18 FDG, [ 18 F]FLT, [ 18 F]FAZA, and 11 C-Methionine Are Suitable Tracers for the Diagnosis and In...

Research Article18FDG [18F]FLT [18F]FAZA and 11C-Methionine AreSuitable Tracers for the Diagnosis and In Vivo Follow-Up ofthe Efficacy of Chemotherapy by miniPET in Both MultidrugResistant and Sensitive Human Gynecologic Tumor Xenografts

Gyoumlrgy Trencseacutenyi1 Tereacutez Maacuteriaacuten1 Imre Lajtos1 Zoltaacuten Krasznai2 Laacuteszloacute Balkay1

Mikloacutes Emri1 Paacutel Mikecz1 Katalin Goda2 Gaacutebor Szaloacuteki2 Istvaacuten Juhaacutesz34

Eniky Neacutemeth1 Tuumlnde Miklovicz1 Gaacutebor Szaboacute2 and Zoaacuterd T Krasznai5

1 Department of Nuclear Medicine University of Debrecen PO Box 63 Nagyerdei Korut 98 Debrecen 4012 Hungary2Department of Biophysics and Cell Biology University of Debrecen Nagyerdei Korut 98 Debrecen 4012 Hungary3 Department of Dermatology University of Debrecen Nagyerdei Korut 98 Debrecen 4012 Hungary4Department of Surgery and Operative Techniques Faculty of Dentistry University of Debrecen Nagyerdei Korut 98Debrecen 4012 Hungary

5 Department of Obstetrics and Gynecology University of Debrecen Nagyerdei Korut 98 Debrecen 4012 Hungary

Correspondence should be addressed to Terez Marian marianpetdotehu

Received 16 January 2014 Revised 14 August 2014 Accepted 28 August 2014 Published 18 September 2014

Academic Editor Yi-Xiang Wang

Copyright copy 2014 Gyorgy Trencsenyi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Expression of multidrug pumps including P-glycoprotein (MDR1 ABCB1) in the plasma membrane of tumor cells often resultsin decreased intracellular accumulation of anticancer drugs causing serious impediment to successful chemotherapy It hasbeen shown earlier that combined treatment with UIC2 anti-Pgp monoclonal antibody (mAb) and cyclosporine A (CSA) isan effective way of blocking Pgp function In the present work we investigated the suitability of four PET tumor diagnosticradiotracers including 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) 11C-methionine 31015840-deoxy-31015840-[18F]fluorothymidine (18F-FLT)and [18F]fluoroazomycin-arabinofuranoside (18FAZA) for in vivo follow-up of the efficacy of chemotherapy in both Pgp positive(Pgp+) and negative (Pgpminus) human tumor xenograft pairs raised in CB-17 SCID mice Pgp+ and Pgpminus A2780ADA2780 humanovarian carcinoma and KB-V1KB-3-1 human epidermoid adenocarcinoma tumor xenografts were used to study the effect of thetreatment with an anticancer drug doxorubicin combined with UIC2 and CSA The combined treatment resulted in a significantdecrease of both the tumor size and the accumulation of the tumor diagnostic tracers in the Pgp+ tumors Our results demonstratethat 18FDG 18F-FLT 18FAZA and 11C-methionine are suitable PET tracers for the diagnosis and in vivo follow-up of the efficacyof tumor chemotherapy in both Pgp+ and Pgpminus human tumor xenografts by miniPET

1 Introduction

The miniPET technique is a well-established noninvasivemethod to detect tumors and follow up the effect of therapyof tumors using PET tumor diagnostic tracers The PETtumor diagnostic tracers like 11C-metionine 2-[18F]fluoro-2-deoxy-D-glucose (18FDG) and (31015840-deoxy-31015840-[18F]fluoro-thymidine) (18F-FLT) are good candidates to measure theeffect of the therapy 18FDG an analog of glucose the most

commonly used PET radiotracer in clinical oncology allowsvisualization of the changes in the glucose metabolic rate intumors [1ndash3] 18FDG undergoes phosphorylation by hexok-inase but can not pass through the rest of glycolysis and itremains trapped in the cell Increased cell proliferation isone of the main features of cancer cells 18F-FLT is used as aPET tracer for visualization of cell proliferationThe trappingof 18F-FLT was demonstrated with the uptake of thym-idine analogue after phosphorylation by thymidine kinase 1

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 787365 10 pageshttpdxdoiorg1011552014787365

2 BioMed Research International

(TK1) in the S phase of cell cycle [4 5] Another tumor dia-gnostic PET tracer is the 11C labelled methionine applied forthe follow-up of the amino acid transport and metabolism inthe tumor [6] Radiolabeled nitroimidazole derivatives (eg[18F]fluoroazomycinarabinofuranoside (18F-FAZA) and [18F]-fluoromisonidazole (18F-MISO)) validated markers to detecthypoxia in cancer cells Nitroimidazoles labeled with 18F iso-tope can undergo an oxygen-reversible single-electron reduc-tion under hypoxic conditions forming reactive oxygen radi-cals that subsequently bind covalently tomacromolecular cel-lular components and are trapped in the intracellular spaceconsequently they are suitable tracers for in vivo detectionof hypoxia in tumors Hypoxia in tumor seems to be animportant prognostic factor of chemotherapy response [7]

Multidrug resistance (MDR) seems to be the most widelyobserved mechanism in clinical cases of chemotherapyresistance This phenomenon is often associated with theoverexpression of certain ATP binding cassette transportersincluding P-glycoprotein (Pgp coded by the MDR1 gene)which is an ATP dependent active efflux pump that is able toextrude a large variety of chemotherapeutic drugs from thecells [8 9]

The conformation sensitive UIC2 mouse monoclonalantibody inhibits Pgpmediated substrate transportHoweverthis inhibition is usually partial and its extent is variable sinceUIC2 binds only to 10ndash40of all Pgpmolecules present in thecell membrane [10 11] It has been shown earlier in in vitroand ex vivo experiments that the combined administrationof UIC2 antibody and certain substrates or modulatorsused at low concentrations increases the antibody bindingleading to a near complete Pgp inhibition thus providing anovel specific and effective way of blocking Pgp function[11 12] Krasznai et al [12] also demonstrated that the com-bined treatment with UIC2 antibody and Pgp modulatorseffectively blocked the function of Pgp in ovarian carcinomacells in vitro and the effect could be followed by using tumordiagnostic tracers 99mTc-MIBI and 18FDG

Ovarian cancer is the secondmost common cancer of thefemale genital tract but accounts for over half of all deathsrelated to gynecologic neoplasms [13] Anthracycline antibi-otics have been used for more than 40 years in the treatmentof gynecologic tumors in the first line in monotherapy or incombination with other drugs [14 15]

