Abstract BRIEF ARTICLE. Xin-Hong He, Wen-Tao Li, Ya-Jia Gu, Bao-Feng Yang, Hui-Wen Deng, Yi-Hua Yu, Wei-Jun Peng

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1 Online Submissions: doi: /wjg.v19.i World J Gstroenterol 2013 July 14; 19(26): ISSN (print) ISSN (online) 2013 Bishideng. All rights reserved. BRIEF ARTICLE Metbonomic studies of pncretic cncer response to rdiotherpy in mouse xenogrft model using mgnetic resonnce spectroscopy nd principl components nlysis Xin-ong e, Wen-To Li, Y-Ji Gu, Bo-Feng Yng, ui-wen Deng, Yi-u Yu, Wei-Jun Peng Xin-ong e, Wen-To Li, Y-Ji Gu, Bo-Feng Yng, Wei- Jun Peng, Deprtment of Rdiology, Fudn University Shnghi Cncer Center, Shnghi , Chin ui-wen Deng, Yi-u Yu, Shnghi Key Lbortory of Functionl Mgnetic Resonnce Imging, Physics Deprtment, Est Chin Norml University, Shnghi , Chin Author contributions: e X performed the mjority of the experiments; Li WT, Gu YJ, Yng BF, Deng W nd Yu Y provided vitl regents nd nlyticl tools, nd were lso involved in editing the mnuscript; Peng WJ designed the study nd wrote the mnuscript. Supported by The Medicl Imgeology Specil Purpose Foundtion of Cncer ospitl/cncer Institute Fudn University, No. YX Correspondence to: Wei-Jun Peng, MD, Deprtment of Rdiology, Fudn University Shnghi Cncer Center, No. 270, Dong n Rod, Shnghi , Chin. weijunpeng378@gmil.com Telephone: Fx: Received: December 25, 2012 Revised: Mrch 22, 2013 Accepted: April 27, 2013 Published online: July 14, 2013 Abstrct AIM: To investigte the metbolic profiles of xenogrft pncretic cncer before nd fter rdiotherpy by high-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy (RMAS 1 NMR) combined with principl components nlysis (PCA) nd evlute the rdiotherpeutic effect. METODS: The nude mouse xenogrft model of humn pncretic cncer ws estblished by injecting humn pncretic cncer cell SW1990 subcutneously into the nude mice. When the tumors volume reched 800 mm 3, the mice received vrious rdition doses. Two weeks lter, tumor tissue sections were prepred for running the NMR mesurements. 1 NMR nd PCA were used to determine the chnges in the metbolic profiles of tumor tissues fter rdiotherpy. Metbolic profiles of norml pncres, pncretic tumor tissues, nd rdition- treted pncretic tumor tissues were compred. RESULTS: Compred with 1 NMR spectr of the norml nude mouse pncres, the levels of choline, turine, lnine, isoleucine, leucine, vline, lctte, nd glutmic cid of the pncretic cncer group were incresed, wheres n opposite trend for phosphocholine, glycerophosphocholine, nd betine ws observed. The rtio of phosphocholine to cretine, nd glycerophosphocholine to cretine showed noticeble decrese in the pncretic cncer group. After further evlution of the tissue metbolic profile fter tretment with three different rdition doses, no significnt chnge in metbolites ws observed in the 1 NMR spectr, while the inhibition of tumor growth ws in proportion to the rdition doses. owever, PCA results showed tht the levels of choline nd betine were decresed with the incresed rdition dose, nd conversely, the level of cetic cid ws drmticlly incresed. CONCLUSION: The combined methods were demonstrted to hve the potentil for llowing erly dignosis nd ssessment of pncretic cncer response to rdiotherpy Bishideng. All rights reserved. Key words: igh-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy; Principl components nlysis; Pncretic cncer; Rdiotherpy Core tip: In the present study, for the first time to our knowledge, high-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy nd principl components nlysis were combined to highlight metbolite profiles of pncretic cncer fter rdiotherpy, 4200 July 14, 2013 Volume 19 Issue 26

