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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.
'Corresponding
0033-7587/94$5.00

72

?1994 byRadiationResearchSociety.
All rights
ofreproduction
inanyform
reserved.

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SUBCELLULAR 10BLOCALIZATION IN GLIOSARCOMA

73

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.

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

75

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

...*

i!

77

"

.
P

A"

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

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