uncertainty and deficiency remain to be explored (1). Problems encountered in radiation dosimetry remain a challenge (2). Preradiationtherapy dosimetry methods havebeen developing intensively since 1991 so that the absorbed dose received by the bone marrow, the dose-limiting organ, can be more accurately predicted (3). No clearcut relationship had been reported, however, betweenresponse and the whole-body radiation dose, which is itselfcontingent on the total activity administered.

Only mean tumor absorbed doses of 10 Gy or morewould provide both significant and prolonged clinical effects (4). Tumor dosimetry, however, is still rather rudimental)' because of the difficulty to determine targetedfunctioning volumes accurately with conventional imagingtechniques (CF or MRI), especially when there is bonemarrow involvement (25). In addition, external detectionand quantificationof tumor radioactivitymay be distortedby backgroundactivity. Therefore the use of the conjugateview technique and, more recently, pretherapy administration of the positron-emitting tracer [@I]MIBG-labeledvariant to predict the actual [‘31I]MIBGmean absorbeddose appears promising (6@7).

The tumoricidaleffects of radiolabeled MIBG are contingent on two main factors: (1) the tumor's capacity totake up and retain the tracer and (2) its intratumoral patternof biodistribution. Attempts have already been made toimprove the first factor; gamma-interferon and retinoicacid have been shown to increase mIBG accumulation invitro, although no clinical trials investigating such pharmacological intervention have been reported (8@9). With respect to the second factor, it is necessary to determine the

best isotope to attach to MIBO, or alternative halogenatedanalogs, taking into account tracer biodistribution in thetarget, so that every tumor cell will be effectively irradiated. Bmmine-76-metabmmobenzylguanidine (MBBG)was recently proposed to explore sympathetic innervationin the heart (10). This 7@Br-labeledMIBO analog appears topossess adequate parameters for imaging and metabolic

Theheterogeneftyof tumoruptakeis likalyto substantiallylimitthe effectivenessof metalodobenz@4guank1ine(MIBG)therapy.This study was done to establish whether metabromobenzylguanidine(MBBG)can target neuroendocrinetumorsand toprovideintratumorbiodistributionand uptake informationin corn_son to MIBG.Mthods: MBBGandMIBGtumoruptakeandkinedcstudieswereperformedinexperimentalPC-12pheochromocytornagrownin nudemice.lntratumordistributionstudieswereperformedusingaUtoradkgraphyandsecondary @nmassspectrornetiy(SIMS)m@roscopy,becausethe lattertechniquecan detectand potentiallyquantifyboth drugsconcomitantlywithinthe sametumorspecimen.Results: MBBGuptakeinPC12tumors was early (2 hr) and intense (80% lDIg). RetentionvalueswereSimilarfor bothdrugs24 hr postinjection.At thecellularlevel,MBBGmostlyaccumulatedin the cytosol.At themulticellular level, cells exhibited staining, but in many areas,SIMS images of both drugs were not spatially correlated.Conclusion:MBBGtargetedexperimentalpheochromocytornaefficlantlywithhighearlyuptakevalues.Bromine-76-MBBGisapromising means of imaging and quantifyingtumor uptake withPET.BothdrugswerelOcaliZedInthecytosol,butthecorrelationbetweenthetwodistributions,asassessedbythevaluesof thestandardizedlocalconcentrations,was weak althoughsignificantmulticellularly.

Key Words: secondaryion mass spectroscopymicroscopy;metabromobenzylguanidine;metalodobenzylguanidine;pheothrornocytorna@metabolicradiotherapy

J NucI Med 1995; 36:859-866

fter more than a decade of worldwide experiencewith metabolic radiotherapyof neural crest tumors usinglabeled metaiodobenzylguarndine(MIBG), many areas of

ReceivedJul. 6, 1994;revisIonacosç*edNov.23, 1994.Forcorrespondenceorreprirdsoon@ DrJérômeClam,DepwtmentofNu

dear Medk@lneA, NedcerHospll@,149ruede Sèvres,75743P@isCedes15,Fr@on.

859SIMS Assessment of MBBGand MIBGin Pheochromocytoma •Clerc at al.

Assessing Intratumor Distribution and Uptakewith MBBG Versus MIBG Imaging andTargeting Xenografted PC12-PheochromocytomaCell LineJérômeClerc, Karine Mardon, HervéGalons, Christian Loc'h, Jean Lumbroso, Pascal Merlet, Jiarong Zhu,Josette Jeusset, AndréSyrota and Philippe Fragu

Eqi4pe de Microscopie lonique, INSERM, Institut Gustave Roussy, Vdkjuij France; Laboratofre de Chinzie O,@ansque2,Unive,@ritéRenéDescartes-Paris V, Pwic, France; Se,vice Hospitalier FredEric Joliot, DRIPP-CEA, O,@ray,France; andService de MédecineNucléaire, Institut Gustave Roussy, Vil1eJUi@France

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radiotherapy and its ability to target bioamine receptorpositive tumors was evaluated. Both drugs were administered to nude mice xenografted with the PC12 rat pheochromocytoma, a cell line known to actively store MIBG invitro. In addition, as data on tracer biodistribution areneeded for microdosimetric evaluation, we capitalizedupon a unique possibility offered by secondary ion massspectrometiy (SIMS), namely intratumor mapping andquantification of MBBG using MIBG as the referencetracer. SIMS microscopy is a microanalytical techniquebased on two principles in physics: the emission of secondal), ions following the bombardment of a specimen by aprimary ion beam and mass spectrometry (11). With SIMS,

it is theoretically possible to visualize biodistribution ofany stable or radioactive ion species within biological material and quantify the signal using an internal standard.Based on our findings with the PC12 experimental pheochromocytoma xenografted into mice, MBBG appears tobe a promising tracer for the work-up of neural-crest tumors.

