Purpose: Pancreatic ductal adenocarcinoma (PDAC) is anaggressive and lethal disease that develops relatively symptom-free and is therefore advanced at the time of diagnosis. Theabsence of early symptoms and effective treatments has createda critical need for identifying and developing new noninvasivebiomarkers and therapeutic targets.
Experimental Design: We investigated the metabolism of apanel of PDAC cell lines in culture and noninvasively in vivowith 1H magnetic resonance spectroscopic imaging (MRSI) toidentify noninvasive biomarkers and uncover potential meta-bolic targets.
Results:We observed elevated choline-containing compoundsin the PDAC cell lines and tumors. These elevated choline-con-taining compoundswere easily detectedby increased total choline
(tCho) in vivo, in spectroscopic images obtained from tumors.Principal component analysis of the spectral data identifiedadditional differences in metabolites between immortalizedhuman pancreatic cells and neoplastic PDAC cells. Molecularcharacterization revealed overexpression of choline kinase(Chk)-a, choline transporter 1 (CHT1), and choline transport-er–like protein 1 (CTL1) in the PDAC cell lines and tumors.
Conclusions: Collectively, these data identify new metaboliccharacteristics of PDAC and reveal potential metabolic targets.Total choline detected with 1H MRSI may provide an intrinsic,imaging probe–independent biomarker to complement existingtechniques in detecting PDAC. The expression of Chk-a, CHT1,and CTL1may provide additional molecular markers in aspiratedcytological samples. Clin Cancer Res; 21(2); 386–95. �2014 AACR.
IntroductionPancreatic cancer is the fourth leading cause of cancer death in
the United States and leads to approximately 227,000 deaths peryear worldwide (1). Early-stage pancreatic cancer is clinicallysilent, and most patients presenting with symptoms attributableto pancreatic cancer have advanced disease. These symptomsbecome apparent after the tumor invades surrounding tissues ormetastasizes to distant organs, resulting in abdominal or mid-back pain, obstructive jaundice, and weight loss (1). Most pan-creatic cancers are histologically classified as pancreatic ductaladenocarcinoma (PDAC; ref. 2), and have a 5-year survival rate ofonly 6% (3). The poor prognosis of PDAC arises from a combi-nation of late-stage diagnosis and limited response to chemo-therapy and radiotherapy, both of which are partly due to the
strong desmoplastic stroma that limits delivery of diagnosticimaging probes and therapeutic agents (4). Similarities in theclinical behavior and imaging features of PDAC and chronicpancreatitis further complicate the detection of PDAC (5). Theunavailability of targeted agents for this disease has also signif-icantly impeded effective treatment.
Although inroads are being made in developing molecularimaging probes such as plectin-1–targeted peptides to detectPDAC (6), these have not been clinically translated. There is anurgent need for noninvasive clinically translatable biomarkers ofPDAC.
Magnetic resonance spectroscopy (MRS) is being evaluated inthe diagnosis of several solidmalignancies such as brain, prostate,and breast cancer (7). An elevation of total choline (tCho) is ametabolic hallmark in the spectra of cancers imaged with 1HMRS(7). tCho, observed as a single peak in vivo, consists of threecholine-containing metabolites, which can be resolved throughhigh-resolution proton spectroscopy into phosphocholine (PC),glycerophosphocholine (GPC), and free choline (Cho). IncreasedPC in tumors is the primary cause of the elevated tCho and is dueto an overexpression of the choline kinase (Chk)-a gene as well asincreased expression of choline transporters (8–10). Chk cata-lyzes the phosphorylation of Cho using ATP, as a phosphatedonor, to produce PC. An elevation of choline uptake by cholinetransporters, followed by phosphorylation by choline kinase, canalso increase endogenous PC (11). Increased expression of thehigh-affinity choline transporter CHT1with a Km of approximate-ly 2mmol/L, also called solute carrier family 5member 7 (SLC5A7;ref. 12), has been previously observed in breast cancer cells (10).
