Preliminary 19F-MRS Study of Tumor Cell Proliferation with...

Research ArticlePreliminary 19F-MRS Study of Tumor Cell Proliferation with31015840-deoxy-31015840-fluorothymidine and Its Metabolite (FLT-MP)

In Ok Ko12 Ki-Hye Jung1 Mi Hyun Kim1 Kyeung Jun Kang1

Kyo Chul Lee1 Kyeong Min Kim3 Insup Noh4 Yong Jin Lee1 Sang Moo Lim5

Jung Young Kim1 and Ji-Ae Park1

1Division of RI-Convergence Research Korea Institute of Radiological amp Medical Sciences Seoul Republic of Korea2Convergence Institute of Biomedical Engineering and Biomaterials Seoul National University of Science amp TechnologySeoul Republic of Korea3Division of Medical Radiation Equipment Korea Institute of Radiological amp Medical Sciences Seoul Republic of Korea4Department of Chemical amp Biomolecular Engineering Seoul National University of Science amp Technology Seoul Republic of Korea5Department of Nuclear Medicine Korea Institute of Radiological amp Medical Sciences Seoul Republic of Korea

Correspondence should be addressed to Jung Young Kim jykimkiramsrekr and Ji-Ae Park jparkkiramsrekr

Received 11 May 2017 Accepted 17 July 2017 Published 26 September 2017

Academic Editor Giancarlo Pascali

Copyright copy 2017 In Ok Ko et alThis is an open access article distributed under the Creative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The thymidine analogue 31015840-deoxy-31015840-[18F]fluorothymidine or [18F]fluorothymidine ([18F]FLT) is used to measure tumor cellproliferation with positron emission tomography (PET) imaging technology in nuclear medicine FLT is phosphorylated bythymidine kinase 1 (TK1) and then trapped inside cells it is not incorporated into DNA Imaging with 18F-radiolabeled FLTis a noninvasive technique to visualize cellular proliferation in tumors However it is difficult to distinguish between [18F]FLTand its metabolites by PET imaging and quantification has not been attempted using current imaging methods In thisstudy we successfully acquired in vivo 19F spectra of natural or nonradioactive 31015840-deoxy-31015840-fluorothymidine ([19F]FLT) and itsmonophosphate metabolite (FLT-MP) in a tumor xenograft mouse model using 94T magnetic resonance imaging (MRI) Thispreliminary result demonstrates that 19F magnetic resonance spectroscopy (MRS) with FLT is suitable for the in vivo assessment oftumor aggressiveness and for early prediction of treatment response

1 Introduction

Tumor cell proliferation is a useful prognostic indicator oftumor aggressiveness and proliferation may be evaluatedto monitor and predict the response to antitumor therapyTumor cells and tissues with a high proliferation rate requirea high rate of DNA synthesis [1ndash5] Radiolabeled thymidineanalogues are standard biomarkers forDNA synthesis and aregenerally used in nuclearmedicine One thymidine analogue[11C]-labeled thymidine is well known as a radiotracerfor positron emission tomography (PET) studies of tumorcell proliferation and DNA synthesis [6ndash9] However theshort physical half-life (20min) of [11C]-thymidine and itsrapid biodegradation are practical limitations to its use

[4 10] Consequently the use of [18F]-labeled 31015840-deoxy-31015840-fluorothymidine ([18F]FLT) PET imaging to assess prolifer-ation in various tumors has been reported in preclinical andclinical studies [11ndash13] [18F]FLT in the cell is phosphorylatedby the enzyme thymidine kinase 1 (TK1) producing [18F]FLTmonophosphate ([18F]FLT-MP) [18F]FLT-MP can then besequentially phosphorylated to form [18F]FLT diphosphate([18F]FLT-DP) and [18F]FLT triphosphate ([18F]FLT-TP)which are metabolically trapped inside cells and are notincorporated into DNA (Figure 1) [14] Li et al demon-strated that metabolites of intracellular FLT during in vitrocell growth could be accurately measured with a liquidchromatography-tandem mass spectrometry (LC-MSMS)assay [15] However this technique is considered a restrictive

HindawiContrast Media amp Molecular ImagingVolume 2017 Article ID 3981358 7 pageshttpsdoiorg10115520173981358

