(3.3.6)--脑科学与影像新技术.pdf
MRI biomarkers identify the differential response of glioblastomamultiforme to anti-angiogenic therapyShahrzad Jalali,Caroline Chung,Warren Foltz,Kelly Burrell,Sanjay Singh,Richard Hill,and Gelareh ZadehBrain Tumor Research Center,Hospital for Sick Children,Toronto,Canada(S.J.,K.B.,S.S.,G.Z.);Radiation Medicine Program,PrincessMargaret Cancer Centre,University Health Network,Toronto,Canada(C.C.,W.F.);Department of Radiation Oncology,Universityof Toronto,Toronto,Canada(W.F.,R.H.);Ontario Cancer Institute,Princess Margaret Cancer Centre,and Department of Medical Biophysics,Universityof Toronto,Toronto,Canada(R.H.);Division of Neurosurgery,University of Toronto and Toronto Western Hospital,University HealthNetwork,Toronto,Canada(G.Z.)Corresponding Author:Gelareh Zadeh,MD,PhD,FRCSC,Toronto Western Hospital,Division of Neurosurgery,399 Bathurst Street,West Wing 4thFloor,Toronto,Ontario M5T 2S8(gelareh.zadehuhn.ca).Background.Although anti-angiogenic therapy(AATx)holds greatpromise for treatment of malignant gliomas,itstherapeutic efficacyis not well understood and can potentially increase the aggressive recurrence of gliomas.It is essential to establish sensitive,non-invasive biomarkers that can detect failure of AATx and tumor recurrence early so that timely adaptive therapy can be instituted.We investigated the efficacy of MRI biomarkers that can detect response to different classes of AATxs used alone or in combinationwith radiation.Methods.Murine intracranial glioma xenografts(NOD/SCID)were treated with sunitinib,VEGF-trap or B20(a bevacizumab equivalent)alone or in combination with radiation.MRI images were acquired longitudinally before and after treatment,and various MRI para-meters(apparent diffusion coefficient,T1w+contrast,dynamic contrast-enhanced DCE,initial area under the contrast enhance-ment curve,and cerebral blood flow)were correlated to tumor cell proliferation,overall tumor growth,and tumor vascularity.Results.Combinatorial therapies reduced tumor growth rate more efficiently than monotherapies.Apparent diffusion coefficient wasan accurate measure of tumor cell density.Vascular endothelial growth factor(VEGF)-trap or B20,but not sunitinib,resulted in sig-nificant reduction or complete loss of contrast enhancement.This reduction was not due to a reduction in tumor growth or micro-vascular density,but rather was explained by a reduction in vessel permeability and perfusion.We established that contrastenhancement doesnot accuratelyreflect tumor volume orvasculardensity;however,DCE-derived parameterscan be used as efficientnoninvasive biomarkers of response to AATx.Conclusions.MRI parameters following therapy vary based on class of AATx.Validation of clinically relevant MRI parameters for indi-vidual AATx agents is necessary before incorporation into routine practice.Keywords:anti-angiogenic therapies,glioblastoma multiforme(GBM),MRI biomarker.Glioblastoma(GBM)is the most aggressive human cancer,with amedian survival ranging from 1218 months from time of diag-nosis and a nearly 100%local recurrence rate following maximaltherapy.1,2One very important step in managing GBM is identify-ing noninvasivemethodsthatpredictresponse to therapyand de-tect early tumor recurrence so that early adaptive treatmentmeasures can be instituted.The histopathological hallmark of malignant transformation toa GBM is the presence of vascular proliferation.3It isthis highly vas-cularized nature of GBMs that has resulted in significant interest inanti-angiogenic therapy(AATx)to control tumor progression andrecurrence.4Vascularendothelialgrowthfactor(VEGF)isthecentralfactor in regulating GBM neovascularization;therefore,strategiesthat inhibit VEGF have been explored the most.5Bevacizumab(Avastin),a humanized monoclonal antibody against VEGF-A,iscurrently accepted as therapy for recurrent GBMs,6and there issome consideration to its use upfront as a primary line of therapyat the time of diagnosis.7Another VEGF inhibitor,VEGF-trap,anengineered VEGF receptor that selectively binds to and inhibits“free”VEGF,has also been shown to be effective in preclinical mod-els of GBM.8,9More recent approaches have explored alternateclasses of AATx compounds including multi-tyrosine kinaseReceived 4 November 2013;accepted 19 February 2014#The Author(s)2014.Published by Oxford University Press on behalf of the Society for Neuro-Oncology.All rights reserved.For permissions,please e-mail:.Neuro-OncologyNeuro-Oncology 16(6),868879,2014doi:10.1093/neuonc/nou040Advance Access date 23 April 2014868 by guest on August 13,2015http:/neuro-oncology.oxfordjournals.org/Downloaded from Fig.1.Growth rate of intracranial GBM xenografts in response to 2 anti-angiogenic therapies(AATx)used as monotherapy or as combinatorial therapywith radiation therapy(RT).(A)Mouse intracranial xenografts were generated using GSC-1(i)and U87(ii)cell lines,and changes in tumor volume werecompared longitudinally between these tumor models.