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Personalized Medicine and Imaging

A Systematic Comparison of 18F-C-SNAT toEstablished Radiotracer Imaging Agents for theDetection of Tumor Response to TreatmentTimothyH.Witney, Aileen Hoehne, Robert E. Reeves,Ohad Ilovich, MohammadNamavari,Bin Shen, Frederick T. Chin, Jianghong Rao, and Sanjiv S. Gambhir

Abstract

Purpose: An early readout of tumor response to therapythrough measurement of drug or radiation-induced cell deathmay provide important prognostic indications and improvedpatient management. It has been shown that the uptake of18F-C-SNAT can be used to detect early response to therapy intumors by positron emission tomography (PET) via amechanismof caspase-3–triggered nanoaggregation.

Experimental Design: Here, we compared the preclinical util-ity of 18F-C-SNAT for the detection of drug-induced cell death toclinically evaluated radiotracers, 18F-FDG, 99mTc-Annexin V, and18F-ML-10 in tumor cells in culture, and in tumor-bearing micein vivo.

Results: In drug-treated lymphoma cells, 18F-FDG, 99mTc-Annexin V, and 18F-C-SNAT cell-associated radioactivity correlat-ed well to levels of cell death (R2 > 0.8; P < 0.001), with no

correlation measured for 18F-ML-10 (R2 ¼ 0.05; P > 0.05). Asimilar pattern of response was observed in two human NSCLCcell lines following carboplatin treatment. EL-4 tumor uptakeof 99mTc-Annexin V and 18F-C-SNAT were increased 1.4- and2.1-fold, respectively, in drug-treated versus na€�ve control animals(P < 0.05), although 99mTc-Annexin V binding did not correlate toex vivo TUNEL staining of tissue sections. A differential responsewas not observed with either 18F-FDG or 18F-ML-10.

Conclusions: We have demonstrated here that 18F-C-SNAT cansensitivelydetectdrug-inducedcell death inmurine lymphomaandhuman NSCLC. Despite favorable image contrast obtained with18F-C-SNAT, the development of next-generation derivatives, usingthe same novel and promising uptake mechanism, but displayingimproved biodistribution profiles, are warranted for maximumclinical utility. Clin Cancer Res; 21(17); 3896–905. �2015 AACR.

IntroductionThe early assessment of drug-induced tumor cell death is of

great prognostic value—enabling rapid selection of the mostefficacious treatment using a personalized medicine approach.In the clinic, currentmethods todetect response rely onmeasuringchanges in tumor size under the guidelines of the ResponseEvaluation Criteria in Solid Tumors (RECIST; ref. 1). Thisapproach, however, lacks sensitivity, andmany weeks may elapsebefore there is evidence on imaging of tumor volume shrinkage.

Cell death, through apoptotic, necrotic or alternate pathways(2), is the surrogate endpoint for most anticancer therapies andtreatments (3, 4). Given the inadequacy of current techniques formonitoring drug response, there have been considerable efforts todevelop new techniques to detect and image treatment efficacythrough measurements of tumor cell death (recently reviewed inref. 5). As drug-induced cell death manifests itself in manydifferent forms, numerous divergent imaging strategies have been

used, targeting a range of both direct and indirect biomarkers.Direct imaging biomarkers include caspase-3, whose activationcommits the cell to programmed (apoptotic) cell death (6, 7), andplasma membrane depolarization (8, 9), which occurs down-stream of caspase-3 (10) or is exposed as a result of membranerupture during necrosis (8). Indirect methods include measure-ments of cell metabolism (11, 12), the release of enzymes fromintracellular compartments (13, 14), or the increased diffusion ofwater that results from loss of cellularity (15).

Radiotracer imaging techniques have shown great promise forthe noninvasive detection of cell death. Early alterations inglucose metabolism following therapy, characteristic of bothstressed and dying cells, have been detected by 2-18F-fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET)in a range of tumor types (16–19). Furthermore, a draft frame-work has been proposed for the routine use of 18F-FDG-PET forresponse-assessment in the clinic (20). In addition to 18F-FDG,the small-molecule radiotracer 2-(5-fluoropentyl)-2-methylmalonic acid (18F-ML-10) has shownutility for cell death imagingpreclinically (21, 22), with first-in-man trials recently reported(23). Out of all the currently available cell death imaging probes,99mTc-HYNIC-labeled Annexin V (99mTc-Annexin V) has been themostwidely studied, having beenused to image drug-induced celldeath inhumanheadandneck squamous cell carcinoma (24) andtumors of the breast, lymphoma, and lung (25). 99mTc-Annexin Vbinds to phosphatidylserine (PS), a temporally stable biomarkerexposed following either apoptotic or necrotic cell death.99mTc-Annexin V, however, has failed to progress beyond phaseII/III clinical trials due to its poor biodistribution profile (26).

Department of Radiology, Stanford University, Stanford, California.

T.H. Witney and A. Hoehne contributed equally to this article.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Sanjiv S. Gambhir, James H Clark Center, StanfordUniversity, 318 Campus Drive, E153, Stanford, CA 94305. Phone: 1-650-725-6175; Fax: 1-650-723-8649; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-3176

�2015 American Association for Cancer Research.