2 e X et l. 1 NMR nd metbonomic studies of pncretic cncer by nlyzing the correltion between rdiotherpy effect nd metbolic chnge, nd optimizing the therpeutic scheme. The results showed tht metbolic profile chnges of pncretic cncer fter rdiotherpy were closely correlted with therpeutic effect. The outcome of the study is both interesting nd beneficil to pthologicl reserch, erly dignosis, nd therpy evlution of pncretic diseses. e X, Li WT, Gu YJ, Yng BF, Deng W, Yu Y, Peng WJ. Metbonomic studies of pncretic cncer response to rdiotherpy in mouse xenogrft model using mgnetic resonnce spectroscopy nd principl components nlysis. World J Gstroenterol 2013; 19(26): Avilble from: URL: DOI: INTRODUCTION Pncretic cncer is mlignnt tumor with very poor prognosis, nd surgery hs been considered s the only rdicl therpy. owever, bout 85% of newly dignosed cses hve developed distnt metstsis, nd only 5%-25% of pncretic hed cncer nd less thn 10% of pncretic body cncer cn be treted with surgicl excision, nd the postopertive recurrence rte is high. Therefore, rdition therpy hs become the predominnt tretment method for loclly dvnced pncretic cncer [1-3]. Therpeutic evlutions of rdiotherpy re minly: remission from the symptoms of pin nd jundice, solid tumor size nd its survivl time, nd the lck of specific trgeted method. During the lst three decdes, there hs been ongoing mgnetic resonnce spectroscopy (MRS) reserch in mlignnt diseses. These studies provided vluble dt on the biochemistry nd metbolism of tumors, nd on the effects of nutrients, hormones, nd growth fctors [4,5]. The mechnisms of ction of nticncer drugs nd the cquired resistnce to these gents were delineted [6,7]. MRS ws lso used for monitoring the response to therpy [8,9]. igh-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy (RMAS 1 NMR) is well-recognized technique in metbonomics studies in vitro, by which biopsy or postmortem smples of intct tissues re spun t the mgic ngle, resulting in significnt improvement in the resolution of the spectrum obtined for some of the line-brodening fctors, such s dipole dipole interctions nd chemicl shift nisotropy, nd mgnetic field inhomogeneities re verged out [10,11]. This pproch requires miniml smple preprtion nd, unlike convenient 1 NMR spectroscopy of tissue extrcts, both queous nd lipid-soluble metbolites cn be observed simultneously in situ. In ddition, informtion bout the metbolic environment of the tumor cn lso be obtined. Therefore, RMAS 1 NMR hs proved to be n efficient method for studying wide vriety of cncers, including brest cncer [12], cervicl cncer [13], kidney cncer [14], prostte cncer [15], mlignnt lymph nodes [16], nd liposrcom [17] of nimls nd humns. owever, so fr, there re very few metbonomic studies in cncer therpeutics by the ppliction of RMAS 1 NMR. RMAS 1 NMR spectr obtined from tissues reflect the dynmic biologicl systems nd processes tht contribute to the overll metbolic sttus of n orgnism. It is not possible to isolte the effects of ny single metbolite signl in spectrum nd, furthermore, the mnul nlysis of even smll number of such spectr is lborious nd complex tsk. Therefore, metbonomists utilize dt reduction nd multivrite nlysis techniques, such s principl components nlysis (PCA), to fcilitte utomted NMR pttern recognition [18,19]. Moreover, our previous study demonstrted tht using 1 NMR nd PCA could discriminte pncretic cncer from chronic pncretitis ccurtely [20]. In the present study, RMAS 1 NMR nd PCA were combined to highlight metbolite profiles of pncretic cncer fter rdiotherpy, in order to nlyze