Limitations of the dorsal skinfold window chamber model in evaluating anti-angiogenic therapy during early phase of angi...

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Title:
Limitations of the dorsal skinfold window chamber model in evaluating anti-angiogenic therapy during early phase of angiogenesis
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English
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Biel, Nikolett M.
Lee, Jennifer A.
Sorg, Brain S.
Siemann, Dietmar W.
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Bi Med Central ( Vascular Cell)
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Abstract:
Background: Angiogenesis is an essential process during tumor development and growth. The murine dorsal skinfold window chamber model has been used for the study of both tumor microvasculature and other vascular diseases, including the study of anti-angiogenic agents in cancer therapy. Hyperspectral imaging of oxygen status of the microvasculature has not been widely used to evaluate response to inhibition of angiogenesis in early tumor cell induced vascular development. This study demonstrates the use of two different classes of anti-angiogenic agents, one targeting the Vascular Endothelial Growth Factor (VEGF) pathway involved with vessel sprouting and the other targeting the Angiopoietin/Tie2 pathway involved in vascular destabilization. Studies evaluated the tumor microvascular response to anti-angiogenic inhibitors in the highly angiogenic renal cell carcinoma induced angiogenesis model. Methods: Human renal cell carcinoma, Caki-2 cells, were implanted in the murine skinfold window chamber. Mice were treated with either VEGF pathway targeted small molecule inhibitor Sunitinib (100 mg/kg) or with an anti-Ang-2 monoclonal antibody (10 mg/kg) beginning the day of window chamber surgery and tumor cell implantation. Hyperspectral imaging of hemoglobin saturation was used to evaluate both the development and oxygenation of the tumor microvasculature. Tumor volume over time was also assessed over an 11-day period post surgery. Results: The window chamber model was useful to demonstrate the inhibition of angiogenesis using the VEGF pathway targeted agent Sunitinib. Results show impairment of tumor microvascular development, reduced oxygenation of tumor-associated vasculature and impairment of tumor volume growth compared to control. On the other hand, this model failed to demonstrate the anti-angiogenic effect of the Ang-2 targeted agent. Follow up experiments suggest that the initial surgery of the window chamber model may interfere with such an agent thus skewing the actual effects on angiogenesis. Conclusions: Results show that this model has great potential to evaluate anti-VEGF, or comparable, targeted agents; however the mere protocol of the window chamber model interferes with proper evaluation of Ang-2 targeted agents. The limitations of this in vivo model in evaluating the response of tumor vasculature to anti-angiogenic agents are discussed. Keywords: Angiogenesis, Angiopoietin-2, Anti-angiogenic therapy, Dorsal skinfold window chamber model, Vascular endothelial growth factor
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Biel et al. Vascular Cell 2014, 6:17 http://www.vascularcell.com/content/6/1/17; Pages 1-11
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doi:10.1186/2045-824X-6-17 Cite this article as: Biel et al.: Limitations of the dorsal skinfold window chamber model in evaluating anti-angiogenic therapy during early phase of angiogenesis. Vascular Cell 2014 6:17.