2 Contrast Media amp Molecular Imaging

FLT FLT-MP

N

NH

O

O

O

HO

F

H3C

N

NH

O

O

OO

F

PHO

O

H3C

Ominus

Figure 1 Chemical structures of FLT and FLT-MP

method which is only used for in vitro drug screening at earlystages19F magnetic resonance imaging (19F MRI) and spec-

troscopy (MRS) represent a promising in vivo quantitativeimaging technique [16ndash18] The nonradioactive isotope 19Fhas a 100 natural abundance with 83 sensitivity of 1HThenegligible background signal of endogenous 19F in biologicalsystems provides an extremely high signal-to-nose ratioand exceptional sensitivity making 19F MRIMRS an idealmodality to monitor in vivo biochemical changes in specificenzyme activity cell tracking and migration hypoxia andquantitative neovascular responses [19 20]

In this study we monitored TK1 activity by quantifyingFLT and FLT-MP in vivo using 19F MRIMRS Our aim wasto develop and validate a suitable 19F MRIMRS imagingbiomarker for cellular proliferation in tumors

2 Results and Discussion

To detect the locations of FLT and FLT-MP we investigatedthe 19F MRS of compounds containing TFA (minus765 ppm) asa reference material Figures 2(a) and 2(b) show that thespectra of FLT and FLT-MP were observed at minus1762 ppmand minus1754 ppm respectively the values were consistent withthe NMR data (Figures S1 and S2 in Supplementary Mate-rial available online at httpsdoiorg10115520173981358)Figure 2(c) shows 19F MR images of phantoms containing25 50 100 and 200mM of FLT demonstrating that thesignal intensity of 19F MR images corresponded with FLTconcentration in phantoms (Figure 2(d)) Figure 2(e) showsthe spectrum of a mixture of FLT and FLT-MP which werewell separated at minus1762 ppm and minus1752 ppm respectivelyFigure 2(f) shows the 19F MRS of the mixture here theformer was FLT-MP and the latter was FLT Because thearea ratios of the spectra for the former and latter wereapproximately 60 and 100 respectively the findings wereconsistent with the concentrations in the mixture of FLT-MP(60mM) and FLT (100mM)

We investigated whether the 19F NMR or 19F MRS signalof intracellular FLT-MP produced as an FLT metabolitecould be detected in vitro In the first group of cells that werenot washed both FLT and FLT-MP were clearly observed in

the 19F NMR spectra although the FLT-MP peak was veryweak (Figure S3) However the signal for FLT in the cells wasvery strong and the concentration of FLT was 167mM Incontrast an FLT-MP peak in the first group of cells was notobserved in the 19F MRS only an FLT peak was observed(Figure S4)

Figure 3 shows the 19F NMR spectra of washed cells inthe second group as a function of time Both intracellularFLT and FLT-MP were clearly observed at minus1752 ppm andminus1745 ppm respectively Because the extra FLT was washedout the FLT signal exhibited a moderate level relative to thecells in the first group Although the extra FLT was washedout the presence of FLT demonstrated that FLT and itsmetabolites were reversible in the cell [2] The amounts ofintracellular FLT-MP and FLT were therefore inconsistentover time A relative ratio of FLT to FLT-MP here demon-strated the on-going phosphorylation of different spectra invarious tumors unlike PET technology

No peak was observed in the 19F MRS signal for intra-cellular FLT-MP formed in the second group of cells becauseof its low concentration These results demonstrated thata typical FLT concentration of 167mM is required for invivo detection by 19F MRS As previous reports in vivo 19FMR imaging is generally used for the high concentration of89mM due to the low sensitivity of that [21]

To chemically confirm the accuracy of quantitation andmetabolite detection by 19F MRS an HPLC assay was per-formed Figure 4 shows HPLC chromatograms for FLT (Rt71min) and FLT-MP (Rt 20min) the concentration wasapproximately 1 120583g120583L The in vitro HPLC chromatogram ofthe second group of cells demonstrated that FLT metabolismresulted in FLT-MP FLT-DP and FLT-TP production (Fig-ure 4(c))

We then investigated the in vivo 19F MR signals forFLT and FLT-MP More precisely we aimed to observethat the appearance of the FLT-MP signal is caused bymetabolism of FLT in vivo Figure 5(a) shows anatomical 1HMR images of a MCF-7 tumor in a mouse and the voxelof interest in the tumor for 19F MRS In the 25min afterinjection of FLT (200mM 100 120583L) a slight 19F signal wasobserved at minus17599 ppm (Figure 5(b)) corresponding withthe location of FLT After 90min the 19F signal was observedat minus17508 ppm (Figure 5(c)) Judging from the results of the