In this paper using an animal model we demonstratethat theminiPET technique combined with tumor diagnosticradiopharmaceuticals is suitable for the detection of eitherPgp expressing or Pgp nonexpressing tumors and it can beapplied for in vivomonitoring of the effect of tumor therapy

2 Materials and Methods

21 Cell Lines Drug sensitive (Pgp+) cell lines and their non-sensitive (Pgpminus) counterparts were used in the experimentsHuman epidermoid (cervix) carcinoma cell linesmdashKB-V-1 (Pgp+) KB-3-1 (Pgpminus)mdashand human ovarian carcinomacell linesmdashA2780AD (Pgp+) A2780 (Pgpminus)mdashwere grown asmonolayer cultures at 37∘C in a 95 humidified air 5 CO

2

atmosphere The cell lines were maintained in 75 cm2 flasks

in Dulbeccorsquos modified Eaglersquos medium (DMEM) containing45 gL glucose and supplemented with 10 heat-inactivatedfetal bovine serum (FBS) 2mM L-glutamine and 25 120583MmLgentamicinThe KB-V-1 cells were cultured in the presence of180 nMvinblastine and theA2780ADcells were culturedwith2 120583M doxorubicin until use The viability of the cells used inour experiments was always higher than 90 as assessed bythe trypan blue exclusion test

22 Laboratory Animals Twenty-four (10- to 12-week-old)pathogen-free B-17 severe combined immunodeficiency(SCID) female mice were used in this study Animals werehoused under pathogen-free conditions in air conditionedrooms at a temperature of 26 plusmn 2∘C with 50 plusmn 10 humidityand artificial lighting with a circadian cycle of 12 h The dietand drinking water (sterilized by autoclaving) were availablead libitum to all the animals The Principles of LaboratoryAnimal Care (National Institute of Health) were strictlyfollowed and the experimental protocol was approved by theLaboratory Animal Care and Use Committee of the Univer-sity of Debrecen

23 Animal Model and Study Design SCID mice wereinjected subcutaneously (sc) with KB-3-1 (15 times 106 cells in150 120583L PBS) cells on the left andKB-V-1 (3times 106 cells in 150 120583LPBS) cells on the right side Other groups of SCID mice wereinjected sc with A2780 (3 times 106 cells in 150 120583L PBS) cells onthe left and A2780AD (45 times 106 cells in 150 120583L PBS) cells onthe right side On each side the animals received two injec-tions of the same cell line one in the shoulder and one in thethigh to double the tumor numbers per animal in order tolimit the number of animals and to maximize the number oftumors imaged Preliminary experiments showed that incontrast to the Pgpminus cells the tumor formation from Pgp+cells has a slower kineticsThis differencewas equalized by theinjection of different cell numbers Four days after the injec-tion mice were treated with doxorubicin (DOX) (5mgkg)combined with UIC2 monoclonal antibody (5mgkg) andcyclosporine A (CSA) (10mgkg) Tumor growth was asse-ssed by caliper measurements every two days by the sameexperienced researcher The tumor size was calculatedusing the following formula (largest diameter times smallestdiameter2)2 [16]

24 Radiotracers The glucose analog (18FDG) was synthe-sized and labeled with the positron-decaying isotope 18Faccording to Hamacher et al [17] The radiosynthesis of thethymidine analog (18F-FLT) was performed according to thepublished method by Grierson and Shields [18] 11C-Methionine was synthesized as described by Mitterhauseret al [19]The 18F-labeled nitroimidazole compound fluoroa-zomycin arabinoside (18FAZA) was performed according tothe published method by Piert et al [7]

25 Small Animal PET Imaging Using RadiopharmaceuticalsAfter the implantation 18FDG 11C-methionine and 18F-FLTscans were repeated at different time points Prior to PET

BioMed Research International 3

mice were fasted overnight On the day of PET imaging micewere prewarmed to a body temperature of 37∘C and this tem-peraturewasmaintained throughout the uptake and scanningperiod to minimize the brown fat visualization Mice wereinjected via the tail vein with 50 plusmn 02MBq of 18FDG 81 plusmn06MBq of 11C-methionine 45plusmn02MBq of 18F-FLT or 55plusmn05MBq of 18FAZA 20min after 11C-methionine or 40minafter 18FDG or 18F-FLT or 120min after 18FAZA tracer injec-tion animals were anaesthetized by 3 isoflurane with a ded-icated small animal anesthesia device 20min static single-frame PET scans were acquired using a small animal PETscanner (miniPET-II Department of Nuclear MedicineDebrecen) to visualize the tumors On the same animal the11C-methionine and 18FDG and 18F-FLT scans were madewithin 4 days

The miniPET-II scanner consists of 12 detector modulesincluding LYSO scintillator crystal blocks and position sen-sitive PMTs [20] Each crystal block comprises 35 times 35 pinsof 127 times 127 times 12mm size Detector signals are processed byFPGA based digital signal processing boards with embeddedLinux operating system Data collection and image recon-struction are performed using a data acquisitionmodule withEthernet communication facility and a computer cluster ofcommercial PCs Scanner normalization and random correc-tion were applied on the data and the images were recon-structed with the standard EM iterative algorithm The voxelsize was 05 times 05 times 05mm and the spatial resolution variesbetween 14 and 21mm from central to 25mm radial dis-tances [20] The system sensitivity is 114

26 PET Data Analysis Radiotracer uptake was expressedin terms of standardized uptake values (SUVs) and tumorto muscle (TM) ratios Ellipsoidal 3-dimensional volume ofinterest (VOI) was manually drawn around the edge of thetumor xenografts activity by visual inspection using Brain-Cad software (httpwwwminipetcthu) The standardizeduptake value (SUV) was calculated as follows SUV = [VOIactivity (BqmL)][injected activity (Bq)animal weight (g)]assuming a density of 1 gcm3The TM ratios were computedas the ratio between the mean activity in the tumor VOI andin the background (muscle) VOI

27 Whole-Body Autoradiography For whole-body autora-diography the implantation of tumor cells was carried out asdescribed above On the 16th day after epidermoid carcinomaand on the 25th day after ovarian carcinoma cell implantationtumor-bearing mice were anaesthetized and the radioligands18F-FLT (45 plusmn 02MBq in 150120583L saline) or 18FDG (55 plusmn02MBq in 150120583L saline) or 18FAZA (55plusmn05MBq in 150 120583Lsaline) were injected via the tail vein Animals were euth-anized 60min after the administration of 18FDG or 18FLTand 120min after 18FAZA injection with 300mgkg pen-tobarbital (Nembutal) Each animal was embedded in 1carboxymethylcellulose solution After being frozen in liquidnitrogen 60120583m thin cryostat sections (Leica CM 3600cryomacrotome Nussloch Germany) were cut in the coronalplane Sections were exposed to phosphorimaging plates (GE