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© 2014 Biel et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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RESEARCHOpenAccess Limitationsofthedorsalskinfoldwindow chambermodelinevaluatinganti-angiogenic therapyduringearlyphaseofangiogenesis NikolettMBiel 1* ,JenniferALee 2 ,BrianSSorg 3 andDietmarWSiemann 1,4 Abstract Background: Angiogenesisisanessentialprocessduringtumordevelopmentandgrowth.Themurinedorsal skinfoldwindowchambermodelhasbeenusedforthestudyofbothtumormicrovasculatureandothervascular diseases,includingthestudyofanti-angiogenicagentsincancertherapy.Hyperspectralimagingofoxygenstatus ofthemicrovasculaturehasnotbeenwidelyusedtoevaluateresponsetoinhibitionofangiogenesisinearlytumor cellinducedvasculardevelopment.Thisstudydemonstratestheuseoftwodifferentclassesofanti-angiogenic agents,onetargetingtheVascularEndothelialGrowthFactor(VEGF)pathwayinvolvedwithvesselsproutingand theothertargetingtheAngiopoietin/Tie2pathwayinvolvedinvasculardestabilization.Studiesevaluatedthetumor microvascularresponsetoanti-angiogenicinhibitorsinthehighlyangiogenicrenalcellcarcinomainduced angiogenesismodel. Methods: Humanrenalcellcarcinoma,Caki-2cells,wereimplantedinthemurineskinfoldwindowchamber.Mice weretreatedwitheitherVEGFpathwaytargetedsmallmoleculeinhibitorSunitinib(100mg/kg)orwithananti-Ang-2 monoclonalantibody(10mg/kg)beginningthedayofwindowchambersurgeryandtumorcellimplantation. Hyperspectralimagingofhemoglobinsaturationwasusedtoevaluateboththedevelopmentandoxygenationof thetumormicrovasculature.Tumorvolumeovertimewasalsoassessedoveran11-dayperiodpostsurgery. Results: ThewindowchambermodelwasusefultodemonstratetheinhibitionofangiogenesisusingtheVEGF pathwaytargetedagentSunitinib.Resultsshowimpairment oftumormicrovasculardevelopment,reducedoxygenation oftumor-associatedvasculatureandimpairmentoftumorv olumegrowthcomparedtocontrol.Ontheotherhand,this modelfailedtodemonstratetheanti-angiogeniceffectoftheAng-2targetedagent.Followupexperimentssuggestthat theinitialsurgeryofthewindowchambermodelmayinterferewithsuchanagentthusskewingtheactualeffectson angiogenesis. Conclusions: Resultsshowthatthismodelhasgreatpotentialtoev aluateanti-VEGF,orcomparable,targetedagents; howeverthemereprotocolofthewindowchambermodelinterfereswithproperevaluationofAng-2targeted agents.Thelimitationsofthis invivo modelinevaluatingtheresponseoftumorvasculaturetoanti-angiogenic agentsarediscussed. Keywords: Angiogenesis,Angiopoietin-2,Anti-angiogenictherapy,Dorsalskinfoldwindowchambermodel, Vascularendothelialgrowthfactor *Correspondence: nmolnar@ufl.edu 1 DepartmentofPharmacologyandTherapeutics,UniversityofFlorida CollegeofMedicine,CancerandGeneticsResearchComplex,2033Mowry Rd.,Gainesville,FL32610,USA Fulllistofauthorinformationisavailableattheendofthearticle VASCULAR CELL 2014Bieletal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated. Biel etal.VascularCell 2014, 6 :17 http://www.vascularcell.com/content/6/1/17

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Introduction Angiogenesis,theformationofnewbloodvesselsfrom pre-existingones,isanessentialprocessintumordevelopmentandgrowth[1].Angiogenesiscanbeseparated intotwokeysteps,(a)vasculardestabilizationmediated bytheAngiopoietin/Tie2axisand(b)endothelialcellactivationheavilyinfluencedbytheVascularEndothelial GrowthFactor(VEGF)anditsreceptorVEGFR-2[2,3]. TheuseofVEGFaxistargetedanti-angiogenicagentshas becomeastandardofcareinmanytumorsettingsinthe lastdecade.However,theirclinicalefficacyhasbeenlimited[4,5].Recently,anewclassofanti-angiogenicagents targetingAngiopoetin-2(Ang-2)hasemergedinhopesto circumventlackofresponseorresistanceseenwithVEGF targetedagents[6].Currentlymanyagentsarebeingevaluatedbothinpreclinicalandclinicalsettingsincluding targetsnotmentionedabove[4,6]. Thedorsalskinfoldwindowchamberiscommonly usedtoevaluatethemicrovasculatureinvarioussettings invivo .Themodelinvolvessurgicalimplantationofatitaniumwindowontoexposedmicrovasculatureonthe dorsalskinofmice[7,8].Thismethodhasbeenusedto evaluateangiogenesisinavarietyofprocessessuchas endometriosisandtumordevelopment;itwasalsoused toevaluatetheearlyphasesofpreclinicaldevelopment ofBevacizumabinthe1990s[9-14].Hyperspectralimagingofhemoglobinsaturationhasallowedforthe evaluationofoxygenationandhypoxia,oxygentransport dynamicsandcharacterizationoftheabnormalvascular physiologyandacuteoxygenfluctuationswithinthe tumormicrovasculature[15-18].Tumormicrovascular responsetovarioustherapiessuchasradiotherapy,vasculardisruptingagentsandsicklederythrocyteshasalso beenevaluatedusingthisimagingsystem[19-21].Applicationsotherthanthetumormicrovasculaturehavealso beenexploredforexamplethecharacterizationofarteriovenousmalformationi nhereditaryhemorrhagic talengiectasia,theformationofspontaneousandinduced A 0 10 20 30 40 50 60 70 80 90 Bright Field HbSat Bright Field HbSat Day 1 Day 6 Day 8 Day 1 Day 6 Day 8 B Figure1 Humanrenalcellcarcinoma,Caki-2,tumorgrowthinthewindowchamber.