Contrast Media amp Molecular Imaging 3

minus76546

minus80 minus100 minus120 minus140 minus160 minus180

minus176254

(a)


minus76515

minus175411

(b)

25mM 50mM 100mM 200mM(c)

R2= 09981

50 100 2001500

FLT (mM)

0

5

10

SNR

15

20

25

(d)


minus76534

minus175250

minus176152

(e)

minus175 minus180

minus175247

(60 mM) (100 mM)

minus175998

FLT-MP FLT

(f)

Figure 2 Typical coil-localized 19F spectra of (a) FLT and (b) FLT-MP containing TFA as a reference (c) 19F MR images of phantomscontaining 25 50 100 and 200mM FLT (d) Signal intensity in 19F MR images of FLT phantoms as a function of FLT concentration (1198772 =0998) (e) Typical coil-localized 19F spectrum of a phantom containing a mixture of FLT (100mM) and FLT-MP (60mM) (f) 19F MRSspectrum of a phantom containing a mixture of FLT (100mM) and FLT-MP (60mM) The area ratio of FLT (100mM) to FLT-MP (60mM)is approximately 100 to 60

phantom study this signal represented FLT-MP despite beingvery weak

Experimentally speaking it was very difficult to accu-rately perform chemical shift imaging (CSI) of FLT and FLT-MP Our studies however first detected a remarkable 19FMR signal in the tumors of living mice thereby observingthe metabolism of FLT by 19F MRS in vivo Understandingthe metabolism of FLT in a tumor-bearing mouse modelmay help us associate metabolism with PET data from[18F]FLT a commonly used radiopharmaceutical in nuclearmedicine [18F]FLT is a good tracer of cell proliferation forassessment of tumor aggressiveness and early prediction oftreatment response [12] PET technology high sensitivity andthe radiation of positron-emitting radioisotopes can easilypermeate tissues making PET a powerful molecular imagingmodality tomonitor the progression of cancer [22] However

PET alone cannot readily distinguish between [18F]FLT and[18F]FLT-MP Specifically it is very difficult to simultaneouslyidentify metabolites in vivo by kinetic analysis of FLT-PETimages [8] In that respect our results show that 19F MRSis a noninvasive and practical way to identify biomoleculesin vivo including fluorine atoms it may thus be utilized tocomplement other imaging tools such as PET

MRIMRS is also a promisingmolecular imagingmethodfor cancer theragnostics [23 24] For example 13CMRIMRSstudy of hyperpolarized [13C]pyruvate and its metabolite([13C]lactate) could be recently used to measure earlyresponses to therapy and the utilization of metabolite levelshas been studied in clinical practice [25ndash27] The hyper-polarized 13C compounds however have restriction on themetabolism studies of DNA synthesis due to a time limit ofhyperpolarization The results of the present study though


K4

7

3 3

7

K3

8 8

K2

K1

K5Cell only

(c) Cell

FLT-MP

(b) FLT-MP

FLT(a) FLT

7

6

5

4

3

2

1109

100

100

100

100

078

064

081

Cell + FLT 60min

Cell + FLT 120 min

minus1720 minus1780 minus1790minus1710 minus1760 minus1770minus1740minus1730 minus1750

(ppm)

Cell + FLT 30min

(e) Cell + FLT 30min

Cell + FLT 5min

(d) Cell + FLT 5min

(g) Cell + FLT 120 min

(f) Cell + FLT 60 min

Figure 3 19F NMR spectra of MCF-7 cell suspensions treated with FLT (01mg1 times 107 cells) as a function of time (dndashg) The quantificationin a relative ratio of FLT to FLT-MP was indicated Typical spectra of (a) FLT (minus1754 ppm) (b) FLT-MP (minus1744 ppm) and (c) MCF-7 cellswithout FLT addition

preliminary demonstrate that detection of [19F]FLT and itsmetabolite using 19F MRS might provide a novel avenue forcancer theragnostics