Healthcare Piscataway NJ USA) For anatomic correspon-dence true color images of the sections were also obtainedby a transparency scanner (Epson Perfection 1640 EPSONDeutschland GmbH Meerbusch Germany) Autoradiogra-phy and transmission images were overlaid to fuse the func-tional and anatomical information For phosphorimage anal-ysis of selected sections the ImageQuant 50 (GE HealthcarePiscataway NJ USA) image analyzing software was used

28 Tumor Sample Preparation 16 days after epidermoid car-cinoma and 25 days after ovarian carcinoma cell implantationmice were euthanized with an overdose of pentobarbital andtumorswere dissected from the animals Tumor samples wereembedded in cryomatrix and snap-frozen in liquid nitrogenSamples were stored at minus80∘C in an ultralow temperaturefreezer

29 Immunohistochemistry Immunohistochemistrywas per-formed on 5 120583m thick frozen tumor sections Frozen sectionswere dried at room temperature and fixed in precooledacetone (minus20∘C) for 10min Sections were then washed andincubated with 03 H

2O2in methanol for 20min to quench

endogenous peroxidase activity After blocking the nonspe-cific binding with 1 BSA for 20minutes sections were incu-bated for 60min at room temperature with mouse anti-Pgpmonoclonal antibody UIC2 (10 120583gmL) After two washeswith PBS anti-mouse EnVision Detection Systems DAB(Dako Denmark) was used to visualize the primary anti-bodies and sections were counterstained with haematoxylinNegative controls were obtained by omitting the primaryantibody

210 Flow Cytometric Measurements Formaldehyde (1 inPBS) prefixed cells were centrifuged at 500timesg for 5min andwashed twice with 1 PBSBSA To visualize the Pgp expres-sion cells (1 times 106 cellsmL) were incubated with 10120583gmL ofUIC2 mAb in PBS containing 1 BSA (PBSBSA) at 37∘C for40min After two washes with ice-cold PBS the cells wereincubated with rabbit anti-mouse Alexa 488 secondaryantibody (10 120583gmL A488-GaMIgG Invitrogen CA) at 4∘Cfor 30min Negative controls were obtained by omittingthe primary antibody A Becton-Dickinson FACScan flowcytometer (Becton-DickinsonMountainView CA)was usedto determine fluorescence intensities Emission was detectedthrough a 540 nmbroadband interference filter forAlexa 488Cytofluorimetric data were analyzed by BDIS CELLQUEST(Becton-Dickinson) or FloWin (written by Drs M Emri andL Balkay Department of Nuclear Medicine University ofDebrecen) software

211 Data Analysis Data are presented as mean plusmn SEM of atleast three independent experiments The significance wascalculated by Studentrsquos 119905-test (two-tailed) The level of signif-icance was set at 119875 le 005 unless otherwise indicated

3 Results

31 Flow Cytometric Studies Flow cytometric analysesshowed a remarkably high expression of Pgp in the Pgp+ cells


(a) (f)

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A2780 Pgpminus A2780AD Pgp+ KB-3-1 Pgpminus KB-V-1 Pgp+

Figure 1 Histopathological analysis of tumor xenografts Autopsy images show the human ovarian (a) and human epidermoid carcinoma(f) tumor xenografts 25 and 16 days after tumor cell implantation respectively Microscopic images of HampE staining ((b) A2780 Pgpminus (c)A2780AD Pgp+ (g) KB-3-1 Pgpminus (h) KB-V-1 Pgp+) and UIC2 mAb-DAB immunostaining ((d) A2780 Pgpminus (e) A2780AD Pgp+ (i) KB-3-1Pgpminus (j) KB-V-1 Pgp+) of xenograft tumor sections Bar 50120583m magnification times200

The ratio of the relative mean fluorescence intensities of thePgp+Pgpminus ovarian carcinoma (A2780ADA2780) and theepidermoid adenocarcinoma (KB-V-1KB-3-1) cell line pairswas 127 plusmn 23 and 100 plusmn 18 respectively

32 Histopathological Studies SCID mice were injected intheir left and right sides with Pgpminus (A2780 KB-3-1) and Pgp+(A2780AD KB-V-1) cells respectively No morphologicaldifferences were seen between the congruous Pgpminus and Pgp+tumors upon histopathological analysis of harvested tumorsby haematoxylin-eosin (HampE) staining (Figures 1(b) 1(c)1(g) and 1(h)) Tumor cells implanted into the SCIDmice retained their Pgp+ or Pgpminus phenotype as proved byimmunostaining of Pgp Immunoperoxidase detectionshowed strong positive staining in Pgp+ tumor cell mem-branes (Figures 1(e) and 1(j)) in contrast no staining wasvisible in Pgpminus tumors (Figures 1(d) and 1(i))

33 Impact of Combined Treatment on Tumor Volume Fourdays after the sc injection of tumor cells mice were treatedwith a combination of UIC2monoclonal antibody DOX andCSA Other groups of animals (tumor-bearing control(untreated)) were injected with PBS The growth rate of theovarian carcinoma tumors (A2780 Pgpminus andA2780ADPgp+)was slower (doubling time 4 days) than that of the epi-dermoid carcinoma tumors (KB-3-1 Pgpminus and KB-V-1 Pgp+doubling time 3 days)The growth rates of the treated tumorswere compared to that of the untreated tumors in both thecases of the Pgp+ and the Pgpminus ones In contrast to the control(untreated) tumors where exponential growth was observed

the combined treatment inhibited the tumor growth(Figure 2) In case of the human ovarian carcinomaxenografts from day 10 significant differences (on day 10 119875 le005 on day 25 119875 le 001) were observed between the treatedand untreated A2780AD and A2780 tumor volumes Theseresults were similar to the experiments with the humanepidermoid carcinoma xenografts where we found signifi-cant differences in the volume (from day 8 119875 le 005from day 14 119875 le 001) of the control and treated tumors(Figure 2(b))

34 miniPET Imaging of Combined Treated and UntreatedTumor Xenografts For in vivo visualization of the effectof the combined treatment on tumors 18FDG and 18F-FLTminiPET scans were performed at different time points andSUVmean SUVmax TMmean and TMmax ratios werecalculated (Figure 3) Fifteen to twenty days after A2780 PgpminusandA2780ADPgp+ cell implantation control (untreated) andcombined treated tumor-bearing mice (6 mice 24 tumors)received 18FDG and 18F-FLT Control tumors demonstratedhigh 18FDG and 18F-FLT uptake (Figure 3(a) left) Quantita-tive image analysis showed significant differences (119875 lt 0001)between the 18FDG avidity of treated Pgp+ (SUVmean =033 plusmn 003 and SUVmax = 054 plusmn 006) and untreated Pgp+(SUVmean = 162 plusmn 025 and SUVmax = 296 plusmn 06) tumorsand also between the treated Pgpminus (SUVmean = 056 plusmn 003and SUVmax = 094 plusmn 007) and untreated Pgpminus (SUVmean= 143 plusmn 017 and SUVmax = 251 plusmn 03) tumors By takingthe TM ratios the differences between the treated anduntreated tumors were also significant (119875 lt 0001) The