(A) Titaniumchambersaresurgicallyimplantedin nudemiceandtumorcellsinjected(210 4 )subcutaneouslyintothewindow. (B) Tumorcellinducedmicrovasculardevelopmentinthewindow chamberovertime.Hyperspectralimagingofhemoglobinsaturationprovideoxygenationstatusoftumormicrovasculature. Biel etal.VascularCell 2014, 6 :17 Page2of11 http://www.vascularcell.com/content/6/1/17

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microvascularthrombosisandocclusionsandimmune celllocalizationandtissuedamageinparticlebased vaccines[18,22,23]. Anti-angiogenicagentsarecommonlyevaluated invivo usingeithermatrigelplugorintradermalassaysaswell ashistologicalassessmentsofthenumberandfunction oftumorassociatedvasculature[24].Todate,themurine dorsalskinfoldwindowchambermodelcombinedwith hyperspectralimaginghasnotbeenwidelyutilized.This modelallowsfortheevaluationoftumorresponseto anti-angiogenicagentsandassessesnotonlythevascular densityofthetumorbutalsoitsoxygenationstatus.Real timeassessmentoftumorvasculatureatmicrovessel resolutionhastremendouspotentialtoanswerseveral importantquestionsregardingaspectsofvascularresponse toanti-angiogenicssuchasoxygenationstatusofthevasculature.ThecurrentstudyevaluatedbothaVEGFand Ang-2targetedapproachontheinductionofearlyhuman renalcellcarcinomacellindu cedangiogenesis.Limitations ofthismodelinthisparticularsettingquicklybecame apparentandarediscussedhere. Materialsandmethods Reagents MouseAng-2ELISAkit(MBS728992)waspurchased fromMyBioSource(SanDiego,CA).MECA-32(ratantimouseCat#120501)waspurchasedfromBioLegend (SanDiego,CA).NG2(rabbitanti-mouseCat#AB5320) wasobtainedfromMillipore(Temecula,CA).AlexaFluor 488(donkeyanti-rabbit)andAlexaFluor594(donkey anti-rat)werepurchasedfromInvitrogen(GrandIsland, NY).VectaShieldmountingmediumwithDAPIwaspurchasedfromVectorLabsInc.(Burlingame,CA).TissueTekOCTCompoundwaspurchasedfromSakuraFinetek (Torrance,CA).2-methylbutanewasobtainedfrom ThermoFisherScientific(Waltham,MA). B 0 0 10 20 30 6 7 8 9 10 11 12 Control anti-VEGF ** Days Tumor volume (mm 3 ) A 0 10 20 30 40 50 60 70 80 90 100% Background BF Hb BF Hb Sunitinib BF Hb Control BF Hb Day 1 Day 5 Day 6 Day 8 Day 1 Day 5 Day 6 Day 8 Control Sunitinib Figure2 VEGFinhibitioninCaki-2cellinducedangiogenesis. MicebearingwindowchamberswithCaki-2tumorsweretreatedwithVEGF inhibitor(Sunitinib). (A) Tumorvolumeand (B) tumormicrovascularresponsetoSunitinibwasevaluatedcomparedtocontrol.Median+90/10 percentile;control(n=8),Sunitinib(n=7)(combinationoftwoindependentexperiments).**,p<0.01,Mann – Whitney U -Test. Biel etal.VascularCell 2014, 6 :17 Page3of11 http://www.vascularcell.com/content/6/1/17

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Cellculture Thehumanclearcellrenalcellcarcinoma,Caki-2,cellline wasoriginallyreceivedfromDr.SusanKnox(Stanford University).Caki-2wasgrowninDulbecco ’ smodified minimumessentialmedium(D-MEM,Invitrogen,Grand Island,NY)supplementedwith10%FBS(Invitrogen, GrandIsland,NY),1%penicillin-streptomycin(Invitrogen, GrandIsland,NY),and1%200-mmol/L L -glutamine (Invitrogen,GrandIsland,NY).Cellswerekeptat37C, 5%CO 2 Drugpreparation KetamineandxylazinewerepurchasedfromWebster Veterinary(Devens,MA)andpreparedinsterilesaline. Theanti-Ang-2monoclonalantibodywaskindlyprovidedbyMedImmune,LLC.Thestocksolution(5mg/ml) wasdilutedtotheworkingdose(10mg/kg)insodiumcitratebuffersolution.Stocksolutionswerekept at 80Candworkingconcentrationsat4C.TheVEGFR smallmoleculeinhibitorSunitinibwasobtainedfrom LCLaboratories(Woburn,MA)andstoredat 20C. WorkingdoseofSunitinibwaspreparedfresheveryday bymakingstockanddiluentbuffersofcitricacid