3 Conclusion

In this study FLT and its metabolite were measured for thefirst time in an in vivo mouse model using 19F MRS Thisresult showed that 19F MRS is suitable for the purpose of invivomonitoring of specific drugs including radiopharmaceu-ticals and their metabolites In addition the findings of thisstudy may support the clinical use of 19F MRIMRS for thequantification and monitoring of the cellular proliferation incancer and to assess the effectiveness of responses to therapyFurther studies are needed to improve the 19F MRS and CSItechniques for in vivo detection

4 Materials and Methods

41 Chemicals All reagents were purchased from commer-cial sources and the following agents were FLT and FLT-MP (Research Center FutureChem Co Ltd Seoul Korea)and trifluoroacetic acid (TFA) (Sigma-Aldrich St LouisMO)

42 High-Performance Liquid Chromatography (HPLC) Thelocations of compound were confirmed by analytical HPLC

using an Atlantis C18analytical column (5 120583m 30 times 150mm)

with 10 EtOH in water (vv) as the mobile phase at a flowrate of 04mLminThe retention times (119877

119905) for FLT and FLT-

MP were 71min and 2min respectively

43 Cell Culture The MCF-7 human breast cancer cell lineexpressing the HSV-tk gene was maintained in RPMI-1640medium supplemented with 10 FBS 1 antibiotics and100 120583gmL of G418 (Invitrogen) Cultures were maintained ina 37∘C incubator with 5CO

2 and themediumwas changed

every 3 daysFor 19F MRS MCF-7 cells were plated and 5 times 107

cells were suspended in 500120583L of serum-free RPMI mediumcontaining FLT (167mM) before being incubated at 37∘C fordifferent time periods (5min 30min 60min and 120min)The cells were divided into two groupsThe first group of cellswas not washed and was used for 19F NMR and 19F MRSThe second group of cells was washed three times with PBSscraped from the plate centrifuged at 1000 rpm for 3minand then collected for use in 19F NMR 19F MRS and HPLCanalyses

For HPLC the pellets were resuspended in PBS to a finalvolume of 1mL andwere then lysed by three cycles of freezingand thawing the lysates were centrifuged at 14000 rpm for5min at 4∘CThe supernatant was used for HPLC analysis

FLT and FLT-MP were extracted from the samples aftergrowth for 5min 30min 60min or 120min by three cycles


800400 600 1000 1200200000

(Minutes)

0000

0010

0020

0030

0040

0050

0060

0070

0080

0090

0100

(AU

)

0110

0120

0130

0140

0150

0160

0170

(a)

800200000 600400 12001000

(Minutes)

0000

0005

0010

0015

0020

0025

0030

0035

(AU

)

0040

0045

0050

0055

0060

0065

0070

0075

0080

0085

(b)

0000

0001

0002

0003

0004

0005

0006(AU

)

0007

0008

0009

0010

0011

0012

0013

0014

200 800000 400 600 12001000

(Minutes)

(c)

Figure 4 HPLC chromatograms of (a) FLT (Rt 71min) (b) FLT-MP (Rt 20min) and (c) MCF-7 cells treated with FLT

of freezing and thawing After centrifugation (14000 rpm for5min at 4∘C) the samples comprising a 90 10 mixture ofsupernatant D

2O were placed in 5mm nuclear magnetic

resonance (NMR) tubes for data acquisition

44 NMR The 19F NMR measurements were conducted at28∘C on a Bruker 400-MHz NMR spectrometer equippedwith a 5-mmBBFOprobeThe experimental parameters wereas follows pulse angle 90∘ (1832 120583sec) repetition rate 1 sec172 K data set 2000 scans All 19F data were processed usingTopSpin and analyzed with MestReNova

45 Animals All animal experiments were conducted incompliance with the Guidelines for the Care and Use ofResearch Animals under protocols approved by the KoreaInstitute of Radiological and Medical Sciences (KIRAMSrsquo)Animal Studies Committee

MCF-7 tumor cells (106 cellsmL) suspended in RPMIserum-depletion media were inoculated into the subcuta-neous tissue (sc) of female BALBc nude mice (6 weeks20ndash25 g of body mass)Themice were anesthetized with 15

isoflurane Tomonitor the formation of FLT-MP 1HMRI and19FMRSwere performed after intravenous bolus injections ofFLT (200mM 100 120583L)

