Distinguishing Inflammation from Tumor and Peritumoral ...activity was tested by adding...

Imaging, Diagnosis, Prognosis

Distinguishing Inflammation from Tumor and Peritumoral Edemaby Myeloperoxidase Magnetic Resonance Imaging

Anne Kleijn1,4, John W. Chen2, Jason S. Buhrman1, Gregory R. Wojtkiewicz2, Yoshiko Iwamoto2,Martine L. Lamfers4, Anat O. Stemmer-Rachamimov3, Samuel D. Rabkin1, Ralph Weissleder2,Robert L. Martuza1, and Giulia Fulci1

AbstractPurpose: Inflammation occurs routinely whenmanaging gliomas and is not easily distinguishable from

tumor regrowth by current MRImethods. The lack of noninvasive technologies that monitor inflammation

prevents us to understand whether it is beneficial or detrimental for the patient, and current therapies do

not take this host response in consideration. We aim to establish whether a gadolinium (Gd)-based agent

targeting the inflammatory enzyme myeloperoxidase (MPO) can selectively detect intra- and peritumoral

inflammation as well as glioma response to treatment by MRI.

Methods: We carried out serial Gd-bis-5-HT-DTPA (MPO-Gd) MRI before and after treating rodent

gliomas with different doses of oncolytic virus (OV) and analyzed animal survival. The imaging results were

compared with histopathologic and molecular analyses of the tumors for macrophage/microglia infiltra-

tion, virus persistence, and MPO levels.

Results: Elevated MPO activity was observed by MRI inside the tumor and in the peritumoral cerebrum

at day 1 post–OV injection, which corresponded with activation/infiltration of myeloid cells inhibiting OV

intratumoral persistence. MPO activity decreased, whereas tumor size increased, as the virus and the

immune cells were cleared (days 1–7 post–OV injection). A 10-fold increase in viral dose temporally

decreased tumor size, but augmented MPO activity, thus preventing extension of viral intratumoral

persistence.

Conclusions: MPO-Gd MRI can distinguish enhancement patterns that reflect treatment-induced

spatiotemporal changes of intratumoral and intracerebral inflammation from those indicating tumor

and peritumoral edema. This technology improves the posttreatment diagnosis of gliomas and will

increase our understanding of the role of inflammation in cancer therapy. Clin Cancer Res; 17(13);

4484–93. �2011 AACR.

Introduction

Management of brain tumors induces inflammatoryresponses that interfere with tumor imaging and monitor-ing the treatment course. Inflammation may also influencethe outcome of the therapy in 2 opposite ways. It can leadto tumor control by killing cancer cells and establishing ananticancer immunity (1–8) or to tumor promotion by

participating in glioma reoccurrence and progression (9–17). It is thus important to establish a noninvasive imagingtechnique that monitors intracerebral inflammation anddistinguishes it from tumor to understand the clinical andphysiologic consequences of this host response and toefficiently diagnose the outcome of cancer treatments thatenhance or inhibit local inflammation.

Oncolytic viruses (OV) present a great potential for thetreatment of malignant gliomas, due to their capacity toreplicate in situ and reach peripheral invasive cancer cells.However, OVs are very immunogenic and, despite theirreplication capacity, they are rapidly cleared from thetumor by inflammatory cells that engulf virus-infectedcancer cells (18–23). Because OV-induced inflammationis rapid and precisely localized, it is an optimal model toestablish techniques for in vivo imaging of intracerebralinflammation during glioma treatment.

Myeloperoxidase (MPO) is an inflammatory enzymepresent in myeloid cells (neutrophils, microglia, andmacrophages). It is secreted during inflammation byactivated, proinflammatory subsets of these cells (24).MPO utilizes hydrogen peroxide to catalyze the formation

Authors' Affiliations: 1Brain Tumor Research Center, 2Center for SystemsBiology and Center for Molecular Imaging Research, and 3Department ofPathology, Massachusetts General Hospital, Boston, Massachusetts;4Department of Neurosurgery, Erasmus MC, Rotterdam, the Netherlands

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

A. Kleijn and J.W. Chen contributed equally to the project.

Corresponding Author: Giulia Fulci, Brain Tumor Research Center, Mas-sachusetts General Hospital, Simches Research Building, CRPZN-3800,185 Cambridge St., Boston, MA 02114. Phone: (617) 643-3431; Fax: (617)643-3422; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-0575

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(13) July 1, 20114484

Research. on April 1, 2021. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 10, 2011; DOI: 10.1158/1078-0432.CCR-11-0575

http://clincancerres.aacrjournals.org/

of reactive oxygen species that kill pathogens, covalentlymodify lipids, cause local damage, and further activate theinflammatory cascade (24, 25). Gd-bis-5-HT-DTPA (MPO-Gd) is a molecular MRI agent that reports MPO activitywith high specificity and sensitivity (26–31). This agent hasbeen validated in vivo to evaluate MPO activity and inflam-mation in atherosclerosis (32), experimental autoimmuneencephalomyelitis (33), stroke (26), and myocardial ische-mia (28). Imaging of MPO activity is possible because of aprolonged gadolinium (Gd) enhancement caused byMPO-mediated oxidation of the Gd-chelating agent that inducesits polymerization and trapping in the tumor mesh (26, 27,30, 34). Therefore, immediately after MPO-Gd adminis-tration, the MRI highlights areas of vessel leakage in thetumor and allows measurement of tumor size whereasprolonged enhancement observed 1 to 2 hours after injec-tion of the agent reflects MPO activity.We have investigated the possibility of using MPO-Gd

MRI to analyze intratumoral and intracerebral inflamma-tion during glioma treatment with OV and tested whethersuch inflammation was associated with improved thera-peutic response. To do this, we examined the patterns ofMPO-Gd MRI contrast enhancement in 2 different rodentglioma models (the rat D74-HveC and the mouse CT-2Agliomas) treated with different doses of the oncolyticherpes simplex virus hrR3 (35) and compared the imagingresults with the extent of intratumoral/intracerebral infil-tration/activation of inflammatory cells, viral load, MPOlevels, and animal survival.Our results indicate that MPO-Gd MRI reports the

in vivo spatiotemporal evolution of the OV-inducedinflammatory response and provides a powerful tool tounderstand the role of inflammation during glioma OVtreatment.

Materials and Methods

Cells, tumor implantation, and treatmentD74/HveC rat glioma cells (36) were grown in

Dulbecco’s modified Eagle’s medium (DMEM) supple-mented with 10% fetal calf serum (FCS) and 7.5 mg/mLblasticidin S (Calbiochem; EMD Biosciences Inc.); CT-2Amouse glioma cells (provided by Dr. Thomas Seyfried,Boston College; ref. 37) were grown in DMEM with 10%FCS.