monohydrateandsodiumcitratedihydrateatpH6.8 and3.2respectively.A1:7stocktodiluentsolutionwas made(~pH3.3)andacidifiedtopH1.0,Sunitinibwas dissolved,andthenthesolu tionwasadjustedtopH3.5. WorkingconcentrationofSunitinibwaskeptatroom temperature. Windowchambersurgeryandtumorinitiation All invivo procedureswereconductedinagreement withaprotocolapprovedbytheUniversityofFlorida InstitutionalAnimalCareandUseCommittee.DorsalskinflapwindowchambersurgerieswerecarriedoutaspreviouslydescribedbyMoyandcolleagues[7].Briefly,female athymicnu/numice(HarlanLaboratories,Indianapolis,IN) weresurgicallyimplantedwithatitaniumwindowchamber onthedorsalskinflap.Duringthesurgicalproceduremice wereanesthetizedviaintraperi toneal(IP)injectionofketamine(100mg/kg)andxylazine(10mg/kg).Humanrenal cellcarcinoma,Caki-2,tumorwasinitiatedinthewindow chamberduringsurgerybyinjectingcells(210 4 in10 l volume)subcutaneouslyinthedorsalskinflappriortoplacinga12mmdiameternumber2roundglasscoverslip (ErieScientific,Portsmouth,NH)overtheexposedskin. Postsurgicalprocedure,animalswerehousedinanenvironmentalchambermaintainedat33Cand50%humidity withstandard12hrlight/darkcyclesfortheremainderof thestudy. Treatmentofwindowchambertumors MiceweretreatedwithSunitinib(100mg/kg)dailyviaoral gavageorwiththeanti-Ang-2antibody(10mg/kg)every 3daysviaIPinjection,startingthedayofwindowchamber surgery/tumorinitiationuptoday11post-surgerywhen micewereeuthanized.Duringthestudy,tumorsweremeasuredeverydayusingcalipersandtumorvolume(mm 3 ) wascalculated,assumingthatthetumorvolumewas 0 100 0 50 100 150 82 34 Average number of vessels/tumor 0 100 Sunitinib (mg/kg) Figure3 TumorvasculatureafterVEGFinhibition. Immunohistochemicalanalysisoftumorsatstudyendpoint.Line,median;*,p<0.05; Mann – Whitney U -Test.Representativeimagesofthemedianofeachgroup.Red,MECA.ImagestakenwithZeissAxioplanImaging2microscope with20objective;scalebar=140 m. Biel etal.VascularCell 2014, 6 :17 Page4of11 http://www.vascularcell.com/content/6/1/17

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halfofanellipsoid,usingthefollowingequation:tumor volume=[ /6d 1 d 2 height].StatisticalsignificancewasdeterminedusingtheMann – Whitney U -Test atp<0.05. Hyperspectralimaging Thespectralimagingsystem,imageacquisition,and imageprocessingmethodshavepreviouslybeendescribed[7,15].Briefly,windowchambertumorswere imageddailyusingaZeissAxioImagermicroscope(Carl Zeiss,Inc.,Thornwood,NY)with100-Wtungstenhalogen lamp,CCDcamerathermoelectricallycooledto 20C (DVCCo.,Austin,TX;Modelno.1412AM-T2-FW)and C-mountedliquidcrystaltunablefilter(LCTF)(CRIInc., Woburn,MA).TuningoftheLCTFandimageacquisition withtheCCDcamerawasautomaticallycontrolledwith LabVIEW8software(NationalInstrumentsCorp.,Austin, TX).Vascularhemoglobinsaturationmeasurementsand imageswerecreatedfromthespectralimagedata,using Matlabsoftware(TheMathworksInc.,Natick,MA). Duringimagingmicewereplacedonaheatedplatformandanesthetizedwith1-2%isofluorane(Webster Veterinary,Devens,MA)inair. Immunohistochemistry Elevendayspostsurgerymicewereeuthanizedwith euthasol(0.01ml/g)(WebsterVeterinary,Devens,MA), titaniumwindowchamberswereremovedandtumors werefreshfrozeninOCTandmethylbutane.Tumors weresectionedat5 mthicknessusingLeicaCM3050S cryostat(LeicaMicrosystemsInc.,BuffaloGrove,IL); sectionswereplacedonsuperfrostplusgoldslides (ThermoFisherScientificInc.,Waltham,MA)andkept at 80Cuntilimmunohistochemicalstaining.Tissue sectionswereacetonefixedfor10min,blockedin2% normalhorseserum,andincubatedovernightat4C withMECA-32andNG2primaryantibodies,atroom temperaturewithsecondaryantibodiesAlexaFluor488 0 10 20 30 40 50 60 70 80 90 100% Background BF Hb Control Anti-Ang-2 BF Hb BF Hb BF Hb 0 0 10 20 30 40 6 7 8 9 10 11 12 Control anti-Ang-2 Days Tumor volume (mm 3 ) B A Day 1 Day 5 Day 6 Day 8 Day 1 Day 5 Day 6 Day 8 Figure4 Ang-2inhibitioninCaki-2cellinducedangiogenesis. MicebearingwindowchamberswithCaki-2tumorsweretreatedwithAng-2 inhibitor(monoclonalantibody). (A) Tumorvolumeand (B) tumormicrovascularresponsetotheAng-2inhibitorwasevaluatedcomparedto control.Median+90/10percentile;control(n=8),anti-Ang-2(n=7)(combinationoftwoindependentexperiments). Biel etal.VascularCell 2014, 6 :17 Page5of11 http://www.vascularcell.com/content/6/1/17

