Bennett BD SIMS II boron in tissue duoplasmatron

Subcellular Localization of p-Boronophenylalanine-Delivered Boron-10 in the Rat 9L Gliosarcoma: Cryogenic Preparation In Vitro and In Vivo Author(s): Brian D. Bennett, Jonathan Mumford-Zisk, Jeff A. Coderre and George H. Morrison Source: Radiation Research, Vol. 140, No. 1 (Oct., 1994), pp. 72-78 Published by: Radiation Research Society Stable URL: http://www.jstor.org/stable/3578570 .

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RADIATION RESEARCH140, 72-78 (1994)

Boron-10 Localization ofp-Boronophenylalanine-Delivered Subcellular In Vitro and In Vivo intheRat9L Gliosarcoma: Preparation Cryogenic JeffA. Coderretand George H. Morrison' Brian D. Bennett,*JonathanMumford-Zisk,* Ithaca, New York 14853; and tMedical Department, *Departmentof Chemistry,Cornell University, Brookhaven National Laboratory,Upton,New York 11973

to 10B(n,cx)7Li may require estimationof microdosimetry, B. D., Mumford-Zisk, Bennett, J.,Coderre,J.A. and Morria 7Li particles have shortranges of about since the and son,G. H. SubcellularLocalizationofp-Boronophenylalaninein 5-9 soft tissue.

Monte Carlo simulationspredictthat pm DeliveredBoron-10in the Rat 9L Gliosarcoma:Cryogenic the of cell probability killingis enhancedby nuclearlocalIn VitroandIn Vivo.Radiat.Res.140,72-78(1994).

Preparation ization (5). Therefore,precise determinationof 10Bat the forion subcellularlevel is needed to testthe simulation in vitrocryogenic A well-characterized preparation predictions of whichminimizes redistribution microscopic isotopeimaging, and to understandfullyhow BNCT killstumortissue.Here, of diffusible species,was used to determinethe distribution cellsincubated withtheboronneu- we apply ion microscopyand cryogenicsample preparaboronin GS-9Lgliosarcoma tion to boron imaging in cultured and subcutaneous troncapturetherapyagent,p-boronophenylalanine (BPA). At agent, the subcellularlevel,boronfromBPA distributes relatively GS-9L gliosarcomaforthe BNCT tumor-targeting within cell.

Boron from BPA was the p-boronophenylalanine (BPA). homogeneously glioma The ion microscope is a directimagingsecondaryion eliminatedrapidly,indicatingthatmostis unbound.Thus a to diffusion Removal mass spectrometer.Samples underhighvacuum are bomartifact. largepool ofboronis susceptible in microdosi- barded by an energeticprimaryion beam thatcauses emisofthisartifact increasesthedegreeofconfidence subcellular distri- sion of secondaryions. The secondaryions are extracted metricresultsinferred fromthehomogeneous of in boron subcutaneous bution.The ion microscopic imaging and transferred mass spectromethrougha double-focusing in situwas achievedin ratstreatedwithBPA. ter.The transfer tumorscryofixed are thus a two-dimensionstigmatic; optics BoronsignalsfromBPA wereadequateto imagemicrodistribuion imageis formedwhichis projectedonto al mass-filtered tionsat the1-pmresolution level.As in thein vitrocase,boron did not localize discretelyat the subcellularlevel. However, a sensitivearraydetector.Digital images are capturedand boronheterogeneity was seenat thetissuelevel.Physiologicallystored for furtheranalysis off-line.Therefore, the ion valid cellularpotassiumand sodiumlevelswereseen,which microscopicimage is a two-dimensionalmap of the distriartifact.Futuretissue butionof a specificelementor isotope on the sample surdemonstrates minimizedredistribution studiesdesignedto correlateion microscopic boronimagesto face.It is capable ofimagingelementsor isotopesin thelow arefeasibleusingcryogenic structure microscopic sampleprepa- ppmrangewith0.5-1.0 pmlateralresolution(6). rationandionmicroscopy.

