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Hndaw Pblshn CopoatonT Scntfc Wold JonalVolm 2013, Atcl ID 796029, 12 pashttp://dx.do.o/10.1155/2013/796029

Research Article18F-Fluorothymidine-Pet Imaging of GlioblastomaMultiforme: Effects of Radiation Therapy onRadiotracer Uptake and Molecular Biomarker Patterns

Sanjay Chandrasekaran,1 Andrew Hollander,2 Xiangsheng Xu,2 Joseph L. Benci,2

James J. Davis,2 Jay F. Dorsey,2 and Gary Kao2

1 University o Washington School o Medicine, A-300 Health Sciences Center, Box 356340, Seattle, WA 98195, USA2 Department o Radiation Oncology, University o Pennsylvania, 8-087 RC, 3400 Civic Center Boulevard, Building 421,

Philadelphia, PA 19104, USA

Cospondnc shold b addssd to Gay Kao; [email protected]

Rcvd 31 Janay 2013; Accptd 25 Fbay 2013

Acadmc Edtos: A. Bttab, K. Hmann, and A. Moabto

Copyht 2013 Sanjay Chandaskaan t al. Ts s an opn accss atcl dstbtd nd th Catv Commons AttbtonLcns, whch pmts nstctd s, dstbton, and podcton n any mdm, povdd th onal wok s poplyctd.

Introduction. PE man s a sl clncal tool o stdyn tmo posson and tatmnt cts. Convntonal 18F-FDG-PE man s o lmtd slnss o man Globlastoma Mltom (GBM) d to hh lvls o lcos ptakby nomal ban and th sltant snal-to-nos ntnsty. 18F-Floothymdn (FL) n contast has shown poms oman GBM, as thymdn s takn p pntally by polatn clls. Ts stds w ndtakn to nvstat thctvnss o 18F-FL-PE n a GBM mos modl, spcally a adaton thapy (R), and ts colaton wth slbomaks, ncldnpolaton andDNA dama.Methods. Nd/athymc mc wth hmanGBM othoas w assssd bymcoPE man wth 18F-FDG and 18F-FL. Pattns o tmo PE man w thn compad to mmnohstochmsty andmmnooscnc o maks o polaton (K-67), DNA dama and pa (H2AX), hypoxa (HIF-1), and anonss(VEGF). Results. W confmd that 18F-FL-PE ptak s lmtd n halthy mc bt nhancd n th ntacanal tmos. Odata th dmonstat that 18F-FL-PE man slly cts th nhbton o tmo by R and colats wth chans nbomak xpsson. Conclusions. 18F-FL-PE man s a pomsn tmo man modalty o GBM, ncldn assssn Rcts and bolocally lvant bomaks.

1. IntroductionGloblastoma Mltom (GBM) s a hhly assvcanc that xpncs a hh at o cnc dsptcombnaton thapy [1]. T s snfcant nd o tmoman modalts o bomakscapabl o dntyn alysta tmos, posttatmnt chans, and sns o tmoposson and cnc o GBM [2]. Poston mssontomoaphy (PE) s an man modalty wdly sdn oncoloy o clncal stan, monton o tatmnt

cacy, and ollowp o dsas cnc [3, 4]. 18F-

Floodoxylcos (18F-FDG) PE, whch mass clllalcos mtabolsm as a ncton o th nzym hxoknas,

s th most common clncally tlzd PE modalty. How-v, o man o hh-ad ban tmos lk GBM, 18F-FDG-PE man has lmtd tlty. T hh basln lvlo lcos ptak wthn th ban slts n a low snal-to-nos ato andobscd tmo man [5, 6]. Consqntly,sval cnt pclncal and clncal stds hav nvst-atd th s o altnatv PE adotacs, ncldn 18F-

Floothymdn (18F-FL) and 11C-Mthonn (11C-ME),o man o GBM [68].