1JHU ICMICProgram,DivisionofCancer ImagingResearch,TheRussellH. Morgan Department of Radiology and Radiological Science, TheJohns Hopkins University School of Medicine, Baltimore, Maryland.2Sidney Kimmel Comprehensive Cancer Center, The Johns HopkinsUniversity School of Medicine, Baltimore, Maryland. 3Departments ofPathology and Translational Molecular Pathology, UT MD AndersonCancer Center, Houston Texas.
M.-F. Penet and T. Shah contributed equally to this article.
Corresponding Author: Zaver M. Bhujwalla, Johns Hopkins University School ofMedicine, 208C Traylor Building, 720 Rutland Avenue, Baltimore, MD 21205.Phone: 410-955-9698; Fax: 410-614-1948; E-mail: [email protected]
Another choline transporter, choline transporter–like protein 1(CTL1) with a Km of approximately 68 mmol/L, was found to beoverexpressed in human lung and colon carcinoma cells (13, 14).
Here, we have characterized the metabolic profile, especiallycholine phospholipid metabolism, in a panel of PDAC cell lines,and tumor xenografts derived from these PDAC cells, implantedsubcutaneously or orthotopically in the pancreas, using 1H mag-netic resonance spectroscopic imaging (MRSI) and MRS. Signif-icantly elevated PCand tChowere observed in thePDAC cell lines,compared with an immortalized non-neoplastic pancreatic cellline. Increases in PC and tCho observed in PDAC cells wereparalleled in tumors derived from the PDAC cells, with elevatedtCho observed in metabolic images from in vivo tumors. Molec-ular characterization of these cells and tumors identified over-expression of Chk-a, CHT1, and CTL1 as the primary causes ofincreased tCho and PC. These data identify tCho as a potentialintrinsic biomarker to detect PDAC noninvasively with 1H MRspectroscopic metabolic imaging. Enzymes in choline phospho-lipid metabolism pathway, such as Chk-a, may provide potentialtherapeutic targets for PDAC treatment.
Materials and MethodsCell lines and tumor implantation
Eight PDAC cell lines and one immortalized pancreatic cell linewere used in the study. Details about the cell lines are provided
in Table 1. BxPC3 and Panc1 were obtained from ATCC(American Tissue Culture Collection). Pa02C, Pa03C, Pa04C,Pa09C, Pa20C, and Pa28C have been previously described (15).Pa02CandPa03Cwerederived from livermetastases, Pa04C fromlung metastasis, and BxPC-3, Panc-1, Pa09C, Pa20C, and Pa28Cwere derived fromprimary adenocarcinomas. For comparison,weused human pancreatic nestin expressing (HPNE) cells fromATCC. HPNE cells were derived from non-neoplastic humanpancreas that stably express human telomerase reverse transcrip-tase (hTERT) after transduction with a retroviral expression vectorcontaining the hTERT gene. Panc1, BxPC3, Pa03C, Pa04C, Pa20C,and Pa28C cells were cultured in DMEM (Sigma) with 10% FBS,100 units/mL penicillin, 100 mg/mL streptomycin, 25 mmol/Lglucose, and 4 mmol/L glutamine. Pa02C and Pa09C were cul-tured in RPMI-1640 (Sigma) with 20% FBS, 100 units/mL pen-icillin, 100 mg/mL streptomycin, 12.5 mmol/L glucose, and2 mmol/L glutamine. hTERT-HPNE cells were cultured accordingto the protocol recommended by ATCC. The base medium wasa combination of 75% DMEM without glucose (Sigma), withadditional 2mmol/L glutamine, 1.5 g/L sodiumbicarbonate, and25%M3Base medium (Incell Corp.). To make complete growthmedium, 5% FBS, 10 ng/mL human recombinant epidermalgrowth factor, 5.5 mmol/L glucose, and 750 ng/mL puromycinwere added to the base medium. DMEM and RPMI havecomparable concentrations of choline chloride (�0.021–0.028 mmol/L).