The ion microscope'sinherenthighsensitivity forlight elementsmakesitwell suitedforboronimaging.Othersubmicrometerboron imagingtechniques include a-particle INTRODUCTION trackautoradiography2 (7, 8) and electronspectroscopic Boron neutroncapturetherapy(BNCT) is a binaryradi- imaging(9, 10). Electronspectroscopicimaginghas inherationtherapyforcancerthatcombinesselectivedeliveryof entlyhighresolutioncapabilityand mayprovidethe sensi10B to the tumorwithnonionizingneutronradiation.Cell tivityforlightelementsnot foundin electronmicroprobe killingis caused by the heavy charged particlesreleased microanalysis. fromthe 10B(n,a)7Li neutroncapturereaction.These particles have a high relative biological effectivenessin vitro (1, 2) and in vivo (3, 4). Relatingradiobiologicalend point 2G. Solores,High resolutionalpha trackautoradiographyand biological studiesof boron neutroncapturetherapy.Ph.D. Thesis,Department of Nuclear Engineering,Massachusetts Instituteof Technology,Cambridge,MA, 1991.

author.

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SUBCELLULAR 10BLOCALIZATION IN GLIOSARCOMA

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forcell attachment andgrowth. ofionmicroscopy to quantitative biological sideofthesiliconpieceis thesubstrate Application cells and 9.6-pm-diameter beads were seeded at Suspended spacer of extensive imaging required development sampleprepato immobilizedif- 2 x 105cellsand 1.2 x 104 beadsper35-mmdish.At 70% confluency, rationmethods.We use cryofixation thecellsweresubjectedto threetreatments, control(no addedboron), ofice crystals that BPA additionand fusiblespeciesandreducetheformation BPA addition FortheBPA withsubsequent washing. can cause morphological nutrient mediumwas aspiratedand replacedwithmedium changes(11, 12). Samplespre- treatments, mustbe vacuumcompatible 1'0B-enriched and, containing (Boron (95 atom%)-L-p-boronophenylalanine paredforionmicroscopy The BPA treatment Biologicals, Raleigh,NC) at 20 pgboron/ml. period the and forquantitative chemical work, physicalcompositwocyclesof tionof thespecimenshouldhave a limitedeffecton ion was 6 h. Afterthisuptakeperiod,one groupunderwent andmediumrefreshment followed indrug-free byincubation aspiration Cell cul- medium microscopic images(i.e. limitedmatrixeffects). for20 min.All incubations werein a humidified 37?C/5%CO2 arerel- atmosphere. turesthatarefreeze-fractured andthenfreeze-dried The samegrowth mediumwasusedforall treatments with of effects matrix effects are additionofBPA as indicated. ativelyfree matrix (13). Since followedthemethodofChandraetal. (11).

Cryogenic preparation inthiscellculture homolimited relatively cryopreparation, dishandexcess silicon is removed fromthetreatment the Briefly, piece geneousmatrixspeciessuchas carbondo notexhibition mediumis wickedaway.Anotherpieceofsiliconis on top,and placed haveled toa rel- thissandwichis Thesedevelopments imageheterogeneity.

Freon-22 plungedrapidlyintoliquidnitrogen-cooled ativesensitivity-based schemeforboronand slush.The frozensandwichis transferred to liquidnitrogen and pried quantification ions usingcarbonas an internalstandard open witha razorblade. Freeze-fracture producescells in whichthe physiological outerleafletoftheapicalmembrane is removedon thetoppieceofsilifor varsubcellular boron localization Quantitative (14,15). foranalysis.Most compartments ious deliveryagentsand cell lines has been reported con,thusexposingtheintracellular theextracellular mediumis removedwiththeupperhalfimportantly, (16, 18) usingthismethod.Also, muchfocushas been membrane, whicheliminates grosscontamination.

For example,ion calciumimaging. placedon quantitative Tumor stor- TheSubcutaneous studiesincellculturehavedemonstrated microscopic inF-344ratsbyinjection Solid tumors were subcutaneously age ofcalciumin theGolgiapparatus(19) and subcellular of5 X 106GS-9Lcellsproduced in0.1mlofgrowth medium.

Tumorswerepalpacalciumexchange kinetics (20). ble 4-5 daysafterinoculationofthecellsand had a volume-doubling ofanimaltissuesforionmicroscope timeofapproximately Samplepreparation 2 days.Tumorswereusedon day11 afterimplanin situ withcopper-jawed uses cryofixation pliersto tation when they were 100-200 mg in size. Ninety-five analysis percent BPA was complexedto fructose to increaseitssolubility were 10B-enriched limitredistribution artifact(20). Thincryosections foundtoadherewelltoan indiumsubstrate, a fiat (25). The BPA-fructosecomplex (800 mg/kg)was administered providing in 4 ml saline.After6 h theratswereanesthetized intraperitoneally analysis(21). Also, usingketamine(100mg/kg) sampleamenableto ion microscopic andxylazine(20 mg/kg).