18F-FL, a nclosd adotac, has md as a sa

and pomsn tool to b sd n conjncton wth 18F-FDG-PE o stdyn assv and polatv tmoslk GBM [9, 10]. Tymdn ptak and mtabolsm q

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Tymdn Knas-1 (K-1) actvty, whch s lvatd n thnomal cllla S-phas and noplastc tsss [11]. Addton-ally, nlk cabon-basd thymdn, 18F-FL has dmon-statd hh spcfcty o K-1 and dcd ncopoaton

nDNA[12]. 18F-FL-PE man hasbn shown n svalclncal stds to colat wth cllla polaton and

tmo posson n hh-ad loma, as dtmnd byth K-67 polaton ndx [1315]. Fthmo, Chn tal. dmonstatd that ncasd adotac ptak n hh-ad loma was assocatd wth dcd patnt svval

[13]. 18F-FL-PE man has also bn shown to colatwth tatmnt spons n clncal stds o bast cancand hh-ad loma [8, 16]. Smla stds hav also bnpomd n oth canc typs ncldn lymphoma andso tss sacoma [1719]. W wshd to stdy th cts

o adaton on 18F-FL-PE man o GBM tmos andscondaly cts on maks lvant to hypoxa, anon-ss, and DNA dama and pa. Sch data cold povdaddtonal ctcal nomaton abot tatmnt spons and

dsas posson.

2. Materials and Methods

2.1. Radiotracers. 18F-FL and 18F-FDG w ppad at thUnvsty o Pnnsylvana Small Anmal Iman Facltys(UPENN SAIF) PE Cnt cycloton (Japan Stl Woks;Modl BC 3015; okyo, Japan).

2.2. Cell Culture. U251 GBM cll lns w nndto stably xpss n oscnt potn and lcas(GFP-LUC) sn pGnFM anscptonal RpotLntvctos pchasd om Systm Boscncs (Montan

Vw, CA, USA) and sotd twc va ow assstd cllsotn (BD FACSVanta SE wth DVa sowa, San Jos,CA, USA at th Unvsty o Pnnsylvana Flow Cytomtyand Cll Sotn Cnt. Clls w own n DlbccosModfd Eal Mdm (DMEM), spplmntd wth 10%tal bovnsm and 1% stptomycn/pnclln antbotcsand mantand at 37C n 5% CO

2.

2.3. Mouse umor Model. All anmal stds w con-dctd accodn to potocols appovd by th Unvsty oPnnsylvana Instttonal Anmal Ca and Us Commtt(IAUCAC). Nd/athymc NC (n/n) mal mc whosd fv p ca n a cntalzd small anmal aclty.

Mc w chckd contnosly o ood halth, popwond haln at th scal st, and nondomaton o thcanm by th tmo.

2.3.1. Heterotopic Flank umors. Sx-wk-old mal nd/athymc mc w njctd sbctanosly and blatallyon th a anks wth 2,000,000 U251-GFP-LUC GBM cllsn 100 L o 1 : 1 mda/Matl (BD Boscncs, San Jos,CA, USA). T xpsson o GFP and lcas nabldtwo ndpndnt mthods to dstnsh th hman tmoclls om th host mos clls. Fst, mc w ansthtzdby njcton (IP) o ktamn (140 m/k) and xylazn(10 m/k). Upon cvn no spons to a to-pnch tst,

a 100 L sspnson o clls, 1x phosphat bd solton(PBS), and Matl w njctd at a st spfcal to thscapla blatally and spfcal and mdal to th hp

jonts blatally [20]. A th pocd, th mc wtnd to th cas and obsvd ntl th ansthsahad clad and sbsqntly montod on a daly bass o

halth, nomal bhavo, and vsal tmo owth.

2.3.2. Orthotopic Intracranial umors. Sx-wk-old nd/athymc mc w njctd ntacanally wth 200,000 U251-GFP-LUC clls. Fst, mc w ansthtzd by IP njc-ton wth ktamn (120m/k) and xylazn (10 m/k).Addtonally, mc cvd mloxcam (5 m/k) thoh IPnjcton n th popatv and postopatv pod.