Male Fischer 344 rats (Taconic Farms Inc.) and C57BL/6Jmice (National Cancer Institute, Frederick, MD) were keptaccording to the guidelines of the Subcommittee onResearch Animal Care of MGH. Tumors were implantedusing stereotaxy as described (36). Seven days after implan-tation, 105 or 106 plaque forming units (pfu) of hrR3 orequivalent volume of phosphate buffer (PBS) were injectedin the tumor by using the same procedure and stereotacticcoordinates as described for the cancer cells.

LacZ and MPO gene expression analysisTotal RNA from the anterior quarter of the tumor-bearing

cerebrum was extracted with the RNeasy Lipid Tissue Kit(Qiagen), from animals treated with virus (106 or 105 pfu)or PBS (5 mL) for 1, 3, or 6 days. Three animals for eachtreatment group/virus dose/time point were used. cDNAwas synthesized using theOmniscript Reverse TranscriptaseKit (#205113; Qiagen) and random primers (Invitrogen).TaqMan PCR was carried out with the ABI Prism 7000 HTSequence Detection System and TaqMan PCR Master Mix(Applied Biosystems). PCR mix included a 25-mL aqueoussolution containing each primer at 0.9 mmol/L, the probe at200 nmol/L, and 2 mL of diluted cDNA. The PCR programincluded 1 cycle of 2minutes at 50�C, 1 cycle of 10minutesat 95�C, and 40 cycles comprising 15 seconds at 95�C,followed by 1 minute at 60�C. 18S rRNA was used asinternal control. Relative quantification of gene expressionwas calculated as 2DCt_lacZ or MPO/2DCt_r18S, where DCt ¼difference between the numbers of cycles needed to reachsaturation for the same gene in 2 different treatment groups.Primers used were as follows: LacZ forward-tgttgccactcgctt-taatgat, reverse-actcgccgcacatctgaact, probe-6FAM-cgctgtac-tggaggc-TAMRA; 18S rRNA forward-gggcccgaagcgtttact,reverse-ttccctagctgcggtatcca, probe-6FAM-caaagcaggcccga-gccgc-TAMRA; MPO forward-tggctggagtgcgatcttc, reverse-cgtgcatctgccagtttgag, probe-6FAM-tgaggccgatactgtc-TAMRA.

ImmunohistochemistryRodent brains from animals treated with 105 pfu of virus

or 5 mL PBS for 1, 3, or 9 days (3 animals/treatment group/time point) were frozen in an isopentane dry-ice bath andsectioned through the entire tumor volume. Every fifthsection was collected for analysis. Tissue slides were fixedin ice-cold acetone and stained as follows. After blockingendogenous proteins and peroxidases with serum-freeprotein block (#X0909) and peroxidase-blocking reagent(#002428; DAKO-Cytomation), sections were incubatedfor 1 hour at room temperature with the following primary

Translational Relevance

Glioma management induces intracerebral inflam-mation undistinguishable from tumor regrowth withcurrent MRI technologies. This poses a recurrent diag-nostic dilemma that prevents the ability of the oncol-ogists to provide the patients with a suitable treatmentplan in a timely fashion. Moreover, it is accepted thatinflammation influences the outcome of the treatment,but the role of this host response remains controversial,being described to control as well as promote tumorrecurrence and progression. Because of the lack ofmeans to monitor inflammation in vivo, this hostresponse is not taken in consideration by current thera-pies, thus preventing further understanding of its clin-ical significance. Herein we address this diagnosticproblem and establish a molecular MRI technology thattracks the spatiotemporal evolution of peri- and intra-tumoral inflammation while monitoring gliomaresponse to treatment. Because this technology utilizesa stable gadolinium-based compound, it has a strongtranslational potential.

Molecular MRI of Intracerebral Inflammation

www.aacrjournals.org Clin Cancer Res; 17(13) July 1, 2011 4485




antibodies (all antibodies were from Serotec, except forMPO antibody which was from Neomarkers): mouse anti-rat CD68 (MCA 341R) and CD163 (MCA 342R), rat anti-mouse F4/80 (MCA497RT), and rabbit anti-human MPO(Ab-1). For bright-field staining, the slides were then incu-bated with a horseradish peroxidase–conjugated (HRP)secondary antibody (ECL anti-mouse IgG, NA931V, andanti-rat IgG, NA935V; Amersham Biosciences Ltd.; andanti-rabbit IgG BA-1000; Vector Laboratories), a LiquidDAB Substrate Chromogen system used for detection(#K3465; DAKO-Cytomation), and hematoxylin for coun-terstaining. For fluorescent staining, the slides were incu-bated with an anti-rat IgG-FITC secondary antibody(Jackson Immunology) and mounted with propidiumiodide (PI) Vectashield mounting medium H-1300 (VectorLaboratories).

The paraffin-embedded human brain sections weredeparaffinized, rehydrated, and treated with 1% SDS beforestaining. The rabbit anti-humanMPO (Ab-1; Neomarkers),mouse anti-human CD68, and CD163 (AbD SerotecP34810 and Q86VB7) were used with the same secondaryantibodies as described earlier.

Intratumoral viral spread was analyzed by detectingb-galactosidase activity with X-gal (5-bromo-4-chloro-3-inodolyl-b-D-galactopyranoside; Sigma-Aldrich).

Spectrophotometric assay for MPO activityThe anterior quarter of the tumor-containing cerebrum

treated with 105 pfu of virus or 5 mL of PBS for 1, 3, or6 days (5 animals/group/time point) was extracted,weighed, and homogenized in 50 mmol/L potassiumphosphate buffer at pH 6.0. After centrifugation, the pelletwas resuspended in cetyltrimethylammonium bromidebuffer (Sigma-Aldrich), homogenized again, and soni-cated. After 3 cycles of freeze–thaw–sonication, the celllysates were centrifuged and the supernatants were col-lected. Protein concentration was measured with the BCAProtein Assay Kit (#23225; Pierce, Thermo Fischer Scien-tific). The protein extracts were then used to measure MPOactivity by detecting Amplite ADHP (AAT Bioquest) oxida-tion through spectrophotometry at 535 nm. The units ofactivity were computed according to the following formula:activity (U/mL) ¼ (DOD � Vt � 4)/(E � Dt � Vs), whereDOD is the change in absorbance, Vt is the total volume, Vsis the sample volume, E is the extinction coefficient¼ 3.9�104 mol�1 L s�1, and Dt is the change in time. The resultingactivity was normalized to 1mg of protein or 1mg of tissueto derive the specific activity. The specificity of the MPOactivity was tested by adding 4-aminobenzyoic acid hydra-zide to the protein lysates before the spectrophotometricreading (38).