Skinwascarefully freeze-dried liverorintestine sectionsdis- resectedto exposethetumorwithoutdamaging indium-mounted thetumorbloodsupply. withrespectto microanatomyIn situcryofixation effect oftumorsensuedas described below.The ratswere playedlimitedmatrix ofketamine/xylazine. bylethalinjection (21). Using thispreparation,vitaminD was shownto sacrificed increasethetransport ofthe"44Ca traceracrosstheintestinal Tumor Cryopreparation ofSubcutaneous villiandintolaminapropria(22). The animalworkhereis In situcryofixation ofthesubcutaneous with tumorwasperformed ifboroncanbe imagedin copper-jawed to reducediffusion ofphyspartofa pilotstudyto determine plierscooledinliquidnitrogen tumors atboronconcentrations iologicalionsandunboundboron(20,21).A recesswascutintothecopapplicabletoBNCT. In the presentstudyBPA-deliveredboronis imaged per plates(jaws) so thata 1-mmdead space remainedbetweenthe and bya knifeedgeto aid cutting in GS-9Lgliomagrownincul- plates.The dead spaceis surrounded with1 gmlateralresolution removalof the tissue.The recesswas used to reducecompressiontureand subcutaneously.

In both,diffusion artifact was induceddistortion ofthesampleduringthefreezing process.Cryosecand sodium showntobe limited concentraandfreeze-drying themethodof usingpotassium tioning, mounting proceededfollowing were takenon a cryostat tionsas thereference forthequalityofcryopreservation of Sod et al. (21), except2-pmcryosections -309C insteadof an ultracryothenativestate(11,23). The highabsoluteboronconcen- (Reichert,model855C Cryocut-4-II)-at microtome.

Sectionswereeitherpressedintoindiumforionmicroscopy tration andrelatively subcellular distribution homogeneous or mountedon a glassslideandstainedwithBasicFuschinandMethylwe foundforBPA-delivered boronimplygoodmicrodosi- eneBlue.Thestained sections wereinspected to determine ifmicroscopmetric studiesshouldprovidemicrodis- ic anatomy wascompromised orcryosectioning. efficacy.

Continuing bythecryofixation tributions correlated totissueanatomy andphysiology.

Ion Microscopy

was performed witha CamecaIMS-3f.A mass-filIon microscopy MATERIALS AND METHODS teredprimary tunedto a 02? beam,acceleratedbya 10-keVpotential, The GS-9Lratgliomacelllinewasderivedfroma tumorinducedby currentof 100 nA and 50-pmdiameter,was rasteredover a 250 x N-nitrosourea (24) and maintainedin DMEM (Dulbecco's ModifiedEagle's 250 pm2area to inducesecondaryion emission.Secondaryion optics weresetto achieve1 pmresolution.

Thesesettings were:150pmtransfer with10%fetalbovineserum(Gibco).

Medium, Gibco)supplemented 1.8 mmfieldapertureand a optics,60 pmcontrastaperturediameter, Cryopreparation of GS-9L Cell Culture 130eV secondary ion energywindow.The secondary ionimageis proGS-9L cells were grownon fourto five 1-cm2pieces of polished sili- jectedontoand amplified screen bya microchannel plate/fluorescent The microchannel con restingon the bottomof a 35-mmPetridish (Corning).The polished detector. plategainusedoverallwasbetween70 and

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74

BENNETT ETAL.