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Figure 2 Impact of combined treatment on tumor growth Pgp+ and Pgpminus treated tumorsrsquo volumes were compared to Pgp+ and Pgpminus control(untreated) tumors in tumor-bearing animals Treatments began 4 days after tumor cell inoculations (a) Impact of combined treatment onA2780AD Pgp+ and A2780 Pgpminus (6 mice 24 tumors) (b) Impact of combined treatment on KB-V-1 Pgp+ and KB-3-1 Pgpminus tumors (6 mice24 tumors) Statistically significant changes in tumor volume compared to the tumor volume of untreated control are indicated (lowast119875 le 005lowastlowast119875 le 001)

uptake of 18F-FLT was significantly increased at the controltumors compared with that at the combined treated tumors(Figure 3(b) left) The 18F-FLT miniPET imaging and SUVvalues showed the efficiency of the combined treatment

The biodistribution of 18FDG and 18F-FLT on days 10ndash15following tumor inoculation in epidermoid carcinoma bear-ing mice is shown in Figure 3(a) The results of 18FDG and18F-FLT miniPET scans showed significant accumulation inthe untreated control tumors (Figure 3(a) right) in contrastto the combined treated KB-V-1 Pgp+ and KB-3-1 Pgpminustumors

Overall in the treated animals the TM ratios showedno difference in the 18FDG and 18F-FLT uptake between theplace of inoculation and its muscle environment proving thelack of tumor cells No significant differences were observedwith these two radiopharmaceuticals in the SUV valuesbetween Pgp+ and Pgpminus tumors

Fifteen to twenty days after A2780 Pgpminus and A2780ADPgp+ cell implantation control (untreated) and treatedtumor-bearing mice received 11C-methionine Control tum-ors showed high 11C-methionine uptake (Figure 4(a)) Quan-titative image analysis demonstrated that there were notabledifferences between the 11C-methionine avidity of the treatedPgp+ (SUVmean = 043plusmn002 and SUVmax = 083plusmn003) anduntreated Pgp+ (SUVmean= 135plusmn046 and SUVmax= 220plusmn06) tumors and also between the treated Pgpminus (SUVmean =045 plusmn 003 and SUVmax = 084 plusmn 006) and untreated Pgpminus(SUVmean = 135 plusmn 08 and SUVmax = 240 plusmn 15) tumors

The 11C-methionine miniPET imaging (Figure 4(b)) andSUV values also showed the efficiency of the combinedtreatment in case of the epidermoid tumor xenografts 10ndash15days after KB-3-1 and KB-V-1 tumor cell implantation thequantitative image analysis showed notable differences

between the 11C-methionine uptake of the treated Pgp+(SUVmean = 049 plusmn 003 and SUVmax = 084 plusmn 002) anduntreated Pgp+ (SUVmean= 152plusmn 043 and SUVmax= 243plusmn068) tumors and also between the treated Pgpminus (SUVmean =049 plusmn 003 and SUVmax = 076 plusmn 005) and untreated Pgpminus(SUVmean = 148 plusmn 046 and SUVmax = 255 plusmn 10) tumors

35 Assessment of Heterogeneity in Tumor Metabolism UsingRadiopharmaceuticals The metabolic heterogeneity of con-trol tumors was investigated on the same animal by 18FDG(22 days after tumor cell injection) 18FAZA (23 days aftertumor cell injection) and 18F-FLT (24 days after tumor cellinjection) usingminiPET scanner and autoradiography tech-niques Control tumors demonstrated heterogeneous 18F-FLT uptake with some areas of moderate uptake surroundedby areas of intense uptake indicating the proliferation oftumor cells We found that the 18FDG positive tumor areasand the 18F-FLT negative areas overlapped one another The18FAZA uptake of these areas was high After the quantita-tive analysis of the miniPET images the differences in themetabolic activity of the investigated tumors are shown on arepresentative figure (Figure 5) By taking the SUVmeanvalues we found the following 18FDG avid areas 120 plusmn 027not 18FDG avid areas 079 plusmn 013 18F-FLT avid areas 267 plusmn035 not 18F-FLT avid areas 215 plusmn 019 18FAZA avid-hypoxic-areas 070 plusmn 008 not 18FAZA avid-not hypoxic-areas 053 plusmn 007

4 Discussion

Xenograft model and the miniPET technique are recentlycommonly usednoninvasivemethods in preclinical studies to


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Figure 3 Effect of combined treatment on the 18FDG and 18F-FLT uptake of Pgp positive and negative human ovarian (left side) andepidermoid carcinoma (right side) tumors (a) 18FDG and 18F-FLT miniPET images of control and combined treated Pgpminus (left side blackarrows) and Pgp+(right side red arrows) tumor-bearing mice (coronal sections) No tumors can be visualized in the treated animals (b)The SUVmean and SUVmax and the mean and maximum TM ratios are displayed (bars represent mean plusmn SEM) Statistically significantdifferences in comparison to combined treated tumors are indicated (lowast119875 le 005 lowastlowast119875 le 001 and lowastlowastlowast119875 le 0001)


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Figure 4 Effect of combined treatment on the 11C-methionine uptake of Pgp positive and negative human ovarian (a) and epidermoidcarcinoma (b) tumors 11C-Methionine miniPET images of control and treated Pgpminus (left side black arrows) and Pgp+(right side red arrows)tumor-bearing mice (coronal sections) No tumors can be visualized in the treated animals

18FDG 18FAZA 18F-FLT 18F-FLT autoradiography Photogram

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Figure 5miniPET andwhole-body autoradiography images from the same control tumor-bearing (ovarian carcinoma) SCIDmouse (upper)Lower panels demonstrate the tumor heterogeneity using different radiopharmaceuticals on a representative Pgp+ tumorWhite arrows Pgpminustumors red arrows Pgp+ tumors Color bars regard to the PET images only

detect and follow up the effect of therapy of humantumors using PET tumor diagnostic radiopharmaceuticalslike 18FDG 18F-FLT 18FAZA and 11C-methionine

It has been demonstrated earlier by Goda et al [11] andKrasznai et al [12] in their in vitro and ex vivo experimentsthat the combined application of the Pgp specific antibodyUIC2 and cyclosporine A is a successful strategy to overcomePgp-mediated multidrug resistance