Onc confmd to b nconscos by to-pnch, achmos ndvdally was placd n a stotactc had am(Stoltn, Wood Dal, IL, USA). Onc placd n th sto-tactc am, th opatv aa was dcontamnatd sn

btadn solton wth a cotton tp applcato. All s-cal pocds w pomd sn atoclavd o stlnstmnts n a clan nvonmnt. Fst, a 1 cm mdlnscalp ncson was mad and th bma was vsalzd.Nxt, a small b hol was mad thoh th skll sn aDml dll 0.5 mm anto and 2 mm latal to th bmaas pomd by Bamann t al. [21]. 200,000 clls n 10 Lo 1xPBS w dawn nto a Hamlton syn. Tn, thsyn was nstd nto th am o th stotactc systmwth th 30G ndl placd dctly ov th nwly catdb hol. T syn was nstd thoh th b hol3.3 mm om th skllsac. Clls wnjctd ata low at(appoxmatly 0.5 L/mn) n odto mnmz th pss

catd at th njcton st. Upon complton o th njcton,th ndl was movd, th b hol was covd wthbon wax, and th ncson was closd sn cyanoacylat.A opaton, th mc w tnd to th cas andobsvd ntl th ansthsa had clad and sbsqntlymontod on a daly bass o halth, nomal bhavo, andtmo owth.

2.4. Mouse umor Imaging

2.4.1. Bioluminescence Imaging (BLI). T mc wth tmosndwnt wkly BLI to obsv tmo owth and qanty

tmo sz [22]. BLI was condctd statn 1 wk atmo njcton and contnd on a wkly bass n acntally shad IVIS Lmna II In Vivo Iman Systm(Calp L Scncs; Hopknton, MA, USA) accodn toth UPENN SAIF dlns. Fst, th mc w ans-thtzd as dscbd abov (s Scton 2.3.1) and con-fmd by natv to-pnch tst. Ts was ollowd bydosal, sbctanos njcton o 60 L o USP ad D-lcn (D-Lcn potassm salt; Gold Botchnoloy;St. Los, MO, USA). Iman was condctd 5 mn aD-lcn njcton ov a pod o 30 mn to ctvlydtmn th maxmm lmnscnc ntnsty. Ron oIntst (ROI)dtmnaton and analyss w condctd

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0

0.5

1

1.5

18F-FDG 18F-FLT

Injecteddose(%)

Intracranial uptake o FDG versus FLT

(a)

Sagittal: midline Axial Coronal

18

F-FDG-PET

18

F-FLT-PET

Dorsal

Dorsal

Dorsal

Dorsal

Ventral Ventral

VentralVentral

CaudalCaudal

CaudalCaudal

Right

Right

Right

Right

Lef

Lef

Lef

LefCranial Cranial

CranialCranial

(b)

Figure 1: T nomal ban shows ntns ptak o FDG bt not FL. (a) Compason o ntacanal % njctd dos (%ID) o 18F-Floodoxylcos (18F-FDG) and 18F-Floothymdn (18F-FL) adotacs n halthy nd/athymc mc. 18F-FDG ptak s at

than18

F-FL n ntact mos ban. (b) Vsal compason o18

F-FDG-PE and18

F-FL-PE man n nd/athymc mc o compasono ntacanal adotac ptak (yllow aows).

wth th Lvn Ima v4.0 sowa (Calp L Scncs;Hopknton, MA, USA). Sowa-natd sac ad-

anc masmnts (photons/sc/cm2/stadan) w sdto calclat Flx

maxvals (photons/scond) n od to

dtmn tmo sz. mos w allowd to ow wthot

ntvnton ntlFlxmax

vals appoachd 1109 (1+10).

2.4.2. Radiation Terapy. Mc tmos wth Flxmax

val-

s on th od o 1 1 0

10

photons/sc (46 wksa njcton) w ansthtzd va IP njcton wthktamn (140 m/k) and xylazn (10 m/k). Ty thnw ndvdally adatd wth snl-dos nlatal 16Gay (Gy) adaton to th ht sd tmo n a dpatmnt-ownd ndctonal anto-to-posto bam X-Ray nt(Schnman Elctoncs (Gants Pass, OR), modl A-9002-100) wth ll body lad shldn xcpt at th mplantdtmo st and w thn sacfcd 46 wks lat.

2.4.3. MicroPE/CImaging. Expmntaland contol mc

w sbsqntly ollowd by sal 18F-FL-PE man.