the correltion between rdiotherpy effects nd metbolic chnges, nd to optimize the therpeutic scheme. The study hs n importnt impliction for reference guides on therpeutic evlution by nucler mgnetic resonnce spectroscopy on pncretic cncer in vivo. MATERIALS AND METODS Animls nd experiment schedule Six- to eight-week-old femle nude mice were obtined from the Plnned Prenthood Reserch Institute, Shnghi, People s Republic of Chin. All nimls in this study were housed under pthogen-free conditions nd mintined in ccordnce with the guidelines of the Committee on Animls of the Second Militry Medicl University, Shnghi, Chin. umn pncretic cncer cell line SW1990 in mid-log-growth phse ws hrvested by trypsiniztion. Single-cell suspensions ( cells in 0.1 ml BSS) were injected subcutneously into the nude mice. The tumors were mesured every 4 d with cliper, nd the dimeters were recorded. Tumor volume ws clculted by the formul: 2 b/2, where nd b re the two mximum dimeters. When tumors reched 2.0 cm 2.0 cm, the durtion of survivl ws recorded nd the mouse euthnized. For the rdiotherpy experiment, when the tumor volume reched 800 mm 3, the mice were divided into four groups. Group A mice were used s untreted controls. Groups B, C, nd D received 10, 20, nd 30 Gy rdition doses, respectively. Tumor size ws mesured s described bove. Two weeks lter, tumor tissue sections were prepred for histologicl tests or for running the NMR mesurements. NMR spectroscopy RMAS 1 NMR experiments were crried out using DRX-500 spectrometer (1 frequency t Mz; 4201 July 14, 2013 Volume 19 Issue 26

3 e X et l. 1 NMR nd metbonomic studies of pncretic cncer Tumor volume (mm 3 ) Untretment 10 Gy 20 Gy 30 Gy Dys fter tretment Figure 1 Effect of rdiotherpy on the growth of humn pncretic tumor in nude mouse. Mice received subcutneous injection of SW1990 cells. When the tumor volume reched bout 800 mm 3, the mice were divided into four groups. Group A mice were used s untreted controls. Groups B, C, nd D received 10, 20, nd 30 Gy rdition doses, respectively. Tumor size ws mesured for two weeks. P < 0.05 vs the untreted group. Bruker Biospin, Rheinstetten, Germny). Tissue smples were rinsed three times with D2O nd plced into 4-mm zirconium oxide MAS rotor with drops of D2O (deuterium lock reference). Spectr were cquired t K using single-pulse nd CPMG pulse sequences, both with wter presturtion during the relxtion dely of 2 s. CPMG pulse sequence ws pplied s T2 filter to suppress signls from the molecules with short T2 vlues (such s mcromolecules nd lipids) using totl TE of 320 ms. The min prmeters used for 1 NMR spectr were: SW = 15 kz; TD = 64 k; NS = 256; nd MAS rte = 5 kz. Spectrl ssignments were confirmed by 2-dimensionl 1-1 TOCSY nd 1-1 COSY (dt not shown), together with vlues obtined from the literture [10,21]. The stbility of tissue smples ws evluted by repeting 1-dimensionl NMR experiment fter overll cquisition. No biochemicl degrdtion ws observed for ny of the tissue smples. Principl components nlysis Spectrl dt were phsed nd bseline-corrected using XWINNMR (Bruker Biospin). All FID were multiplied by n exponentil function equivlent to 0.3-z line brodening fctor prior to Fourier trnsformtion. Ech RMAS 1 NMR spectrum ws segmented into 211 regions of equl width (0.04 ppm) over the region , nd the signl intensity in ech region ws integrted using AMIX version 3.6 (Bruker, Biospin). The region ws removed to eliminte bseline effects of imperfect wter sturtion. Prior to PCA, ech integrl region ws normlized by dividing by the sum of ll integrl regions for ech spectrum [12,14]. In order to exclude the effects of lipids