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and594for1hr.Tissuesectionswereimagedwitha ZeissAxioplan2imagingmicroscope(CarlZeiss,Inc., Thornwood,NY)withEXFOX-Cite120lightsource (LumenDynamicsGroupInc.,Ontario,Canada).Images weretakenwithaRetigaEXiFastdigitalCCDcamera (QImaging,BritishColumbia,Canada)andprocessedin OpenLab5software(PerkinElmerInc.,Waltham,MA); RhodamineforMECA-32/AlexaFluor594,FITCforNG2/AlexaFluor488andDAPIfilterswereused.Vessel countswereobtainedbytakinguptotenrandomfields/ tumorat20objective,countingthenumberofvessels ineachrandomfield.Thenumberofperi-endothelial cellcoveredvesselswascountedinthetumorperiphery foreachtumor.Statisticalsignificancebetweencontrol andtreatedgroupswasdeterminedusingtheMann – Whitney U -Testatp<0.05. Serumangiopoietin-2levels Bloodfromthetailveinwasdrawnondays3and5post surgeryfrommice(2mice/group)that(1)didnothave surgery(baseline),(2)thatunderwentsurgeryand(3)ones thatunderwentsurgeryandinoculatedwithtumorcells. About100 lofbloodpermousewascollectedandplaced onicefor2hrtoletthebloodclot.Bloodwasthencentrifugedfor15min,4Cat1000g.Serumwascollectedand storedat 80Cuntilanalysis.SerumAng-2levelswere calculatedusingamouse-specificAng-2ELISAkit.Based oncompanyrecommendation,themanufacturer ’ sprotocol wasalteredtoload30 lofeitherstandardsorserumand 50 lof3.3folddilutedconjugatein0.9%NaClperwell andincubatefor2hrat37C;manufacturersprotocolwas otherwisefollowed. Intradermalangiogenesisassay All invivo procedureswereconductedinagreement withaprotocolapprovedbytheUniversityofFloridaInstitutionalAnimalCareandUseCommittee.Female athymicnu/numicewereinjectedintradermallywith 10 5 Caki-2cellsin10 lvolumeatfoursitesontheventralsurface.Beginningthedaypriortotumorcellinjection,miceweretreatedwitheitherdailyoralgavageof Sunitinib(100mg/kg)orIPinjectionoftheanti-Ang-2 antibody(10mg/kg)every3daysuptosixdayspost tumorcellinoculation.Miceweretheneuthanized,tumorsmeasuredviacalipersandtumorvolume(mm 3 ) calculated,assumingthetumorvolumetobeanellipsoid,usingthefollowingequation:tumorvolume= /6 d 1 d 2 height.Skinflapswerethenremovedandvesselsgrowingintotumornoduleswerecountedusinga LeicaMZ16FdissectingmicroscopewithLeicaKL1500 LCDfiberopticilluminator(LeicaMicrosystemsInc., BuffaloGrove,IL)at2.5xoriginalmagnification.ImageswerecapturedwithaRetigaEXiFast1394digital CCDcamera(QImaging,BritishColumbia,Canada)and OpenLab5software(PerkinElmerInc.,Waltham,MA). Statisticalsignificancebetweencontrolandtreated groupswasdeterminedusingtheMann – Whitney U -Test atp<0.05. 0 10 0 50 100 82 76 0 10 Figure5 TumorvasculatureafterAng-2inhibition. Immunohistochemicalanalysisoftumorsatstudyendpoint.Line,median;*,p<0.05; Mann – Whitney U -Test.Representativeimagesofthemedianofeachgroup.Red,MECA.ImagestakenwithZeissAxioplanImaging2microscope with20objective;scalebar=140 m. Biel etal.VascularCell 2014, 6 :17 Page6of11 http://www.vascularcell.com/content/6/1/17

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Results Humanrenalcellcarcinoma,Caki-2,growthinthe windowchamber Renalcellcarcinomaisahighlyvascularizeddisease andanti-angiogenicagentsarecurrentlyusedasboth firstandsecondlinetreatmentsinpatients.Caki-2 cells,aVHLmutant,highlyvascularandaggressively growingcellline,wereimplantedintothewindow chamber(Figure1A).Inductionoftumorcellinduced angiogenesiswasseenatdays5 – 6posttumorcell implantationatwhichpointthetumorsquicklyexpandedandbecameheavilyvascularizedwith50-80% hemoglobinsaturationinthemicrovasculatureatday8 (Figure1B). VEGFinhibitionimpedestumorandvesselgrowthin Caki-2tumorsinthewindowchamber Caki-2tumorsgrewrapidlyandexpandedtheirmicrovasculaturegreatlyovertimewithincreasedoxygenation levelswithinthetumormass.Treatmentofmicewith theVEGFinhibitorledtoasignificant5.2-foldreduction intumorvolume(p<0.01)(Figure2A)aswellasadramaticimpairmentoftumorvasculatureandoxygenation (Figure2B).Immunohistochemicalanalysisoftumor vasculatureatstudyendpointrevealeda2.4-foldreductioninvesselnumberofthetreatedgroupscomparedto control(p<0.0001)(Figure3). Ang-2inhibitiondoesnotaffectCaki-2tumorgrowthin thewindowchamber TreatmentofmicewiththeAng-2inhibitordidnot showimpairmentoftumorgrowthorvasculardevelopment(Figure4).However,immunohistochemicalanalysisoftumorvasculatureatstudyendpointrevealed a1.1-foldreductioninthetreatedgroupscompared tocontrol(p<0.05)(Figure5 ).Furthermore,analysis ofthevascularstructurerevealeda4-foldincreasein thenumberofvesselsthatmaintainedpericytecoverageinthetreatedgroupscomparedtocontrol(p<0.05) (Figure6). 0 10 0 50 100 150 200 250 21 86 anti-An g -2 (m g /k g ) C 0 10 MECA/ NG2/ DAPI A B MECA/ NG2/ DAPI MECA/ NG2/ DAPI # of pericyte covered vessels Figure6 VascularstructureafterAng-2inhibition. Immunohistochemicalanalysisofvascularstructureatstudyendpoint. (A) Normal vasculaturewithpericytecoverage. (B) Tumorvasculaturewithoutpericytecoverage.Scalebar,46 m. (C) Ang-2inhibitionledtoincreased numberofvesselsthatmaintainedpericytecoverage.Line,median;*,p<0.05;Mann-Whitney U -Test.Representativeimagesofeachgroup. Whitearrowsshowpericytecoveredvessels;yellowarrowshowsvesselswithoutpericytecoverage.Red,MECA(endothelium);green,NG2 (pericyte);blue,DAPI.ImagestakenwithZeissAxioplanImaging2microscopewith20objective;scalebar=140 m. Biel etal.VascularCell 2014, 6 :17 Page7of11 http://www.vascularcell.com/content/6/1/17

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VEGFandAng-2inhibitionintheintradermal angiogenesismodel ToevaluatetheinhibitionofboththeVEGF/VEGFR andAng-2/Tie2pathwaysintheabsenceofsurgicalinstallationofdorsalskinfloldwindowchambersanintradermalassaywasused.InhibitionoftheVEGF/VEGFR pathwaywithSunitinib(100mg/kg)ledtothereductionofbothtumorandvesselgrowthby43(p<0.001) (Figure7A)and2.5-fold(p<0.0001)(Figure7B)respectivelycomparedtocontrol.Treatmentofmicewiththe anti-Ang-2antibody(10mg/kg)ledtoareductionofboth tumorandvesselgrowthby3.1(p<0.001)(Figure7C)and 1.6-fold(p<0.0001)(Figure7 D)respectivelycomparedto control. Surgeryassociatedwiththewindowchambermodelled toanincreaseinserumAng-2levels ThereleaseofAng-2fromendothelialcellsasawound healingresponsehasbeenpreviouslynoted[25,26].The circulatingAng-2levelsweredeterminedinmicethat underwentsurgery.ResultsshowthatmicethatunderwentsurgeryhadanincreasedlevelofAng-2intheir circulationcomparedtocontrolmicethatdidnotreceivesurgery(Figure8).Furthermore,thepresenceof 0 100 0 2 4 6 8 ** Tumor volume (mm 3 ) 0 10 0 2 4 6 8 ** anti-Ang-2 (mg/kg) Tumor volume (mm 3 ) 0 10 0 40 80 120 63 39 *** anti-An g -2 (m g /k g ) Vessel count 0 100 0 40 80 120 63 25 *** Vessel count Control Sunitinib Control A C B D Sunitinib (mg/kg) Sunitinib (mg/kg) anti-Ang-2 Figure7 Ang-2andVEGFinhibitionintheintradermalassay. MicewereinjectedintradermallywithCaki-2renalcellcarcinomacellsand treatedwitheithertheAng-2inhibitor(10mg/kg)orSunitinib(100mg/kg)beginningthedaypriortotumorcellinoculation.Tumorvolume (A,C) andthenumberoftumorcellinducedbloodvessels (B,D )weredeterminedattheendofa7dayperiod.Bar,meanwithSEM(n=12); line,median(n=12);**,p<0.01;***,p<0.0001;Mann – Whitney U -Test. Biel etal.VascularCell 2014, 6 :17 Page8of11 http://www.vascularcell.com/content/6/1/17

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tumorcellsinmicethatunderwentsurgeryfurtherincreasedtheAng-2levelsinthecirculationcomparedto micethatonlyunderwentsurgery. Discussion Vascular-targetedagentsthatinhibittheformationof newbloodvesselsfrompre-existingoneshavebecomea standardofcareinseveralcancersettingsoverthelast decade[4,27].Acohortofpatientsdonotrespondor stoprespondingtotreatmentwiththecurrentlyFDA approvedanti-angiogenicagents.Asaconsequencenew pathwaysintheangiogenicprocesshavebeenexploited astherapeutictargets[4,6]. Themurinedorsalskinfoldwindowchambermodel hasbeenpreviouslyusedtostudyandunderstandthe microvasculatureofnotonlytumorsbutotherdiseases aswell[7-17,19,21-23].Recentlytheuseofhyperspectralimaging,toevaluatevascularoxygenationstatus