(B)U87 xenografts were treated with AATxs(sunitinib or VEGF-trap),RT alone,or AATx+RT.Tumor volumes were measured using T2w-MRI images on days 7,10,14,17,and 21 post tumor implantation.Line graphs(i,ii)are representativeof relative tumor volume normalized to baseline tumor(day 7)in control and experimental groups.Bar graphs(iii)compare the relative tumorJalali et al.:MRI biomarkers for Anti-angiogenic therapy in GBMNeuro-Oncology869 by guest on August 13,2015http:/neuro-oncology.oxfordjournals.org/Downloaded from inhibitors such as sunitinib,10which has been shown to reduce gli-oma tumor growth in GBM xenografts.11,12Despite the attractive mechanistic approachof AATx therapy ina highly vascularized tumor,clinical experience to date suggeststhat AATx drugs have not significantly increased the survival ofGBM patients.Bevacizumab,despite demonstrating initial clinicalimprovements and radiological response,has shown transientbenefits,and GBM recurrence is inevitable,often with a muchmore invasive phenotype.1315Use of VEGF-trap has been asso-ciated with some radiological response,but without any signifi-cant improvement in recurrent GBM.16Similarly,sunitinib hasfailed to show benefits in improving progression-free survival inGBMs.17The ability to detect recurrence early,prior to a more aggres-sive phenotype,is critical for initiating alternative targeted ther-apies.Furthermore,it is now believed that the efficacy of AATxcan be potentiated if it is combined with standard chemotherapy,radiation therapy(RT),or other targeted therapies.18,19Establish-ing noninvasive biomarkers that can accurately detect the re-sponse to different classes of AATx is important and may beuseful for scheduling combinatorial therapy.Multiparametric MRI provides a promising noninvasive strat-egy to characterize cellular and vascular properties of tumorsin addition to defining response to chemotherapy,RT,and AATxtherapies.2022For instance,parameters derived from perfusionMRI have been used to assess response to AATx therapies inpatients diagnosed with GBM.23Diffusion MRI parameters haveshown value in predicting survival outcome in patients with re-current GBM who are treated with bevacizumab.24However,todate there has been no clinical or preclinical studies that directlycompare various MRI parameters in response to different classesof AATx.Clinical studies are limited in their ability to obtain repeatmultiple biopsies at the time of perceived tumor recurrence.Assuch,preclinical studies provide the advantage of making directcomparison between specific MRI parameters and tumor charac-teristics in response to various classes of AATx.In this study,wefocused on establishing MRI parameters that are indicative oftumor cellularity,growth,and vascularity in response to AATxwhen administered as monotherapy or in combination with RTand evaluating these MRI parameters across different classesof AATxs in preclinical models of GBM.Findings from this studycould be used to inform directed questions in the clinical settingusing cohorts of patients followed serially with frequent MRI im-aging that compares clinical outcome and tumor progressionwithMRIanddynamiccontrast-enhanced(DCE)-basedparameters.Materials and MethodsCells and TransfectionWe used 2 different GBM cell lines for this study.One,an estab-lished human glioma stem cell line U87(ATCC,Rockville)and theother,a glioma stem cell line(GSC-1)obtained from a humanGBM tumor specimen at The University of Texas MD AndersonCancer Center(Houston),as previously described.25To generatestable U87 or GSC-1 cell lines with VEGF-trap expression,cellswere transfected with VEGF-trap using a PiggyBac transposonsystem(provided by Dr.Nagy),as previously described.26Intracranial Xenograft Models of Glioblastoma MultiformeAll animal experiments were carried out according to institutionalAnimal Care Committee guidelines.Intracranial xenografts weregenerated,as described previously.27Seven days post U87 cellimplantation and 21 days post GSC-1 cell implantation,weused baseline MRI to ensure equal tumor sizes prior to institutingtreatment.We ensured successful intracranial expression ofVEGF-trap in-vivo by imaging the sectioned brain ex-vivo(Supple-mentary Fig.S1).Anti-angiogenic TherapyIn order to allow direct one-to-one comparison of the effects ofAATx with a sufficient number of animals for each group,we lim-ited the time window of the comparison to 10 days of treatment(days 717),starting 1 week following tumor cell implantation.Each treatment arm included 810 mice repeated in duplicate,and the AATxs evaluated here included sunitinib,VEGF-trap,andB20,which is the mouse equivalent of bevacizumab or Avastin(Genentech).Sunitinib(Pfizer)was prepared as previouslyreported,28and a dose of 40 mg/kg/day was administered byoral gavage.Mice implanted with U87:VEGF-trap cells weregiven 3 mg/mL doxycycline(Sigma)in drinking water.B20 wasadministered intraperitoneally(150 mg/mouse)every 3 days.The onset of treatment remained the same for all AATxs.Tostudy the