Cells were cultured in standard cell culture incubator condi-tions at 37�C in a humidified atmosphere containing 5% CO2.Subcutaneous tumors were generated by inoculating 2� 106 cellssuspended in 0.05mLofHanks balanced salt solution in theflankof severe combined immunodeficient (SCID) male mice. Ortho-topic implantation was performed as previously described (16).Viable tumor pieces of approximately 1 mm3 harvested fromsubcutaneous tumors were implanted into the pancreas of anes-thetized male SCID mice via a subcostal left incision of approx-imately 1 cm. All surgical procedures and animal handling wereperformed in accordance with protocols approved by the JohnsHopkins University Institutional Animal Care and Use Commit-tee, and conformed to the Guide for the Care and Use of Labo-ratory Animals published by the NIH.
In vivo MRSIAnesthetized tumor-bearing mice were imaged at 4.7T for
subcutaneous tumors or at 9.4T for orthotopic tumors usingBruker Biospec MR scanners (Bruker Biospin Corp.). Body tem-perature of the animals was maintained in the magnet by athermostat-regulated heating pad. Localized 1H MR spectra wereacquired using a 15-mm diameter home-built solenoid coilplaced around the subcutaneous tumor or a 25-mm Bruker(Bruker Biospin Corp.) volume coil placed around the torso of
Table 1. Details of pancreatic cell lines studied
Cell line Source Tissue derivation Carcinoma type Stage Sex
Pancreatic ductal adenocarcinoma (PDAC) is an aggressiveand lethal disease that develops relatively symptom-free and istherefore advanced at the time of diagnosis. The poor prog-nosis of PDAC is due to a combination of late-stage diagnosisand limited response to chemotherapy and radiotherapy,arising in part from the strong desmoplastic stroma that limitsdelivery of diagnostic imaging probes and therapeutic agents.The absence of early symptoms and effective treatments hascreated a critical need for identifying and developing newnoninvasive biomarkers and therapeutic targets.
Our results have identified aberrant choline metabolism aswell as differences in lactate and glutamate in human pancre-atic cancer cells and xenografts. These results create much-needed new possibilities to detect pancreatic cancer using 1Hmagnetic resonance spectroscopy that merit rapid investiga-tion in human subjects. Metabolic targets in choline phos-pholipid metabolism and in glutaminolysis and glycolysismay provide novel treatments for a disease that has severelylimited treatment options.
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the orthotopic tumor-bearing mice. Spectra from a 4-mmthick central slice localized within the tumor were acquired witha 16-mm field of view (FOV) for subcutaneous tumors, or 32mmFOV for orthotopic tumors, and zero-filled to an in-plane spatialresolution of 1 mm � 1 mm. Spectra were acquired with 8scans per phase encode step, an echo time (TE) of 272 ms forsubcutaneous tumors, and 135 ms for orthotopic tumors, and arepetition time (TR) of 1,059 ms for subcutaneous tumors, and1,500 ms for orthotopic tumors, using a 2D-CSI (chemical shiftimaging) sequence with VAPOR (VAriable Pulse power andOptimized Relaxation delays) water suppression (17). Theseacquisition parameters were optimized to obtain a good tChosignal from the tumors in less than 35minutes. Reference 2D-CSIimages of the unsuppressed water signal were acquired from thetumorswith a TEof 20ms, a TRof 1,059ms, and 2 scans per phaseencode step, with all other parameters remaining the same.Quantitativemaps of tChowere generated from the spectroscopicimages using unsuppressed water signal as an internal reference(18).Quantitativemetabolicmapswere generated using in-houseIDL programs. For orthotopic tumors, single voxel PRESS1H spectra were also acquired with a TE of 67.5 ms, a TR of1,500 ms, and voxel size of 5 � 5 � 5 mm3.