throughhemoglobinsaturationabsorption,allowedfor moreindepthevaluationofmicrovascularresponseto, amongothers,vascular-targetedagentsandtheireffect onthemicrovasculature[20].Thedorsalskinfoldwindowchambermodelwithhighresolutionhyperspectralimaging,toourknowledge,hasnotbeenwidely usedtoevaluateanti-angiogenictherapyontheearly initiationofangiogenesis anddevelopmentoftumors. Ferraraandcolleagueshaveusedthewindowchamber modelwithbasicimagingtechniquestodemonstrate theanti-angiogeniceffectsofBevacizumabinpreclinicaldevelopment[11-14].T odate,therearenodatain theliteratureusingthismodeltoevaluateAng-2targetedagents. Inthepresentstudy,twodifferentclassesofantiangiogenicagentswereevaluated.SunitinibisasmallmoleculetyrosinekinaseinhibitorthattargetstheVEGFR1-3 andPDGFRandhasbeenFDAapprovedin2006asfirst linetreatmentinmetastatickidneycancer[28].TheantiAng-2monoclonalantibodywasusedastheAng-2targetedagent.ResultsshowthatCaki-2cellsinjectedat 210 4 cellsinitiateangiogenesis5 – 6daysposttumorcell injection(Figure1).WhenCaki-2tumorbearingmice weretreatedwiththeVEGFinhibitorduringan11day periodthegrowthofthetumorwassignificantlyinhibited by5.2-foldcomparedtountreatedtumors(Figure2A). Thedevelopmentoftumormicrovasculatureoftreated micewassignificantlyimpairedwithsparseandpoorly oxygenatedvessels(Figure2B).Immunohistochemicalanalysisoftumorsatendpointfurtherdemonstratedthe significantlyimpairedvasculatureoftreatedmicewitha 2.4-foldreductioninthenumberofvesselscompared tocontroltumors(Figure3).Theresultsclearlyshow thatthewindowchambermodelandhyperspectralimagingcanbeausefultooltoevaluateagentstargeting theVEGFpathwayandtheresponseofthetumorand microvasculaturetosuchtreatment.Resultsnicelydemonstratenotonlytheinhibitionofvasculardevelopmentbut alsothepooroxygenationofthemicrovasculaturethatthe tumorpossesses. Ontheotherhand,theresultsshowadifferenttumor responsetotheAng-2inhibitor.Micetreatedwiththe 0 400 500 600 700 800 900 1000 1100 Normal Surgery Surgery + tumor Serum Ang-2 (pg/ml) No surgery (normal) Surgery only Surgery + tumor Figure8 Ang-2serumlevelsinmice. LevelsofAng-2inthecirculationweredeter minedformicethatunderwentwindowchamber surgery /+tumorcellinjectionandcomparedtobasallevels(nosurgery). Biel etal.VascularCell 2014, 6 :17 Page9of11 http://www.vascularcell.com/content/6/1/17

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antibodydidnotshowanysignificantimpairmentof tumorgrowth(Figure4A)comparedtountreatedtumorsnordiditseemtohaveaneffectonthevascular densityoroxygenationofthevessels(Figure4B).These resultswereratherpuzzlingatfirstbutimmunohistochemicalanalysisrevealedaslightbutsignificantreductioninthevesselnumber(Figure5)aswellasahighly significant4-foldincreaseinpericytecoveredvesselswhen tumorsweretreatedwiththeAng-2inhibitor(Figure6). Furthermore,previousresultsshowedasignificantantiangiogeniceffectoftheAng-2inhibitor,whichistheoppositeresponsethatisseeninthewindowchambermodel [29].Therefore,theintradermalassaywasrepeatedwith boththeVEGFandAng-2inhibitorstocloselymirrorthe experimentconductedinthewindowchambermodel. ResultsfromtheintradermalassayshowthatVEGF inhibitionledtoasignificant43-(Figure7A)and2.5fold(Figure7B)reductionintumorvolumeandvessel numberrespectivelycomparedtocontrolcorrelatingwith theresultsseeninthewindowchambermodel.Contrary toresultsseenwiththeAng-2inhibitorinthewindow chamber,theantibodyledtoasignificant3.1-(Figure7C) and1.6-fold(Figure7D)reductionintumorvolumeand vesselnumbercomparedtocontrol.Similartumorvolume andvesselresultswiththeVEGFtargetedagentbutopposingresultswiththeAng-2targetedagentledtoquestionsaboutthedifferencebetweenthetwomodels.In general,thesoledifferencebetweentheintradermalassay andwindowchambermodelistheinitialsurgerythatis involvedwiththewindowchambermodel.Reviewofthe basicbiologyoftheAng-2andVEGFaxisinphysiological responsetoinjuryledtothehypothesisthatperhapsthe surgeryinvolvedwiththewindowchambermodelleadsto therapidreleaseofAng-2fromdamagedendothelialcells whentheskinflapiscuttoexposethevasculatureonthe