immediate effects of eliminating VEFG-trap on physio-logical characteristics of tumor vasculature,the supply of doxy-cycline was stopped between days 17 and 21 post intracranialimplantation of the U87:VEGF-trap cells.The tumors wereallowed to grow until the mice became moribound and wereeuthanized according to animal care protocols.Radiation TherapyA single fraction of 8 Gy radiation was delivered at day 8 followingintracranial injection using a cone-beam CT image-guided smallanimal irradiation system(XRT225Cx,Precision X-Ray,Inc),inwhich mice were positioned in an in-house,custom-built stereo-tactic immobilization device,as previously described.29,30Magnetic Resonance ImagingMRI was performed with a 7 Tesla Biospec 70/30(Bruker Corpor-ation),using the B-GA12 gRTient coil insert and 7.2 cm innerdiameter,linearlypolarizedvolumeresonatorcoilforvolume between all treatment groups within the 10-day treatment window.(C)Apparent diffusion coefficient(ADC)maps were generated fromDWI-MRI images.Bar graphs represent percentage change in ADC values from normal contralateral brain region.(D)Bar graphs represent thenumber of Ki67 positive tumor cells(i)and tumor cell density at day 17 post intracranial injection(ii).In all cases,values represent mean+SE of 810 independent experiments per each time point.Significant differences from control are represented as*P,.05,*P,.01,and*P,.0001.Jalali et al.:MRI biomarkers for Anti-angiogenic therapy in GBM870 by guest on August 13,2015http:/neuro-oncology.oxfordjournals.org/Downloaded from Fig.2.Change in contrast enhancement of GBM xenografts using contrast MRI images in response to anti-angiogenic therapies(AATx)and AATx+radiation therapy(RT).(A)MRI images examining contrast enhancement of U87 and GSC-1 intracranial xenografts.Representative T2w,and T1w+contrast images demonstrate MRI differences between U87 and GCS-1 tumors,with GSC-1 showing a more invasive tumor but with less contrastenhancement than U87(i).MRI images acquired on day 17 following U87 tumor implantation(10 days following treatment with AATx,RT orAATx+RT).T2w images outline tumor region and T1w+contrast plus DCE images represent contrast enhancement in different experimental groups(ii).(B)Bar graphs represent extent and percentage change in contrast enhancement of the tumor region compared with adjacent normal brain onT1w+contrast MRI images.Values are shown as mean+standarderror,and significant differences between eachtreatment arm and control group aredepicted as*P,.05 and*P,.01.(C)Line graphs show relative changes in tumor volume(measured on T2w-MRI)and contrast enhancement(measured on T1w+contrast MRI)for control and each treatment arm.There is a lack of correlation between tumor growth and change in contrastenhancement.Jalali et al.:MRI biomarkers for Anti-angiogenic therapy in GBMNeuro-Oncology871 by guest on August 13,2015http:/neuro-oncology.oxfordjournals.org/Downloaded from radiofrequency transmission,as detailed previously.29Multipara-metric MRI was used to detect real-time changes in physiologicalproperties ofatumor inresponse to single-agent orcombinatorialtherapy with AATx at days 7,10,14,and 17 after intracranialtumor implantation.The acquisition of multiparametric MRIimages and analysis of cerebral blood flow(CBF)are describedin Supplementary Material.Magnetic Resonance Image AnalysisMIPAV software(National Institutes of Health)was used to ana-lyze the MRI images.Tumor region of interest(ROI)on T2-weighted(T2w)images was manually defined and used tomeasure the tumor volume.For contrast enhancement analysis,the percentage increase in contrast enhancement of the tumorROI on T1-weighted(T1w)+contrast images was normalized toadjacent nontumor region.Apparent diffusion coefficient(ADC)and dynamic contrast enhanced(DCE)maps were generatedfrom diffusion(DWI)or DCE-MRI images.Tumor ROIs on T1w+contrast or T2w images were transposed to corresponding diffu-sion and DCE maps.For diffusion analysis,percentage change inADC value of tumor,as compared with similar contralateral re-gion,was calculated based on evidence of improved motion sen-sitivity.29Contrast enhancement of the regions that wereoverlapping with ventricles was excluded from analysis.For DCEanalysis,tumor ROIand arterial input functionwereselected,pro-pagated to all time points on each slice,and used forextraction ofcontrast enhancement.Average contrast enhancement data andDCE tool v1.04(www.TheDCETool.ca)were used to calculate theinitial area under the contrast enhancement curve(iAUC).Theconcentration of contrast agent in tumor region over time wascalculated using modified Tofts model kinetic analysis.ImmunostainingImmunostaining was performed on cryopreserved frozen brainsections of control and treated animals using anti-human pri-mary antibodies including anti-CD31(1:500,BD Pharmingen),anti-Ki67(1:25,Dako),anti-angiopoietin-1(Ang-1)(1:100,SantaCruz Biotechnology),and anti-angiopoietin 2(Ang-2)(1:100,Santa Cruz Biotechnology)per manufacturers protocol.