K

N

screenwereacquiredwitha Thomson 80%. Imagesofthefluorescent Ltd.camdevice(CCD) ina Photometrics, CFS-TH7882charge-coupled erahead,modelCH220.Fourteen-bit digitized imagesweretransferred cameracontroller, modelCC220,to an AppleMacbythePhotometrics timeswere:in viaGPIB interface. intoshIlci computer Imageacquisition cell culture,0.2 s for39K,0.2 s for23Na,60 s for40Ca, 30 s for12C,and 0.3 s for39K,0.3 s for23Na,60 s for4Ca, 60 s 180s for10B;inrattumor, for12C,and 120s forI'oBand "B. Sixteen-bit digitalimageprocessing on the MacintoshusingDIP-StationLight(Hayden was performed Note that Image ProcessingGroup,Boulder,CO) imagingsoftware. video displayand printing are limitedto 8-bitprecision.The imagenormalizes16-bitimagedata to 8-bitfordisplay. processingsoftware Potassium andsodiumimageswereacquiredforequal timesso thatrelaandsodiumconcentrations couldbe shownqualitatively tivepotassium is madepossiblein the8-bitgamut in the8-bitgamut.Thiscomparison thesodiumimageso thatthesodiumand potassium byrenormalizing pixelvalue.Thisis validsincetherelaimageshavethesamemaximum factors ofpotassium andsodiumareverysimilar tivesensitivity (14). The servesas a morphological marker calciumlocalization (11). cytoplasmic wasassessedforthein vitroandin vivosamples.Under Background conditions no signalwas detectedat m/z10 in thein vitroconimaging to the10Bsignal trols.In in vivosamplestherewas a 10% contribution isofromnaturally sourcesofboron,i.e. diet.The 1'B and"11B occurring Ifboronintake wereusedto calculatethisbackground. topeabundances bytheanimalarisesonlyfromnaturalsources,thesignalsfrom"'B and "B wouldbe 20 and 80% ofthetotalsignal,respectively.

Fortunately, 1oBwouldbe a boronis low in thediet;otherwise occurring naturally ratstheisoofthetotal10Bsignal.In ourl'B-BPA-treated majorfraction 36%. topeabundanceofioBwas 64% ofthetotaland "B contributed 1oBthatcontributes to total10Bis The amountof naturally occurring 20% ofthe"B abundance.In thiscase thatis equal to 10% ofthetotal "oBsignal.Thus 90% of the10Bsignalcomesfrom'oB-BPA,and 10B "oBdistributions imagesfromthistissuerepresent'oB-BPA-delivered withlittlebackground contamination. incellculture as described ofelements wasperformed Quantification inthis effects arelimited byAussereretal. (14). As statedabove,matrix so complexcalibration methodsarenot well-characterized preparation, forboron,calionmicroscope relative factors Briefly, sensitivity required. elementcarbon cium,potassiumand sodiumwithrespectto thematrix ofionmicroscopy andinductively couaredetermined bythecorrelation analyses of cellular pled plasma atomic emission spectrometry Sincematrix effects are notdetectablein thiscellculture homogenates. matrix andhomohomogeneous species,suchas carbon, cryopreparation, FIG. 1. Ion microscopic preparedGS-9L imagesfromcryogenically andsodium, givenearly homogegeneousionicspecies,suchas potassium period). nousintracellular ionsignals(13,23). As a result, therawimagespresent- gliomacellsincubatedwithBPA in vitro(beforeelimination Sodium(Na) was to concentration. is directly proportional assessment ofthesubcellular distribution. Brightness ed providea usefulqualitative in this normalizedto potassium(K) to showthehighK/Naratio.Nucleiare ofboronin tissuewasnotattempted Absolutequantification lowcalciumconassessmentof possible seenin thecalcium(Ca) imageas regionsofrelatively pilot study.Rather,we made a preliminary Areasin the and the cell a centration, doughnut-like appearance. give in variations ion which would cause concentrayield, matrix-dependent calciumimagethatare blottedout weresaturatedin the8-bitgamut intheionimages(26). tion-independent changesinbrightness (display),but notin the 16-bitgamut(CCD). BoronfromBPA was imagedas theenriched10Bisotope.Also, theion imageofthematrix reference carbonis given.Bar = 10 pm.

RESULTS

...

Ik:T

Microdistribution of BPA-Delivered Boron in Cultured GS-9L Glioma Cells

image,one can see the changed.Lookingat thepotassium in BPA-treated boundaryof each cell in the fieldof view.The sodium The subcellularboron distribution to providea qualitative to potassium GS-9Lgliomacellsis showninFig.1,alongwiththepotas- imagewasnormalized The sodiumlevel is The cells assessmentof the cryopreparation. sium,sodium,calciumand carbondistributions. levelineachcell.The high muchlowerthanthepotassium wereexposedto 20 pg'oB/mlas L-p-boronophenylalanine ofthe ratioindicatesgoodpreservation All imagesinFig. 1 potassium/sodium (95% l?B) for6 h and thencryofixed. stateofthelivecellpriorto cryofixashowthesamegroupofcells;onlythemassselectionwas nativephysiological

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SUBCELLULAR 'B LOCALIZATION IN GLIOSARCOMA 120

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of boronforcellsbeforeand aftera 20-minelimination wetweightconcenperiodis giveninFig.2. The estimated * Nucleus was79.4Vgboron/g trationin thecytoplasm 100 (SEM = 4.3) BPA-freetreatment caused The 20-min beforeelimination. in both the in boron concentration a substantial reduction 80 was nucleus and cytoplasm.The boron concentration and nuclear reducedby46% and 39% in thecytoplasmic differThere was no respectively. significant compartments, and nuclearboronconcentraence betweencytoplasmic tionsbeforeelimination.