MATERIAL AND METHODS

Cells and Experimental TumorsTheratpheochromocytomacell linePC12was shownto store

dopamine, norepinephrine and more recently MIBG (12). Thisdrug is first taken up at the membrane level through the neuronspecificuptake-i mechanism,whichis energy-dependentand saturatesat 1O_6M. Oncein thecell, MIBGis storedinspecializedgranules, particularly numerous in pheochromocytoma, through aso called uptake-3 system (4). This system requires a proton pumpand is inhibited by reserpine. The cells were propagated in aRPMI 1640 medium containing 15% horse serum and 5% fetal calfserum plus antibiotics. Several series of6—8-wk-old pre-irradiated(5Gy)nudemice(SPSwissnu/numice,10miceperseries)wereinjectedsubcutaneouslyin the flankswith approximately3 x iO@cells. Small tumors (50—200mg) were obtained about 3 wk later.

RadlOpharmaCeutlcalsThesynthesisandpharmacologicalpreparationof[76Br]MBBG

has been reportedelsewhere (13). Briefly, ammonium(76Br)bromide is produced by the irradiationof natural arsenic with a30-MeV(3He)ionbeam.Then,[76Br]-MBBGis preparedby heteroisotopicexchangebetweenthe [76Br]-BrNH4and the coldiodineatomsof MIBGusinga@ assistedsubstitutionreaction.Bromine-76-MBBG is separated from the iodo precursor byHPLC andfinally dissolvedin salineand filtered onto a 0.22-smMillipore membrane. The radiopharmaceutical is produced in a65%radiochemicalyieldwithaspecificactivityof2OMBq/nmole.

Thesynthesisof stableMBBGwas requiredforSIMSexperiments because the weight of the injected amounts of the radioactive brominatedanalogwas clearlybelow the detection sensitivityof the SIMS microscope. The synthesis of MBBG sulfate wasachieved after condensation of 3-bromobenzylaminehydrochloride (3.34 g, 15 mmole) with 2-methyl-2-thiopseudourea sulfate(5.16g,30mmole)inthepresenceofdimethylfonnamide(DMF),which was foundto facilitatethe reaction.Briefly, a mixtureof thetwo precursors in 20 ml DMF was heated at 100°Cfor 4 hr. Aftercooling, the solvent was distilled under vacuo. The resulting solidwasdissolvedin150mlof isopropanololandleftovernightat4°C.Thewhite crystalswere isolatedby ifitrationandrecrystallized

fromisopropanolol:2.25,yield:48%.Specificspectraof thefinalmolecule were obtained: IR(KBr):2200—3300cmi, 1640 cm1;1H-NMR(DMSO-d6,TMS) ö:3.95 (s, 2H), 7.31 (m, 2H), 7.51(dd, 1H), 8—9.2(s, large, 5H). The crystal was weighed, resuspended in saline and sterilized as above.

The MIBG,[127I]@4ffiG(1300j.@g/400@tl),used in the SIMSexperiments was kindly provided by CIS-Bio International (Gifsur-Yvette,France),aswellas [@I]MIBG(@-O.4MBq/nmole)forautoradiographic purposes. Iodine-123-MIBG was used forgammacameraimagingand countingpurposesat a specfficactivity of about 0.05 MBq/nmole.

ProtocolsBromine-76-MBBG uptake by the tumors and various organs

wasdetermined2, 4, 8, 12,19and24hrafterinjectionof about74kBq in the tail vein. Parallel uptake experiments were performedwith [‘@I]MIBG8 and24 hrafterintraperitonealinjectionof about1100—1850kBq. In all cases,the sampleswere weighed,radioactivity was measured in a gamma well-counter and results wereexpressed as a percentageof the injected dose per gram(%ID/g).

Macro@c ImagingWhole Mouse Body Autoradiography with [76Br]MBBG.

Ethyl-ether anesthetized animals who had received 1110 kBq[76Br]MBBGwere frozen,8 hr postinjectionin an ice-isopentanesolution (—75°C)and then placed into a cold solution of carboxymethylcellulose(CMC-1.5%)to permitroughsectioning(20pm) in a ciyomicrotome. Precooled adhesive tape was applied onthe tissue slice to remove sections that were further dried andexposed for 10 days on autoradiographic films. Films were develo_, analyzedandcolor-codedusingacomputerizeddensitometnc system.