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Despite the promise of noninvasive imaging techniques for theassessment of tumor cell death, no imaging agent has yet beenaccepted into routine clinical practice. This has prompted thedevelopment of a new generation of agents designed to monitorresponse. One such radiotracer, 18F-C-SNAT, has been shown inpreliminary studies to detect early response to therapy in tumorsby PET via a novel mechanism of caspase-3–triggered cyclizationand nanoaggregation (Supplementary Fig. S1; ref. 7). Here, wedirectly compared the performance of 18F-C-SNAT to establishedradiotracers, 18F-FDG, 99mTc-Annexin V, and 18F-ML-10 (Fig. 1),for the noninvasive detection of cell death. We found that allradiotracers except 18F-ML-10 were able to sensitively measuredrug-induced cell death in cell culture.However, only 18F-C-SNATwas able to measure treatment response in vivo. Together, ouranalysis suggests that 18F-C-SNAT may have utility for the detec-tion of drug-induced cell death in the clinic.

Materials and MethodsCell culture

EL-4murine lymphoma cells and PC-9 humanNSCLC (ATCC)were grown in complete RPMI medium (Life Technologies; 11.1

mmol/L glucose), supplemented with 10% fetal calf serum, 100U/mLpenicillin, and 100mg/mL streptomycin. A549 cells (ATCC)were grown in DMEM medium (Life Technologies; 25 mmol/Lglucose) with 10% fetal calf serum, 100 U/mL penicillin, and100 mg/mL streptomycin. All cells were propagated at 37�C in ahumidified atmosphere containing 5% CO2. EL-4 cells could notbe authenticated as they were of non-human origin, with A549and PC-9 cells purchased from an authenticated cell biobank justprior to publication.

RadiotracersRadiosynthesis of 18F-FDG, 99mTc-Annexin V, 18F-ML-10, and

18F-C-SNAT was based on previously described methodology, asdetailed in the Supplementary Data.

Cell uptake studiesEighteen to 20 hours after either etoposide (Sigma-Aldrich;

15 mmol/L) or vehicle treatment (DMSO), EL-4 cells were splitinto two fractions for analysis: (i) assessment of radiotracer celluptake and binding. (ii) Cell death determination by flow cyto-metry. For cell uptake studies, 106EL-4 cellswere incubated for 10,30, or 60 minutes in fresh RPMI (1 mL) at 37�C with respectiveradiotracer todetermineuptake kinetics. PC-9 andA549 cellswereseeded at 105 cells per well of a 6-well plate 24 hours prior tocarboplatin treatment (Sagent; 200 mmol/L; 72 hours), as previ-ously described (27). For all cell uptake experiments, the radio-tracers were added at the following concentrations and specificactivities: 18F-FDG, 0.185 MBq/mL, <0.38 � 0.08 mmol/L, >2.6GBq/mmol; 99mTc-Annexin V, 10 nmol/L, �0.185 MBq/mL, 21.1� 2.2 GBq/mmol; 18F-ML-10, 1.48 MBq/mL, <0.18 mmol/L, >7.4GBq/mmol; 18F-C-SNAT, 1.48 MBq/mL, 0.015 � 0.007 mmol/L,123.2 � 61.4 GBq/mmol. Following incubation for the allottedtime, adherent cells were trypsinized (0.25% trypsin; 1mmol/LEDTA) and harvested by centrifugation (1,200 � g; 3 minutes;4�C). Detached cells present in the media before trypsinizationwere retained and pooled with the trypsinized cells. Suspensioncells were collected by centrifugation (1,200� g; 3minutes; 4�C),and all cells were washed three times with ice-cold Hank's Buff-ered Salt Solution (HBSS; 1 mL; 1,200� g; 3 minutes; 4�C), withthe final cell pellet lysed in 200 mL RIPA buffer (Thermo FisherScientific Inc.). One hundred microliters of cell lysate was trans-ferred to counting tubes and decay-corrected radioactivity wasdetermined on a gamma counter (Cobra II Auto-Gamma counter,Packard Biosciences Co.). The remaining lysate was frozen andused for protein determination following radioactive decay using

Figure 1.Chemical structure and molecular weight for 18F-FDG, 99mTc-Annexin V, 18F-ML-10, and 18F-C-SNAT. The radioisotope is shown in bold type.

Translational Relevance

Patients with similar tumor types frequently respond dif-ferently to the same therapy. An early assessment of treatmentresponse in individual patients would allow rapid selection ofthe most effective treatment. Therapy response in the cliniccurrently relies on measurements of tumor size, which isineffectual as an early response marker. In response, a numberof positron emission tomography (PET) and single photonemission computed tomography (SPECT) radiotracers havebeen developed to noninvasively measure therapy-inducedcell death in a range of human cancers. Despite early promise,none of these imaging agents havemade it into routine clinicalpractice. The development of new radiotracers to assessresponse to therapy is required if the goal of personalizedmedicine for the treatment of cancer is to be realized. Here, wedesigned a novel caspase-3–triggered cyclization and nanoag-gregation radiotracer with improved sensitivity for the detec-tion of cell death in comparison with current gold standard inmolecular imaging.

Detection of Tumor Cell Death by Radionuclide Imaging

www.aacrjournals.org Clin Cancer Res; 21(17) September 1, 2015 3897




a bicinchoninic acid (BCA) 96-well plate assay (Thermo FisherScientific Inc.). Ten microliters of standards from the originalnonwashed cell/media suspension was counted to quantitate thepercentage of radiotracer uptake/binding in cells.

To correlate cell-associated radioactivity with levels of drug-induced cell death,mixtures of vehicle and etoposide-treated EL-4cells were prepared 18 to 20 hours after either vehicle or drugaddition. Mixtures contained 0%, 25%, 50%, 75%, and 100% v/vetoposide-treated cells in 6-mL total volume, with the remainingvolume made from vehicle-treated cell suspension. One millioncells were subsequently collected for cell death analysis by flowcytometry, with the remaining cells used to assess cell-associatedradioactivity 60 minutes after radiotracer addition, as describedabove.