nd concentrte on the impcts of LMW metbolites in the CCM region, PCA ws gin done for 1 CPMG NMR spectr over the rnge , ech 0.04 ppm wide. PCA ws used to clculte new, smller set of orthogonl vribles from liner combintions of the intensity vribles, while retining the mximum vribility present within the dt. These new vribles re the derived principl components, nd the distribution of their vlues (scores) permits the simple visuliztion of seprtion or clustering between smples. The weightings (lodings) given to ech integrl region in clculting the principl components llows for the identifiction of those spectrl regions of gretest influence to the seprtion nd clustering nd, hence, the deduction of biomrkers of pncretic cncer. Sttisticl nlysis Continuous vribles re expressed s men ± SD. Sttisticl nlysis of dt ws done by Student s t test using SigmPlot softwre. Differences were considered sttisticlly significnt t P < RESULTS Rdiotherpy of humn pncretic tumor-bering nude mouse One week fter SW1990 tumor cell inocultion, tumor size ws mesured nd tumor volume recorded weekly. All 32 nude mouse models generted tumor tissues, nd the success rte of model construction ws 100% (32/32). Tumor volume in the control group (untreted), nd the three groups which were given 10, 20, nd 30 Gy rdition re shown in Figure 1. The trnsplnted tumor volume before tretment ws 0.8 cm 3 on verge, incresing with breeding period in the control group. Compred with the control group, the tumor volume of the tretment groups reduced significntly, with the most obvious being the 30 Gy dose tretment group. These dt showed tht rdiotherpy could effectively suppress the growth of pncretic cncer in the nude mice. The chnges in the morphologicl levels re expected to be ccompnied with observble chnges in the tissue biochemicl composition which cn be ccessed with RMAS 1 NMR spectroscopy ex vivo. Metbolic profiles of norml pncres nd pncretic tumor tissues Using 1 NMR spectroscopy, components such s Cho, turine (Tu), betine (Bet), glutmic cid (Glu), glycerophosphocholine, nd choline phosphte (GPC + PC), cetic cid (Ace), lnine (Al), nd lctic cid (Lc) were detected nd identified in the norml pncres nd isolted trnsplnted tumor tissues in the nude mouse by their spectrum peks. The literture ws referred to before (18-20) nd 2-D spectrum estimtion (J-res, COSY, TOCSY) (Figure 2A). Score plots of PCA bsed on 1 NMR spectr were performed on 8 norml nd 8 tumor smples, in which the spectr region ws = , nd the miniml region = 0.04 (Figure 2B). As shown in the loding plots, the min fctors tht differentited the smples were , , , , , nd , which were con July 14, 2013 Volume 19 Issue 26

4 e X et l. 1 NMR nd metbonomic studies of pncretic cncer A (c) GPC PC GPC PC (d)? Al G TMAO&Bet () Tu Lc Gly Cho G Lc Cr Cr Glx Al Vl Ile/Leu (b) B Scores PC1, PC Norml 0.25 Lodings PC1, PC PC PC Cncer PC1 Score plot () PC1 Score plot (b) Figure 2 igh-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy spectr of norml pncres nd trnsplnted pncretic tumor (500 mz). A: Norml pncres (); Trnsplnted pncretic tumor (b); Amplified dt from spectr region (c); Amplified dt from spectr region (d). For pek ssignments, see list of bbrevitions used; B: Principl Component Anlysis to compre the metbolic profiles between norml pncres nd pncretic cncer bsed on the high-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy spectr. Pnels () nd (b) re score nd loding plots. : Norml pncres; : Pncretic cncer. sistent with wht ws observed in Figure 2A, corresponding to the residul lipid, Lc, Al, Cho compound, Tu, nd unknown chemicls. As is well-known, bsolute concentrtion