Nuclear Imaging. Five mice injected with [‘@I]MIBGunderwent scintigraphicstudies 10, 19 and 24 hr postinjection. Eachanimal was anesthetized three times with ketamine (15 @g/g)andimages were acquired on a DSX gamma camera (Sopha Medical,Buc, France)equippedwitha high-resolution,parallel-holecollimator. An aliquot (370 kBcijof the administeredtracerwas alsoimaged as an internal standard and tumor uptake was determinedwith ROI method with background correction for extratumor activity estimated on the tumor-free flank. Finally, 24-hr MIBGscintigraphic uptake in tumors was compared to the values obtamed through direct gamma well counting as described above.Two additional mice bearing the nontargeted neuroblastoma IGRN-835 were used as controls (14).

MIcroscopicImagIngMIBG Micmautoradiography. Two animals received about

1850 kBq of [‘@I]MIBGand were killed 24 hr later. Chemicalsample processing (see below) was performed and 2-jan sectionsof thespecimenweremountedon slides,coatedwithAmershamLM1 emulsion, exposed for 8-20 days and finally stained withhematoxylin-eosin.

SIMSAnt4ysLr.Stable MBBGwas injected into the animalsintraperitoneallyby escalatingdose increments(0, 100,200,400,600 pg). In addition, 400 @tgof [127I]@4ffiQwere concomitantlyinjected into mice, who received 200-400 @gof MBBG. All animalswere killed24hr later.

Firative Procedures, Embeddàtg and Sectioning. Sample processing was performed using either a chemical or a cryo-procedureas previouslydescribed.Withthe chemicalprocedure,whichcannot prevent drug diffusion, fragments are fixed in glutaraldehydeandparaformaldehydeincacodylatebufferandfinallyresin

860 The Journalof NudearMedicine•Vol.36 •No. 5 •May1995

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Tissues 4hr 8hr l2hr 24hr

Tumor 32.6±3.3@ 53.3±4.8

TABLE IBk@disthbution(%ID/g)of [ThBrIMBBGandE1nsIIMIBGin NudeMiceXenograftedwiththe PC12PheochromocytornaCellUne*

‘82.3±7221.6 ±4.O@5.12 ±0.58

1.8 ±2.93±0.54

1.5±O24'@2.11 ±0.241.36 ±0. 18@'3.19±0.671.45±O.3O@18.7±4.35.@±o.93t0.75±0.110.32± O.@

202 ±3.719.4 ±5.40.93±0.320.45 ±0. lS@0.87±0.150.47 ±0.05?0.54±0.100.48±0.020.79±0.380.66±0.146.5±1.53.9±1.6?0.3 ±0.07

0.18± 0.037

*VaIuesarepresentedinboldfsceforMBBGandinitalicsforMIBG.tp < 0.05. MBBG and MIBG uptake values were compared usingthe Bonferroni'st-testwlth a rink ofo.05. Uptake kinetic studies were performed

atalltimesforMBBG(boldchar@ers)butoolyat8and24hrpostinjedionfOr(1@MIBG.Al mioehedPC12xenograftsobtalneddlrectlyfromtumorcells.ActivItiesusedfortheseexpedrnentsweretypicallyinthe rangeof 75 kBqof (@B,]MBBG(specificactMty 20 MBq/nmole)and750 kBqof[1mIJMIBG(specificactMty@0.05 MBajnmola).DitlerencesinearlyuptakevaluesmayreflectdifferencesInthe routesofadminlatration,intraperitoneallyfor MIBGandIntravenou&yfor MBBG.

4.34±0.67

2.95±0.30

2.73 ±0.37

1.54±0.48

6.20±0.55

0.51 ±0.05

Heart

Lungs

Liver

S

Adrenals

Muscle

2.30±0.66

1.72±0.30

1.33±0.34

2.31 ±0.66

121±2.5

0.76±0.19

embedded.The cryomethods,whichallowin situ immobilizationof diffusibledrugs,consistinhigh-speedfreezing(5000Ksec1) inliquidpropan,cryo-substitutionin acetone(183K)andciyo-embedding (213K) in Lowicryl K11M (Polysciences Ltd., Eppetheim, Germany ). Semi-thin sections (3 pm)were deposited onultrapuregoldholders for SIMSanalysis.

SIMSMkroscopy Imaging.Descriptionof the IMS-3Fmicroscope (Cameos, Courbevoie, France) has been reported elsewhere(15). Briefly,a 5—15-nAprimarycesiumion beamis focased on the surface of the resin-embeddedspecimen to sputterandpossibly ionize the atoms located in the superficiallayers (1—5am). The secondary ions, characteristic of the atomic compositionof the analyzed area, are focused, energy-filteredandseparatedina mass spectrometer. The ion images are finallydisplayedusing ahighly sensitive camera and a dedicated microcomputer system(16).All imagesareacquiredwith highmassresolution(W@.M>2000) to guarantee signal specificity because cluster ions are emitted close (<0.07 uma) to the ions of interest (17). SIMS analysisprogressively erodes the specimen at a destruction rate of about1.5 nm/secandprovidesimageswith a 500-nmresolutionfor adetection sensitivity of less than 1 ppm for halogens. The tissuestructure can be visualized through the mapping ofthe polyatomicion @CN, the nuclei of the cells through 31P, whereas drugbiodistributionis delineatedthrough detection of 1271_(MIBG)and @Bror 81BC (MBBG) because bromine has two stableisotopes of similar abundance: 7@BC (50.57%) and 81Br(49.43%).