However,a smallbutsignificant was cytoplasmic preference seen afterBPA elimination = 37.1 Vg boron/g, SEM = 3.3, vs Bnucleus = (Bcytoplasm 20 reducSEM = 2.2). Giventhesubstantial 29.6pg boron/g, BPA eliminaseenduring tionintheoverallboroncontent tion,we concludethata largepool ofboronis in a rapidly 0 20 0 boundto intracellular exchangeableform,notcovalently timein BPA-free medium,min in either The wetweightconcentration macromolecules. mediboron-free without to with or exposure compartment fromthenucleus umis abovelevels FIG. 2. Quantitative analysisofboronelimination inBNCT (27). tobe effective thought and cytoplasmof GS-9L cells afterchangingto BPA-freemedium. difference betweencytoplasmic and nuclear Microdistribution There is no significant Boronin ofBPA-Delivered case. A smallbutsignificant differboronlevelsin thezeroelimination GS-9L Tumor Subcutaneous afterthe20-min eliminaencewasfoundbetweennucleusandcytoplasm decreaseinboronconcentration GS-9Ltumors tion(P < 0.05).Therewasa substantial insitu,6 h afterintraperiwerecryofixed inboththecytoplasm and nucleusafterthe20-minelimination period tonealinjection to BPA whichwascomplexed of 800 mg/kg boronwasreduced46%. Bars= SEM. (P < 0.01).Cytoplasmic Withthisadministration forincreasedsolubility. fructose tumor.3 thebulkboronlevelis about90pgboron/g is showninFig.3. Morphological A stainedcryosection level.Nucleiandcytois intactat themicroscopic ratio integrity tion.Quantitative analysisgivesa potassium/sodium as wellas a bloodvesselcontaining ofcryoprepared cell plasmare discernible of7:1,whichis comparable to a variety methodprovided sodi- red blood cells.Thus the cryofixation linesstudiedin ourlaboratory.

The lowintracellular distortion. um,withouthighsodiumaroundthecells,demonstrates sampleswithminimal elementalimages ion microscopic thatthefreeze-fracture processremovestheextracellular High-magnification distributheelemental Extra- aregiveninFig.4. As incellculture, mediumthatresidedabove theplasmamembrane. sectionarevalidatedbytherelacellularmediumis also removedfromareas surroundingtionsinthiscryoprepared ratioobservedintheintracelsodiumimageshowstherelativelytively thecells.Thenormalized highpotassium/sodium and sodium lularcompartment. lowintracellular sodium.If thehighextracellular Quantitative analysisofpotassium this ratioof4:1.Although were not removed,the sodiumimage would be much sodiumgivesa potassium/sodium The wide dynamicrangeof the CCD camera ratiois lowerthanthatmeasuredforGS-9L cellsin vitro, brighter.

The homogeneous matrixele- thetissuedoes notshowtheexcessivecalciumaccumulaallowssuchdiscrimination. ionicspecies,K' and tionassociatedwithdamagedorinjuredcells(23). ment,carbon,and thehomogeneous marker The use ofcalciumas an intrinsic ion imagesignals,whichis Na+,givenearlyhomogeneous morphological intissueis morediffidiscrimination consistent withthelackofmatrixeffects foundin thiscell forcytoplasm/nucleus secSincethe2-pmcryostat cultthanintheculture culture system. cryopreparation (13). thanthediameterofa single betweencytoplasm tionstakenhereare thinner discrimination Clearmorphological ofthesectionplanethatinterandnucleusis providedinthecalciumimage.The concen- cell,one mustfindportions ofthesamecell.The lowerthanin sectboththenucleusand cytoplasm tration ofcalciuminthecellnucleusis visibly whichis characterized seeninvitro, ineachcell.Theabovedescription, thecytoplasm highintra- cellularcalciumpattern ratiosand lownuclearcalcium, bylownuclearcontentsurrounded cellularpotassium/sodium byhigherlevelsin the with canbe foundin animaltissuepreparations a good freeze-fracture characterizes (12,24). cytoplasm, preparation ratio.Thislocalizationpatternis inthesecryofixed cellsis a highpotassium/sodium theboronlocalization Therefore, towhentheywerealive. unaltered essentially compared The boronimageinFig. 1 showsthatboronhas a relain eachcellat thisspatial distribution tivelyhomogeneous subcellular distribution resolution.