In our experiments we used similar treatment carriedout on mouse xenografts Our aim was to test the efficacyof the combined treatment on drug sensitive and resistanthuman ovary and cervix cancer xenograft models by usingin vivo medical imaging technique (miniPET) and the mostcommonly applied tumor diagnostic radiopharmacons(18FDG 18F-FLT and 11C-metionine) in the visualization ofgynecological tumors

The tumor cells were inoculated at four places in each ani-mal in order to provide self-control experiment and reducethe necessary number of experimental animalsThe presence

of Pgp in the tumors was proved by immunohistochemistry(Figure 1)

The A2780 and A2780AD human ovarian tumors grewslower than the epidermoid adenocarcinoma tumors (KB-3-1KB-V-1) (Figure 2) The treatment of the tumors started fourdays after the inoculation using a dose of 5mgkg doxoru-bicinThe size of the tumors was small (2ndash5mm3) but visuallyobservable Lee et al [21] performed efficacy studies usingBALBc mice bearing C-26 colon carcinoma tumors In theirstudy they applied dendrimer-DOX in which the DOX wasattached bymeans of a stable carbamate bond in an equivalentof 20mgkg doxorubicin in a single dose 8 days after tumorimplantation which caused complete tumor regression and100 survival Kratz et al [22] published similar results theyobserved in both male and female mice an LD50 of doxoru-bicin 12mgkg Graf et al [23] have shown that tumor growthwas inhibited by a single dose of doxorubicin ranging from25 120583g to 200120583g in xenograft lymphoma tumors Kim et al[24] treated the human ovarian A2780DOX carcinoma


xenografts with 10mgkg DOX three times at 3-day intervalswithout any effect of the MDR resistant tumor growth

In our work we diagnosed the presence and followed thegrowth of multidrug sensitive and resistant tumors as wellas the efficacy of the combined treatment with three tumordiagnostic tracersThe results show that the 18FDG (a glucosemetabolic tracer) 11C-methionine (tracer for amino acidtransport and protein synthesis) and the 18F-FLT (a prolif-eration tracer) can be effectively used for monitoring boththe Pgp+ and the Pgpminus tumors The accumulation of thetracers could be followed by definite SUV values although the18F-FLT SUV values were higher than that of the 18FDG(Figure 3) Ong et al [25] called attention to the fact that thePET tracer accumulation can be different in in vitro exper-iments with tumor cells and in xenografts made from thesame cells in vivo therefore the pharmacon uptake should betested Xenografts in vivo can be less sensitive to PET tumordiagnostic tracers than cell lines in vitro In our experimentsin both theA2780 and theA2780AD aswell as theKB-3-1 andKB-V-1 Pgp+ and Pgpminus cell line pairs just as the xenograftsmade with these cell lines all three tumor diagnostic tracers(18FDG 18F-FLT and 11C-methionine) could be well usedJensen et al [5] used ovarian cancer (A2780) xenograftsin nude mice and theymdashsimilarly to our resultsmdashmeasuredhigher 18F-FLT than 18FDG uptake On the other hand theymeasured approximately 50 lower SUVmean and SUVmaxvalues compared to our results The differences can beexplained by the different experimental protocols and the bio-logical differences between the nude and SCID mice Eben-han et al [26] used KB-3-1 cervix carcinoma xenograftmodel(in nude mice) and found that the xenografts showed low18FDG SUV and were better visualized by 18F-FLT It is inagreement with other studies reporting that for assessing theearly response to anticancer treatment 18F-FLT was supe-rior to 18FDG [5] However other studies found higher18FDG uptake compared to 18F-FLT accumulation in severalxenograft models [27 28] In our experiments both tracersshowed good visualization of the Pgp+ and Pgpminus tumors andthe efficacy of the treatment18FDG the most commonly used PET radiotracer in

tumor diagnostics allows visualization of the changes in theglucose metabolic rate in tumors [3] Since Pgp is a transportATPase its activity may increase the ATP demand of the cellsexpressing it at high level [12] The increased energy demandof the cancer cells manifests in higher glucose metabolismsthat can be measured by 18FDG accumulation but a numberof factors can affect the FDG uptake in tumors [1 3 29 30]

The different proliferation activity of the different tumorsas well as the variance in the tumor volume sufferinghypoxiamay result in further differences in the 18FDGuptake(Figure 5) Similarly to our results several authors report het-erogeneity in the 18FDG uptake of different tumors [31 32]The experimental model used in our experiments providesexactly the same extracellular conditions (free glucose con-centration in the blood injected radiotracer dose anaes-thetizing procedure etc) since both of the Pgp+ and the Pgpminustumors grow in the same mice therefore the measured

differences in the accumulation of the radiopharmacon showthe intrinsic characters of the tumors

In our experiments we initiated the treatment of thetumors four days after the inoculation of the cells when thegynecologic tumors were still rather small We aimed to dev-elop a model analogous to the clinical situation when afterremoving the tumor by surgery a systemic therapy is appliedto hinder the development of multidrug resistant primary ormetastatic tumors

Our results show that the above described combinedtreatment is an effectivemethod for the chemotherapy of bothPgp+ and Pgpminus human ovarian carcinoma and epidermoidadenocarcinoma tumors growing in mice and the efficacy ofthe treatment can be followed by miniPET using 18FDG 18F-FLT and 11C-methionine radiotracers In addition 18FAZA isa suitable tracer to detect the hypoxia of the tumor in xeno-grafts Using multitracer miniPET 18FDG 18F-FLT and18FAZA analyses also help to detect the metabolic hetero-geneity of the tumors

Abbreviations

CSA Cyclosporine ADOX Doxorubicin18FAZA [18F]Fluoroazomycin-arabinofuranoside18FDG 2-[18F]Fluoro-2-deoxy-D-glucose18F-FLT (31015840-Deoxy-31015840-[18F]fluorothymidine)mAb Monoclonal antibodyMDR Multidrug resistancePBS Phosphate buffered salinePET Positron emission tomographyPgp P-GlycoproteinPgp+ P-Glycoprotein positivePgpminus P-Glycoprotein negative

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Gyorgy Trencsenyi and Terez Marian contributed equally tothis study

Acknowledgments

This work was supported by Szodoray grant from theUniversity of Debrecen (recipients are Z T Krasznai andK Goda) The work was also supported by OTKA GrantsPD75994 K72762 NK101337 NK101680 and TAMOP-422A-111KONV-2012-0023 The authors also thank TamasNagy and Judit P Szabo for the technical assistance

References

[1] R L Wahl ldquoTargeting glucose transporters for tumor imagingldquosweetrdquo idea ldquosourrdquo resultrdquo Journal of Nuclear Medicine vol 37no 6 pp 1038ndash1041 1996


[2] E M Rohren T G Turkington and R E Coleman ldquoClinicalapplications of PET in oncologyrdquo Radiology vol 231 no 2 pp305ndash332 2004