46 MRI All 1HMRI and 19FMRIMRS data were acquiredwith a 94T animal MRI system and 20mm surface coil(370ndash420MHz) (Agilent Technologies USA)1H MR images were acquired with a fast spin echo

sequence using the following settings repetition time (TR)2500ms echo time (TE) 25msmatrix 256times 256 field of view(FOV) 5 times 5 cm2 slice thickness 2mm gap 0mm averages 2and scan time 2min 45 sec19FMR images of phantomwere acquired with a gradient

echo sequence using the following settings TR 100ms TE40ms matrix 64 times 64 FOV 5 times 5 cm2 slice thickness 2mmgap 0mm averages 1200 and scan time 2 h 8min19FMRS of phantoms and in vivo data were acquiredwith

Point-REsolved Spectroscopy (PRESS) using the followingsettings TR 3000ms TE 15ms voxel size 5 times 5 times 5mm3averages 512 and scan time 25min


(a) (b)

(c)

Figure 5 In vivo 19FMR spectrum in amouse tumormodel FLT (200mM 100 120583L) was injected into tail veins (a) Anatomical 1HMR imagesof the mouse were obtained using fast spin echo sequence with the voxel of interest in the tumor (5 times 5 times 5mm3) Time-course of 19F MRspectra at (b) 25min after injection (ai) (minus1759 ppm) and (c) 90min ai (minus17508 ppm) 19F MRS data were acquired with Point-REsolvedSpectroscopy (PRESS) using TR 3000ms TE 15ms averages 512 scan time 25min

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

This research was supported by a grant of the Korea Instituteof Radiological and Medical Sciences (KIRAMS) funded bythe Ministry of Science ICT amp Future Planning Republic ofKorea (no 171104553917110454150461-2017)

References

[1] J L Schwartz Y Tamura R Jordan J R Grierson and K AKrohn ldquoMonitoring tumor cell proliferation by targeting DNAsynthetic processes with thymidine and thymidine analogsrdquoJournal of Nuclear Medicine vol 44 pp 2027ndash2032 2003

[2] J R Bading and A F Shields ldquoImaging of cell proliferationstatus and prospectsrdquo Journal of Nuclear Medicine vol 49supplement 2 pp 64Sndash80S 2008

[3] V W Hu G E Black A Torres-Duarte and F P Abramsonldquo3H-thymidine is a defective tool with which to measure rates

of DNA synthesisrdquoThe FASEB journal official publication of theFederation of American Societies for Experimental Biology vol16 no 11 pp 1456-1457 2002

[4] S Nimmagadda and A F Shields ldquoThe role of DNA synthesisimaging in cancer in the era of targeted therapeuticsrdquo Cancerand Metastasis Reviews vol 27 no 4 pp 575ndash587 2008

[5] L M Kenny E O Aboagye and PM Price ldquoPositron emissiontomography imaging of cell proliferation in oncologyrdquo ClinicalOncology vol 16 no 3 pp 176ndash185 2004

[6] D Christman E J Crawford M Friedkin and A P WolfldquoDetection of DNA Synthesis in Intact Organisms withPositron-Emitting [Methyl-11C]Thymidinerdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 69 no 4 pp 988ndash992 1972

[7] T Tahara Z Zhang M Ohno et al ldquoA novel 11C-labeledthymidine analog [11C]AZT for tumor imaging by positronemission tomographyrdquoEJNMMIResearch vol 5 124 no 1 2015

[8] D A Mankoff A F Shields J M Link et al ldquoKinetic analysisof 2-[11C]thymidine PET imaging studies Validation studiesrdquoJournal of Nuclear Medicine vol 40 no 4 pp 614ndash624 1999

[9] J Toyohara M Okada C Toramatsu K Suzuki and T IrieldquoFeasibility studies of 41015840-[methyl-11C]thiothymidine as a tumor


proliferation imaging agent in micerdquo Nuclear Medicine andBiology vol 35 no 1 pp 67ndash74 2008

[10] DAMankoffA F ShieldsMMGraham JM Link J F Earyand K A Krohn ldquoKinetic analysis of 2-[carbon-11]thymidinePET imaging studies Compartmental model andmathematicalanalysisrdquo Journal of Nuclear Medicine vol 39 no 6 pp 1043ndash1055 1998

[11] A F Shields ldquoPET imaging with 18F-FLT and thymidineanalogs promise and pitfallsrdquoThe Journal of Nuclear Medicinevol 44 no 9 pp 1432ndash1434 2003