The quantitative 3j. Coderre,unpublishedresults. analysis mCytoplasm

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76

BENNETT ETAL.

highextracellularsodiumwas probablysputteredaway tuning.Overall,theelementaldistribuduringinstrument withthefindings tionsin thetumortissueare consistent incultured cells.

DISCUSSION include Recentadvancesin 10B(n,c)'Li microdosimetry Thusthe and simulations. bothanalyticaldeterminations given physicaldose to thecell nucleuscan be determined level.The physical boronlocalizationat themicrometer mustbe dose calculatedfromtheboronmicrodistribution end pointbeforethe correlatedto a validradiobiological The effectiveness ofBPA BNCT effect canbe understood. in BNCT has notyetbeen correlatedwitha subcellular It is localizationpatternobtainedbydirectmeasurement. and knownthatBPA deliversboronto gliomaselectively intracerebral thatBPA-basedBNCT controls gliosarcomas ofGS-9Lcellsclonedexvivo (28-30).Also,thelowsurvival in a FIG. 3. Lightmicrograph showingmorphological preservation tumors irradiated afterBPA administration from rat brain oftheGS-9L rattumorafterinsitucryofixation.

Notethe cryosection relatedto uptakeby is directly thatBNCT activity redbloodcellsin thecenterofthemicrograph. indicates bloodvesselcontaining Bar = 20 pm. gliomacells(31). Here,we foundthatborondeliveredto withBPA showsno discrete GS-9Lcellsbya 6-hincubation inthecytoplasm ornucleus.The boronis elimilocalization medifromthecellswhenplacedindrug-free natedrapidly is aboutthesameinboththecytoplasm inthecalciumimagebythearrows.

Thisstructur-um.The reduction identified localwithonlya smallpreferential is notfoundforboroninthesameareas,indicat- andnucleus, al pattern cytoplasmic thata at thesubcel- izationafterelimination. boronlocalization suggests Rapidelimination ingthatthereis no discrete oftheboronexistsin a rapidlyexchangeable lularlevel.These subcellularcalciumand borondistribu- largefraction The artifact. withthosefoundin cultured intracellular tionpatternsare consistent poolthatis pronetoredistribution redistribution methodusedhereeliminates betweenthedis- cryopreparation GS-9Lcells.Note,however, thedifference results to microdosimetric thusaddingconfidence tributions ofpotassium andboronat thetissuelevel.Potas- artifact, For fromthesubcellular distribution. cells thatmaybe inferred siumdoes notvaryfromone regionto thenextwithin a subcellularfractionation is comparison, visiblein thefieldofview,whereassomeheterogeneity studyreporteda boron BPA-delivered content for is due to verylownuclearboron theboronheterogeneity seenforboron.Whether boron in the Given cellular differences at thecellularlevelcannotbe ascer- (1.05% oftotal functional nucleus)(32). a homogeto structure orcelltype thenuclearvolumeof32% usedin thatreport, tainedfromthisimage.Correlation of32%. nuclear content in result a much neous in futurework.Of interest is considereda prerequisite gives higher than ademore boron can be We found absolute is that boron thispilotstudy BPA-delivered concentrations, imaged in and subcutaneous GS-9L cultured for at 1 pmresolution viaionmicroscopy.

BNCT, quate is indicatedwhen Favorablemicrodosimetry havebeenshownto be limited gliosarcoma.

Althoughmatrixeffects is combinedwiththeuniform in intestineand liverin samplespreparedby theabove theabsoluteconcentration Thisis ofboronin thenucleusandcytoplasm. technique,theremaybe unknowneffectsin the GS-9L distribution of BPA-based effect with the known curative consistent levels tumor.It is wellknownthatintracellular potassium rat9L gliosarcoma are higherthansodium.Further, potassiumand sodium BNCT ofintracerebral (28-30). deterin thecytoplasmor nucleus. do notlocalize specifically Quantitative microdosimetry usingexperimentally is in in which mined microdistributions these features.