Both 18F-FDG and 18F-FL mcoPE and mcoC man

w condctd at th UPENN SAIF laboatoy. Fo ank

tmos, 18F-FL-PE mcoPE/C was condctd poto adaton thapy (R) and 5 and 13 days (1 wkand 2 wks) a R. Mc w mantand on a 13% soan and oxyn ansthsa systm (VtEqp Inc;Plasanton, CA) own at a at o 1.0 lt/mn. Rado-tac was dlvd va a latal tal vn njcton wth amaxmm o 200 C500 C and a 60 mn ptak tm.Anmal tmpat was mantand sn a wam a soc

and sbsqntly montod (Vtoncs; Wst Laaytt, IN,USA). McoPE man was pomd on a Phlps Md-cal Systm (Clvland, OH, USA) small anmal manmachn (A-PE). Small anmal mcoC was sbsqntlypomd on an Imk scann (CI Molcla ImanInc. (now Smns; Malvn, PA)) wth a spatal solton

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Surace radiance

Fluxmax = 1 106

Fluxmax = 1 109

1 109

1.6 105

(a)

Time (weeks afer injection)

0 2 4 6 8

Meanfluxmax

0+00

1+09

2+09

3+09

(b)

0

0.5

1

1.5Intracranial FLT uptake

Week 7Week 1

Injecteddose(%)

(c)

Sagittal: midline Axial Coronal CT sagittal: midline

Dorsal

Dorsal Dorsal

Dorsal Dorsal

Dorsal

Ventral

Ventral

Ventral

Ventral

Ventral

Ventral

Caudal Caudal Caudal

CaudalCaudalCaudal

Right

Right

Right

Right

Lef

Lef

Lef

Lef

Cranial Cranial Cranial

Cranial Cranial Cranial

Week1

Week7

(d)

Figure 2: Othotopc bantmos a adly mad by FL-PE. (a) Vsal compason o Bolmnscnc Iman (BLI) sac adanc(photons/sc/cm2/stadan) n othotopc ntacanal tmos wth BLI Flx

max(photons/scond)vals o1106 (1+6)and 1109 (1+9).

(b) Idntfcaton o an xponntal owth pattn n ntacanal tmosbasd on Flxmax

masmnts ( = 5). (c) Compason o %ID ontacanal 18F-FL %ID at 1 and 7 wks a njcton. (d) Vsal compason o 18F-FL ptak n ntacanal tmos basd on Flx

max

vals o1 106 and 1 109 (wks 1 and 7, spctvly). mo vsalzd at 7 wks (yllow aows).

dos n mos body, tmo, and ban w obtand va

mltplcaton o th ava ROI voxl val and sz (mm3)sn opn-soc Amd Mdcal Iman Data Exam-n (http://www.amd.soco.nt). C bon and tss

constcton w condctd wth opn-soc OsXDcom Vw (http://www.osx-vw.com).

2.5. Histological Analyses

2.5.1. issue Harvesting. Mc w sacfcd wth an ov-dos o ktamn and xylazn and psd va a ca-dac catht wth 1xPBS and 10% Fomaln. mos wmovd and stod at 4C ovnht n 10% Fomaln andplacd n a 30% scos solton, thn ozn n cooldIsopntan (2-Mthylbtan) and stod at 80C.

2.5.2. Immunohistochemistry. T tss sampls w sc-tond nto sal 10 m slcs sn a Mcom cyostat

(TmoScntfc; Walldo, Gmany) and placd on mco-scop slds (Tmo Fsh Scntfc; Waltham, MA, USA).Slds w l at oom tmpat ovnht and wamdo 30mn at 37C po to antn tval. Slds w

mmsd o 15 mnts n ctat b ttatd to pH 6.0at 95C, nsd wth 1xPBS, and mmban pmablzatonwas pomd sn ton-X 100 (Sma-Aldch; St. Los,MO, USA). Sampls w stand o K-67 (BD Boscncs;San Jos, CA, USA), VEGF (Abcam; Cambd, MA, USA),and HIF-1 (Santa Cz; Santa Cz, CA, USA) sn anAB nzym ABC kt (Vcto Laboatos; Blnam, CA,USA). Slds w ncbatd wth th pmay antbody o60 mn at oom tmpat, nsd, and ncbatd wthsconday antbody o 60 mn at oom tmpat. ABnzym was addd wth sbsqnt DAB Poxdas Sb-stat (Vcto Laboatos; Blnam, CA, USA). Samplsw fxd va mmson n an thanol dlton ss, fnal

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0 5 20

Control

RT

12Surace

radiance

Radiance afer-RT (days)

1 1010

1 109

(a)

0 5 10 15 20

Control

RT

Days afer treatment

BLI fluxmax (photons/s)