MR spectroscopy of dual-phase extractsCell and tumor extracts were obtained using a dual-phase
extraction method with methanol/chloroform/water (1/1/1;ref. 19). Briefly, approximately 1.5 � 107 cells were harvestedby trypsinization, washed twice with saline, and pooled into aglass centrifuge tube. Cell pellets were suspended in ice-coldmethanol, vigorously vortexed, and kept on ice for 10 minutes.Next, 4 mL of chloroform and 4mL of ice-cold water were added,and the solution was vortexed. To obtain tumor extracts, tumorswere freeze-clamped and ground to powder. Ice-cold methanolwas added, and the tumor extract samples were homogenized.Chloroform and ice-cold water were finally added. Cell andtumor extract samples were kept at 4�C overnight for phaseseparation. Samples were then centrifuged for 30 minutes at15,000 g at 4�C to separate the phases. The water/methanolphase containing the water-soluble metabolites was treated withchelex (Sigma) for 10 minutes on ice to remove divalent cations.The chelex beads were removed through filtration. Methanol wasthen removed by rotary evaporation, and the remaining waterphase was lyophilized and stored at �20�C. Water-solublesamples were dissolved in 0.5 mL of D2O (Sigma) containing3-(trimethylsilyl) propionic-2,2,3,3,-d4 acid (TSP; Sigma) as aninternal concentration standard (sample pH of 7.4). Fullyrelaxed 1H MR spectra of the extracts were acquired on a BrukerAvance 500 spectrometer operating at 11.7 T (Bruker BioSpinCorp.) using a 5-mm HX inverse probe, and the followingacquisition parameters: 30� flip angle, 6,000 Hz sweep width,9.5 s repetition time, time-domain data points of 32K, and 128transients (19). Spectra were analyzed using Bruker TOPSPIN 2.1software (Bruker BioSpin). Integrals of the metabolites of interestwere determined and normalized to the number of cells and tothe tumor weight, respectively. To determine concentrations,metabolite peak integration values from 1H spectra were com-pared with the internal standard. Metabolites were estimatedfrom at least three experimental samples from each cell line toobtain averaged values. Statistical significance was evaluatedusing the Student t test. P values � 0.05 were consideredstatistically significant unless otherwise stated.
Principal component analysis of MR spectral dataSingle pulse MR spectra obtained from pancreatic cell lines
were subjected to multivariate principal component analysis(PCA). Raw spectral data were processed using TOPSPIN 2.1,and regions between 0.5 and 9.0 ppmwere exported as raw datapoints using Bruker AMIX software (Version 3.8.7; ref. 20). Theresulting data matrices with normalized integral values wereexported into Microsoft Office Excel 2010. The region from 4.5to 5.2 ppm was excluded from the analysis to remove theresidual signal of HOD and water. The data obtained werenormalized by dividing each integral of the segment by thetotal area of the spectrum to compensate for differences inoverall metabolite concentration between individual samplesthat can arise due to dilution errors. The whole matrix wasimported into Matlab environments (MATLAB R2012b,8.0.0.783; The MathWorks, Inc.) for spectral alignments. Ico-shift algorithm for spectral alignments was used to remove thesmall chemical shift drifts in the spectra arising because of pH/ionic variations (20). The matrix was further imported to "TheUnscrambler X" Software package (Version 10.0.1; CAMOSoftware Inc.) for PCA.
Immunoblot of cell and tumor extractsProteins were extracted from cells, from freeze-clamped sub-
cutaneous or orthotopic tumors, and from normal mousepancreas, using RIPA lysis buffer fortified with a proteaseinhibitor cocktail, dithiothreitol, phenylmethylsulfonyl fluo-ride, sodium orthovanadate, and sodium fluoride (Sigma).Protein lysate from a normal human pancreas was obtainedfrom OriGene (OriGene). Protein concentration was estimatedusing the Bradford Bio-Rad protein assay Kit (Bio-Rad). Approx-imately 60 to 75 mg of total protein were resolved on 10% SDS-PAGE, transferred onto nitrocellulose membranes, and probedfor 2 hours at room temperature with an in-house human-specific rabbit polyclonal Chk-a antibody (dilution 1:100;ref. 19), a mouse and human cross reactive rabbit polyclonalCHT1 antibody (dilution 1:2,000; Cat. No. NBP1-62339;Novus Biologicals), or a human and mouse cross reactivepolyclonal CTL1 antibody (1:1,000; Cat. No. TA315247; Ori-Gene). GAPDH was used as a loading control and detected witha human and mouse cross reactive monoclonal antibody (dilu-tion 1:10,000; Cat. No. SAB1405848; Sigma). Immunoblotswere developed using the SuperSignal West Pico chemilumi-nescent substrate Kit (Thermo Scientific). Because we used ahuman-specific rabbit polyclonal Chk-a antibody, we per-formed quantitative real-time polymerase chain reaction(qRT-PCR) of normal mouse pancreas using primers designedfor mouse Chk-a. Total RNA was isolated from tumor samplesusing the QIAshredder and RNeasy Mini Kit (Qiagen). cDNAwas prepared using the iScript cDNA synthesis Kit (Bio-Rad).cDNA samples were diluted 1:10 and real-time PCR was per-formed using IQ SYBR Green supermix and gene-specific pri-mers in the iCycler real-time PCR detection system (CFX-96Connect; Bio-Rad). All primers were designed using Beacondesigner software 7.8 (PREMIER Biosoft). The expression oftarget RNA relative to the housekeeping ribosomal gene 18s(using corresponding mouse and human primers) was deter-mined based on the threshold cycle (Ct) as DCt ¼ Ct of targetgene – Ct of 18s. The 18s Ct values (mean � SEM) for normalmouse pancreas and for orthotopic tumors were 10.1 � 0.06(n ¼ 3) and 9.9 � 0.08 (n ¼ 12), respectively.