ThereThe ionmicroscopic tissue, anatomy corimagesdisplay will related to boron if do not obscure the native matrix concentration, providetheultimate fore, effects, present, The is readily answersto themicrodosimetry-end elementallevelswithinthetissue.One artifact pointrelationship. shows that ion here in tissue Removal of the extracellular matrix however. detected, reported pilot study in a relevant boron can BPA-delivered to cellular beam is the microscopy image veryrapidcompared by primary matrixmusthave tumortissue.In the in vivoGS-9L modelwe see boron material.In thiscase theextracellular levelandsomeheterogeneat thesubcellular The homogeneity consistedofmostlywaterwithdissolvedelectrolytes.

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SUBCELLULAR 'B LOCALIZATION IN GLIOSARCOMA

...*

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oftheGS-9L rattumor.The ratwas treatedwiththeBPA-fructose FIG. 4. Ion microscopic complex cryosection imagesfroma freeze-dried to potassium(K) to showthehigh is directly to concentration. beforeinsitucryofixation.

Sodium(Na) was normalized Brightness proportional itis difficult to finda partofthetwo-dimensional a complexmixture ofcellularcompartments.

K/Naratio.This2-pmslicecontains Therefore, image motif seenin vitro; thecytoplasm andadjacentnucleusofthesamecell.Arrowspointto thecalcium(Ca) nuclear/cytoplasm i.e., planethatintersects levelsofcalcium.The arrowsinthe thataresurrounded thearrowsinthecalciumimagepointto subcellular byhigher regionsoflowcalciumcontent boronimageserveas registration marksthatpointto thesamearea. The discretelocalizationseen forcalciumis notevidentforboronin these are homogeat thetissuelevel,butpotassium and sodium(notseenbecauseofthenormalization) regions.The boronshowssomeheterogeneity neous. Bar = 10 pm.

couldnotoccuron thesamescaleas theresoat thetissuelevelmay redistribution ityat thetissuelevel.Heterogeneity be due to cell type-dependent technique. uptakeofBPA. Definitive lutionlimitoftheimaging to celltypevia stained correlation ofboronaccumulation illumination ofthe adjacentsectionsis requiredforfurther ACKNOWLEDGMENTS microdosimetry-end pointrelationship. to This workwas supportedby DOE grantDE-FGO2-91ER61138 forboronmicrolocaliza-G.H.M. andDOE contract To date,analytical development DE-ACO276CH00016to J.A.C.The Cornell to estimate microdosime-NIH/NSFDevelopmentalResourceforBiophysicalImagingand Optotionhaslaggedbehindourability As boronimaging electronicswasusedforcell culturework.We also thankDr. Subhash trybasedon MonteCarlocalculations.

Smithfortechnical adviceon cryosectioning. reachhighersensitivity andspatialresolution,ChandraandChristina technologies In this becomesevenmoreimportant. samplepreparation wasusedto ensurethat Received:November24,1993;accepted:June17,1994 workcryogenic samplepreparation

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BENNETT ET AL.

78

17. B. D. Bennett,X. Zha, I. Gay and G. H. Morrison,Intracellular boronlocalizationand uptakein cell culturesusingimagingsecH. G. BornerandB. Larsson,Therelative 1. D. Gabel,R. G. Fairchild, forneutron (ionmicroscopy) capture ondaryionmassspectrometry cellsoftheneutron inV79 Chinesehamster forcancer.Biol.Cell74,105-108(1992). biologicaleffectiveness therapy Radiat.Res. 98, 307-316 capturereactionin boronand nitrogen. andW. W. Webb,CalciE. P. W. Kable,G. H. Morrison 18. S. Chandra, (1984). mammalian cells intheGolgiapparatus ofcultured umsequestration and K. Kanda,M. Ichihashi T. Matsuzawa, 2. H. Fukuda,T. Kobayashi, andionmicroscopy. confocalmicroscopy revealedbylaserscanning on RBE ofa thermal beamandthe'0B(n,ot)7Lireaction Y. Mishima, J.CellSci.100,747-752(1991). culturedB-16 melanomacells. Int. J. Radiat.Biol. 51, 167-175 Subcellular 19. S. Chandra,W. A. Aussererand G. H. Morrison, imag(1987). incultured cellswithionmicroscopy.

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