Meanfluxmax

3+10

6+10

9+10

1.2+11

1.5+11

0+00

(b)

0

2

4

6

8

10

A B

Mouse

Beore RT

Afer RT

Ratioof%ID(RT)/%ID

(control)

Change in 18F-FLT uptake in

RT versus control tumors

(c)

Figure 3: Radaton thapy lads to poond owth nhbton o ank tmos. (a) BLI o blatal ank xnoas at 0, 5, 12, and 20 daysollown nlatal 16 ay (Gy) adaton thapy (R). Rdcd sac adanc notd n R tmos. (b) Compason o chans n BLIFlx

maxvals n contol ( = 3) and R ( = 3) tmos. Rdctons n Flx

maxsn by 5 days a R. (c) Compason o chan n %ID o

18F-FL ptak n R vss contol tmos ( = 2) bo R and 2 wks a R. R tmos dmonstat dcasd ptak at 2 wks.

mmson n xyln, and sald wth Cytosal (TmoScntfc/Rchad-Allan Scntfc; Waltham, MA, USA).

2.5.3. Immunofuorescence. ss sampls w ppad asdscbd abov (s Scton 2.5.2). Sampls w stando K-67 (BD Boscncs), VEGF (Abcam), HIF-1 (Santa

Cz), and H2AX (Mllpo; lcma, CA, USA) snan ABC kt (Vcto Laboatos, Blnam, CA, USA)Slds w ncbatd wth th pmay antbody and thnncbatd wth an AlxaFlo 594 conjatd (Invto-n/Lchnolos; Calsbad, CA, USA). sconday ant-body. Slds w montd sn Montn Mdm wthDAPI (Vcto Laboatos). Sampls w analyzd sn aoscnc mcoscop (Nkon Eclps E-2000U).

3. Results and Discussion

3.1. Intracranial MicroPE/C Imaging. o compa th pat-

tns o ptak o 18F-FDG o 18F-FL n halthy nd/

athymc mc, th mc ndwnt mcoPE man60 mn ollown tal vn njcton wth th adotac.Intacanal adotac ptak was qantfd basd on% njctd dos (%ID). 18F-FDG ptak nto th nomal

ban was >4x hh than 18F-FL ptak (%ID 1.43 vss

0.33, spctvly) (F 1(a)). 18F-FDG ptak was n-

om thohot th ntacanal spac whn vwd n thcoonal, axal, and sattal plans, whas 18F-FL ptakwas vsally ndtctabl (F 1(b)). Ts slts ndcat

that th s a hh backond snal wth 18F-FDG that s

not psnt o 18F-FL. Concntaton o both adotacswthn th bladd was notd to b compaabl (data notshown). Sch man valdats that 18F-FL ptak wthn

th halthy ban s mnmal compad to convntonal 18F-

FDG and ssts that ntacanal 18F-FL ptak woldncas wth tmo psnc.

W ths compad ptak pattns o18F-FDG and 18F-FL n ntacanal tmos. Halthy nd/athymc mc ( =5) w ntacanally njctd wth U251-GFP-LUC loma

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1000

100

Voxeluptakevalue

Dorsal

Ventral

Caudal

Caudal

Right

Right

Right

LefLef

Lef

Cranial

CranialCoronal

Axial

(a)

1000

100

Voxeluptakevalue

Dorsal

Ventral

Caudal

Caudal

Right

Right

LefLef (control)

Lef

Cranial

Cranial

(RT)

(RT)

Right (RT)

Coronal

(control)

(control)Axial

(b)

Figure 4: FL-PE man dmonstats R-ndcd chans n tmo cos. (a)18

F-FL-PE o blatal ank tmos po to nlatal16 Gy R. Not th smla pattn o adotac ptak blatally n coonal and axal vws (wht aows). 3D C/bon man o anksac anatomy s shown. (b) 18F-FL-PEo blatal ank tmos2 wks a nlatal 16 Gy R. Unq vaatons n adotacptakasn. Not dcasdadotac n contoltmo co (wht aows); nom ptak n R tmos (yllow aows). 3D C/bon mano ank sac anatomy s shown.