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Clin Cancer Res; 21(2) January 15, 2015 Clinical Cancer Research388
Levels of Chk-a, CHT1, and CTL1 varied across the PDAC celllines but were clearly higher than in non-neoplastic HPNE cells(Fig. 1A). The HPNE cells showed no detectable levels of Chk-aexpression and a low level ofCHT1.Of the cancer cell lines, Pa04Cand Pa28C had the lowest levels of Chk-a. Representative high-resolution 1H MR spectra of the choline-containing metabolitesregion are shown in Fig. 1B. Consistent with the nondetectableChk-a expression and a low level of CHT1, the lowest levels ofcholine-containing metabolites were observed in the HPNE cellscompared with the 8 PDAC cell lines investigated. Quantitativedata for choline-containing metabolites obtained from water-soluble cell extracts are shown in Fig. 1C. Significantly higher PC
and tCho levels were observed in all the PDAC cell lines comparedwith the non-neoplastic HPNE cells. Among the cancer cells,Pa03C and Pa20C showed the highest PC levels. PC levels werethe lowest in Pa04C and Pa28C cancer cells, consistent with thelowest Chk-a in these cells. There were no clear differences in thecholine-containing compounds based on stage, or derivationfrom primary or metastatic cancers. Neither GPC nor Cho levelswere consistently associated with PDAC.
Results from PCA are presented in Fig. 2. The scattered PCAscore plot (Fig. 2A) shows a clustering of non-neoplastic HPNEpancreatic cells versus the pancreatic cancer cell lines.MultivariatePCA of the spectral data identified increases in lactate, acetate,glutamate, creatine, tCho, and myo-inositol, and decreases inalanine and aspartate in the cancer cells compared with the non-neoplastic cells (Fig. 2B).
Figure 1.A, representative immunoblots from apanel of pancreatic cell linesshowing Chk-a, CHT1, and CTL1expression with GAPDH as loadingcontrol. B, representative high-resolution 1H spectra of the choline-containing compounds regionobtained from HPNE and PDAC celllines. C, quantitative data forcholine-containing compounds in apanel of pancreatic cell lines. Valuesrepresent mean � SEM, n ¼ 4.� , P < 0.05 HPNE vs. all.
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Tumor metabolismIncreases of PC and tCho observed in the cells were reflected in
the 1HMR spectra of subcutaneous tumor xenograft extracts (Fig.3A). Consistent with the cell data, additional metabolites such asalanine, creatine, phosphocreatine, lactate, and total creatinewereobserved in the tumor spectra. The patterns of these metaboliteswere different between the tumors. Harvested Panc1 tumors werecharacterized by the highest level of lactate, PC, and tCho, Pa04Cby the highest level of alanine, and BxPC3 and Panc1 had thehighest level of creatine and total creatine (Fig. 3B).