Detection of cell death in vitroApoptosis and necrosis in cell mixtures containing 0% to 100%

drug-treated cells were visualized by flow cytometry using amethod adapted from ref. 12, in parallel to cell uptake studies.Fluorescein isothiocyanate (FITC)–Annexin V (BioLegend) incombination with 7-amino-antinomycin D (7-AAD; BioLegend)were used for cell death determination. Early apoptotic cells weredefined as cells positively stained for FITC–Annexin V but not7-AAD, with both late apoptotic and necrotic classified as cellspositive for both stains. FlowJo (v.7.6.5; Tree Star, Inc.) was usedfor analysis.

In vivo tumor modelsAll experimental procedures involving animals were approved

by Stanford University Institutional Animal Care and Use Com-mittee. EL-4 tumor cells (5 � 106 in 100 mL PBS) were injectedsubcutaneously on the back of female C57BL/6 mice (aged6–8 weeks; Charles River Laboratories) and grown to approx-imately 150 mm3, over 6 days. Tumor dimensions were mea-sured daily using a caliper, with tumor volumes calculated bythe equation: volume ¼ (p/6) � a � b � c, where a, b, and crepresent three orthogonal axes of the tumor (27). At day 6,animals were size-matched and either treated with clinicallyformulated etoposide (Topsar, Novaplus; 70 mg/kg) or leftuntreated. Treatment–response analysis was performed 24 hoursafter drug treatment, 7 days after tumor cell implantation. In aseparate cohort of mice, the effect of drug-treatment on tumorvolume was followed up to 96 hours after therapy. For all otherexperiments, animals were culled 25.5 hours after therapy andtissues excised for analysis.

Imaging studiesPET imaging scans were carried out on a docked Siemens

Inveon PET/CT scanner (matrix size, 128 � 128 � 159; CTattenuation-corrected; nonscatter corrected), following a bolusi.v. injection of approximately 10 MBq of the radiotracer intotumor-bearing mice (n ¼ 3–4 per group). Dynamic scans wereacquired in list mode format over 90 minutes, as described indetail in the Supplementary Data.

Biodistribution studiesApproximately 10 MBq of radiotracer was injected via the tail

vein of anesthetized EL4 tumor-bearing C57BL/6 mice (n ¼ 4–8per group). These mice were maintained under anesthesia andwarmed to 37�C to replicate imaging conditions. At 90 minutesafter radiotracer injection, animals were sacrificed by exsangui-

nation via cardiac puncture. For all animals, tumors were excisedimmediately upon death, weighed, and rapidly placed in 10%formalin (Fisher Scientific) for fixation. Tumor and tissue radio-activity was subsequently determined on a gamma counter(decay-corrected; Cobra II Auto-Gamma counter, Packard Bios-ciences Co.). Predefined 3.7 MBq standards (250 mL) were alsocounted for data normalization to injected dose. Data wereexpressed as percent injected dose per gram of tissue (%ID/g).

ImmunohistochemistryFormalin-fixed tumors were embedded in paraffin, sectioned

into 5-mm thick slices and placed on microscope slides accordingto standard procedures (Histo-Tec Laboratory). Sections weretaken at regular intervals across the entire tumor volume in orderto capture previously described heterogeneity of EL-4 tumors(28). Sections were stained with either hematoxylin and eosin(H&E; Histo-Tec) or with a colorimetric terminal deoxynucleoti-dyl transferase–mediated dUTP nick end labeling (TUNEL) assayusing a kit by Promega (DeadEnd), according to the manufac-turer's instructions, with each section digitized using a NanoZoo-mer 2.0RS whole-slide scanner (Hamamatsu). The percentage ofTUNEL-positive cells was measured across the entire tumor sec-tion using ImageJ (v.1.47; NIH, Bethesda, Maryland). Six sectionswere analyzed per tumor, with three tumors evaluated per treat-ment group (na€�ve or drug-treated) for each radiotracer studied.TUNEL positivity was compared with the corresponding tumoruptake values previously determined by biodistribution.

Statistical analysisData were expressed as mean � SD, with the exception of

tumor volume measurements, which were presented as mean�SEM. Statistical significance was determined using the Student ttest (Microsoft Excel). For correlation analysis, linear regres-sion, statistical significance, and 95% confidence levels weredetermined using Prism software for Mac OS X (v.6.0e; Graph-Pad Software). Differences between groups were consideredsignificant if P � 0.05.

ResultsMeasurements in cellsTime course of radiotracer accumulation. EL-4 murine lymphomacells rapidly undergo caspase-3–mediated cell death followingetoposide treatment (12). By 18 hours after therapy, cell deathhad reached 48.9% � 6.7% in comparison with 2.6% � 1.4% invehicle-treated cells, as determined by flow cytometry (Fig. 2A; P <0.001;n¼4).Temporal 18F-FDG,99mTc-AnnexinV, 18F-ML-10,and18F-C-SNAT uptake/cell binding was measured at 18 to 20 hoursafter drug treatment to permit the comparison of their uptakekinetics (Fig. 2B). 18F-FDG cell uptake increased linearly with timein vehicle-treated cells, rising from 3.1% � 1.2% radioactivity/mgprotein at 10 minutes after radiotracer addition, to 10.7% � 2.2%radioactivity/mg protein by 60 minutes. In comparison, 18F-FDGuptake in drug-treated cells at 60 minutes was 2.0% � 0.7%radioactivity/mg protein, 5.3-fold lower than vehicle controls(n ¼ 4; P ¼ 0.0008). Conversely, high 99mTc-Annexin V bindingto drug-treated cells was evident by just 10minutes after radiotraceraddition, at 110.4% � 4.0% radioactivity/mg protein, with signif-icantly lower binding measured in vehicle controls (6.0% � 1.9%radioactivity/mg protein; P < 0.001; n ¼ 3). By 60 minutes afterradiotracer addition, 99mTc-Annexin V binding was 24.3-fold

Witney et al.