[12] W Chen T Cloughesy N Kamdar et al ldquoImaging proliferationin brain tumors with 18F-FLT PET comparison with 18F-FDGrdquoJournal of Nuclear Medicine vol 46 no 6 pp 945ndash952 2005

[13] A F Shields J R Grierson B M Dohmen et al ldquoImagingproliferation in vivo with [F-18]FLT and positron emissiontomographyrdquoNatureMedicine vol 4 no 11 pp 1334ndash1336 1998

[14] E T McKinley G D Ayers R A Smith et al ldquoLimits of [18F]-FLT PET as a Biomarker of Proliferation in Oncologyrdquo PLoSONE vol 8 no 3 Article ID e58938 2013

[15] W Li M Araya M Elliott et al ldquoMonitoring cellular accumu-lation of 3-deoxy-3-fluorothymidine (FLT) and its monophos-phate metabolite (FLT-MP) by LC-MSMS as a measure of cellproliferation in vitrordquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 879 no 28pp 2963ndash2970 2011

[16] J Ruiz-Cabello B P Barnett P A Bottomley and J W MBulte ldquoFluorine (19F) MRS and MRI in biomedicinerdquo NMR inBiomedicine vol 24 no 2 pp 114ndash129 2011

[17] R B van Heeswijk Y Pilloud U Flogel J Schwitter and MStuber ldquoFluorine-19 magnetic resonance angiography of themouserdquo PLoS ONE vol 7 no 7 Article ID e42236 2012

[18] I Tirotta V Dichiarante C Pigliacelli et al ldquo(19)F magneticresonance imaging (MRI) from design of materials to clinicalapplicationsrdquo Chemical Reviews vol 115 no 2 pp 1106ndash11292015

[19] X Yue Z Wang L Zhu et al ldquoNovel 19F activatable probefor the detection of matrix metalloprotease-2 activity byMRIMRSrdquo Molecular Pharmaceutics vol 11 no 11 pp 4208ndash4217 2014

[20] C Gonzales H A I Yoshihara N Dilek et al ldquoIn-vivo detec-tion and tracking of T cells in various organs in a melanomatumor model by 19F-fluorine MRSMRIrdquo PLoS ONE vol 11 no10 Article ID e0164557 2016

[21] W Yu Y Yang S Bo et al ldquoDesign and Synthesis of FluorinatedDendrimers for Sensitive 19F MRIrdquo Journal of Organic Chem-istry vol 80 no 9 pp 4443ndash4449 2015

[22] K A Krohn D A Mankoff and J F Eary ldquoImaging cellularproliferation as a measure of response to therapyrdquo Journal ofClinical Pharmacology vol 41 (suppl) pp 96Sndash103S 2001

[23] P Caravan ldquoStrategies for increasing the sensitivity of gadolin-ium based MRI contrast agentsrdquo Chemical Society Reviews vol35 no 6 pp 512ndash523 2006

[24] W Negendank ldquoStudies of human tumors by MRS A reviewrdquoNMR in Biomedicine vol 5 no 5 pp 303ndash324 1992

[25] M J Albers R Bok A P Chen et al ldquoHyperpolarized 13Clactate pyruvate and alanine Noninvasive biomarkers forprostate cancer detection and gradingrdquoCancer Research vol 68no 20 pp 8607ndash8615 2008

[26] J D MacKenzie Y-F Yen D Mayer J S Tropp R E Hurdand D M Spielman ldquoDetection of inflammatory arthritisby using hyperpolarized 13C-pyruvate with MR imaging andspectroscopyrdquo Radiology vol 259 no 2 pp 414ndash420 2011

[27] K R Keshari and D M Wilson ldquoChemistry and biochemistryof 13C hyperpolarized magnetic resonance using dynamicnuclear polarizationrdquo Chemical Society Reviews vol 43 no 5pp 1627ndash1659 2014

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OncologyJournal of



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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


FLT FLT-MP

N

NH

O

O

O

HO

F

H3C

N

NH

O

O

OO

F

PHO

O

H3C

Ominus

Figure 1 Chemical structures of FLT and FLT-MP

method which is only used for in vitro drug screening at earlystages19F magnetic resonance imaging (19F MRI) and spec-

troscopy (MRS) represent a promising in vivo quantitativeimaging technique [16ndash18] The nonradioactive isotope 19Fhas a 100 natural abundance with 83 sensitivity of 1HThenegligible background signal of endogenous 19F in biologicalsystems provides an extremely high signal-to-nose ratioand exceptional sensitivity making 19F MRIMRS an idealmodality to monitor in vivo biochemical changes in specificenzyme activity cell tracking and migration hypoxia andquantitative neovascular responses [19 20]