Quantification with SIMS Microscopy. The secondary ionbeam intensity can be measured with an electron multiplier and isproportionalto the correspondingelemental concentrationand tothe primaryion beam intensity. The primarybeam, however, ispartly repelled by charge effects occurring at the surface of theembedded insulating specimen. Because carbon is present at alarge and ratherhomogeneous concentrationin the specimen, aswellasintheresins,itcanbeusedasaninternalreference.Tissueequivalentresinstandardscontainingincreasingproportionsof

halogens had been synthetized to derive calibration curves between isotopic ratios (ion of interest/carbon reference) and concentrations (18). We used 8 @imwide apertures, which can besuperimposedonto an ‘@CNion image(62jan) and cyclesof 10consecutive 1-sec measurements were acquired. Counting wasperformedon ciyopreparedfragmentsfor the 200-and 400-sginjecteddose levels.

RESULTS

Bromlne-76 MBBG Uptake In PCI2 Xenograft.d MiceTumor and tissue concentrations of both radiopharma

ceuticals are given in Table 1. Tumor tissue had the highestand most prolonged uptake. MBBG strongly accumulatedin tumors, even early after administration(2-4 Kr),with apeak value as high as 80% ID/g at 8 hr postinjection, but asignificantreduction in uptake occurred between 8 and 24hr. Comparisons between the targeting efficacies of MBBGand MIBG must be interpretedwith caution in these cxperiments since molar drug doses and specific activitiesused were veiy differentfor both drugs. In addition, lowerearly uptake values encountered with MIBG (intraperitoneally) as compared to MBBG (intravenously) may partlyreflect the routes of administration.

Macroautoradlography and Gamma Camera ImagesThe whole-body autoradiogram(Fig. 1) displays [76Br]-

MBBG accumulation at the periphery of the tumor. Therewas partialcentral necrosis, which may be overlooked onplanar scintigraphic images. Gamma camera images of apanel of 3 PC-12 mice showed intense accumulation of[123IJMIBG,at anytimepostinjection(Fig. 2). Tumoruptake, as determined from the scans and using the actualweight of the tumor obtained after dissection, was 25.1 ±4.1, 24.2 ±6.6 and 17.5 ±6.5% ID/g 10, 17 and 24 hr

861SIMSAssessment of MBBGand MIBGin Pheochromocytoma•Clercat al.

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S Sd@

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FIGURE 2. Gamma camera imagesof a panel of three PCI2-xenograftednudemiceinjectedwith[1@'I]MIBG(1110kBqadministeredintraperitoneally).Theimage(50kcts,posteriorview)wasacquired10hrpostinjection.Thereisintensetraceraccumulationinthexenografts,readilyvisibleintheflanks(smallarrows).Noradioactivitycanbeseeninthecontrol(C)nontargetedneuroblastoma,IGR-N-835.ThetumorwasmanuallydelineatedusingacobaltpenelI(largearrow).“H―indicatestheheadoftheanimalsand“Sd―theinternalstandard.

originalsites of uptakeandbecause the lateralresolutionofsuch autoradiogramsexceeds 500 run.

SIMS Mapping and Counting of MBBGSIMS always exhibited a specific signal for stable bro

mine, regardless of the tissue section analyzed, the sampleprocessing method applied and the amount of MBBG injected. With chemical sample processing, the tissue structure was well preserved and nucleoli could be visualized onthe @CNion images. With 102-ian wide image fields (Fig.4), many cells appeared positive for MBBG, especially inthe vicinity of the lumen of vessels. With cryopreparedfragmentsand 60-@&mwide fields, MBBG and MIBG weresimultaneously detected. Both drugs were mainly accumulated in the cytosol of the tumor cells (Fig. 5). Althoughboth drugshadratherhomogeneous distributionpatternsinmost of the analyzed areas, which correlates with the tumor model, we found that MBBG was also able to accumulate intensely in several foci of the tumor (Fig. 6). Finally, similar images of MBBG distribution were obtainedusing either @Bror 81Brfor drugdetection, thus providinga powerful internalcontrol for image specificity (Fig. 7).

Local quantification of MBBG uptake, corresponding to2440measurements, is given in Table 2. It appears that thehigher the injected dose the higher the mean local drugconcentration; wide discrepancies, however, were foundamongthe 8-jan wide countingareas, with variations in therange of concentration as large as 80 p@gof drugper mg oftissue. In addition,manylocalconcentrationvalueswere10—80times above the values expected according to radioactive uptake experiments. Finally, we found that the intensities ofthe secondary standardizedbromineion beams,obtained on 102-pin sputtered areas, were highly correlated (r = 0.997, p < 1O@),which is in agreementwith theirsimilarity in abundance.