MRIRats treated with 105 or 106 pfu of virus or 5 mL PBS for 1,

3, or 7 days (3 animals/treatment group/viral dose/timepoint) were anesthetized with isoflurane (2.0% at 2 L/min)and imaged using a 4.7-T, 16-cm bore MRI system (BrukerPharmascan). Rats were imaged 1 day before and 1, 3, and

7 days after injection of hrR3. MR images were acquiredbefore and 2 hours after the intravenous administration ofMPO-Gd, synthesized as previously described (29) fromDTPA-Gd (Magnevist; Berlex Laboratories), and injectedat 0.3 mmol/kg mouse and 0.1 mmol/kg rat, using aT1-weighted rapid acquisition with refocused echoes(RARE) sequence [repetition time (TR)¼ 800milliseconds,echo time (TE) ¼ 13 milliseconds, matrix (MTX) ¼ 192 �192, slice thickness ¼ 1.0 mm, number of excitations(NEX) ¼ 8, field of view (FOV) ¼ 2.5 cm for mouse and3.5 cm for rats). T2-weighted imaging was also carried out(TR ¼ 4217 milliseconds, TE ¼ 60 milliseconds, MTX ¼192 � 192, slice thickness ¼ 0.8 mm, NEX ¼ 8, FOV is thesame as for T1-weighted sequences). Standard DTPA-Gdwas used in control animals to verify that MPO-Gdenhancement was specific for MPO activity.

MRI data analysisRegions of interest (ROI) including the tumor, contral-

ateral brain tissue, and muscle were selected using thefreeware OsiriX (www.oxirix-viewer.com). Contrast-to-noise ratios (CNR) were computed for each ROI accordingto the following formula: CNR ¼ (postcontrast ROIlesion –postcontrast ROImuscle)/SDnoise – precontrast ROIlesion –precontrast ROImuscle)/(SDnoise), where ROIlesion is the ROIof the enhancing areas, and SDnoise is the SD of noisemeasured from an ROI placed in an empty area of theimage. CNRs were normalized by dividing each CNR by thehighest CNR to enable comparison between different ani-mals. Activation ratios (AR) were computed by dividing theCNR of the late phase (75 minutes) over the CNR of theearly phase (first postcontrast images, 5 minutes) toaccount for nonspecific enhancement from leakage. Tumorradius was computed by measuring the maximum trans-verse dimension.

Statistical analysisComparisons between multiple groups were carried out

with 2-sided ANOVA test followed by means comparisonswith post hoc Tukey’s test using the software Prism (Graph-pad Software Inc.). Comparison between 2 groups wascarried out using the Student t test. A value of P < 0.05was considered to be statistically significant. All error barsindicate SEM.

Results

Kinetics of activation and recruitment ofinflammatory cells

We have previously established that treatment of a ratglioma with hrR3 induces intratumoral recruitment ofmature peripheral macrophages (CD68þ/CD163þ) andactivation of brain inflammatory cells (CD68þ/CD163�)that accumulate around the tumor borders (19, 20). To useOV treatment as a model for establishing in vivo imagingtechniques of intracerebral/intratumoral inflammation, weanalyzed the kinetics of CD68þ and CD163þ cells activa-tion/recruitment during treatment of the D74-HveC rat

Kleijn et al.

Clin Cancer Res; 17(13) July 1, 2011 Clinical Cancer Research4486




glioma with hrR3 beginning 1 day post–viral injection to apoint when the animals appeared moribund (6 days fromPBS injection in control animals, and 9 days from viraldosing in treated rats), which was considered as end oftherapy. Because both these inflammatory cells subtypesengulfed hrR3-infected cancer cells (19, 20), we comparedthis kinetic profile with the kinetics of viral clearance.The virus was gradually and completely cleared from the

tumor between day 1 post–viral dosing and the end oftherapy (Fig. 1). The intratumoral viral concentrationmatched with activation/infiltration of CD68þ andCD163þ cells, which were prominent at day 1 post–viraldosing (compared with animals receiving PBS), decreasedat day 3, and were similar to control in moribund animals.Because brain inflammatory cells produce high levels of

MPO (31, 39), we hypothesized that MPO-Gd MRI couldmonitor OV-induced infiltration and activation of CD68þ

and CD163þ cells. To test this hypothesis, we analyzedwhether the kinetics of activation/infiltration of theseinflammatory cells and viral clearance corresponded tochanges in MPO mRNA levels and enzymatic activity(Fig. 2). For this assay, rats with established D74-Hvecgliomas were treated with PBS (controls) or hrR3 andsacrificed at days 1, 3, and 6 after virus injection. We chose6 days as the last time point because of the highmortality ofthe control rats. We found that more than 90%of viral LacZmRNA is cleared from the tumor between days 1 and 3 andthe remainder is cleared by day 6 [undetectable by reversetranscription (RT)-PCR; Fig. 2A]. Accordingly, we observedthe highest levels of MPOmRNA and activity at day 1 post–viral dosing and both decreased significantly by day 3(Fig. 2B–D). No MPO mRNA levels were detected by RT-PCR in control animals and at day 6 after virus injection,however, we could still observe some MPO activity in boththese situations (Fig. 2B and C). The decrease of MPOactivity between days 3 and 6 was not very strong in OV-treated animals and showed a high variability amonganimals, suggesting that intratumoral decrease of macro-

phages is ongoing but not yet fully established as observedwhen rats appeared moribund (Fig. 2C). Because it isimpossible to precisely dissect the tumor tissue from adja-cent cerebrum, these assays do not distinguish betweenintratumoral and peritumoral MPO activities. However,immunohistochemical staining of rat brains harboringgliomas and treated with OV or PBS clearly shows thepresence of MPO in both the intratumoral and peritumoralareas (Supplementary Fig. S1), thus matching the distribu-tion of CD68þ and CD163þ cells observed in Figure 1.

We have previously reported on the similarity of inflam-matory cellular responses in mice and rats carrying humanor syngeneic gliomas treated with different types of OV (19,20, 22, 23). Accordingly, comparison of hrR3 spread in theCT-2A mouse glioma between 6 and 72 hours from OVinjection showed that most of the intratumoral viruseswere cleared within 3 days of delivery, and this matchedwith infiltration of F4/80þ macrophages (Fig. 3A) andincreased MPO mRNA levels and enzymatic activity(Fig. 3B). Moreover, the GBM from a patient treated withthe oncolytic adenovirus ONYX-015 (40) also presentedinfiltration of CD68þ and CD163þ cells (20) expressingMPO (Supplementary Fig. S2).