Altered choline metabolism in subcutaneous tumorsImmunoblot analysis revealed high Chk-a, CHT1, and CTL1
expression in all four xenografts (Fig. 4A). Chk-a and CTL1 werehighest in Panc1 tumors. As shown in Fig. 4B, tCho was detectedin all the subcutaneous tumor xenografts imaged in vivo. Weobserved a heterogeneous distribution of the signal within eachtumor and between each tumor type. Panc1 tumors presentedwith high tCho levels (Fig. 4C), consistent with the tumor extractdata, and with the high Chk-a and CTL1 expression observed inimmunoblots obtained from these tumors.
Altered choline metabolism in orthotopic tumorstCho was detected in vivo in orthotopically implanted tumors
using 2D-CSI (Fig. 5A) and single voxel 1HMRS spectra (Fig. 5B).We compared Chk-a, CHT1, and CTL1 in orthotopic tumors toprotein lysate obtained from normal human pancreas. Similar tothe subcutaneous tumors, increasedChk-a, CHT1, andCTL1wereobserved in the orthotopic tumors comparedwith normal humanpancreas. Also, similar to the observations made in subcutaneoustumors, Panc1 tumors presented with higher levels of Chk-a andCTL1 (Fig. 5C). Normal mouse pancreas had undetectable levelsof CHT1 and CTL1 (see Fig. 4C). Unlike the CHT1 and CTL1antibodies that are mouse and human cross-reactive, the Chk-aantibody was designed against human Chk-a. We therefore char-
acterized Chk-a mRNA in mouse pancreas and in orthotopichuman pancreatic cancer xenografts using primers for mouse andhuman Chk-a, respectively. We observed significantly higherChk-a mRNA in the orthotopic tumors compared with normalmouse pancreas (Fig. 5D).
DiscussionWe observed a consistent increase of PC and tCho, and a
consistently increased expression of Chk-a and CHT1 in a panelof human PDAC cell lines. The changes in choline metabolismwere clearly due to malignant transformation because there wasno dependence on whether these cancer cells originated fromprimary or metastatic tumors. These results identify PDAC as partof the increasing compendiumof cancers that demonstrate alteredcholine metabolism following malignant transformation (7).
Orthotopic pancreatic cancer models have been previouslydeveloped (21) and represent an important advance in the inves-tigation of this disease in compatible microenvironments, espe-cially because expression of green or red fluorescent protein inthese models provides the opportunity for noninvasive opticalimaging of the tumors (22, 23). Althoughmuch of our metaboliccharacterization was performed with subcutaneous tumors, wealso investigated cholinemetabolism inorthotopically implantedpancreatic tumors.
In tumors in vivo, the increased PC and tChowere primarily dueto overexpression of Chk-a, as well as the choline transportersCHT1 andCTL1. Increased expression of CHT1 andCTL1 in othercancers has been previously reported (10, 13, 14). Other cholinetransporters andphospholipases (7)may also have contributed tothe altered choline metabolism and should be further investigat-ed. Chk-a, CHT1, and CTL1 may provide companion diagnosticmolecular markers to detect pancreatic cancers. Patterns of cho-line metabolites, Chk-a, CHT1, and CTL1 observed in culturedcells were not entirely mirrored in the tumors in vivo. In culture,
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Figure 2.A, PCA of the high-resolutionspectral data-derived score plots ofwater-soluble cell extracts, showingclustering differences betweennonmalignant HPNE vs. PDAC celllines based on differences in theiroverall metabolic profile. B, PCAanalysis shows increased lactate,tCho, acetate, glutamate, creatine,myo-inositol, and decreased alanineand aspartate in PDAC cellscompared with nonmalignantHPNE cells.
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viable cells are maintained under carefully controlled conditions.In vivo, however, physiologic conditions such as hypoxia, and theassociated acidic extracellular pH that exist in pancreatic cancers(24), may affect choline phosphorylation and uptake througheffects on Chk-a (25) and transporters (13). Perfusion-limiteddelivery of substrates in poorly vascularized tumor regions willalso influence metabolite concentrations in vivo. In addition,necrosis and acute cell death, the extent of which varies betweentumors, will also reduce levels of metabolites, enzymes, andtransporters, contributing to differences between cells in cultureand tumors in vivo. These factors can also explain variations inChk-a, CHT1, and CTL1 expression within tumor groups, and
between subcutaneous and orthotopic tumors. Despite somevariations, the consistent pattern that emerged was an increaseof tCho and Chk-a, CHT1 and CTL1 in PDAC cells and tumors.