FIGURE 1. Whole-bodyautoradiogramof a mouse bearingthePCI2 pheochromocytomaxenograft injected with. I@Br1MBBG(1110 kBq)8 hr beforedeath.The analyzedimage,fscilltatedby adleplayusingpseudocolors,showsstrongaccumulationof MBBGonlyat the penpheiyof the tumorbecauseits centeris necrotic(arrow).

postinjection, respectively. if one excludes one mouse, forwhich tumor dissection had been incomplete, uptake vatues derived from scanning and tumor counting correlatedclosely (r = 0.96, p < 0.05, n = 6).

Iodlne-125 MIBG ImagIng of Tumors byMlcrOautoradlOgraphy

Due to the strong aflinity of tumors for MIBO, intensestaining was observed after 12 days of exposure (Fig. 3).Most of the cells had accumulated MIBG and exhibited arather homogeneous pattern of distribution at the tissuelevel. At the cellular level, staining mainly affected thecytosol. Some silver grains were also evidenced, however,on cell nuclei because the chemical sample processingmethod used here cannot prevent MIBG diffusion from

... .‘. . ..@ .@ .., .@;

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FiGURE 3. Autoradlographyof PC12 pheochromocytomaxenograftedInto mice lnje@edintraperitonealywith 1850kBq of[1@IJMIBGandkilled24hrlater.Sampleswereexposedfor12daysandcoloredwithhematoxy1k@-eoeln.Accordingtothetumormodel,mostoftheeelsarestained,paIticUIarIythOSeclosetothelumenofvessels.At the CellUlarlevel,however,the silvergr@nsare evidancedbothinthecytosolandinthenudelbecausethechemicalsamplaprocessingmethodwasinadequate(Bar= 5 pm).

862 The Journalof NudearMedicine•Vol.36 •No. 5 •May1995

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FIGURE 4. SIMS im@es of r1BrJMBBGwlthkithe PCI2 xenograftafterInvivoadministrationof200 @gofthedrugandchemIcalsamplaprocesalng.@A)SIMSImageofthe tissuestructureis dehneatedthroughpolyatomic26cN(cytosoland nuclei).(B)Phosphorus@31Ph)SIMSimagemappingofthenucleland(C)SIMSmappingofMBBGthrough81Brdetection,displayedin Inverse

@deoscale.Onthephosphorusimage(B),nucleoliarevisible(smallarrows),butbettermappingofthewholetissuestructureIsobtamedwith26CNdetection.MBBGIntratumor distributionappearsratherhomogeneous due to the chemical sampleprocessing.Notethemarkedstainingofthecellscloseto thevessels.Imagefield:102@hm,bar:10pm.

IGR-N-835 neuroblastoma, and even after a 400-pg injected dose, correspond to local concentration valueswhich are below the detection threshold of the IMS-3F forhalogens(14,19).

MBBG and MIBG intratumor distribution patterns werecrudely homogeneous, but MBBG could also be mapped asrather intense foci of accumulation, especially when theclyopreservation procedure was used. Indeed, the redistribution of stored drugscannot be ruledout duringchemicalsample processing due to passive drug diffusion in solvents. This is why ciyotechniques ought to be used toensure signal specificity and to preserve the actual drugconcentrationlevels requiredfor SIMS quantification(20).The focal pattern of accumulation with MBBG remainsunexplained. This molecule may gain easier access to tumors because it is much smaller and less lipophilic thanMIBG.

Both drugs were shown to accumulate in the cytosol ofthe tumor cells, but a minute signal was also detected forbromine or iodine when these images were superimposedonto the phosphorusimages, which essentially mappedcellnuclei (Fig. 6). Two explanations are plausible here. First,drugs situated in the cytosol but close to the nucleus membrane may be imaged in cell nuclei or at the peripherybecause the IMS-3F only has a lateral resolution of500 am.Second, SIMS analysis functions by successive acquisitions directed at selected ions. The typical acquisitiontimes for 26CN@and 31P are 15-30 sec, whereas durationvalues bordering90 sec are often needed to analyze bro

.@.‘“@..FiGURE 5. SIMS k@es of MBBG andMIBGwithinthePCI2 xenograftafterInvivoadministrationof 400pg of the drugsandsamplepro@ withtheayetecMques

.@ The phosphonis lm@e @) maps tumor caN‘. nucleiwhichappearveryIrregular.BethstebleMIBG(B)andMBBG(C)exhibItedhomogeneousIntratumordistributionthatcorrelateswith the tumormodeLThereare,however,distributionsthatcannotbesuper

:@ knposed.Imagefield:60 pin.C

SIMS Assessment of MBBG and MIBG in Pheochrornocytorna•Clam at al. 863

DISCUSSION

The use of transplantabletumor models endowed withthe same specific uptake and storage systems as thoseencountered in chromaflin tissue and related tumors is aprerequisite to reliably mimic traceruptake by tumor cellsin humans. Indeed, most human tumors had ischemic andnecrotic regions with potentially decreased radiosensitivityand in which radionucide accessibility may be critical (2).The PC12 pheochromocytoma tumor model was considered appropriate for in vivo pharmacological studies because its avidity for MIBG was retained at a high level afterxenografting.