Altogether, these data indicate that the inflammatoryresponse induced by OV is associated with MPO mRNAlevels and enzymatic activity. This phenomenon is notspecies-specific and suggests the possibility to imageinflammation in vivo with MPO-Gd MRI.

MPO-Gd MRI during OV treatmentWe determined whether MPO-Gd MRI could detect the

kinetics of CD68þ and CD163þ cells activation/infiltrationobserved by immunohistochemistry and distinguish theareas of inflammation from the bulk tumor tissue. Weimaged rats carrying D74-HveC tumors 1 day previraldosing for tumor baseline signal, and 1, 3, and 7 dayspost–hrR3 injection (Fig. 4). Each imaging sessionincluded analysis of tumor size (we measured the diameter

Figure 1. Correlation of hrR3clearance with activation/infiltration of CD68þ and CD163þ

immune cells in the rat D74-HveCglioma. Column 1 (LacZ),decrease of viral intratumoralspread [b-galactosidase staining(LacZ): blue] between days 1 and 9post–hrR3 injection (black arrowsindicate persisting OV clones).Columns 2 and 3, CD68þ cells(brown) in the tumor andperitumoral area (black arrows) inanimals receiving hrR3 (CD68-hrR3) or PBS (CD68-PBS).Columns 4 and 5, CD163þ cells(brown) localized exclusivelyinside the tumor of hrR3- andPBS-treated animals (CD163-hrR3 and CD163-PBS). For eachtime point, adjacent slides areshown.

4: CD163-hrR31: LacZ 2: CD68-hrR3

3 mm

Day 9 Day 9 Day 6 Day 9 Day 6

3 mm3 mm3 mm3 mm


3 mm3 mm3 mm3 mm3 mm


3 mm3 mm3 mm3 mm3 mm

3: CD68-PBS 5: CD163-PBS






of the region enhancing 5 minutes after injection of theMPO-Gd contrast agent) and quantification of the MPOactivation ratio (determined by analyzing the persistence ofthe contrast 75 minutes after injection of MPO-Gd). Wecould not image the animals after day 7 postviral dosingbecause of a rapid health decline.

Before viral dosing (day 0), the MPO-Gd enhancementfaded over time during the imaging session, indicatinglow MPO activity (Fig. 4A). At days 1 and 3 post–viraldosing, the MPO-Gd contrast decreased comparablyslower inside the tumor center and increased in theparenchyma surrounding the tumor (Fig. 4A). Calcula-tion of the MPO activation ratio from MPO-Gd MRIshowed a 3-fold increase between days 0 and 1 and atrend of decrease from day 1 to 7 (Fig. 4C), whichmatched with the MPO activity measured through theenzymatic assays (Fig. 2C). At day 7, most of the enhance-ment in the center of the tumor faded 75 minutes afterMPO-Gd injection (Fig. 4A and C) and few CD163þ

macrophages were detected by histopathology in thesetumors (Supplementary Fig. S3). However, there waspersistence of MPO-Gd contrast at the periphery betweenthe tumor and the brain parenchyma and histopathologyrevealed accumulation of CD68þ cells in the same regions(Supplementary Fig. S3). MRI carried out at day 1 afterOV treatment, using standard DTPA-Gd as contrast agent,indicated rapid leakage of this agent from the tumortissue and adjacent brain (Fig. 4B and D). These resultsshow that MPO-Gd contrast enhancement comes from

MPO secreted by 2 distinct cell populations, inside andsurrounding the treated tumor, reflecting a difference inthe host response to OV therapy.

Comparison of images obtained through MPO-Gd MRIwith those obtained using dextran-coated iron oxide par-ticles (MION), which are incorporated by phagocytes andconfer superparamagnetic properties to these cells (41), onanimals treated with hrR3 for 3 days indicates that these 2technologies for in vivo imaging of immune cells overlaponly partially. MION imaging did not detect the peritu-moral area of inflammation visible through MPO-Gd MRIbut had a broader distribution inside the tumor (Supple-mentary Fig. S3).

Analysis of tumor size (Fig. 4E) indicated tumor growthbetween days 0 and 1. This growth was slower betweendays 1 and 3 post–hrR3 injection and became pronouncedagain between days 3 and 7. The retardation of tumorgrowth observed between days 1 and 3 is likely due to theoncolytic activity of the virus and was not observed inanimals treated with PBS. Notably, the overall trend oftumor size change after virotherapy, as reported on thediameters measured by MRI at 5 minutes after delivery ofthe contrast agent, is opposite of the trend of in vivo MPOactivity measured from delayed images (75 minutes post–MPO-Gd injection; cf. Fig. 4C and E), underscoring thatthis technology can distinguish areas of inflammation fromthe tumor tissue.

We then imaged and measured hrR3-mediated inductionof MPO activity with MPO-Gd MRI in mice carrying the

LacZ

mR

NA

fold

indu

ctio

n 1.2

A B

C D

1.0

0.8

0.6

0.4

0.2

0.0

ctrl d1 d3 d6

Day 3 or 6 vs. day 1: P < 0.0001

MP

O m

RN

A fo

ld in

duct

ion 1.2

1.0

0.8

0.6

0.4

0.2

0.0

ctrl d1 d3 d6

Day 3 or 6 vs. day 1: P < 0.0001

MP

O a

ctiv

ity/m

g of

pro

tein

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4ctrl d1 d3 d6

Day 6 vs. day 1 post-OV: P < 0.0325

MP

O a

ctiv

ity/m

g of

tiss

ue

0

5

10

15

20hrR3 + ABAH

PBS

hrR3

*P = 0.0075

*

**

**P = 0.009

Figure 2. Kinetics of intracerebralviral LacZ and host MPO mRNAlevels and of MPO activity. A andB, decrease in viral LacZ and hostMPO mRNA levels between days(d) 1 and 6 post–hrR3 injection(n¼ 3). The mRNA levels at day 1were arbitrary considered as 1,and the folds of decrease at day 3and day 6 from day 1 werecalculated. At each time point, wealso collected RNA from animalstreated with PBS, but no LacZ orMPO mRNA was detected in thiscontrol (ctrl) group and is indicatedwith a dotted flat line at 0. C,average MPO activity (n ¼ 5) inexcised rat brains bearing gliomatreated with hrR3 (continuedcurve) or PBS (dotted flat line) for1, 3, and 6 days (significantdifference only between day 6 andday 1 post–hrR3 injection, and atdays 1 and 3 between PBS- andhrR3-treated animals, P < 0.0001).The specificity of the assay forMPO activity was established in apreliminary experiment byinhibiting MPO with4-aminobenzyoic acid hydrazide(ABAH; ref. 38) before thespectrophotometric reading (D).