In this study we monitored TK1 activity by quantifyingFLT and FLT-MP in vivo using 19F MRIMRS Our aim wasto develop and validate a suitable 19F MRIMRS imagingbiomarker for cellular proliferation in tumors

2 Results and Discussion

To detect the locations of FLT and FLT-MP we investigatedthe 19F MRS of compounds containing TFA (minus765 ppm) asa reference material Figures 2(a) and 2(b) show that thespectra of FLT and FLT-MP were observed at minus1762 ppmand minus1754 ppm respectively the values were consistent withthe NMR data (Figures S1 and S2 in Supplementary Mate-rial available online at httpsdoiorg10115520173981358)Figure 2(c) shows 19F MR images of phantoms containing25 50 100 and 200mM of FLT demonstrating that thesignal intensity of 19F MR images corresponded with FLTconcentration in phantoms (Figure 2(d)) Figure 2(e) showsthe spectrum of a mixture of FLT and FLT-MP which werewell separated at minus1762 ppm and minus1752 ppm respectivelyFigure 2(f) shows the 19F MRS of the mixture here theformer was FLT-MP and the latter was FLT Because thearea ratios of the spectra for the former and latter wereapproximately 60 and 100 respectively the findings wereconsistent with the concentrations in the mixture of FLT-MP(60mM) and FLT (100mM)

We investigated whether the 19F NMR or 19F MRS signalof intracellular FLT-MP produced as an FLT metabolitecould be detected in vitro In the first group of cells that werenot washed both FLT and FLT-MP were clearly observed in

the 19F NMR spectra although the FLT-MP peak was veryweak (Figure S3) However the signal for FLT in the cells wasvery strong and the concentration of FLT was 167mM Incontrast an FLT-MP peak in the first group of cells was notobserved in the 19F MRS only an FLT peak was observed(Figure S4)

Figure 3 shows the 19F NMR spectra of washed cells inthe second group as a function of time Both intracellularFLT and FLT-MP were clearly observed at minus1752 ppm andminus1745 ppm respectively Because the extra FLT was washedout the FLT signal exhibited a moderate level relative to thecells in the first group Although the extra FLT was washedout the presence of FLT demonstrated that FLT and itsmetabolites were reversible in the cell [2] The amounts ofintracellular FLT-MP and FLT were therefore inconsistentover time A relative ratio of FLT to FLT-MP here demon-strated the on-going phosphorylation of different spectra invarious tumors unlike PET technology

No peak was observed in the 19F MRS signal for intra-cellular FLT-MP formed in the second group of cells becauseof its low concentration These results demonstrated thata typical FLT concentration of 167mM is required for invivo detection by 19F MRS As previous reports in vivo 19FMR imaging is generally used for the high concentration of89mM due to the low sensitivity of that [21]

To chemically confirm the accuracy of quantitation andmetabolite detection by 19F MRS an HPLC assay was per-formed Figure 4 shows HPLC chromatograms for FLT (Rt71min) and FLT-MP (Rt 20min) the concentration wasapproximately 1 120583g120583L The in vitro HPLC chromatogram ofthe second group of cells demonstrated that FLT metabolismresulted in FLT-MP FLT-DP and FLT-TP production (Fig-ure 4(c))

We then investigated the in vivo 19F MR signals forFLT and FLT-MP More precisely we aimed to observethat the appearance of the FLT-MP signal is caused bymetabolism of FLT in vivo Figure 5(a) shows anatomical 1HMR images of a MCF-7 tumor in a mouse and the voxelof interest in the tumor for 19F MRS In the 25min afterinjection of FLT (200mM 100 120583L) a slight 19F signal wasobserved at minus17599 ppm (Figure 5(b)) corresponding withthe location of FLT After 90min the 19F signal was observedat minus17508 ppm (Figure 5(c)) Judging from the results of the


minus76546


minus176254

(a)


minus76515

minus175411

(b)

25mM 50mM 100mM 200mM(c)

R2= 09981

50 100 2001500

FLT (mM)