Images of both MBBG and MIBG intratumoruptakewere constantly obtained with the IMS-3F. Although bothdrugs were taken up within a few hours after injection,

SIMS experiments were performed24 hr later. Indeed, theamountof drugtaken up at 24 hr is a more reliablegauge ofcumulated activity, and thus at mean absorbed dose levels.The 200- and 400-pg of stable drugs injectedyielded bloodconcentration levels borderingon 1 @iM24 hr postinjection(0.34%ID/g of blood and for MBBG), a value known toauthorize significant passive diffusion in tumor cells. Because a concentration gradient exists between the bloodand tumor cells, however, blood concentrations overestimate the amounts of drug which effectively reach the Uimor. In addition, the PC12 xenografts had very high specific uptake values compared to those of nontargetedtissues. Overall, intratumorMBBG or MIBG levels, whichmay result from passive diffusion, such as in the nontarget

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4I

‘- -U.

. @, I@ ‘@

@,‘

,.@ mineoriodine,somuchsothatadepthof100—300nmmayseparate the first and last sputtered plans which ultimatelyproduce the final ionic image. This phenomenon causes afurther reduction of ten or so nanometers in the lateralresolutionof the composite image resultingfromthe superimposition of the computerized elementary ionic images.Most of these problems will be solved with the new generationof SIMS microscopes which offerparalleldetectionand a lateralresolution attaining50 am (21). These micro

@ scopes are ten times more sensitive than the IMS-3F whose

halogen detection threshold is 0.5 p@g/gfor biological materials with a spatial resolution of below 1 @m.

Local quantification, standardized with the 12Cseconda!), current, was first achieved on 8-nn wide areas, a sizewell adapted for most problems of cell biology. Countingon cell-scaled areas not only led to a wide range of localconcentration values, but also to overestimations compared to the expected mean values derived from radioactive counting. A similar phenomenon, albeit limited, hasbeen reportedin the countingof freshly organifiediodine inthyroid fofficles (22). This may be because very differentmolar drug doses are considered, ranging from microgramsin SIMS analyses to picogramsin radioactiveexperiments.This also indicates that the sputtered volumes (mg tissue)involved in SIMS analysis are very small and may or maynot correspond to sites of drug accumulation. To obtaindata more adaptable for multicellular dosimetry, SIMSanalysis was also performed on 102-jim wide fields. Unfortunately, we were unable to obtain standardized MBBGlocal concentration values in our analysis of these largerfields because the standard calibration curves had onlybeen derived for small volumes of tissue-equivalent resin

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FIGURE 6. SIMS imagesof MBBG and MIBG withinthe PC12xenograftafterin vivoadministraNonof 400pg of the drugsandsampleprocessingw@iclyotechniques.Thephosphorusimageindicatesthetissuestructure(A).Here,[1@IJMIBGusratherhomogeneoualydistributed(B)comparedto MBBG,whichshowsfod ofaccumulation(Cand D). Becausebrominehastwo stableisotopesof similarabundance, ftis possible to map MBBGthroughthe detectbnof [@BrJMBBG(C)or[@1Br]MBBG(D).Bothimagesareverysimilarand thus pro@Adea powerfulinternalcontrolfor spedflcftydetec@on.TheminutedUferencesobservedbetween(C)and(D)areduetothefactthatthetwoSIMSimagesareactuallyseparatedbyapproximately50nm.Imagefield:60pm.

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$ -@FiGURE 7. SIMS imagesof supenmposedelemental @nimagesin pseudocoicrscorrespondrngto a zoomeddetailview of the lowercornerinFigure5.Thenuclelaredisplayedingreen(3@P).(Left)MIBGisimagedinred-to-yellowandMBBGinblue.Notethatmostofthesignal is e@@idencedin the pennudear areas, but that MIBG and MBBG distilbutionsare not supedmposable.(Right)MBBG mappingobtainedthroughthedetectionof“@Br(red)orof81@(blue).

864 TheJournalofNudearMedicine•Vol.36•No.5 •May1995

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InjecteddoseNOrmalIZed “@BrcountsDrug concentration(pg drugkngtissue)(p9)N (79Br/12C)x iO'3mean ±s.d.range

TABLE 2SIMSQuantificationofLocalConcentrationofMBBGwfthinPC-12Tumors

2007303.38 ±4.37.88 ±10.1(0—75.6)2004704.23±6.09.92 ±14.3(0.3-80.2)2008002.64±I .766.02 ±3.84(0-17.84)4002307.66±6.7118.4 ±16.1(2.76-65.3)

SIMS quantificationof the drugIs derivedfrom measurementsof secondary @Brion beam Intensity.This currentis standardizedby thecorresponding12@@ Ionbeamintensityandthedrugconcentrationis thenderivedfromcalbationcuivesdeterminedforthesamenOrmalIZed@nicratio(@Br/@C)countedintissue-equivalentresin.N isthenumberofmeasurementsforeachcountlasting1sec.TwodoselevelsofinjectedMBBGarepresented.Allmeasurementsaremadeonctyopreparedfragments.ThenOrmalIzedIonicratiovalueof<0.002correspondsto an undetectablelocalconcentration. _______________________________________

and were only available for bromine-to-carbonratios over0.002. A comparison of the local concentrations of bothdrugs was nonetheless obtained by measuring the normalized secondary bromine and iodine ion beams (@Br/'2Cversus 1vI/12C)corresponding to the sputtering of these102-1tan wide areas. Both emissions were moderately correlated (r = 0.3 to 0.5, n = 1200measurements, p < 10@)forall analyzed areas, suggesting that uptake mechanisms atthe cellular level, though highly specific, only partiallyaccount for global uptake in xenografts.