Kleijn et al.





CT-2A glioma (Fig. 5). Also in this case, we detected a spatio-temporal change in the enhancement pattern between theearly and delayed images, which differentiated tumor frominflammation. We found that increased in vivo MPO activ-ity reported by molecular MRI following viral treatmentmatched with the infiltration of F4/80þ monocytic cells(Fig. 3) and with increased MPO activity measured throughenzymatic assays on excised tumors (Fig. 3), suggesting thatthis technology is applicable to different species.

Increased viral dose augments the inflammatoryresponse and does not allow prolonged viralpersistenceTo further match the MPO-Gd contrast enhancement

with changes in virus-induced inflammation, we comparedrats treated with 2 different doses of hrR3 (105 and 106

pfu). Increasing viral load at the time of injection did notchange the time frame with which the virus is completelycleared; that is, at 7 days, no virus was detected throughRT-PCR in either treatment group (data not shown). Oneday after virus injection, there was 10-fold difference inLacZ between these 2 treatment groups that related to thedifference in viral dose, but the MPO mRNA levels weresimilar (Fig. 6A and B). At day 3, the difference in LacZ

mRNA between these 2 treatment groups was only about2-fold and the group receiving the higher viral dose hadincreased MPO mRNA (Fig. 6A and B). This correspondedto increased MPO activity measured by MPO-Gd MRI(Fig. 6C) and smaller tumor size (Fig. 6D).

Discussion

We have shown that the myeloid cells activated in thebrain and infiltrating the tumor upon OV treatment induceMPO, thus allowing MRI of their inflammatory activitythrough a gadolinium-based molecular imaging agent (27,29). Our data indicate that the kinetics of intracerebral/intratumoral inflammation, verified by histopathology ofCD68þ and CD163þ cells in a rat orthotopic gliomamodeltreated with an oncolytic herpes simplex virus, correspondsto the kinetics of MPO activity measured on excised brainsby an enzymatic assay. These changes can be tracked in vivothrough MPO-Gd MRI. The specificity of the contrastinduced by MPO-Gd was proved by comparing MPO-GdMRI with standard DTPA-Gd MRI. Moreover, previouslypublished works have shown that there is no detectableMPO-Gd MRI contrast in mice knocked out for MPO,thus emphasizing the specificity of this MRI agent (26, 28,

Figure 3. Intratumoral hrR3persistence, macrophageinfiltration, and MPO levels in themouse CT-2A glioma. A, decreaseof intratumoral hrR3 (blue)between 6 and 72 hours post–OVinjection, and infiltration of F4/80þmacrophages (green) into thetumor (red, PI) treated with hrR3 orPBS for 72 hours. The imagesshow almost the whole tumorarea, which occupies the entirephotographic field. B, MPOmRNA(fold induction in hrR3 versus PBS;n ¼ 3) and enzymatic activity(n¼ 5) in brains with CT-2Agliomas treated with hrR3 or PBS.

2 mm

2 mm

6 h post-OV

A

B

3 d post-OV

1 mm 1 mm

OVOV

1 mm

PBS

1 mm

PBS

hrR3 intratumoral spread F4/80+ macrophages Tumor PI staining

0

4

8

12

16

20

hrR3PBS

**M

PO

act

ivity

/mg

of ti

ssue

**P = 0.030

**P = 0.035

OV + ABAH0

2

4

6

8

MP

O m

RN

A in

duct

ion

fold

**P = 0.001

PBS hrR3






31). The levels of viral LacZ and host MPO mRNAsdisplay similar kinetics. However, whereas MPO mRNAis only detectable during the acute phase of this inflam-matory response, baseline MPO activity is always present.This could be due to a general low abundance of MPOmRNA molecules or to a dual MPO regulation system:

genetic and enzymatic (42). Because MPO-Gd MRI isreproducible in mouse and rat gliomas established inthe respective syngeneic animal model and because wehave shown that activation of CD68þ and CD163þ cells isnot virus-, tumor-, or species-specific, and that these cellsinfiltrate the tumor of patients treated with OV (19, 20,

5 minA

B

75 min

Day 0 Day 0

Day 1 Day 1

5 min 75 min

Day 3 Day 3

Day 7 Day 7

5 min Day 1 75 min Day 1

TumorPeriphery

MP

O a

ctiv

atio

n ra

tio

0 2 4 6 8

0.0

0.5

1.0

C

D

E

Day

Peripheral MPO day 3 vs. day 0: P = 0.024Peripheral MPO day 7 vs. day 0: P = 0.023Tumor MPO day 1 vs. day 0: P = 0.046

0.0

0.5

MP

O a

ctiv

atio

n ra

tio

Tumor Periphery

MPO-Gd

Gd

PBS OV

0.00

0.01

0.02

0.03

0.04

*

0 2 4 6 80.2

0.3

0.4

0.5

0.6

MT

D (

cm)

OVPBS

Day

Tum

or s

ize

slop

e(d

ay 1

to d

ay 7

)

P = 0.039

Figure 4. MPO-Gd MRI duringoncolytic virotherapy of ratgliomas. A, MRI scans of 1 rat withD74-HveC tumor pre- (day 0) andpost–hrR3 injection (days 1, 3 and7) carried out 5 and 75 minutesafter MPO-Gd injection. The blackcircles indicate the tumor areadetermined from the scans at 5minutes post–MPO-Gd injection;black arrows indicate areas ofenhancement in peritumoral area;at day 1 and day 3, theenhancement reaches areas distalfrom the tumor, whereas at day 7 itis concentrated at the tumorborder. B, MRI scans of 1 rat atday 1 post–hrR3 injection for 5 and75 minutes postinjection ofDTPA-Gd. C, kinetics of intra- andperitumoral MPO-Gdenhancement in 3 rats treated withhrR3 (P values for significanttemporal differences areindicated). D, comparison ofMRI-calculated MPO activationrate in animals imaged withMPO-Gd or with DTPA-Gd at1 day post–OV injection.E, kinetics of tumor growth(maximum tumor radius ¼MTD) inrats treated with PBSor OV.

Kleijn et al.