Clin Cancer Res; 21(17) September 1, 2015 Clinical Cancer Research3898




higher in drug-treated versus vehicle-control cells, at 173.2% �15.7% radioactivity/mg and7.1%�2.4% radioactivity/mg, respec-tively (P < 0.001; n¼ 3). Nodifference in EL-4 cell uptake in treatedversus control cells was detectable with 18F-ML-10 over the 60minutes time course (P > 0.05; n¼ 3). Cell-associated radioactivityat 60 minutes in etoposide-treated cells was 3 orders of magnitudelower than 99mTc-Annexin V, at 0.10% � 0.04% radioactivity/mg.18F-C-SNAT retention indrug-treated cells followed an initial phaseof slow uptake for the initial 30 minutes, followed by a rapidincrease in the rate of retention over the proceeding 30 minutes.There was no change in 18F-C-SNAT cell uptake in vehicle controlcells over the 60 minutes time course. At 60 minutes, 18F-C-SNATcell uptake was 4.5-fold higher in drug-treated cell in comparisonwith vehicle controls, at 1.5%� 0.2% radioactivity/mg and0.3%�0.1% radioactivity/mg, respectively (P < 0.001; n ¼ 3).

Correlation to cell death. Radiotracer uptake and EL-4 cell bindingwere further compared with levels of drug-induced cell death inculture. Here, different ratios of drug-to-vehicle–treated cells werecombined to create a large dynamic range of dying cells, fromapproximately 3% to 80% of the total cell population (Fig. 3). Asboth early and late apoptotic/necrotic cells were present in thedrug-treated samples used to prepare the sample series (Supple-mentary Fig. S2), it was impossible to determine the contributionof each mechanism of death to cell uptake using this method.Instead, cell-associated radioactivity was compared with cellsidentified as positively stained by FITC–Annexin V, defined hereas "dying cells." Both 99mTc-Annexin V and 18F-C-SNAT cell

uptake/binding significantly correlated with levels of cell death(R2 ¼ 0.985 and 0.924, respectively; P < 0.001), with 18F-FDGuptake negatively correlated to death (R2 ¼ 0.842; P < 0.001).There was no statistically significant correlation found for 18F-ML-10 (R2 ¼ 0.053; P > 0.05). It is important to note that 99mTc-Annexin V was correlated to a fluorescent Annexin V derivativeused for the assessment of cell death, a potential limitation of thiscomparison.

Monitoring therapy response in NSCLC. To explore whether theradiotracer uptake results in EL-4 lymphoma cells were gener-alizable to other tumor cell lines taken from solid tumors, wenext assessed radiotracer uptake in either vehicle or carboplatin-treated human PC-9 and A549 NSCLC cells; a tumor type wellknown for its relative resistance to common chemotherapeutics(29). Seventy-two hours of carboplatin treatment inducedextensive cell death in both lines (Supplementary Fig. S3A). InPC-9 and A549 cells, drug treatment resulted in a significant69.8% (P < 0.001; n¼ 3) and 43.9% (P¼ 0.01; n¼ 3) reductionin 18F-FDG uptake 60 minutes after radiotracer addition (Sup-plementary Fig. S3B). For 99mTc-Annexin V, radioactivity wasincreased 5- and 4-fold in PC-9 and A549 cells, respectively incomparison with vehicle control cells (P < 0.001; n ¼ 3).Similarly, 18F-C-SNAT was increased 3.8-fold (P ¼ 0.03) and2.9-fold (P < 0.001) in PC-9 and A549, respectively (n ¼ 3). For18F-ML-10, no significant increase observed in drug-treated cellsversus vehicle for either PC-9 or A549 cells, in agreement withdata obtained for EL-4 cells.

Figure 2.Time course of radiotracer uptake and retention in etoposide- and vehicle-treated EL-4 cells. A, flow cytometric analysis of vehicle or drug-treated (15 mmol/Letoposide; 18 hours) EL-4 cells. Cell death was detected with FITC–Annexin V (l Ex/Em ¼ 488/535) and 7-AAD staining (l Ex/Em ¼ 488/647). Q1 ¼ viable;Q2¼ early apoptotic; Q3¼ late apoptotic/necrotic. B, temporal changes in cell-associated radioactivity in 18 hours vehicle or etoposide-treated cellsweremeasuredwith 18F-FDG, 99mTc-HYNIC-Annexin V, 18F-ML-10, and 18F-C-SNAT. Mean � SD (n ¼ 3-4/group). � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Monitoring treatment response in tumorsWe next evaluated the specificity of each radiotracer to nonin-

vasively measure tumor cell death in vivo. Subcutaneous EL-4tumors grew rapidly in C57BL/6 mice, detectably by day 4 andreaching approximately 150 mm3 6 days after implantation(Fig. 4A). On day 6, mice were size matched and either treatedwith drug or left untreated. Drug treatment resulted in a significant40%reduction in tumorvolumeafter24hours, reduced from159.8� 26.3 mm3 to 95.3 � 16.0 mm3, whereas the tumor volumeincreased from 145.3 � 23.2 mm3 to 256.4 � 36.3 mm3 in na€�veanimals (80% increase; P < 0.001; n¼ 7/group). At this time point,drug-treated tumors were characterized by loss of tumor cellularity,immune cell infiltration (Fig. 4B), and extensive DNA damagethroughoutmultiple slices of the tumor, determinedbyhistochem-ical staining (Fig. 4C). In na€�ve tumor-bearing animals, DNAdamage was limited to the peripheral margins of the tumor. By96 hours after therapy, tumors had shrunk to 42.8 � 7.2 mm3,growing to 393.4 � 23.2 mm3 in na€�ve animals (Fig. 4A).