SIMS promises to be a powerful tool in the fieldof tracerdevelopment. Compared to quantitative autoradiography,which has the advantage of being widely available, onlySIMS is able to map and quantify multiple stable drugssimultaneously. In addition, not all radioactive isotopes areable to provide autoradiogramswith acceptable sensitivityor lateral resolution, and, even when they are suitable,their use in humans is problematic for obvious ethical andlegal reasons. Finally, because of filteringby mass spectrometry, all SIMS images have the same lateral resolution, irrespective of the physical decay properties of theanalyzed ions under study. Depth-counting profiles havealready been obtained with SIMS in biological specimens,and three-dimensionalimagingis under development. Thistechnique is complementary to fluorescent confocal microscopy, which is particularly interesting since it providesthree-dimensionalimages of the distributionof monoclonalantibodies directed at tumor antigens or receptors.

Some of the partial responses with MIBG therapy arelikely to reflect dramatic heterogeneity in dose distribution,as previously demonstratedin experimentalneuroblastoma(19), because 90% of the dose is delivered within 815 @mofa point source with ‘@‘I.Among the alternative halogensthat can be attached to benzylguanidines, 76Brhas favorable physical characteristics for both imagingand therapy.Moreover,PET emissionmay permitaccurateexternalquantification of tumor compared to that expected withSPECT or planar images, with the added advantage ofbeing able to provide better spatial resolution (10).

MBBG has interesting properties for therapy since itdecays through two highly energetic beta emissions (3.1

and 3.6 MeV), which account for particles with pathlengths ten times the values reached with ‘@‘I.This propcity is particularly needed when the objective of internalradiotherapyis tokilltumornodulesabove5mm,andevenmore so to obtain tumor shrinkage by administeringthetracer de novo, i.e., as first-line therapy (23). Indeed, ininternalradiotherapy,the probabilityof tumorcure is maximal for a critical tumor size, which depends on the meanrangeofthe particlesemitted. This size has been calculatedto be of approximately 2 mm for experimental sphericaltumor models when ‘@‘Iis used (24). The physical half-life

(16.2 Kr)of 76Bris acceptable for therapy and sufficientlylong to permit delivery from distant cyclotron facilities.Finally, no unwanted thyroid irradiation will occur withthis drugbecause bromine, unlike iodine, is not trappedbythis gland.

CONCLUSION

It is noteworthy that the rangeof 76Brin tissue could beof interest to guide the surgeon duringsurgery and permitthe selective detection of labeled tissues from the surfaceto a few millimeters in depth because intraoperativebetaprobes are insensitive to gamma radiation(25).

We now know that MBBG will reduce the heterogeneityof dose distribution.The next step is to evaluate whetherhuman tumors will take up MBBG and determine bonemarrow toxicity, the dose-limiting factor with MIBG.

ACKNOWLEDGMENT

The authorsthankMs. L. Saint-Angefor editingthe manuscript.

REFERENCES

1. ShapiroB.Summaiy,conclusions,andfuturedirectionsof[―I]metaiodo.benzylguanidinetherapyinthe treatmentofneural crest tumors.JNudBiolMed 1991;35:357—363.

2. Shapiro B, Sisson JC, Wieland DM, Ct al. Radiopharmaceutical therapy ofmalignantpheochromocytomawith [“I]metaiodobenzylguanidine:resultsfrom ten years of experience. INuci Bid Med 1991;35:269-276.

3. Fielding SL, Flower MA, Ackeiy DM, Kemshead J, Lashford LS, LewisU. The treatment of resistant neumblastomawith “I-MIBG:alternativemethodsof dose prescription.RadiotherapyOncol 1992;25:73-76.

4. Smets LA, Rutgers M. Modeistudies on metaiodobenzylguanidine (MIBG)

865SIMS Assessment of MBBG and MIBG in Pheochromocytoma•Clerc at al.

by on January 28, 2018. For personal use only. jnm.snmjournals.org Downloaded from

uptake and storage: relevancefor ‘31I.@@4@ffi@fl@@sm@of neuroblastoma.INuciBiolMed 199135:191-194.

5. Krempf M, Lumbroso J, Mornex R, et al Treatment of malignant pheochromocytomawith [‘3'!Jmetaiodthenzylguanidine:a French multicenterstudy.INuci BIOIMed1991;35:284-287.

6. ShulkinBM,SissonJC,KoralKF,ShapiroB,WangX, JonhsonJ.Conjugate-view gamma camera method for estimating tumor uptake of iodine131-meta-iodobenzylguanidine.INuciMed 1988;29542-548.