22, 23) and secrete MPO, there is high translationalpotential for this technology. This is strengthened bythe lower toxicity of MPO-Gd than that of many clinicallyapproved MRI agents (30).An important advantage of MPO-Gd MRI is its ability to

distinguish between inflammation and tumor. Inflamma-tion decreases between days 1 and 7 post–viral dosing,whereas the tumor size increases. Moreover, comparison ofanimals treated with 2 different doses of virus shows

stronger inflammation and smaller tumors for animalsreceiving the highest viral dose. Indeed, even thoughOVs induce other oxidases in cancer cells, MPO-Gd isspecific to MPO, which is expressed only in myeloid cells.Current MRI strategies cannot distinguish between inflam-matory areas from tumor regrowth. This is a recurrentdiagnostic dilemma that prevents the ability of the oncol-ogists to provide the timeliest and most suitable treatmentplan to the patient. Thus, this MRI technology can solve acritical diagnostic problem for the treatment of braintumors. To establish the broad diagnostic applicability ofthis technology, it is important to test its capacity to detectinflammation during other therapeutic strategies such asradiation, immunotherapies, and treatment with anti-inflammatory and antiangiogenic drugs.

Attempts to image intracerebral inflammation by MRIwere previously made using MIONs (41), which detect allphagocytic cells. However, even though this strategy candetect OV-induced intratumoral macrophages (19, 20), itdoes not enhance the peritumoral inflammation, nor pro-vides information on tumor size. Conversely, MPO-GdMRI can identify spatiotemporal changes of active inflam-matory cells infiltrating into the cancer and in the brainparenchyma surrounding the tumor. Fusion of MION andMPO-Gd MRI scans in the same tumor model suggests thatthese 2 strategies detect 3 different cellular subsets: (i)intratumoral phagocytic cells not making MPO; (ii) peri-tumoral MPO-secreting cells that are not phagocytic; and(iii) intratumoral phagocytic cells that also produce MPO.It is not clear whether the peritumoral cells are notenhanced by MION MRI because MIONs do not reachthese cells or because they are not phagocytic. We havepreviously published that CD68þ microglia surroundingthe tumor infiltrates into the tumor region and engulfs theinfectious OV (20), suggesting that MIONs do not diffuseinto the peritumoral parenchyma. However, the phagocyticactivity of these cells may be acquired only after theirinfiltration into the tumor, indicating that the different

Figure 5. MPO-Gd MRI in themouse CT-2A glioma. A, MRIscans 5 and 75 minutes afterMPO-Gd injection in 1 mouse withCT-2A tumor 1 day before (day 0)and 3 days post–hrR3 injection.B, quantification of MPOactivation rate in 3 mice beforeand after treatment.

Day 0

Day 3

5 min

5 min

Day 0 75 min

Day 3 75 min

0.0

0.2

0.4

0.6

0.8

1.0

1.2

A B

MP

O a

ctiv

atio

n ra

tio

Day 0 Day 3

**P = 0.003α = 0.05

0

2

4

6

8

10

A B

C D

106 pfu105 pfu

106 pfu105 pfu

106 pfu105 pfu

106 pfu105 pfu

LacZ

mR

NA

fold

indu

ctio

nM

PO

act

ivat

ion

ratio

Day 1 Day 3 Day 1 Day 3

*P < 0.0001**P = 0.0013

MP

O m

RN

A fo

ld in

duct

ion

MT

D (

cm)

0.00.51.01.52.02.53.03.5

*P = 0.0025

0.0

0.2

0.4

0.6

0.8

*

*P = 0.019

*

**

*

*

0.0

0.2

0.4

0.6

0.8

Day 1 Day 3 Day 1 Day 3

*P = 0.046*

Figure 6. Treatment diagnosis in animals receiving different doses ofhrR3. A and B, changes in mRNA levels for the viral LacZ and host MPOgenes between animals receiving 105 pfu of hrR3 (considered as baselinelevel 1) and those receiving 106 pfu of virus at 1 and 3 days after treatment(n ¼ 4). C and D, differences in MPO activation rate and tumor radiusmeasured through MPO-Gd MRI at 1 and 3 days post–hrR3 injection at105 or 106 pfu (n ¼ 3).






cellular subsets enhanced by MION and MPO-Gd MRI arethe same cells at different stages of inflammatory activa-tion. Further comparison and combination of these ima-ging technologies will allow a deeper understanding of thedistinct role of individual innate immune cells in theinflammatory responses (31, 39, 43–46).

Imaging inflammation is also useful in ways other thansimply recognizing it in a recurring tumor after a specifictreatment. As our understanding of cancer biology advances,increased relevance is being given to this host defenseresponse. Even though the role of inflammation in theultimate therapeutic outcome is unclear (being describedboth as controlling and as promoting tumor recurrence andprogression), it is accepted that it influences tumor treatmentoneway or the other. It is therefore crucial to establish in vivotechniques that allow understanding of the relationshipbetween inflammationand theoutcomeof tumor treatment.Demand for these techniques is further emphasized by thecurrent trend of examining drugs that modulate immuneprocesses, such as COX-2 inhibitors and nonsteroidal anti-inflammatory drugs, as anticancer agents. Finally, inflam-mation poses a crucial dilemma in OV treatment. Eventhough it is established that host innate immunity is detri-mental for OV lytic activity, inflammatory cells can alsokill cancer cells and synergize with OV in tumor treatment(47, 48). Virotherapies aimed at increasing and suppressingOV-induced immunity were studied and both strategiespresented positive results (47, 48). In this respect, our dataindicate that the increase in the viral dose temporallyaugments the infiltration/activation of MPOþ cells. Eventhough the higher viral dose transiently decreases the tumor

size, it is impossible to establish from these data whetherthe temporal lysis of the tumor is mediated by OV orimmune cells alone or by the combination of these 2 factors.Further studies comparing MPO-Gd MRI and MION MRIduring OV treatment in the presence of anti-inflammatoryor proinflammatory agents will bring more insights intothe role that different innate immune cells may have in thetherapeutic outcome.

In conclusion, MPO-Gd MRI will strongly improve ourdiagnostic ability of the effects of cancer treatment on thetumor versus tumor microenvironment. Imaging inflam-mation during proinflammatory and immunosuppressivetherapies will allow understanding of its role in cancertreatment and on side effects of current therapies.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by the National Cancer Institute (R21-CA135526 to G. Fulci), the National Institute for Neurological Disorders(R01-NS070835 and R01-NS072167 to J.W. Chen, R01-NS032677 to R.L.Martuza, and P30-NS045776 to S. Rabkin), and the National Heart, Lungand Blood Institute (K08-HL081170 to J.W. Chen) at the NIH.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 1, 2011; revised April 26, 2011; accepted April 29, 2011;published OnlineFirst May 10, 2011.

References1. Auf G, Carpentier AF, Chen L, Le ClancheC, Delattre JY. Implication of

macrophages in tumor rejection induced by CpG-oligodeoxynucleo-tides without antigen. Clin Cancer Res 2001;7:3540–3.