Tumor uptake and radiotracer biodistribution. 18F-FDG, 99mTc-Annexin V, 18F-ML-10, and 18F-C-SNAT tumor uptake, illustratedin Fig. 4D, and ex vivo biodistribution profiles, summarized inSupplementary Table S1, were measured in EL-4 tumor-bearinganimals 90 minutes after radiotracer injection in eitherdrug-treated or na€�ve animals. 18F-FDG, 99mTc-Annexin V, and18F-ML-10 pharmacokinetics were dominated by renal excretion.In addition, high heart and kidney retention were measuredwith 18F-FDG and 99mTc-Annexin V, respectively. 18F-C-SNATelimination was through both the renal and hepatobiliary routes.18F-ML-10 radioactivity had almost completely cleared from allorgans by 90 minutes. For drug-treated mice, 18F-FDG was sig-nificantly higher than na€�ve animals in liver, small intestine, andlarge intestine, but lower in muscle (P < 0.05). Bone-associated

radioactivity was significantly higher in treated versus na€�veanimals with 99mTc-Annexin V (P < 0.05).

Ex vivo g-counting of excised tumors revealed a significantincrease in tumor-associated radioactivity in drug-treated versusna€�ve tumor-bearing mice for both 99mTc-Annexin V and 18F-C-SNAT, at 1.4- and 2.1-fold, respectively (P < 0.05; n ¼ 4–7 mice/group). There was no change in 18F-FDG or 18F-ML-10 tumoruptake and retention following drug treatment 90 minutes afterinjection of radiotracer (Fig. 4D and Supplementary Table S1). Apositive tumor-to-muscle ratiowasmeasuredwith all radiotracersand treatment conditions, whereas a positive tumor-to-bloodratio was only measured with 18F-FDG at 90 minutes after-radiotracer injection (Supplementary Table S2).

Correlation of radiotracer binding to histology. The specificity of theradiotracers to image tumor response to therapy was achievedthrough correlation of their retention with TUNEL staining, anindependent marker of drug efficacy, and the current clinicalgold standard (Fig. 5). EL-4-tumor-bearing animals injected with18F-C-SNAT showed a good correlation (R2 ¼ 0.778; n ¼ 3animals/group; P ¼ 0.02) between whole tumor mean radioac-tivity at 90minutes after radiotracer injection and TUNEL stainingin histologic sections obtained post mortem, whereas there was nocorrelation for either 18F-FDG, 99mTc-Annexin V, or 18F-ML-10(R2¼ 0.249, 0.231, and 0.001, respectively; n¼ 3 animals/group;P > 0.05).

Radionuclide imaging of tumor cell death. Dynamic PET imageswere acquired from animals with implanted EL-4 tumors(Fig. 6). Similarly to ex vivo biodistribution data, 18F-FDGdistribution was characterized by high uptake in the tumor,heart, and bladder (Fig. 6A). 18F-FDG-PET/CT fusion images,highlighting the tumor xenograft and rendered in 3D, are

Figure 3.Correlation between therapy-inducedcell death and radiotraceraccumulation in cells. Flow cytometricquantitation of cell death wascompared with cell-associatedradioactivity for 18F-FDG, 99mTc-HYNIC-Annexin V, 18F-ML-10, and 18F-C-SNAT in cell mixtures. Lines of linearregression and 95% confidence levels(broken gray lines) are shown. Mean�SD (n ¼ 3 independent experiments).Experiments performed on separatedays are represented by either gray,open, or filled circles. �� , P < 0.001;NS, not significant.

Witney et al.





shown in Fig. 6A, ii (full, uncropped versions are displayed inSupplementary Videos SM1 and SM2). 18F-FDG tumor uptakewas characterized by a steady increase in tumor retention inboth na€�ve and drug-treated animals, with a plateauing ofradioactivity recorded between 60 and 90 minutes. By 90minutes, a significant increase in tumor-associated 18F-FDGuptake was measured in drug-treated animals in comparisonwith na€�ve animals at 15.1 � 1.2%ID/g and 12.3 � 1.1%ID/g,respectively (P ¼ 0.011; n ¼ 4/group). There was no differencein the area under the tumor time–activity curves (AUC)between the two treatment groups (P > 0.05).

Tumor uptake and retention was visible above backgroundin both na€�ve and drug-treated animals with 18F-ML-10 PET(Fig. 6B), with increased uptake noticeable around the peripheryof the tumor (Fig. 6B, ii and Supplementary Videos SM3 andSM4). The kinetic profile of tumor retention varied considerablyfor 18F-ML-10 in comparison with 18F-FDG, as shown by thetumor time–activity curves (TAC). Rapid tumor uptake of 18F-ML-10, peaking at 10 minutes, preceded a slow washout of radioac-

tivity over the remaining 80 minutes. There was no difference inimage-derived tumor uptake values (%ID/g90 or AUC) betweenna€�ve and drug-treated cohorts.

