Biodistribution and PET Imaging. The in vivo experimentalmodel of tumor apoptosis was established by s.c. injection of 38C13 murinelymphoma cells (5,000 cells) on the back of 6- to 8-week-old male C3H/hej mice(Harlan). All animal work was performed by licensed investigators in accor-dance with the United Kingdom’s ‘‘Guidance on the Operation of Animals(Scientific Procedures) Act 1986’’ (HMSO, London, United Kingdom, 1990).When xenografts reached �100 mm3, the mice were injected with cyclophos-phamide (100 mg/kg i.p., n � 3) or vehicle (water, n � 3). At 24 h posttreat-ment, the animals were scanned on a dedicated small animal PET scanner(Siemens Inveon PET module, Siemens Molecular Imaging Inc.) following abolus i.v. injection of �3.7 MBq of [18F]ICMT-11. Dynamic emission scans wereacquired in list mode format over 60 min. The acquired data were then sortedinto 0.5-mm sinogram bins and 19 time frames for image reconstruction,which was done by filtered back projection. Cumulative images of the dy-namic data (0 to 30 min) were iteratively reconstructed (OSEM3D) and used forvisualization of radiotracer uptake and to define the regions of interest (ROIs)with the Siemens Inveon Research Workplace software (three-dimensionalROIs were defined for each tumor). The count densities were averaged for allROIs at each of the 19 time points to obtain a time versus radioactivity curve(TAC). Tumor TACs were normalized to that of whole body at each of the timepoints to obtain the normalized uptake value (NUV). Direct [18F]ICMT-11tumor uptake was assessed subsequent to the PET scan. For this, blood wastaken by cardiac puncture from the animals and tumors were excised. Thetissues were weighted and immediately counted for fluorine-18 radioactivity.Data were expressed as tumor to blood ratio.

Active Caspase-3 and TUNEL Immunohistochemistry Assay. Following PET im-aging studies, tumor tissues were excised, fixed in formalin, embedded inparaffin, sectioned (5 �m slices) and processed for active caspase-3 and DNAdegradation terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) fluorescent detection assays using the Cleaved Caspase-3 (Asp 175)monoclonal antibody (Cell Signaling Technology) coupled with the AlexaFluor 594 goat anti-rabbit (Invitrogen) and the In Situ Cell Death Detection Kit(Roche), respectively. The ProLong Gold Antifade mounting solution (Invitro-gen) containing 4�,6-diamidino-2-phenylindole (DAPI) was added to tissuesections prior coverslips mounting. All assays were performed according to themanufacturer instructions and sections were counterstained with hematoxy-lin and eosin (H&E) staining. Images from two histological sections separatedby 1 mm, 10 random fields per section (at 400� magnification), were capturedusing an Olympus BX51 fluorescent microscope and the staining intensitieswere determined using the ImageJ software [National Institutes of Health(NIH)].

Statistical Analysis. Data were expressed as mean � standard error of themean (SEM) and the significance of comparison between two data sets wasdetermined using Student’s t test (Prism v5.0 software, GraphPad) and definedas significant (*, 0.01 � P � 0.05), very significant (**, 0.001 � P � 0.01),and extremely significant (***, P � 0.001).

ACKNOWLEDGMENTS. We thank R. Levy (Division of Oncology, StanfordSchool of Medicine, CA) for donating 38C13 murine lymphoma cells; N.Nawroly (Flow Cytometry Facility, Imperial College St. Mary’s Medical School,London, U.K.) and W. Gsell (Biological Imaging Center Facility, MRC-ImperialCollege, London, U.K.) for assistance with flow cytometry and PET imaging,respectively; and the Hammersmith Hospital Department of Histopathologyfor the processing (tissue paraffin embedding and sectioning). This work wassupported by Cancer Research U.K.-Engineering and Physical Sciences Re-search Council Grant C2536/A10337 and U.K. Medical Research Council GrantU1200.005.00001.01 (to E.O.A.).

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