2. Castriconi R, Daga A, Dondero A, Zona G, Poliani PL, Melotti A, et al.NK cells recognize and kill human glioblastoma cells with stem cell-like properties. J Immunol 2009;182:3530–9.

3. Chang GH, Barbaro NM, Pieper RO. Phosphatidylserine-dependentphagocytosis of apoptotic glioma cells by normal human microglia,astrocytes, and glioma cells. Neuro Oncol 2000;2:174–83.

4. Friese MA, Steinle A, Weller M. The innate immune response in thecentral nervous system and its role in glioma immune surveillance.Onkologie 2004;27:487–91.

5. Graf MR, Jadus MR, Hiserodt JC, Wepsic HT, Granger GA. Devel-opment of systemic immunity to glioblastoma multiforme using tumorcells genetically engineered to express the membrane-associatedisoform of macrophage colony-stimulating factor. J Immunol1999;163:5544–51.

6. Graf MR, Prins RM, Merchant RE. IL-6 secretion by a rat T9 gliomaclone induces a neutrophil-dependent antitumor response with resul-tant cellular, antiglioma immunity. J Immunol 2001;166:121–9.

7. Nickles D, Abschuetz A, Zimmer H, Kees T, Geibig R, Spiess E, et al.End-stage dying glioma cells are engulfed by mouse microglia with astrain-dependent efficacy. J Neuroimmunol 2008;197:10–20.

8. Wallace PK, Romet-Lemonne JL, Chokri M, Kasper LH, Fanger MW,Fadul CE. Production of macrophage-activated killer cells for target-ing of glioblastoma cells with bispecific antibody to FcgammaRI andthe epidermal growth factor receptor. Cancer Immunol Immunother2000;49:493–503.

9. Condeelis J, Pollard JW. Macrophages: obligate partners fortumor cell migration, invasion, and metastasis. Cell 2006;124:263–6.

10. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860–7.

11. de Visser KE, Korets LV, Coussens LM. De novo carcinogenesispromoted by chronic inflammation is B lymphocyte dependent. Can-cer Cell 2005;7:411–23.

12. Hussain SF, Yang D, Suki D, Grimm E, Heimberger AB. Innate immunefunctions of microglia isolated from human glioma patients. J TranslMed 2006;4:15.

13. Iliopoulos D, Hirsch HA, Struhl K. An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to celltransformation. Cell 2009;139:693–706.

14. Markovic DS, Glass R, Synowitz M, Rooijen N, Kettenmann H.Microglia stimulate the invasiveness of glioma cells by increasingthe activity of metalloprotease-2. J Neuropathol Exp Neurol 2005;64:754–62.

15. Morandi E, Zingaretti C, Chiozzotto D, Severini C, Semeria A, Horn W,et al. A cDNA-microarray analysis of camptothecin resistance inglioblastoma cell lines. Cancer Lett 2006;231:74–86.

16. Morimura T, Neuchrist C, Kitz K, Budka H, Scheiner O, Kraft D, et al.Monocyte subpopulations in human gliomas: expression of Fc andcomplement receptors and correlation with tumor proliferation. ActaNeuropathol (Berl) 1990;80:287–94.

17. Synowitz M, Glass R, F€arber K, Markovic D, Kronenberg G, HerrmannK, et al. A1 adenosine receptors inmicroglia control glioblastoma-hostinteraction. Cancer Res 2006;66:8550–7.

Kleijn et al.





18. Friedman A, Tian JP, Fulci G, Chiocca EA, Wang J. Glioma virother-apy: effects of innate immune suppression and increased viral repli-cation capacity. Cancer Res 2006;66:2314–9.

19. Fulci G, Breymann L, Gianni D, Kurozomi K, Rhee SS, Yu J, et al.Cyclophosphamide enhances glioma virotherapy by inhibiting innateimmune responses. Proc Natl Acad Sci U S A 2006;103:12873–8.

20. Fulci G, Dmitrieva N, Gianni D, Fontana EJ, Pan X, Lu Y, et al.Depletion of peripheral macrophages and brain microglia increasesbrain tumor titers of oncolytic viruses. Cancer Res 2007;67:9398–406.

21. Kurozumi K, Hardcastle J, Thakur R, YangM, Christoforidis G, Fulci G,et al. Effect of tumor microenvironment modulation on the efficacy ofoncolytic virus therapy. J Natl Cancer Inst 2007;99:1768–81.

22. Lamfers ML, Fulci G, Gianni D, Tang Y, Kurozumi K, Kaur B, et al.Cyclophosphamide increases transgene expression mediated by anoncolytic adenovirus in glioma-bearing mice monitored by biolumi-nescence imaging. Mol Ther 2006;14:779–88.

23. Wakimoto H, Fulci G, Tyminski E, Chiocca EA. Altered expression ofantiviral cytokine mRNAs associated with cyclophosphamide'senhancement of viral oncolysis. Gene Ther 2004;11:214–23.

24. Arnhold J, Flemmig J. Humanmyeloperoxidase in innate and acquiredimmunity. Arch Biochem Biophys; 500:92–106.

25. Saran M, Beck-Speier I, Fellerhoff B, Bauer G. Phagocytic killing ofmicroorganisms by radical processes: consequences of the reactionof hydroxyl radicals with chloride yielding chlorine atoms. Free RadicBiol Med 1999;26:482–90.

26. BreckwoldtMO, Chen JW, Stangenberg L, Aikawa E, Rodriguez E, QiuS, et al. Tracking the inflammatory response in stroke in vivo bysensing the enzyme myeloperoxidase. Proc Natl Acad Sci U S A2008;105:18584–9.

27. Chen JW, Pham W, Weissleder R, Bogdanov A Jr. Human myeloper-oxidase: a potential target for molecular MR imaging in atherosclero-sis. Magn Reson Med 2004;52:1021–8.

28. Nahrendorf M, Sosnovik D, Chen JW, Panizzi P, Figueiredo JL, AikawaE, et al. Activatable magnetic resonance imaging agent reportsmyeloperoxidase activity in healing infarcts and noninvasively detectsthe antiinflammatory effects of atorvastatin on ischemia-reperfusioninjury. Circulation 2008;117:1153–60.

29. Querol M, Chen JW, Weissleder R, Bogdanov A Jr. DTPA-bisamide-based MR sensor agents for peroxidase imaging. Org Lett 2005;7:1719–22.

30. Rodriguez E, Nilges M, Weissleder R, Chen JW. Activatable magneticresonance imaging agents for myeloperoxidase sensing: mechanismof activation, stability, and toxicity. J AmChemSoc 2010;132:168–77.