quntifiction for metbolites is difficult in RMAS spectroscopy, nd the metbolite rtios re commonly used for sttisticl nlysis. Tble 1 shows the reltive signl integrls nd signl rtios for some metbolites tht contributed to the clssifiction of norml pncres nd pncretic tumor tissues discussed in the bove sections. Compred to the norml pncres, concentrtions of Ileu, Leu, Vl, Lc, Al, Glu, Tu, Cho, nd some crbohydrtes (G, contined glctose β- possibly due to chrcteristic twin pek t 4.52) incresed reltively in the pncretic tumor smples, while GPC + PC, Bet, GPC/Cre, nd unknown chemicls t 3.66 decresed reltively. The level 4203 July 14, 2013 Volume 19 Issue 26

5 e X et l. 1 NMR nd metbonomic studies of pncretic cncer Tble 1 Reltive integrls nd their rtios from some selected metbolites contributing to the clssifiction of norml pncres nd pncretic tumor tissues of Ace nd PC/Cre showed no significnt chnge. Metbolic profiles of pncretic tumor tissues fter rdiotherpy The metbolic profiles of tumor tissues fter rdiotherpy were lso detected by 1 NMR. As shown in Figure 3A, no significnt metbolic chnges were observed in the 1 NMR spectrum. PCA nlysis ws further conducted on smples in ech group, with the spectrum integrtion region = , nd the miniml region = 0.04 (Figure 3B). In score plots, most of smples in the control group concentrte in the upper left, but overlp prtly with smples in the 10 Gy rdition dose group. A prtil overlp is shown between the 10, 20, nd 30 Gy rdition dose groups, but overll it seems tht the three groups hve left, upper, nd lower distribution trend in terms of scores. Loding plots showed the chnges of Cho-contining compounds, long with Ace nd Bet content mong the three dose groups. Tble 2 shows the reltive signl integrls nd signl rtios for some metbolites tht contributed to the evlution of pncretic tumor tissues response rdiotherpy. Cho content showed significnt difference between the control nd 30 Gy dose groups, s well s the 10 nd 30 Gy dose groups. The Cho content decresed with n increse of rdition dosge. Bet content lso decresed with n increse of rdition dosge. In contrst, Ace content showed positive reltionship with the rdition dosge. DISCUSSION Norml pncres Pncretic tumor P -vlue Metbolites Choline 2.75 ± ± Turine 1.99 ± ± Betine 2.91 ± ± Glutmic cid 0.29 ± ± Alnine 0.60 ± ± Lctte 1.93 ± ± Acetic cid 0.06 ± ± Glycerophosphocholine ± ± phosphocholine Metbolites rtio Glycerophosphocholine/ Cretine Phosphocholine/ Cretine 3.51 ± ± ± ± Although RMAS 1 NMR combined with PCA hs been demonstrted s n efficient method for studying wide vriety of niml nd humn cncers [12-17], this combined method hs not been reported to nlyze the metbolic fetures of cncer response to therpy. ere, for the first time to our knowledge, our findings demonstrte tht pplying this combined method hs the potentil for clinicl ssessment of the pncretic cncer rdiotherpeutic response. Kpln et l [22] conducted 1 NMR nlysis on perchlorte extrct (wter-soluble) of heterotopic trnsplnted pncretic cncer tissue in nude mice. Compred with the norml pncres of nude mice, Tu nd Lc content in trnsplnted tumors incresed, GPC content decresed, nd there ws little chnge in Cho nd PC. owever, in previous studies, some importnt informtion my be missed, nd humn fctors introduced s destructive process in extrction will led to negtive impct on the results, long with poor experimentl repetbility results from different p vlues. Therefore, in this study, 1 NMR combined with PCA ws pplied to the metbolic study on trnsplnted tumor tissues in humn pncretic tumor-bering nude mouse model. This hs voided the error fctor