[3] S Vallabhajosula ldquo 18F-labeled positron emission tomographicradiopharmaceuticals in oncology an overview of radiochem-istry and mechanisms of tumor localizationrdquo Seminars inNuclear Medicine vol 37 no 6 pp 400ndash419 2007

[4] J S Rasey J R Grierson L W Wiens P D Kolb and J LSchwartz ldquoValidation of FLT uptake as a measure of thymidinekinase-1 activity in A549 carcinoma cellsrdquo Journal of NuclearMedicine vol 43 no 9 pp 1210ndash1217 2002

[5] M M Jensen K D Erichsen F Bjorkling et al ldquoEarly detec-tion of response to experimental chemotherapeutic top216with [18F]FLT and [18F]FDG PET in Human Ovary CancerXenografts in Micerdquo PLoS ONE vol 5 no 9 Article ID e129652010

[6] P L JagerW Vaalburg J Pruim E G E DeVries K-J Langenand D A Piers ldquoRadiolabeled amino acids basic aspects andclinical applications in oncologyrdquo Journal of Nuclear Medicinevol 42 no 3 pp 432ndash445 2001

[7] M Piert H J Machulla M Picchio et al ldquoHypoxia-specifictumor imaging with 18F-fluoroazomycin arabinosiderdquo Journalof Nuclear Medicine vol 46 no 1 pp 106ndash113 2005

[8] H Glavinas P Krajcsi J Cserepes and B Sarkadi ldquoThe role ofABC transporters in drug resistance metabolism and toxicityrdquoCurrent drug delivery vol 1 no 1 pp 27ndash42 2004

[9] K Goda Z Bacso andG Szabo ldquoMultidrug resistance throughthe spectacle of P-glycoproteinrdquo Current Cancer Drug Targetsvol 9 no 3 pp 281ndash297 2009

[10] E B Mechetner B Schott B S Morse et al ldquoP-glycoproteinfunction involves conformational transitions detectable bydifferential immunoreactivityrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no24 pp 12908ndash12913 1997

[11] K Goda F Fenyvesi Z Bacso et al ldquoComplete inhibition of p-glycoprotein by simultaneous treatment with a distinct class ofmodulators and the UIC2 monoclonal antibodyrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 320 no 1 pp81ndash88 2007

[12] Z T Krasznai A Toth PMikecz et al ldquoPgp inhibition byUIC2antibody can be followed in vitro by using tumor-diagnosticradiotracers 99119898Tc-MIBI and 18FDGrdquo European Journal ofPharmaceutical Sciences vol 41 no 5 pp 665ndash669 2010

[13] K Kitajima K Murakami S Sakamoto Y Kaji and K Sug-imura ldquoPresent and future of FDG-PETCT in ovarian cancerrdquoAnnals of Nuclear Medicine vol 25 no 3 pp 155ndash164 2011

[14] J T Thigpen J A Blessing P J DiSaia E Yordan L F Carsonand C Evers ldquoA randomized comparison of doxorubicin aloneversus doxorubicin plus cyclophosphamide in the managementof advanced or recurrent endometrial carcinoma aGynecologicOncologyGroup Studyrdquo Journal of Clinical Oncology vol 12 no7 pp 1408ndash1414 1994

[15] G Balbi S Visconti A Monteverde M-A Manganaro and ACardone ldquoLiposomal doxorubicin a phase II trialrdquo Acta Bio-medica de lrsquoAteneo Parmense vol 78 no 3 pp 210ndash213 2007

[16] N AideM Briand P Bohn et al ldquo120572v1205733 imaging can accuratelydistinguish betweenmature teratoma and necrosis in 18F-FDG-negative residual masses after treatment of non-seminomatoustesticular cancer a preclinical studyrdquo European Journal ofNuclearMedicine andMolecular Imaging vol 38 no 2 pp 323ndash333 2011

[17] K Hamacher H H Coenen and G Stocklin ldquoEfficient stere-ospecific synthesis of no-carrier-added 2-[18F]-fluoro-2-deoxy-D-glucose using aminopolyether supported nucleophilic sub-stitutionrdquo Journal of Nuclear Medicine vol 27 no 2 pp 235ndash238 1986

[18] J R Grierson and A F Shields ldquoRadiosynthesis of 31015840-deoxy-31015840-[18F]fluorothymidine [18F]FLT for imaging of cellular prolifer-ation in vivordquo Nuclear Medicine and Biology vol 27 no 2 pp143ndash156 2000

[19] MMitterhauserWWadsak A Krcal et al ldquoNew aspects on thepreparation of [11C]Methioninemdasha simple and fast onlineapproach without preparative HPLCrdquo Applied Radiation andIsotopes vol 62 no 3 pp 441ndash445 2005

[20] I Lajtos M Emri S A Kis et al ldquoPerformance evaluation andoptimization of the MiniPET-II scannerrdquo Nuclear Instrumentsand Methods in Physics Research A Accelerators SpectrometersDetectors and Associated Equipment vol 707 no 36 pp 26ndash342013

[21] C C Lee E R Gillies M E Fox et al ldquoA single dose ofdoxorubicin-functionalized bow-tie dendrimer curesmice bea-ring C-26 colon carcinomasrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 103 no45 pp 16649ndash16654 2006

[22] F Kratz G Ehling H-M Kauffmann and C Unger ldquoAcuteand repeat-dose toxicity studies of the (6-maleimidocap-royl)hydrazone derivative of doxorubicin (DOXO-EMCH) analbumin-binding prodrug of the anticancer agent doxorubicinrdquoHuman and Experimental Toxicology vol 26 no 1 pp 19ndash352007

[23] N Graf K Herrmann B Numberger et al ldquo(18F)FLT issuperior to [18F]FDG for predicting early response to antiprolif-erative treatment in high-grade lymphoma in a dose-dependentmannerrdquo European Journal of Nuclear Medicine and MolecularImaging vol 40 no 1 pp 34ndash43 2013

[24] D Kim E S Lee K Park I C Kwon and Y H Bae ldquoDoxoru-bicin loaded pH-sensitive micelle antitumoral efficacy againstovarian A2780DOX119877 tumorrdquo Pharmaceutical Research vol 25no 9 pp 2074ndash2082 2008

[25] L-C Ong Y Jin I-C Song S Yu K Zhang and P K H Chowldquo2-[18F]-2-deoxy-D-Glucose (FDG) uptake in human tumorcells is related to the expression of GLUT-1 and hexokinase IIrdquoActa Radiologica vol 49 no 10 pp 1145ndash1153 2008

[26] T Ebenhan M Honer S M Ametamey et al ldquoComparisonof [18F]-Tracers in various experimental tumor models by PETimaging and identification of an early response biomarker forthe novel microtubule stabilizer patupilonerdquoMolecular Imagingand Biology vol 11 no 5 pp 308ndash321 2009