clls and ndwnt wkly BLI wth sbsqnt 18F-FL-PE man (F 2(a)). Intacanal tmo owth acossall mc dmonstatd an xponntal owth pattn asmasd by chans n man BLI Flxmax, wth ncasd

polaton byond 4 wks (F 2(b)). 18F-FL-PEman was pomd n slct mc ( = 2), and

ntacanal 18F-FL ptak was qantfd by %ID at wks

1 and 7, whch colatd wth a BLI Flxmax, o 1 106

and 1 1 09, spctvly. Radotac ptak was 4.7xat at 7 wks vss 1 wk (%ID 1.58 vss 0.33,sp.) (F 2(c)). Vsally, adotac ptak was mostappcatd n th la ntacanal tmos (7 wks a

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Control RT

10x 40x 10x 40x

(a)

Control RT

10x 40x 10x 40x

(b)

Figure 5: Radaton thapy lads to nhbton o polaton. (a) Hmatoxyln and Eosn (H&E) stann o contol ank tmos and anktmos cvn 16 Gy R (10x and 40x). (b) Immnohstochmcal stann o polaton mak K-67 o ank tmos as pomdn (a).

njcton). Addtonally, adotac ptak at 7 wks waslocalzd to th tmo st, wth mnmal ptak n thsondn nomal ban tss (F 2(d)). Ts data

dmonstat that 18F-FL concntats pntally wthntmo and sst that adotac ptak colats wthtmo sz.

3.2. Flank umor MicroPE/C Imaging. W wshd tonvstat th ct o adaton thapy (R) on adotacptak. Howv, th lmtd sz o ntacanal tmosn th mc dd not pmt scnt man solton.Addtonally, mc wth ntacanal tmos wth Flx

max

vals at than 1 1 09 w qckly tmnal (datanot shown) and tho nlkly to svv scntlylon a adaton o xtndd ollowp. To, wsotd to ank xnoas o sbsqnt stds.

Expmntal mc ( = 3) wth blatal U251-GFP-LUCank tmos w xposd to a snl nlatal acton o16 Gy R to th ht sd ank tmo and tmo owth o

th adatd and contol nonadatd tmos was sbs-qntly ollowd sally wth BLI (F 3(a)). Flx

maxn

contol tmos possvlyncasd n th wks ollownadaton, whl R tmos dmonstatd dcton n BLIstatn a 1 wk (F 3(b)).

In addton to BLI, 18F-FL-PE man o slctxpmntal mc ( = 2) was pomd bo Rand 1 and 2 wks ollown snl dos 16 Gy R o th

ht sd ank tmo. Dcass n 18F-FL adotacptak n R tmos w notd by 1 wk a R andsstand ntl 2 wks a R. Dcass n %ID n Rtmos at 2 wks and om 23% to 64%. (Spplmn-tal F 1, s Spplmntay Matal avalabl onln

at http://dx.do.o/10.1155/2013/796029) Whn companchans n %ID n R vss contol tmos, R tmosxhbtd snfcant dcton n adotac ptak by 2wks (F 3(c)).

Vsally, 18F-FL-PE man on both coonal and axalman ssts qal adotac nhancmnt btwncontol and tatmnt tmos po to R (F 4(a)).

Uptak pattns ollown adothapy, howv, w d-nt btwn tatd and ntatd tmos (F 4(b)).L sd contol tmos w ond to xhbt pphalnhancmnt wth notabl cntal pallo, sstn hypoxandcd cntal ncoss wth th omaton o psdopal-sads, a wll-chaactzd phnomnon on sn n GBM[23, 24]. Qt stknly, adatd tmos w dmnshdn sz, and th small tmos showd nom adotacptak thohot th tmo mass bt wth dcasdntnsty n compason to contol tmos.

Fo ths plot stds, a snl dos R mn wastlzd to nvstat th cts o R on tmo owth.Sch a tchnq s smla to hypoactonatd stotactc

adosy whch s sd clncally o patnts wth canc[25, 26]. Ts slts n o mos modl ncoa clncalollow-on stds wth actonatd R mns.