involved in complex processes such s tissue extrction. Moreover, due to the ppliction of the 500 mz high-field strength NMR instrument, the spectrum resolution obtined is significntly higher thn tht reported in the literture, with more metbolites being found nd vrition chrcteristics of metbolites embodied more clerly. Consequently, not only did the ccurcy of spectrum pek identifiction improve, but sttisticl nlysis errors were lso reduced. In this study using 1 NMR combined with PCA, pncretic cncer ws shown to hve higher Tu, Ileu, Leu, Vl, Lc, Al, Glu, nd Cho levels reltive to norml pncres, while GPC + PC, nd Bet nd GPC/Cre levels decresed reltively. Compred to the other metbolites, Tu, Lc, nd Al hd the most noticeble differences between norml pncres nd pncretic cncer. Ace nd PC/Cre showed no significnt difference between norml nd pthologicl conditions. The results suggest tht these chnges in the metbolite profile might be used s metbolic mrkers for the erly dignosis of pncretic cncer. Rdiotherpy is locl tretment, nd its ultimte gol is to erdicte tumor cells thoroughly, while protecting norml tissues nd vitl orgns s much s possible [23]. The ppliction of computer tomogrphy simultions nd the three-dimensionl conforml technique in rdiotherpy hs boosted the pncretic trget dosge nd offered better protection for the gstrointestinl trct. Currently, therpeutic evlutions of rdiotherpy re minly: remission from the symptoms of pin nd jundice, solid tumor size nd its survivl time, nd the lck of specific trgeted method [23]. By imging exmintion, tumor size nd contrst gent enhncement were observed to determine tumor ctivity, nd indirectly determine therpy efficcy, lthough lcking strong direct evidence [24-27]. In this study, we use 1 NMR nd PCA to compre pncretic cncer metbolic vrition chrcteristics before nd fter rdiotherpy. Although no significnt metbolic chnges were observed in the 1 NMR spectr, PCA results showed trend of certin chnges mong different dosge groups. We found tht the Ace level ws incresed, which positively correlted with the rdition dose. In contrst, Cho nd Bet levels were decresed, which in July 14, 2013 Volume 19 Issue 26

6 e X et l. 1 NMR nd metbonomic studies of pncretic cncer A () (b) (c) (d) B Scores PC1, PC2 Lodings PC1, PC2 PC PC PC1 Score plot () PC1 Score plot (b) Figure 3 igh-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy spectr of trnsplnted pncretic tumor fter rdiotherpy (500 mz). A: Untreted group(); 10 Gy tretment group (b); 20 Gy tretment group (c); 30 Gy tretment group (d); B: Principl component nlysis to compre the metbolic profiles of the pncretic tumor fter rdiotherpy bsed on the high-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy spectr. Pnels () nd (b) re scores nd lodings plots. : Untreted group; : 10 Gy tretment group; : 20 Gy tretment group; +: 30 Gy tretment group. versely correlted with the rdition dose. Additionlly, other metbolites, including Tu, Ileu, Leu, Vl, Lc, Al, Glu, nd GPC + PC showed no significnt chnge fter rdiotherpy. Thus, these dt suggest tht the chnges in these metbolite profiles might provide reference guide on therpeutic evlution by NMR on pncretic cncer in vivo. Choline-contining metbolites (CCM) hve lredy been chosen s biomrkers in vrious crcinom studies [28,29] ; however, they hve not been mentioned in cncer tretment so fr. CCM levels were shown to increse in most cncer tissues, which were explined s result of high membrne concentrtion during the prolifertion of cncer cells. We found tht Cho level ws reduced in pncretic cncer fter rdiotherpy, suggesting tht prolifertion of cncer cells ws inhibited in response to rdiotherpy. owever, PC nd GPC levels showed no significnt chnge in tumor tissue fter rdiotherpy. This 4205 July 14, 2013 Volume 19 Issue 26