The three noninvasive imaging methods currently used todetect PDAC are ultrasound, CT, andMRI. Endoscopic ultrasoundis the primary method for imaging the pancreas and detectingsmall preinvasive lesions. PDAC often presents as a mass with aloss of normal homogeneous parenchymal echo pattern (26). Thesensitivity of ultrasound is approximately 95% if the lesion ismore than 3 cm. With CT, which is the method of choice forevaluating patients with symptoms that are suggestive of thedisease, lesions larger than 1.5 cm are detected at 100% but
smaller lesions have a sensitivity of 77% and a specificity of 100%(27). The high soft tissue contrast of MRI provides an attractiveoption in staging PDAC (27). The desmoplastic stroma of PDACprevents the delivery of contrast agents resulting in an absence ofhyperintensity in the tumor compared with normal parenchymathat is useful in guiding resection and staging (27).
The pattern of increased choline transporters and Chk-aobserved in several cancers has resulted in the development ofradiolabeled choline analogs as PET imaging tracers to detectcancers through the increased uptake and phosphorylation ofthe tracer (28, 29). Our data suggest that PET imaging ofradiolabeled choline may be used to detect PDAC, providedthere is adequate delivery of the probe, as the desmoplasticstroma in pancreatic cancers may impede its delivery (4). Arecent study detected a relatively high uptake, but a low resi-dence time, of a radiolabeled choline tracer in the pancreas ofnormal volunteers (30), suggesting that PDAC may be detectedusing relatively long-lived radiolabeled choline PET tracers thatallow imaging at later time points to achieve good systemicclearance (30).
1H MRSI has the advantage of detecting intrinsic metaboliccontrast and does not require the delivery of an extrinsic markerfor detection. Most clinical MRI scanners have the ability toperform proton MRS, making the incorporation of proton MRSwith MRI easily achievable. Increased tCho, detected with 1HMRS, is consistently observed in cancer cells but not in nonma-lignant cells (31–33). As a result, tCho detected by 1H MRS isbeing evaluated as a diagnostic and prognostic biomarker inmultiple human cancers (7). These human studies have con-firmed that cancers have increased tCho (34, 35). Previous limita-tions due to motion artifact can now be minimized throughmotion correction in acquisition and processing (36). Mostdiagnostic MRSI studies have focused on lesions with a size of10 mm or greater (34), but the availability of higher field 3Tmagnets that are approved for clinical studies significantlyincreases the sensitivity of spectroscopic detection of tCho incancers (37). Our results are also consistent with a previous studyof tumors derived from Capan-1 human pancreatic cancer cellsthat showed increased signal intensity in the 3.2 ppm region of 1HMR spectra, corresponding to the choline-containing compound
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Figure 4.A, representative immunoblotsshowing Chk-a, CHT1, and CTL1proteins in BxPC3, Panc1, Pa02C, andPa04C tumors, and in tissue obtainedfrom normal mouse pancreas.GAPDH was used as loading control(n ¼ 3). The arrow marks CTL1. B,representative anatomical images ofBxPC3, Panc1, Pa02C, and Pa04Ctumors (top row), and correspondingrepresentative tCho maps (bottomrow). C, tCho concentrations inPa04C, BxPC3, Pa02C, and Panc1tumors (n ¼ 3 per group; � , P < 0.05).
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Clin Cancer Res; 21(2) January 15, 2015 Clinical Cancer Research392
region (38). These results support investigating the use of 1HMRSas a diagnostic technique to complement existing technologies indetecting pancreatic cancer. Ultimately the ability of using 1HMRspectroscopy to detect PDAC will depend upon tCho levels innormal pancreas. Recently, three human studies performed at 3Tin a limited number of subjects have shown the feasibility ofobtaining 1H MR spectra from a voxel placed in the pancreas andin pancreatic cancers using breath-holding to minimize motion-related artifacts (39–41). Although our orthotopic spectroscopicimaging data failed to detect tCho signal from normal pancreas,
studies performed in humans suggest that the tCho signal may berelatively high in normal pancreas (41), depending uponwhetherthe voxel is placed in the head or the body or tail of the pancreas(40). The head of the pancreas had significantly lower tChonormalized to water (40). In these studies, a lower tCho to waterratio was observed in the cancer voxels compared with normalpancreas (40, 41). Differences in water concentration betweennormal and fibrotic tissue, investigating large, potentially necrot-ic, lesions, and the less than optimum signal to noise levels weresome of the limitations identified in these studies (40).