In contrast to what was observed with 18F-ML-10 and 18F-FDG-PET, a clear difference in tumor-associated 18F-C-SNAT uptakebetween treatment groups was measured by PET (Fig. 6C). Indrug-treated mice, background activity was increased when com-pared with na€�ve mice, possibly reflecting the trend towardincreased blood retention observed in biodistribution studies(Supplementary Table S1). In na€�ve mice, tumor uptake wasconfined to the margins (Fig. 6C, ii and Supplementary VideoSM5), corresponding to elevated regions of DNAdamage observedby histochemical staining of excised tumors (Fig. 4C), whereasuniform tumor distribution was evident in tumors of drug-treatedmice (Fig. 6C, ii and Supplementary Video SM6). The profile oftumor uptake varied considerably between drug-treated and na€�veanimals (Fig. 6C, iii). In drug-treated animals, C-SNAT rapidlyaccumulated in tumors, peaking at 10 minutes, with subsequentstabilization of tumor-associated radioactivity. For na€�ve animals,

Figure 4.Characterization of therapy response and radiotracer accumulation in tumors. A, caliper measurements of EL-4 tumor volumes in na€�ve anddrug-treated mice. Measurements were recorded as a function of time after cell implantation, with mice treated on day 6 (red arrow). All subsequentanalysis was made on day 7, 24 hours after therapy (green arrow). Data represent mean � SEM (n ¼ 7–8 mice/group). B, H&E staining ofna€�ve and drug-treated histologic tumor sections. Tumor sections were scanned by a whole-slide scanner, with representative regions of interestselected at �10 and �20 magnification (inset), indicating loss of cellularity and immune cell infiltration following treatment (day 7, 24 hoursafter drug). Scale bar, 100 mm. C, DNA fragmentation (colorimetric TUNEL assay) detection in na€�ve and drug-treated whole tumor sections.Representative false-color sections are shown, acquired at �1.25 magnification (scale bar, 2.5 mm). D, radiotracer accumulation in drug-treatedand na€�ve tumors. Radioactivity was measured by g-counting of excised tumors 90 minutes after radiotracer administration. The values are the meanvalue � SD of at least 4 mice (number of mice shown on graph).






18F-C-SNAT peaked to similar levels 10 minutes after injection,followed by a slowwashout from the tumor over the remaining 80minutes. By 90 minutes after injection, tumor uptake was 2.2 �0.2%ID/g in drug-treated tumors versus 0.8� 0.2%ID/g when leftuntreated, a 2.7-fold increase (P < 0.001, n ¼ 3-4 animals/group).

Dynamic SPECT imaging was not possible with the experimen-tal setup available. Instead, static 60 to 90minutes SPECT imageswere obtained for 99mTc-Annexin V (Supplementary Fig. S4).These images revealed typical 99mTc-Annexin V tissue distribu-tion, with high liver, kidney, and bladder radioactivity, which wasconsistent with biodistribution data. Although tumor uptake wasevident above background, we were unable to distinguishbetween drug-treated tumors and na€�ve controls using qualitativeimaging data alone, reflecting the small differences observed by exvivo g-counting.

DiscussionResistance to chemotherapy andmolecularly targeted therapies

provides a major hurdle for cancer treatment due to the under-lying genetic and biochemical heterogeneity of tumors. An earlyread-out of drug efficacy, through the measurement of tumor celldeath, would provide valuable insights into resistance status,allowing the selection of the most appropriate therapy (or com-bination) for the individual and termination of ineffectual treat-ments at an early stage.

There have been considerable efforts to develop methods tononinvasively measure tumor cell death (recently reviewed inref. 6), but as yet, no cell-death imaging agent has made it intoroutine clinical practice. There is therefore a need to developnew imaging agents for the assessment of therapy response. Anumber of important considerations are required to assess the

suitability for any novel cancer imaging agent: namely, thetemporal and spatial distribution of the imaging biomarker intumors, the sensitivity and specificity of detection, tumor-to-background contrast, and the pharmacokinetic profile ofthe radiotracer. Here, we systematically compared the novelimaging agent, 18F-C-SNAT, to clinically evaluated radiotracers,18F-FDG, 18F-ML-10, and 99mTc-Annexin V, for the detection oftumor cell death. We selected the well-characterized EL-4murine lymphoma model (11–13) for the assessment ofdrug-induced cell death. Lymphomas typically respond wellto first-line therapy in comparison with the majority of solidtumor types (30), accurately reflected by extensive drug-induced cell death observed here in EL-4 cells and in implantedtumors (Figs. 2A and 4A–C, respectively). We then furtherassessed therapy response in two additional NSCLC lines toconfirm that the variability in results between the differentradiotracers was not specific to this one cell line.

In cell culture experiments, EL-4 cell–associated radioactivityfor the different radiotracers varied over 3 orders of magnitudefollowing drug treatment (Fig. 2), highlighting large differencesin their sensitivity and mechanisms to detect cell death.For 99mTc-Annexin V, cell-associated radioactivity was173.2% � 15.7% radioactivity/mg protein at 60 minutes afteraddition of radiotracer, in comparison with just 0.10% �0.04% radioactivity/mg for 18F-ML-10. Furthermore, 18F-ML-10 uptake failed to correlate to drug-induced cell death in thismodel, either in cells or in tumors (Figs. 3 and 5, respectively); apattern also replicated in the two NSCLC cells tested here. Thisis in contrast to previous reports in a preclinical model ofneuronal cell death (26) and in drug-treated tumor cells inculture, in which a tritiated derivative of ML-10 was used (31).It is possible that tissue culture media buffering of extracellular

Figure 5.Correlation of radiotracer uptake tohistologic measure of tumor celldeath. DNA damage, assessed byTUNEL staining of whole tumorsections (n ¼ 6 sections per tumor,3 tumors/treatment group), werecompared with tumor-associated18F-FDG, 99mTc-HYNIC-Annexin V,18F-ML-10, and 18F-C-SNATradioactivity measured in tumors.Radioactivity was measured byg-counting of excised tumors 90minutes after radiotraceradministration (n ¼ 3 tumors/group).Lines of linear regression and 95%confidence levels (broken gray lines)are shown. � , P < 0.05; �� , P < 0.01;�� , P < 0.001.