[27] Y-J Yang J-S Ryu S-Y Kim et al ldquoUse of 31015840-deoxy-31015840-[18F]fluorothymidine PET to monitor early responses to radi-ation therapy in murine SCCVII tumorsrdquo European Journal ofNuclear Medicine andMolecular Imaging vol 33 no 4 pp 412ndash419 2006

[28] L Brepoels S Stroobants G Verhoef T De Groot L Mortel-mans and C De Wolf-Peeters ldquo18F-FDG and 18F-FLT uptakeearly after cyclophosphamide andmTOR inhibition in an exper-imental lymphoma modelrdquo Journal of Nuclear Medicine vol50 no 7 pp 1102ndash1109 2009

[29] A Breier L Gibalova M Seres M Barancik and Z SulovaldquoNew insight into P-Glycoprotein as a drug targetrdquoAnti-CancerAgents in Medicinal Chemistry vol 13 no 1 pp 159ndash170 2013


[30] T A D Smith ldquoInfluence of chemoresistance and p53 status onfluoro-2-deoxy-d-glucose incorporation in cancerrdquo NuclearMedicine and Biology vol 37 no 1 pp 51ndash55 2010

[31] S Chicklore V Goh M Siddique A Roy P K Marsden andG J R Cook ldquoQuantifying tumour heterogeneity in 18F-FDGPETCT imaging by texture analysisrdquo European Journal ofNuclear Medicine and Molecular Imaging vol 40 no 1 pp 133ndash140 2013

[32] D Vriens J A Disselhorst W J G Oyen L-F De Geus-Oeiand E P Visser ldquoQuantitative assessment of heterogeneity intumor metabolism using FDG-PETrdquo International Journal ofRadiation Oncology Biology Physics vol 82 no 5 pp e725ndashe7312012


(TK1) in the S phase of cell cycle [4 5] Another tumor dia-gnostic PET tracer is the 11C labelled methionine applied forthe follow-up of the amino acid transport and metabolism inthe tumor [6] Radiolabeled nitroimidazole derivatives (eg[18F]fluoroazomycinarabinofuranoside (18F-FAZA) and [18F]-fluoromisonidazole (18F-MISO)) validated markers to detecthypoxia in cancer cells Nitroimidazoles labeled with 18F iso-tope can undergo an oxygen-reversible single-electron reduc-tion under hypoxic conditions forming reactive oxygen radi-cals that subsequently bind covalently tomacromolecular cel-lular components and are trapped in the intracellular spaceconsequently they are suitable tracers for in vivo detectionof hypoxia in tumors Hypoxia in tumor seems to be animportant prognostic factor of chemotherapy response [7]

Multidrug resistance (MDR) seems to be the most widelyobserved mechanism in clinical cases of chemotherapyresistance This phenomenon is often associated with theoverexpression of certain ATP binding cassette transportersincluding P-glycoprotein (Pgp coded by the MDR1 gene)which is an ATP dependent active efflux pump that is able toextrude a large variety of chemotherapeutic drugs from thecells [8 9]

The conformation sensitive UIC2 mouse monoclonalantibody inhibits Pgpmediated substrate transportHoweverthis inhibition is usually partial and its extent is variable sinceUIC2 binds only to 10ndash40of all Pgpmolecules present in thecell membrane [10 11] It has been shown earlier in in vitroand ex vivo experiments that the combined administrationof UIC2 antibody and certain substrates or modulatorsused at low concentrations increases the antibody bindingleading to a near complete Pgp inhibition thus providing anovel specific and effective way of blocking Pgp function[11 12] Krasznai et al [12] also demonstrated that the com-bined treatment with UIC2 antibody and Pgp modulatorseffectively blocked the function of Pgp in ovarian carcinomacells in vitro and the effect could be followed by using tumordiagnostic tracers 99mTc-MIBI and 18FDG

Ovarian cancer is the secondmost common cancer of thefemale genital tract but accounts for over half of all deathsrelated to gynecologic neoplasms [13] Anthracycline antibi-otics have been used for more than 40 years in the treatmentof gynecologic tumors in the first line in monotherapy or incombination with other drugs [14 15]

In this paper using an animal model we demonstratethat theminiPET technique combined with tumor diagnosticradiopharmaceuticals is suitable for the detection of eitherPgp expressing or Pgp nonexpressing tumors and it can beapplied for in vivomonitoring of the effect of tumor therapy

2 Materials and Methods

21 Cell Lines Drug sensitive (Pgp+) cell lines and their non-sensitive (Pgpminus) counterparts were used in the experimentsHuman epidermoid (cervix) carcinoma cell linesmdashKB-V-1 (Pgp+) KB-3-1 (Pgpminus)mdashand human ovarian carcinomacell linesmdashA2780AD (Pgp+) A2780 (Pgpminus)mdashwere grown asmonolayer cultures at 37∘C in a 95 humidified air 5 CO

2

atmosphere The cell lines were maintained in 75 cm2 flasks

in Dulbeccorsquos modified Eaglersquos medium (DMEM) containing45 gL glucose and supplemented with 10 heat-inactivatedfetal bovine serum (FBS) 2mM L-glutamine and 25 120583MmLgentamicinThe KB-V-1 cells were cultured in the presence of180 nMvinblastine and theA2780ADcells were culturedwith2 120583M doxorubicin until use The viability of the cells used inour experiments was always higher than 90 as assessed bythe trypan blue exclusion test

22 Laboratory Animals Twenty-four (10- to 12-week-old)pathogen-free B-17 severe combined immunodeficiency(SCID) female mice were used in this study Animals werehoused under pathogen-free conditions in air conditionedrooms at a temperature of 26 plusmn 2∘C with 50 plusmn 10 humidityand artificial lighting with a circadian cycle of 12 h The dietand drinking water (sterilized by autoclaving) were availablead libitum to all the animals The Principles of LaboratoryAnimal Care (National Institute of Health) were strictlyfollowed and the experimental protocol was approved by theLaboratory Animal Care and Use Committee of the Univer-sity of Debrecen

23 Animal Model and Study Design SCID mice wereinjected subcutaneously (sc) with KB-3-1 (15 times 106 cells in150 120583L PBS) cells on the left andKB-V-1 (3times 106 cells in 150 120583LPBS) cells on the right side Other groups of SCID mice wereinjected sc with A2780 (3 times 106 cells in 150 120583L PBS) cells onthe left and A2780AD (45 times 106 cells in 150 120583L PBS) cells onthe right side On each side the animals received two injec-tions of the same cell line one in the shoulder and one in thethigh to double the tumor numbers per animal in order tolimit the number of animals and to maximize the number oftumors imaged Preliminary experiments showed that incontrast to the Pgpminus cells the tumor formation from Pgp+cells has a slower kineticsThis differencewas equalized by theinjection of different cell numbers Four days after the injec-tion mice were treated with doxorubicin (DOX) (5mgkg)combined with UIC2 monoclonal antibody (5mgkg) andcyclosporine A (CSA) (10mgkg) Tumor growth was asse-ssed by caliper measurements every two days by the sameexperienced researcher The tumor size was calculatedusing the following formula (largest diameter times smallestdiameter2)2 [16]