3.3. Eects o Radiation Terapy on Cellular Prolieration and

DNA Damage. T dcasd 18F-FL-PE adotacptak n R tmos compad to contol tmos smdlkly d to dcd tmo cll polaton. o tst thshypothss, w pomd mmnohstochmsty (IHC) oK-67, an stablshd mak o cllla polaton. Mcwth adatd and contol xnoa tmos w sacfcd,and th tmos w sbsqntly havstd and pocssd

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H & E H2AX H2AX

1x 4x 20x

(a) Contol

H & E H2AX H2AX

1x 4x 20x

(b) R

Figure 6: Radaton thapy lads to makd ncas n DNA dama wthn th tatd tmo. (a) H&E stan (1x) and mmnooscntstann w/ DAPI (bl) o Gamma-H2AX (H2AX) (d) (4x and 20x) n contol ank tmos, wth spcfc ocs on tmo co (whtaows). (b) Stann as n (a) n ank tmos a 16 Gy R, wth spcfc ocs on th tmo co (yllow aows) and notabl H2AXxpsson at 20x (bl aows).

o hstoloc xamnaton. Hmatoxyln and Eosn (H&E)stann appad compaabl btwn contol and adatdtmos (F 5(a)). In contast, adatd tmos showdmakdly dcasd K-67 stann (F 5(b)). Tsslts sst that R ld to dcasd tmo polaton,ths ctd by dmnshd K-67 actvty. Smla fndnso dmnshd K-67 a adaton hav also bn notdn th clnc n both astocytoma and oal sqamos cllcacnoma [27, 28].

W also soht to valat dcasd tmo pola-ton sconday to adaton also cooboatd wth nta-

ton o cll dath. H&E stann was compad to Gamma-H2AX (H2AX) xpsson sn mmnooscnc (IF).H2AX s a DNA hston that ndos phosphoylatonon th S139 sd n th psnc o dobl-standdDNA (dsDNA) baks, sch as that ndcd by R. IncasdH2AX snaln s sstv o sccssl R dlvy [2931]. Gat attnton was vn to xpsson lvls wthnth tmo co, vn that GBM s known to xpss ph-notypc and notypc htonty n tmo maks at owthn th tmo co [32, 33]. Contol tmos dmonstatdncasd osn stann wthn th tmo co (F 6(a)).Ts aas also cospond wth dcd H2AX oc (d)and DAPI snaln (bl). Pphal to th tmo co,

mltpl H2AX oc wth cospondn DAPI stann anotd. Sch fndns sst a lack o vabl clls wthnth tmo co, lkly d to tmo ncoss. Smlaly, schncotc oc hav bn dmonstatd to b a hallmak oGBM on both patholoy and adoloy [34].

In adatd tmos, H&E stann also vald la,cntally locatd aas that a composd o a ml omxd hmatoxyln and osn localzaton (F 6(b)). Incompason to contol tmos, IF stds o H2AX (d)xpsson n adatd tmos dmonstatd ncasdbomak xpsson wth postv DAPI (bl) stann o

ncl n wthn th cntal ml. Ts s bst vsalzdat hh solton (20x). Postv H2AX xpsson andpostv DAPI stann n ths cntal aas a sstvo post-adaton cll covy and pa n th sttn oadaton ndcd apoptoss [3537].

3.4. Impact o Radiation Terapy on 18F-FL-PE Imaging,and Markers o Angiogenesis, and Hypoxia. T dcdpolaton o tmo and tmo ncoss may b d atlast n pat to R cts on th tmo vasclat. Ts vdnc that GBM ndos vascla chans latnto hypoxa and anonss, and ths on mpact tmo

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VEGF

18F-FLT axial

18F-FLT coronal

1x 1x

4x4x

HIF-1

(a) Contol

VEGF

18F-FLT axial

18F-FLT coronal

1x 1x

4x4x

HIF-1

(b) R

Figure 7: Dcasd 18F-FL ptak cosponds wth ons o hypoxa and anonss. (a) Compason o axal and coonal vws 18F-FL-PE man sctons n contol ank tmos and mmnohstochmcal (IHC) and mmnooscnt (IF) stann wth DAPI ovascla ndothlal owth acto (VEGF) and hypoxa ndcton acto 1 alpha (HIF-1). Postv VEGF and HIF-1 (d) wth dcdDAPI (bl) s notd (wht and d aows). (b) Compason o18F-FL-PE man sctons and cospondn IHC and IF stann nank tmos 2 wks a 16 Gy R as pomd n (a). Postv tmo VEGF and HIF-1 xpsson wth DAPI snaln a notd (whtand d aows).

owth and tatmnt sstanc. Sch fndns hav bn

vsalzd clncally wth PE man [38]. W thosoht to compa 18F-FL-PE man to IF stann omaks o anonss and hypoxa. Hypoxa s knownto b nvolvd n tmo cnc, chmo- and ado-sstanc, nvason, and dcasd patnt svval [24, 39].W chos to xamn xpsson o vascla ndothlalowth acto(VEGF) andhypoxa ndcton acto-1 (HIF-1), as both hav bn stdd xtnsvly and dmonstatdto b actv n GBM [24, 4043].