7 e X et l. 1 NMR nd metbonomic studies of pncretic cncer Tble 2 Reltive integrls nd their rtios from some selected metbolites contributing to the evlution of pncretic tumor tissues response to rdiotherpy Untreted 10 Gy 20 Gy 30 Gy P -vlue Metbolites Choline 3.99 ± ± ± ± Turine ± ± ± ± Betine 1.58 ± ± ± ± Glutmic cid 0.46 ± ± ± ± Alnine 1.93 ± ± ± ± Lctte 8.30 ± ± ± ± Acetic cid 0.06 ± ± ± ± Glycerophosphocholine + phosphocholine ± ± ± ± Metbolites rtio Glycerophosphocholine/Cretine 2.35 ± ± ± ± Phosphocholine/Cretine 6.22 ± ± ± ± Untreted vs 30 Gy; 2 Untretment vs 20 Gy; 3 Untretment vs 10 Gy. might be explined by blockge of Cho-kinse nd PC trnsferse, or by the consumption of PC through the CDP-Cho pthwy [30,31]. Thus, we my deduce tht incresing Cho nd unchnged PC nd GPC could be used s unique profile of pncretic cncer response to rdiotherpy. Bet dontes methyl groups for the remethyltion of homocysteine to methionine nd dimethylglycine, which supports proper liver nd pncretic function, cellulr repliction, nd detoxifiction rections. Becuse Cho is the precursor of Bet, the decrese of both Bet nd Cho levels in pncretic cncer fter rdition tretment must be interrelted. Interestingly, the Ace level showed no significnt difference between the norml pncres nd pncretic cncer. owever, Ace level drmticlly incresed with the rdition dose. The underlying significnce of this needs to be further investigted. In summry, lthough the number of smples in our study ws limited, the potentil of RMAS NMR for the in vitro investigtion of pncretic disese response to rdiotherpy should not be ignored. The bove results clerly demonstrte tht the metbolic profile chnges of pncretic cncer fter rdiotherpy were closely correlted with therpeutic effect through RMAS 1 NMR nd the PCA combined method. Becuse metbolite chnges observed by RMAS NMR lwys occur before morphologicl chnges investigted by MRIS, RMAS NMR will certinly be beneficil to pthologicl reserch, erly dignosis, nd therpy evlution of pncretic diseses. COMMENTS Bckground Therpeutic evlutions of rdiotherpy re minly: remission from the symptoms of pin nd jundice, solid tumor size nd its survivl time, nd the lck of specific trgeted method. During the lst three decdes, there hs been ongoing mgnetic resonnce spectroscopy reserch in mlignnt diseses. These studies provided vluble dt on the biochemistry nd metbolism of tumors, long with the effects on nutrients, hormones, nd growth fctors. Reserch frontiers igh-resolution mgic ngle spinning proton mgnetic resonnce spectroscopy (RMAS 1 NMR) is well-recognized technique in metbonomics studies in vitro, by which biopsy or postmortem smples of intct tissues re spun t the mgic ngle, resulting in significnt improvement in the resolution of the spectrum obtined for some of line-brodening fctors such s dipole dipole interctions nd chemicl shift nisotropy. Mgnetic field inhomogeneities re lso verged out. This pproch requires miniml smple preprtion nd, unlike convenient 1 NMR spectroscopy of tissue extrcts, both queous nd lipid-soluble metbolites cn be observed simultneously in situ. Innovtions nd brekthroughs Although RMAS 1 NMR combined with principl components nlysis (PCA) hs demonstrted to be n efficient method for studying wide vriety of niml nd humn cncers, this combined method hs not been reported to nlyze the metbolic fetures of cncer response to therpy. ere, RMAS 1 NMR nd PCA were combined to highlight metbolite profiles of pncretic cncer fter rdiotherpy, nd by which the correltion between rdiotherpy effect nd metbolic chnge ws nlyzed, nd the therpeutic scheme optimized July 14, 2013 Volume 19 Issue 26