skinthatisspared.Theangiopoietinaxisisnotonlyinvolvedwiththerapidresponsetovascularinjuryasa woundhealingresponsebutisalsoapro-inflammatory factor[25,26,30-33].Thesurgerytoimplantthewindow chambercouldnotonlyelicitawoundhealingbutalsoa pro-inflammatoryresponse. Tosupportthishypothesisserumsamplesfrommice thathavenotreceivedsurgery,micethatreceivedsurgeryandoneswithsurgeryandtumorcellinjectionwere evaluatedatvarioustimepointsaftersurgery.Results demonstratethatmicethatreceivedsurgeryhad1.4-and 1.5-foldhigherAng-2levelsintheserumcomparedto basallevelsinmicethatdidnotreceivesurgeryatdays3 and5postsurgeryrespectively(Figure8).Furthermore, micethatreceivedsurgeryandwereinjectedwithtumor cellshadaslightlyhigherincreaseof1.6-foldcomparedto baselineatdays3and5postsurgery(Figure8). Inconclusion,themurinedorsalskinfoldwindow chambermodelisavaluablemodeltoevaluatetumor microvasculatureandvascularoxygenationusinghyperspectralimaging.Cautionswiththismodelshouldbe takenwhenassessingthevascularresponsetotherapeuticstrategies.Inthisstudytwodifferentclassesof anti-angiogenicagentswereevaluated.TheAng-2/Tie2 axisisimportantinvasculardestabilizationwhilethe VEGF/VEGFRaxisplaysaroleinendothelialcellactivationtoproliferate,migrateandformnewvessels.While bothaxesareessentialinphysiologicalangiogenesissuch aswoundhealing,theyneverthelesshaveverydifferent roles.EndothelialcellsstoreAng-2inWeibel-Palade Bodiestobeabletoquicklyrespondtoenvironmental changessuchasvascularinjury[34-36]whileVEGFisessentialintheformationofnewvasculature,alaterresponseinwoundhealing[37].Itisclearthatthesurgery involvedwiththewindowchambermodelupsetsthe normalbalanceofAng-2inthemicroenvironmentleadingtoskewedresultstoAng-2inhibition.Thewindow chambermodel,however,isagreattooltoevaluatethe inhibitionofendothelialcellactivationandcouldbeutilizedtorapidlyandeffectivelyevaluateanti-angiogenic agentsinpreclinicalstudies.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions NMBhelpedwiththedesignofthestudy,carriedoutallexperimentand draftedthemanuscript.JALparticipatedinwindowchambersurgeries, hyperspectralimagingandbloodcollectionandeditedthemanuscript.BSS participatedinthedesignofthestudy,helpedwithhyperspectralimaging andeditedthemanuscript.DWSwastheprincipalinvestigator,helped designthisstudyandeditedthemanuscript.Allauthorsreadandapproved thefinalmanuscript. Acknowledgements TheauthorswouldliketothankMedImmune,LLCforkindlyprovidingthe anti-Ang-2monoclonalantibodyandMardaJorgensen(UniversityofFlorida TissueCore)forassistancewithimmunohistochemistry. Financialsupport ThesestudiesweresupportedinpartbyagrantfromtheNationalCancer Institute(PublicHealthServiceGrantCA089655)andNationalInstitutesof Health(T32TrainingGrant5T32CA009126-33). Authordetails1DepartmentofPharmacologyandTherapeutics,UniversityofFlorida CollegeofMedicine,CancerandGeneticsResearchComplex,2033Mowry Rd.,Gainesville,FL32610,USA.2J.CraytonPruittFamilyDepartmentof BiomedicalEngineering,UniversityofFlorida,BiomedicalSciencesBuilding, Gainesville,FL32610,USA.3CancerDiagnosisProgram,DivisionofCancer TreatmentandDiagnosis,NationalCancerInstitute,Rockville,MD20852, USA.4DepartmentofRadiationOncology,UniversityofFloridaCollegeof Medicine,2000SWArcherRoad,Gainesville,FL32610,USA. Received:30March2014Accepted:27July2014 Published:4August2014 References1.FolkmanJ: Tumorangiogenesis:therapeuticimplications. NewEngJMed 1971, 285: 1182 – 1186. 2.AugustinHG,KohGY,T hurstonG,AlitaloK: Controlofvascularmorphogenesis andhomeostasisthroughtheangiopoietin-Tiesystem. NatRevMolCellBiol 2009, 10: 165 – 177.Biel etal.VascularCell 2014, 6 :17 Page10of11 http://www.vascularcell.com/content/6/1/17

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Nat 2005, 438: 932 – 936.doi:10.1186/2045-824X-6-17 Citethisarticleas: Biel etal. : Limitationsofthedorsalskinfoldwindow chambermodelinevaluatinganti-angiogenictherapyduringearly phaseofangiogenesis. VascularCell 2014 6 :17. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Biel etal.VascularCell 2014, 6 :17 Page11of11 http://www.vascularcell.com/content/6/1/17