24 Radiotracers The glucose analog (18FDG) was synthe-sized and labeled with the positron-decaying isotope 18Faccording to Hamacher et al [17] The radiosynthesis of thethymidine analog (18F-FLT) was performed according to thepublished method by Grierson and Shields [18] 11C-Methionine was synthesized as described by Mitterhauseret al [19]The 18F-labeled nitroimidazole compound fluoroa-zomycin arabinoside (18FAZA) was performed according tothe published method by Piert et al [7]

25 Small Animal PET Imaging Using RadiopharmaceuticalsAfter the implantation 18FDG 11C-methionine and 18F-FLTscans were repeated at different time points Prior to PET













3 Results



(a) (f)

(b) (c)

(d) (e)

(g) (h)

(i) (j)









020406080

100120140160180

0 5 10 15 20 25 30Time (days)

Tum

or v

olum

e (m

m3)


lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

(a)

0

20

40

60

80

100

120

140


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Tum

or v

olum

e (m

m3)


(b)









4 Discussion




0

18FD

G18F-

FLT

SUV10

018FD

G18F-

FLT

(a)

0

1

2

3

4

5

6

7

8


TM ratio(max)

0

1

2

3

4

5

6

7

0123456789

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

18FD

G ac

cum

ulat

ion

0

1

2

3

4

5

6

18FD

G ac

cum

ulat

ion

18F-

FLT

accu

mul

atio

n

18F-

FLT

accu

mul

atio

n

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




TM ratio(max)


TM ratio(max)


TM ratio(max)

(b)



5

0

11C-

Met

hion

ine

Control Treated SUV

(a)

5

0

Control Treated SUV

11C-

Met

hion

ine

(b)



SUV10

0

SUV10

0
















Abbreviations






Acknowledgments


References















































3 Results



(a) (f)

(b) (c)

(d) (e)

(g) (h)

(i) (j)









020406080

100120140160180

0 5 10 15 20 25 30Time (days)

Tum

or v

olum

e (m

m3)


lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

(a)

0

20

40

60

80

100

120

140


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Tum

or v

olum

e (m

m3)


(b)









4 Discussion




0

18FD

G18F-

FLT

SUV10

018FD

G18F-

FLT

(a)

0

1

2

3

4

5

6

7

8


TM ratio(max)

0

1

2

3

4

5

6

7

0123456789

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

18FD

G ac

cum

ulat

ion

0

1

2

3

4

5

6

18FD

G ac

cum

ulat

ion

18F-

FLT

accu

mul

atio

n

18F-

FLT

accu

mul

atio

n

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




TM ratio(max)


TM ratio(max)


TM ratio(max)

(b)



5

0

11C-

Met

hion

ine

Control Treated SUV

(a)

5

0

Control Treated SUV

11C-

Met

hion

ine

(b)



SUV10

0

SUV10

0
















Abbreviations






Acknowledgments


References




































(a) (f)

(b) (c)

(d) (e)

(g) (h)

(i) (j)









020406080

100120140160180

0 5 10 15 20 25 30Time (days)

Tum

or v

olum

e (m

m3)


lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

(a)

0

20

40

60

80

100

120

140


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Tum

or v

olum

e (m

m3)


(b)









4 Discussion




0

18FD

G18F-

FLT

SUV10

018FD

G18F-

FLT

(a)

0

1

2

3

4

5

6

7

8


TM ratio(max)

0

1

2

3

4

5

6

7

0123456789

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

18FD

G ac

cum

ulat

ion

0

1

2

3

4

5

6

18FD

G ac

cum

ulat

ion

18F-

FLT

accu

mul

atio

n

18F-

FLT

accu

mul

atio

n

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




TM ratio(max)


TM ratio(max)


TM ratio(max)

(b)



5

0

11C-

Met

hion

ine

Control Treated SUV

(a)

5

0

Control Treated SUV

11C-

Met

hion

ine

(b)



SUV10

0

SUV10

0
















Abbreviations






Acknowledgments


References




































020406080

100120140160180

0 5 10 15 20 25 30Time (days)

Tum

or v

olum

e (m

m3)


lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowastlowast

(a)

0

20

40

60

80

100

120

140


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowast

Tum

or v

olum

e (m

m3)


(b)









4 Discussion




0

18FD

G18F-

FLT

SUV10

018FD

G18F-

FLT

(a)

0

1

2

3

4

5

6

7

8


TM ratio(max)

0

1

2

3

4

5

6

7

0123456789

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

18FD

G ac

cum

ulat

ion

0

1

2

3

4

5

6

18FD

G ac

cum

ulat

ion

18F-

FLT

accu

mul

atio

n

18F-

FLT

accu

mul

atio

n

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




TM ratio(max)


TM ratio(max)


TM ratio(max)

(b)



5

0

11C-

Met

hion

ine

Control Treated SUV

(a)

5

0

Control Treated SUV

11C-

Met

hion

ine

(b)



SUV10

0

SUV10

0
















Abbreviations






Acknowledgments


References





































0

18FD

G18F-

FLT

SUV10

018FD

G18F-

FLT

(a)

0

1

2

3

4

5

6

7

8


TM ratio(max)

0

1

2

3

4

5

6

7

0123456789

10

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowastlowast


lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowast

lowastlowastlowast

18FD

G ac

cum

ulat

ion

0

1

2

3

4

5

6

18FD

G ac

cum

ulat

ion

18F-

FLT

accu

mul

atio

n

18F-

FLT

accu

mul

atio

n

lowastlowastlowast

lowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast

lowast

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast


lowastlowastlowast




TM ratio(max)


TM ratio(max)


TM ratio(max)

(b)



5

0

11C-

Met

hion

ine

Control Treated SUV

(a)

5

0

Control Treated SUV

11C-

Met

hion

ine

(b)



SUV10

0

SUV10

0
















Abbreviations






Acknowledgments


References




































5

0

11C-

Met

hion

ine

Control Treated SUV

(a)

5

0

Control Treated SUV

11C-

Met

hion

ine

(b)



SUV10

0

SUV10

0
















Abbreviations






Acknowledgments


References











































Abbreviations






Acknowledgments


References



































18 FDG, [ 18 F]FLT, [ 18 F]FAZA, and 11 C-Methionine Are Suitable Tracers for the Diagnosis and In...

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