31. Swirski FK, Wildgruber M, Ueno T, Figueiredo JL, Panizzi P, IwamotoY, et al. Myeloperoxidase-rich Ly-6Cþmyeloid cells infiltrate allograftsand contribute to an imaging signature of organ rejection in mice. JClin Invest 2010;120:2627–34.

32. Ronald JA, Chen JW, Chen Y, Hamilton AM, Rodriguez E, Reynolds F,et al. Enzyme-sensitive magnetic resonance imaging targeting mye-loperoxidase identifies active inflammation in experimental rabbitatherosclerotic plaques. Circulation 2009;120:592–9.

33. Chen JW, Breckwoldt MO, Aikawa E, Chiang G, Weissleder R.Myeloperoxidase-targeted imaging of active inflammatory lesions in

murine experimental autoimmune encephalomyelitis. Brain 2008;131:1123–33.

34. Chen JW, Querol Sans M, Bogdanov A Jr, Weissleder R. Imaging ofmyeloperoxidase in mice by using novel amplifiable paramagneticsubstrates. Radiology 2006;240:473–81.

35. Goldstein DJ, Weller SK. An ICP6::lacZ insertional mutagen is used todemonstrate that the UL52 gene of herpes simplex virus type 1 isrequired for virus growth and DNA synthesis. J Virol 1988;62:2970–7.

36. Wakimoto H, Johnson PR, Knipe DM, Chiocca EA. Effects of innateimmunity on herpes simplex virus and its ability to kill tumor cells.Gene Ther 2003;10:983–90.

37. Marsh J, Mukherjee P, Seyfried TN. Akt-dependent proapoptoticeffects of dietary restriction on late-stage management of a phos-phatase and tensin homologue/tuberous sclerosis complex 2-defi-cient mouse astrocytoma. Clin Cancer Res 2008;14:7751–62.

38. Kettle AJ, Gedye CA, Hampton MB, Winterbourn CC. Inhibition ofmyeloperoxidase by benzoic acid hydrazides. Biochem J 1995;308:559–63.

39. Swirski FK, Nahrendorf M, Etzrodt M, Wildgruber M, Cortez-Reta-mozo V, Panizzi P, et al. Identification of splenic reservoir mono-cytes and their deployment to inflammatory sites. Science2009;325:612–6.

40. Chiocca EA, Abbed KM, Tatter S, Louis DN, Hochberg FH, Barker F,et al. A phase I open-label, dose-escalation, multi-institutional trial ofinjection with an E1B-Attenuated adenovirus, ONYX-015, into theperitumoral region of recurrent malignant gliomas, in the adjuvantsetting. Mol Ther 2004;10:958–66.

41. Moore A, Weissleder R, Bogdanov A Jr. Uptake of dextran-coatedmonocrystalline iron oxides in tumor cells and macrophages. J MagnReson Imaging 1997;7:1140–5.

42. Van Der Veen BS, de Winther MP, Heeringa P. Myeloperoxidase:molecular mechanisms of action and their relevance to human healthand disease. Antioxid Redox Signal 2009;11:2899–937.

43. Anderson CF, Mosser DM. A novel phenotype for an activatedmacrophage: the type 2 activated macrophage. J Leukoc Biol2002;72:101–6.

44. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. NatRev Immunol 2005;5:953–64.

45. Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T,Figueiredo JL, et al. The healing myocardium sequentially mobilizestwo monocyte subsets with divergent and complementary functions.J Exp Med 2007;204:3037–47.

46. Sunderk€otter C, Nikolic T, Dillon MJ, Van Rooijen N, Stehling M,Drevets DA, et al. Subpopulations of mouse blood monocytes differin maturation stage and inflammatory response. J Immunol2004;172:4410–7.

47. Prestwich RJ, Errington F, Diaz RM, Pandha HS, Harrington KJ,Melcher AA, et al. The case of oncolytic viruses vs the immunesystem: waiting on the judgment of Solomon. Hum Gene Ther2009;20:1119–32.

48. Stanford MM, Breitbach CJ, Bell JC, McFadden G. Innate immunity,tumor microenvironment and oncolytic virus therapy: friends or foes?Curr Opin Mol Ther 2008;10:32–7.






2011;17:4484-4493. Published OnlineFirst May 10, 2011.Clin Cancer Res Anne Kleijn, John W. Chen, Jason S. Buhrman, et al. Edema by Myeloperoxidase Magnetic Resonance ImagingDistinguishing Inflammation from Tumor and Peritumoral

Updated version

10.1158/1078-0432.CCR-11-0575doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2011/05/26/1078-0432.CCR-11-0575.DC1Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/17/13/4484.full#ref-list-1

This article cites 47 articles, 17 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://clincancerres.aacrjournals.org/content/17/13/4484To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-11-0575http://clincancerres.aacrjournals.org/content/suppl/2011/05/26/1078-0432.CCR-11-0575.DC1http://clincancerres.aacrjournals.org/content/17/13/4484.full#ref-list-1http://clincancerres.aacrjournals.org/cgi/alertsmailto:[email protected]://clincancerres.aacrjournals.org/content/17/13/4484http://clincancerres.aacrjournals.org/

/ColorImageDict > /JPEG2000ColorACSImageDict > /JPEG2000ColorImageDict > /AntiAliasGrayImages false /CropGrayImages false /GrayImageMinResolution 200 /GrayImageMinResolutionPolicy /Warning /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict > /GrayImageDict > /JPEG2000GrayACSImageDict > /JPEG2000GrayImageDict > /AntiAliasMonoImages false /CropMonoImages false /MonoImageMinResolution 600 /MonoImageMinResolutionPolicy /Warning /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 900 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict > /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False

/CreateJDFFile false /Description > /Namespace [ (Adobe) (Common) (1.0) ] /OtherNamespaces [ > /FormElements false /GenerateStructure false /IncludeBookmarks false /IncludeHyperlinks false /IncludeInteractive false /IncludeLayers false /IncludeProfiles false /MarksOffset 18 /MarksWeight 0.250000 /MultimediaHandling /UseObjectSettings /Namespace [ (Adobe) (CreativeSuite) (2.0) ] /PDFXOutputIntentProfileSelector /NA /PageMarksFile /RomanDefault /PreserveEditing true /UntaggedCMYKHandling /LeaveUntagged /UntaggedRGBHandling /LeaveUntagged /UseDocumentBleed false >> > ]>> setdistillerparams> setpagedevice

Date post:	20-Oct-2020
Category:	Documents
Upload:	others
View:	2 times
Download:	0 times

Distinguishing Inflammation from Tumor and Peritumoral ...activity was tested by adding...

Documents