0

5

10

SNR

15

20

25

(d)


minus76534

minus175250

minus176152

(e)

minus175 minus180

minus175247

(60 mM) (100 mM)

minus175998

FLT-MP FLT

(f)







K4

7

3 3

7

K3

8 8

K2

K1

K5Cell only

(c) Cell

FLT-MP

(b) FLT-MP

FLT(a) FLT

7

6

5

4

3

2

1109

100

100

100

100

078

064

081

Cell + FLT 60min

Cell + FLT 120 min


(ppm)

Cell + FLT 30min


Cell + FLT 5min

(d) Cell + FLT 5min





3 Conclusion
















800400 600 1000 1200200000

(Minutes)

0000

0010

0020

0030

0040

0050

0060

0070

0080

0090

0100

(AU

)

0110

0120

0130

0140

0150

0160

0170

(a)

800200000 600400 12001000

(Minutes)

0000

0005

0010

0015

0020

0025

0030

0035

(AU

)

0040

0045

0050

0055

0060

0065

0070

0075

0080

0085

(b)

0000

0001

0002

0003

0004

0005

0006(AU

)

0007

0008

0009

0010

0011

0012

0013

0014

200 800000 400 600 12001000

(Minutes)

(c)














(a) (b)

(c)




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















minus76546


minus176254

(a)


minus76515

minus175411

(b)

25mM 50mM 100mM 200mM(c)

R2= 09981

50 100 2001500

FLT (mM)

0

5

10

SNR

15

20

25

(d)


minus76534

minus175250

minus176152

(e)

minus175 minus180

minus175247

(60 mM) (100 mM)

minus175998

FLT-MP FLT

(f)







K4

7

3 3

7

K3

8 8

K2

K1

K5Cell only

(c) Cell

FLT-MP

(b) FLT-MP

FLT(a) FLT

7

6

5

4

3

2

1109

100

100

100

100

078

064

081

Cell + FLT 60min

Cell + FLT 120 min


(ppm)

Cell + FLT 30min


Cell + FLT 5min

(d) Cell + FLT 5min





3 Conclusion
















800400 600 1000 1200200000

(Minutes)

0000

0010

0020

0030

0040

0050

0060

0070

0080

0090

0100

(AU

)

0110

0120

0130

0140

0150

0160

0170

(a)

800200000 600400 12001000

(Minutes)

0000

0005

0010

0015

0020

0025

0030

0035

(AU

)

0040

0045

0050

0055

0060

0065

0070

0075

0080

0085

(b)

0000

0001

0002

0003

0004

0005

0006(AU

)

0007

0008

0009

0010

0011

0012

0013

0014

200 800000 400 600 12001000

(Minutes)

(c)














(a) (b)

(c)




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















K4

7

3 3

7

K3

8 8

K2

K1

K5Cell only

(c) Cell

FLT-MP

(b) FLT-MP

FLT(a) FLT

7

6

5

4

3

2

1109

100

100

100

100

078

064

081

Cell + FLT 60min

Cell + FLT 120 min


(ppm)

Cell + FLT 30min


Cell + FLT 5min

(d) Cell + FLT 5min





3 Conclusion
















800400 600 1000 1200200000

(Minutes)

0000

0010

0020

0030

0040

0050

0060

0070

0080

0090

0100

(AU

)

0110

0120

0130

0140

0150

0160

0170

(a)

800200000 600400 12001000

(Minutes)

0000

0005

0010

0015

0020

0025

0030

0035

(AU

)

0040

0045

0050

0055

0060

0065

0070

0075

0080

0085

(b)

0000

0001

0002

0003

0004

0005

0006(AU

)

0007

0008

0009

0010

0011

0012

0013

0014

200 800000 400 600 12001000

(Minutes)

(c)














(a) (b)

(c)




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















800400 600 1000 1200200000

(Minutes)

0000

0010

0020

0030

0040

0050

0060

0070

0080

0090

0100

(AU

)

0110

0120

0130

0140

0150

0160

0170

(a)

800200000 600400 12001000

(Minutes)

0000

0005

0010

0015

0020

0025

0030

0035

(AU

)

0040

0045

0050

0055

0060

0065

0070

0075

0080

0085

(b)

0000

0001

0002

0003

0004

0005

0006(AU

)

0007

0008

0009

0010

0011

0012

0013

0014

200 800000 400 600 12001000

(Minutes)

(c)














(a) (b)

(c)




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c)




Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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