7. Ott RJ,TaitD, FlowerMA, BabichJW,LambrechtRM.Treatmentplanningfor ‘31fl@4ffi@3radiotherapyof neural crest tumours using @I.MIBGpositronemissiontomography.BTIRO4IOI199265:787-791.

8. MontaldoP0, CarboneR, PonzoniM, Cornaglia-FerrarisP. Gamma-interferon increases metaiodobenzylguanidineincorporationand retention inhumanneuroblastomacells. CancerRes 199252:4960-49M.

9. lavaroneA, LasordllaA, Seividei1, RiccardiR,MastrangeloR.Uptakeand storage of m-iodobenzylguanidine are frequent neuronal functions ofhumanneuroblastomacell lines. CancerRes 199353:304-309.

10.ValetteH,Loc'hC,MardonK, etaLBrnmine-76-metabrnmthenzylguanidine: a PETradiotracerformappingsympatheticnewesofthe heart.JNuclMed 199334:1739—1744.

11.GalleP.Tissuelocalizationofstableandradioactivenuclidesbysecondaiyion microscopy.INuciMed 198223:52-57.

12. SmetsLA, JanssenM, RutgersM, Ritzen K, BuitenhuisC. Pharmacokinetics and intracellulardistributionof the tumor-targetedradiopharmaceuticel m-iodthanzyl-guanidinein SK-N-SH neuroblastoma and PC12pheochromopcytomacells. Intl Cancer 1991;48:609-615.

13. LOC'H a@, Mardon K, Valette H, BrutescoC, et al. Preparationandpharmacologicalpreparationof [@Br@-meta-bmmthenzylguanidine([@BrFMBBG).NUCIMe4Bid 199421:49-55.

14.Bettan-RenaudL, BayleCh,TeyssierJR,BenardJ.Stabilityofphenotypicandgenotypktraitsduringthe establishmentofahumanneuroblastomacellline, IGR-N-835.Intl Cancer 1989;44:460-466.

15.FraguPh,BiançonC,FourréC,aeroJ,JeussetJ,HalpernS.Howcan

SIMS microscopybe used in biomedicine?MicrobeamAn4@sis1993;2:199-207.

16. OlivoJC,K.hanE, Halpern5, BriançonC, FraguPh, Di PaolaR. Microcomputer system for ion microscopy digital imagingand processing.IMic@wc@çy198956:1O5—114.

17. FourréC,HalpernS, Jeusseti, aercj, FraguPh.Signthcanceofsecondaiyion mass spectrometlymicroscopyfor technetium-99mmappingin leukocytes.INUC1Med 19@233:2162-2166.

18. Jeusset 3, Halpern5, BriançonC, GarciaF, Fragu Ph. Halogenstandardizationby SIMSmicroscopyfor applicationsin biologicalsamples[Abstract]Bid Cell 1992;75:8a.

19. aamJ, HalpernS, FourréC,OmriB,JeussetJ,FraguP.SIMSmicroscopyirnagingoftheintratomorbiodistrilutionofmetaiodthenzylguanidinein thehuman SK-N-SH neuroblastomacell line xenograftedinto nude mice. INuciMed 199334:1565—1570.

20. Hlppe-SanwaldS. Impactof freeze substitutionon biologicalelectronmicroscopy.Mkiuw Res Tech199324:400-422.

21. SlodzianG,DaigneB, GirardF, BoustF, HillionF. Scanningsecondaiyionanalyticalmicroscopywith paralleldetection.Bid Cell 199274:43—50.

22. BriançonC, HalpernS, Jeusseti, FraguPh. Effectsofvarious iodineintakeon iodinethyroidautoregulation.Studywithanalyticalionmicroscopyand129j@IBid Tnice Element Res 1992;32:267-273.

23. HoefnagelCA, De Kraker J, VoütePA, ValdéOlmos PA. Preoperative@ therapy of neuroblastoma at diagnosis

(“MIBGde novo―).INuciBiolMed 199135:248-251.24.AminAE,WheldonTE,O'DonoghueJA,BarretA. RadiObiOIOgIcalmod

clingofcombined targeted131!therapy and total body irradiationfor treatmentofdisseminatedtumorsofdifferingradiosensitivity.IntlRadiatOncolBid Phys 199327:323—330.

25. DaghighianF, MazziottaFC, HoffmanEl, et a!. Intraoperativebeta probe:a device for detectingtissue labeledwith positronor electronemittingisotopesduringsurgeiy.Med P#zys1994;21:153—157.

866 TheJournalof NudearMedicine•Vol.36•No.5 •May1995

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1995;36:859-866.J Nucl Med.   Jeusset, André Syrota and Philippe FraguJérôme Clerc, Karine Mardon, Hervé Galons, Christian Loc'h, Jean Lumbroso, Pascal Merlet, Jiarong Zhu, Josette  Targeting Xenografted PC12-Pheochromocytoma Cell LineAssessing Intratumor Distribution and Uptake with MBBG Versus MIBG Imaging and

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