8 e X et l. 1 NMR nd metbonomic studies of pncretic cncer Applictions The study hs importnt impliction for reference guide on therpeutic evlution by nucler mgnetic resonnce spectroscopy on pncretic cncer in vivo. Peer review The uthors investigted whether metbolic profile chnges of pncretic cncer fter rdiotherpy were closely correlted with therpeutic effect through the RMAS 1 NMR nd PCA combined method. The outcome of the study is interesting nd beneficil to pthologicl reserch, erly dignosis, nd therpeutic evlution of pncretic diseses. REFERENCES 1 Greenlee RT, Murry T, Bolden S, Wingo PA. Cncer sttistics, CA Cncer J Clin 2000; 50: 7-33 [PMID: DOI: /cnjclin ] 2 Wng L, Yng G, Lu X, ung ZJ, Li. Pncretic cncer mortlity in Chin ( ). World J Gstroenterol 2003; 9: [PMID: ] 3 irshberg B, Libutti SK, Alexnder R, Brtlett DL, Cochrn C, Livi A, Chng R, Shwker T, Skrulis MC, Gorden P. Blind distl pncretectomy for occult insulinom, n indvisble procedure. 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Cncer Sci 2007; 98: [PMID: ] 21 Grrod S, umpfer E, Sprul M, Connor SC, Polley S, Connelly J, Lindon JC, Nicholson JK, olmes E. igh-resolution mgic ngle spinning 1 NMR spectroscopic studies on intct rt renl cortex nd medull. Mgn Reson Med 1999; 41: [PMID: ] 22 Kpln O, Kushnir T, Askenzy N, Knubovets T, Nvon G. Role of nucler mgnetic resonnce spectroscopy (MRS) in cncer dignosis nd tretment: 31P, 23N, nd 1 MRS studies of three models of pncretic cncer. Cncer Res 1997; 57: [PMID: ] 23 Shinchi, Tko S, Nom, Mtsuo Y, Mtki Y, Mori S, Aikou T. Length nd qulity of survivl fter externl-bem rdiotherpy with concurrent continuous 5-fluorourcil infusion for loclly unresectble pncretic cncer. Int J Rdit Oncol Biol Phys 2002; 53: [PMID: DOI: /S (01) ] 24 Ishikw, Suzuki Y, Nkym Y, Nkmoto S, Kusb T, Kkinum S, Skt Y, Mitsuhshi N, Niibe. Intropertive rdiotherpy nd bypss surgery for unresectble pncretic cncer. eptogstroenterology 2000; 47: [PMID: ] 25 Cienfuegos JA, Mnuel FA. Anlysis of intropertive rdiotherpy for pncretic crcinom. Eur J Surg Oncol 2000; 26 Suppl A: S13-S15 [PMID: ] 26 Ceh M, vn Tienhoven G, Goum DJ, Veenhof C, Schneider CJ, Ruws EA, Pho SS, González González D. Fesibility nd efficcy of high dose conforml rdiotherpy for ptients with loclly dvnced pncretic crcinom. Cncer 2000; 89: [PMID: ] 27 Ktz M, Bouvet M. Novel gene therpy pproches to 4207 July 14, 2013 Volume 19 Issue 26

9 e X et l. 1 NMR nd metbonomic studies of pncretic cncer pncretic cncer. Int J Gstrointest Cncer 2003; 33: [PMID: ] 28 Loening NM, Chmberlin AM, Zeped AG, Gonzlez RG, Cheng LL. Quntifiction of phosphocholine nd glycerophosphocholine with 31P edited 1 NMR spectroscopy. NMR Biomed 2005; 18: [PMID: ] 29 Cheng LL, Anthony DC, Comite AR, Blck PM, Tzik AA, Gonzlez RG. Quntifiction of microheterogeneity in glioblstom multiforme with ex vivo high-resolution mgicngle spinning (RMAS) proton mgnetic resonnce spectroscopy. Neuro Oncol 2000; 2: [PMID: DOI: / ] 30 Podo F. Tumour phospholipid metbolism. NMR Biomed 1999; 12: [PMID: ] 31 Morvn D, Demidem A, Ppon J, Mdelmont JC. Quntittive RMAS proton totl correltion spectroscopy pplied to cultured melnom cells treted by chloroethyl nitrosoure: demonstrtion of phospholipid metbolism ltertions. Mgn Reson Med 2003; 49: [PMID: DOI: /mrm.10368] P- Reviewers Sun Z, Supiot S S- Editor Zhi L- Editor Rutherford A E- Editor Zhng DN 4208 July 14, 2013 Volume 19 Issue 26

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