Witney et al.





pH may reduce cell binding of 18F-ML-10, whose binding ispurportedly mediated through the acidification of the externalplasma membrane leaflet (31). Experimental evidence linkingapoptosis-specific alterations in asymmetric membrane distri-bution to 18F-ML-10 uptake is limited however, and not sup-ported by our findings in vivo, where the acidic tumor micro-environment likely remains largely un-buffered (13).

Drug treatment induced high levels of DNA damage and lossof cellularity in vivo, characteristic of an apoptotic response.Despite the high levels of cell death, accumulation in EL-4tumors was moderate (<3%ID/g) for all radiotracers except 18F-FDG (Figs. 4C and 6A). A significant decrease in 18F-FDGuptake is indicative of a positive response to therapy, asmeasured here in EL-4 cells (Figs. 2B and 3), as a consequenceof glucose transporter redistribution from the plasma mem-brane to the cytosol (12). Despite levels of drug-induced celldeath correlating excellently with a reduction in 18F-FDGuptake in cells, no response was detectable by g-counting ofexcised tumors ex vivo (Figs. 4D and 5), with a small butstatistically significant increase observed by semiquantitativeimaging parameters in vivo. 18F-FDG uptake is not confined totumor cells, with drug-induced alterations in other constituentsof the tumor microenvironment potentially confounding mea-surements in vivo. For example, infiltration of FDG-avidimmune cells following drug treatment, detected here by H&Estaining (Fig. 4B), may mask tumor cell–specific reductions inglucose metabolism in this immunocompetent murine model,as has been observed elsewhere (32, 33). These data are in linewith the general advice for nuclear medicine physicians to waita minimum of 10 days after chemotherapy before performing18F-FDG-PET scans to bypass potential immune effects (20). Areduction in 18F-FDG in lymphoma patients has, however,been observed just 24 hours after initiation of treatment(34), highlighting a potential limitation of preclinical mousemodels of cancer, where the nature and timing of immuneresponses are known not to precisely match those found in theclinic (35).

Although changes in 18F-FDGuptake act as an indirect measureof treatment response, Annexin V and its labeled derivatives havebeen shown to measure bona fide cell death, through binding toexposed PS with nanomolar affinity (36). Annexin V continues tobe the gold standard for measurements of cell death in vitro. Inculture, 99mTc-Annexin V binding excellently correlated to celldeath, with high sensitivity and specificity in both the lymphomaand two NSCLC cell lines. Despite encouraging performance incell culture, a poor pharmacokinetic profile and low tumorbinding was observed in vivo. The half-life of 99mTc (6 hours)permits imaging at time points over 12 hours after radiotracerinjection. The rapid clearance of 99mTc-AnnexinV fromcirculationand the absence of tissue redistribution just 30 minutes after

Figure 6.Dynamic PET imaging of cell death in vivo. PET image analysis of EL-4 tumorsin na€�ve and drug-treatedmicewas performed for 18F-FDG (A), 18F-ML-10 (B),and 18F-C-SNAT (C). Corresponding axial and sagittal PET-CT images

(60–90 minutes summed activity) of na€�ve and drug-treated EL-4 tumor-bearing mice are shown in panels (i). Arrowheads indicate the tumor,identified from the CT image. (ii) Representative PET-CT (60–90 minutessummed activity) volume rendering technique (VRT) images of tumor-bearing na€�ve and drug-treated mice. Arrows indicate the tumor. (iii) Thetumor TAC representing average counts from a dynamic 90-minute scan 24hours after drug treatment and in na€�ve animals. Data aremean� SD (n¼ 3–4animals per group). � , P <0.05; �� ,P <0.001. B¼ bladder, H¼ heart, L¼ liver,SI ¼ small intestine.






injection (37), however, supports an imaging protocol, wherebyresponse is assessed at 90 minutes after intravenous injection. Atthis time point, 99mTc-Annexin V tumor uptake did not correlatesignificantly to levels of DNA damage (Fig. 5), with amodest 40%increase in radioactivity accumulation following drug treatmentdetectable by average counts in excised tumors. Nonspecifictumor retention of Annexin V in the vascular space has recentlybeen attributed to changes in enhanced permeability and reten-tion (EPR) effects at baseline and after therapy (38). Despiteadvances in both site-specific and PET labeling strategies littlehas been achieved to improve the in vivo profile and tumorbinding of this radiotracer (39).