18F-FL-PE man o contol tmos 2 wks anlatal R dmonstatd a notcabl dcton n ado-tac ptak wthn th tmo co as shown n bothaxal and coonal plans, potntally d to vascla nslt

(F 7(a)). IHC stann pattns o VEGF and HIF-1

(d) colat wth ons o ncasd osn stann, aspvosly shown. Expsson o both bomaks may bmtally latd, as ncasd hypoxa sconday to pol-aton can ndc VEGF podcton as a mans to stmlatanonss and dc hypoxa [23, 34, 44]. IF stannvals smlaly absnt DAPI (bl) stann wth ncasdoscnc o VEGF and HIF-1. Ts spmposd cn-tal psnc o VEGF and HIF-1 n nonadatd tmocos sppots o po hypothss o cntal ncoss [24].

In adatd tmos, 18F-FL adotac ptak was alsodmnshd wthn th tmo co, smla to contol tmos(F 7(b)). IHC stann o VEGF and HIF-1 (d) was

vsally dntcal. IF stann o tmo cos was postv o

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ncasd xpsson o both bomaks wth concomtantpostv DAPI (bl) snaln. Smla ncass n VEGFxpsson a R n GBM hav bn dmonstatd invitro [45]. Smlaly, R s known to ndc hypoxa and toncas HIF-1 xpsson [46, 47]. Addtonally, n C6 atlomas, HIF-1 platon was notd wthn 48 hos and

p to 8 days ollown 8 Gy R [48]. Ts data sst thatncasd bomakxpsson ollown hh dos 16 Gy Rs sconday to onon tmo pa.

4. Conclusions

18F-FL-PE man povds an xctn altnatv to18F-FDG-PE n th stdy o GBM. In o ntacanal

mos modl, w dmonstatd sccssl 18F-FL mano ntacanal GBM othotopc tmos wth adotacspan o th ntact mos ban. Addtonally, 18F-FLptak wthn tmos colatd wth tmo sz.

In htotopc ank xnoas, 18F-FL adotacptak pattns sst adatd tmos dmonstatdtctabl bt lobally dmnshd adotac ptak assn by dcas n %ID. Ts s most lkly d to tmocll dath and dcd postadaton polaton (K-67).Iadatd tmos also showd chans o bomaks odsDNA baks (H2AX), hypoxa (HIF-1), and vasclamodln (VEGF).

Ts data sst that 18F-FL adotac ptak vasd to chans n th GBM tmo mconvonmnt, andthat PE man may adly dnty ths chans. Schdata ncoa th in vivo and clncal stds wth novl

PE adotacs sch as 18F-FL, wth hops o ltmatly

mpovn th man and ca o patnts wth GBM.

Conflict of Interests

T athos dcla that thy hav no conct o ntsts.

Acknowledgments

T athos a atl to m Jnkns, L Shman,Chstna Chapman, and Saa Davs o xpt assstanc.

Ty a atl to Ds. Wafk El-Dy and Akva Mntzo th hlpl commnts. T athos wold lk toacknowld D. Stv Hahn whos nvalabl ncoa-mnt and sppot hlpd mak ths sach possbl. Tyacknowld th Small Anmal Iman Faclty (SAIF) atth Unvsty o Pnnsylvana o sa o th MRI andOptcal/Bolmnscnc Co Faclts. Ts wok was sp-potd by th Natonal Insttts o Halth (RC1 CA145075(G. Kao and J. F. Dosy) and K08 NS076548 (J. F. Dosy)),Bohs Wllcom Ca Awad o Mdcal Scntsts(1006792 (J. F. Dosy)), and Unvsty o WashntonMdcal Stdnts Rsach ann Poam (S. Chan-daskaan).

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