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Figure 5.A, images of tCho overlaid onanatomic images from mice bearingorthotopic Panc1, Pa02C, and Pa04Ctumors. B, corresponding single voxel(5mm3) spectra localized in the centerof the tumor. C, representativeimmunoblots showing Chk-a, CHT1,and CTL1 proteins in normal humanpancreas and in orthotopic BxPC3,Panc1, Pa02C, and Pa04C tumors.GAPDH was used as loading control.D, differences in Chk-a mRNA innormal mouse pancreas, and inorthotopic BxPC3, Panc1, Pa02C, andPa04C tumors, using 18s ribosomalRNA as a reference gene. DCt ¼ Ct ofChk-a – Ct of 18s. Values representmean � SEM, n ¼ 3 per group.� , P < 0.05; ���, P < 0.0005.
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Investigations comparing spectra obtained from normal andmalignant pancreatic regions will require precise placement ofvoxels in viable non-necrotic tumor regions, elimination ofmotion-related effects, and accurate quantitation of metabolites.The use of MRSI that provides a tCho map rather than theplacement of single voxels will address heterogeneities in thepancreas and in pancreatic cancers.
In addition to altered choline metabolism, differences in othermetabolites were identified in PDAC cells compared with HPNEcells, someofwhichwere also observed in the tumor extracts. PCAidentified an increase in lactate, acetate, glutamate, creatine, andmyo-inositol and a decrease in alanine and aspartate in cancercells compared with non-neoplastic pancreatic cells. Tumorextracts also showed differences in alanine, creatine, phospho-creatine, and total creatine between the different tumor types.These results suggest that, in addition to enzymes in cholinephospholipid metabolism, enzymes in glutaminolysis or glycol-ysis may provide new targets to treat PDAC. Studies have shownthe efficacy of targeting Chk-a in preclinical studies of breast(19, 42), colon (43), bladder, lung, and cervical cancers (44). AChk-a inhibitor (TCD-717) is currently undergoing a phase Iclinical trial in patients with cancer (NCT01215864). Decreasedproliferation has been observed in pancreatic adenocarcinoma–derived MIA PaCa-2 cells following glutamine deprivation (45).
In summary, our results have identified aberrant cholinemetabolism due to increased expression of Chk-a, CHT1, andCTL1 as well as differences in metabolites such as lactate andglutamate, in pancreatic cancer cells and tumors. These resultscreate much-needed new possibilities for the detection of pan-creatic cancer using 1H MRS that merit rapid investigation inhuman subjects. Metabolic targets in choline phospholipidmetabolism and in glutaminolysis and glycolysis may provide
novel treatments for a disease that has severely limited treatmentoptions.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design: A. Maitra, Z.M. BhujwallaDevelopment of methodology: D. Artemov, Z.M. BhujwallaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M.-F. Penet, T. Shah, S. Bharti, B. Krishnamachary,F. Wildes, Z.M. BhujwallaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M.-F. Penet, T. Shah, S. Bharti, B. Krishnamachary,A. Maitra, Z.M. BhujwallaWriting, review, and/or revision of the manuscript: M.-F. Penet, T. Shah,D. Artemov, A. Maitra, Z.M. BhujwallaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Y. Mironchik, Z.M. BhujwallaStudy supervision: Z.M. Bhujwalla
AcknowledgmentsThe authors thank Dr. V.P. Chacko for expert technical assistance.
Grant SupportThis work was supported by NIH R01CA136576, R01CA138515,
R01CA73850, R01CA82337, P50CA103175, and P30 CA006973.The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.
Received April 18, 2014; revisedOctober 7, 2014; acceptedOctober 12, 2014;published OnlineFirst November 4, 2014.
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