Given the relatively poor performance of existing radiotra-cers, alternative approaches are required for the measurementof cell death. PET-labeled and fluorescent derivatives of apeptidic caspase-3/7 substrate, C-SNAT and C-SANF, respec-tively, have recently emerged as promising agents for cell deathimaging (7, 40). Specificity of C-SNAT for caspase-3/7 waspreviously shown, and through abrogation of signal in thepresence of a caspase-3 inhibitor, and with a control probecontaining the D-DEVD sequence, resistant to caspase-3 cleav-age (7, 40). Here, 18F-C-SNAT uptake was significantly elevatedbetween 2.9- and 4.5-fold in EL-4 and NSCLC cells followingtreatment. In EL-4 xenograft tumors, 18F-C-SNAT uptake cor-related strongly with the levels of drug-induced DNA damage(R2 ¼ 0.778), with 18F-C-SNAT uptake increased 2.7-fold indrug-treated tumors versus na€�ve controls just 24 hours aftertherapy. This early readout of treatment response was shown tobe an accurate predictor of outcome, assessed by almostcomplete tumor regression 72 hours after therapy (Fig. 4A).At 90 minutes after radiotracer injection, washout of unboundradiotracer in muscle resulted in a positive tumor-to-muscleratio in both drug-treated and na€�ve mice. Retention of theradiotracer in the blood pool combined with relatively lowtumor uptake, however, reduced tumor-to-background con-trast and may confound simple endpoint measurements, par-ticularly for the evaluation of vascular-damaging anticanceragents where radiotracer delivery to the tumor may beimpaired. Of note was the combined hepatobiliary and renalexcretion of 18F-C-SNAT, which may limit assessment of treat-ment response to tumors of the upper thoracic region, forexample, those of the breast, in which background is minimal.We are currently developing second-generation caspase-3–mediated nanoaggregation radiotracers with improved phar-macokinetic properties, and improved signal to backgroundfor enhanced clinical utility.

Not included in this comparison study was the PET-labeledcaspase-3 inhibitor, isatin-5-sulfonamide (18F-ICMT-11),which has shown potential for tumor cell death imaging(6, 27, 41) and has recently progressed to clinical trials (42).Under similar conditions to those described here, however, EL-4 tumor retention of 18F-ICMT-11 was increased by a modest65% in treated versus vehicle control tumors (43), whilesharing the same suboptimal pharmacokinetic profile as 18F-C-SNAT, and displaying lower tumor binding in vivo (41). Apotential limitation of caspase-3–based imaging agents, such as18F-C-SNAT and 18F-ICMT-11, is the transient temporal expres-sion of cleaved caspase-3 following the induction of apoptosis.As a result, the imaging window for the detection of treatmentresponse must be carefully selected for these caspase-3–basedimaging agents (41). In addition, measurements of caspase-3

limits detection to apoptotic mechanisms of death and fails todetect therapy-induced necrosis (27).

In summary, we have systematically compared a range ofradionuclide imaging agents, with diverse mechanisms ofaction, for cell death imaging. Despite promising resultsobtained in isolated adherent and suspension cells in culture,confounding factors in vivo, such as microenvironmentalchanges and radiotracer biodistribution, limited the ability ofboth 99mTc-Annexin V and 18F-FDG to sensitively measure celldeath. The claimed selectivity of 18F-ML-10 to monitor treat-ment response could not be replicated in the EL-4 tumor modelin the current work. Although progress has been made with thedevelopment of novel PET agents, such as 18F-C-SNAT, there isan urgent need to continue to develop new radiotracers thatdisplay an improved pharmacokinetic profile and tumoruptake in order to measure treatment response of tumorslocated in the abdomen and surrounding regions. Further-more, detailed clinical studies will be needed to truly identifya radiotracer that can have utility in monitoring treatmentefficacy.

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

Authors' ContributionsConception and design: T.H. Witney, A. Hoehne, S.S. GambhirDevelopment of methodology: T.H. Witney, A. Hoehne, O. Ilovich, B. Shen,F.T. Chin, S.S. GambhirAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): T.H.Witney, A.Hoehne, R.E. Reeves, B. Shen, F.T. ChinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T.H. Witney, A. Hoehne, F.T. Chin, J. Rao,S.S. GambhirWriting, review, and/or revision of the manuscript: T.H. Witney, A. Hoehne,R.E. Reeves, O. Ilovich, B. Shen, F.T. Chin, J. Rao, S.S. GambhirAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T.H.Witney, A.Hoehne, F.T. Chin, S.S. GambhirStudy supervision: T.H. Witney, S.S. GambhirOther (provided radio-tracers for the studies): M. Namavari

AcknowledgmentsThe authors thank Francis Blankenberg for provision of reagents and

for helpful comments relating to the preparation and evaluation of 99mTc-HYNIC-Annexin V. In addition, the authors thank Gayatri Gowrishankar,Carmel Chan, and Adam Shuhendler for insightful discussion and JohnRonald for assistance with image analysis. The authors also acknowledgethe SCi3 Small Animal Imaging Service Center, which was used to create datapresented in this study, specifically, Tim Doyle for assistance with SPECTacquisition. The SCi3 Small Animal Imaging Service Center is supported byNCI grants 1P50CA114747 (ICMIC P50) and CA124435-02 (Cancer CenterP30). The ML-10 precursor was supplied from GE Healthcare. We would liketo acknowledge funding support from NCI ICMIC P50 CA114747 and theBen and Catherine Ivy Foundation.

Grant SupportThis work was funded byNCI ICMIC P50 114747 and the Ben andCatherine

Ivy Foundation.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 15, 2014; revised April 10, 2015; accepted May 4, 2015;published OnlineFirst May 13, 2015.

Witney et al.





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2015;21:3896-3905. Published OnlineFirst May 13, 2015.Clin Cancer Res Timothy H. Witney, Aileen Hoehne, Robert E. Reeves, et al. to TreatmentRadiotracer Imaging Agents for the Detection of Tumor Response

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