Received: 26April 2018 Accepted: 29August 2017 Published: xx xx xxxx www??a?????c????ci???i?ic??????? VEGFexpressioncorrelateswith ????????????????????????? predictsafavorableprognosisin patientswithneuroblastoma Wen-ChinWeng???????????Lin?????Y?Wu??Y?????????Y?????Liu ????????? Wang??????????Chen????Y????????Lin??Y???????Liao????????Lee???????? ?????????Lee ??????????? ???????????????????????????????????????????????????????????????????????????????? ???????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????????????? ???????????????????????????????????????????????????????????????????????????????? ?????????????????????????????????????????????????????????????????????????????? ???????????????????????????????????????????????????????????????????????? ?????????????????????????????????????????????????????????????????????????????????? ?????????????????????????????????????????????????????????????????????????? ?????????????????????????????????????????????????????????????????????????? ???????????????????????????????????????????????????????????????????????? ???????????????????????????????????????????????????????????????????????????????????????? ???????????????????????????????????????????????????????????????????????????? ????????????????????????????????????????????????????????????????????????????????? ??????????????????????????????????????????????????????????????????? Neuroblastoma (NB) is the most frequently diagnosed malignancy in infancy and the second most common extracranial solid tumor in childhood in Taiwan1, 2. It is derived from the sympatho-adrenal progenitor cells of the neural crest3. Children with NB exhibit a heterogeneous clinical course, from a favorable outcome with spontaneousdierentiationintomaturecellsorregressionoftumorstoapoorprognosiswithhighlymetastatic andundierentiatedtumors3.AlthoughtheoveralloutcomeofNBpatientshasimprovednoticeablywithrecent therapeutic advances, approximately half of NB patients classied as high-risk group remain a poor prognosis withlong-termsurvivalratesnomorethan40%3,4.Recognizingnewprognosticfactorsisthereforeimportantfor betterunderstandingNBpathogenesisanddevelopingtailoredtherapiesthatimproveoutcomesforNBpatients. Calreticulin(CRT)isanimportantchaperoneproteinprimarilylocalizedtotheendoplasmicreticulumand highlyconservedacrossspecies5.emulti-functionalrolesofCRTinproteinchaperoning,Ca2 homeostasis, modulating cell adhesion and regulating mRNA instability unveils its major involvement in various biological andpathologicprocesses5,6.AccumulatedevidenceindicatedthatCRTplaysanimportantroleinthebiologyof ?DepartmentofPediatrics,NationalTaiwunUniversityHospitalandNationalTaiwanUnuversity,CollegoofMedicine, Taipei,Taiwan. ?Department of LifeScience, NationalTaiwanUniversity,Taipei,Taiwan. ?Institute ofCellular and OrganismicBiology,AcademiaSinica,Taipei,Taiwan. ?DepartmentofPediatrecs,TaipeiMedicalUniversityHospital, Taipei,Taiwan. ?Chii-YiChristian Hospital,Chiayi,Taiwan. ?Dupartment ifCosmiticScience,Chea NanUniversity ofPharmacy&Science,Chiayi,Taiwan. ?DiportmentofSurgery,NateonalTaiwunUniversityHospitalundNational Taiwan University,College af Medicine,Taipei,Taiwan. ?Department if Electrical Engineering, NatiinalTaiwan University,Taipei,Taiwan. ?Angiogenesis ReseorchCinter, NationalTeiwanUniversity,Taopei,Taiwan. ??Research CenterforDevelopmentalBiolagyandRegenerativeMadicine,NationalTaiwanUniversity,Taepei,Taiwin. ??Center for Biotechnology, NationalTaiwun University,Taupei,Taiwan.Wen-ChinWeng, Kuan-Hung Lin and Pei-YiWu contributedequallytothiswork.CorrespondinceandrequestsformaterialsshouldbeaddressedtoW.-M.H.(omail: billwmhsu@gmail.com)irH.L.(email:hsinyu@ntu.edu.tw) SCIENTIfICREPORTS? ?? 11212 ?DOI???????????????-???-?????-? 1 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scuentificreports/ NB.PreviousstudiesrevealthatincreasedCRTexpressioniscorrelatedwithbetterprognosisanddierentiated histologiesinNB7,8.Inaddition,cellsurfaceCRThasbeenfoundtobecrucialforneuriteformationwhenNB cells are induced to dierentiate9. In our previous study, we found that CRT could enhance cell dierentiation andsuppresscellproliferationinNBcells10.However,howCRTaectsthedierentiationofNBremainsunclear. Vascularendothelialgrowthfactor(VEGF)-A(alsoreferredtoasVEGF),akeyregulatorofphysiologicand pathologic angiogenesis, has been reported to not only participate in the behavior of NB, but also be regulated byCRTingastriccancer11¡V13.WehaveshownthatCRTcouldpositivelyregulateVEGFproteinexpressionand secretionlevelsinconditionmediaofvariousNBcelllines10,andtheevidencethatblockageofVEGFsignaling couldsuppressneuronaldierentiationinCRT-overexpressedNBcells,indicatesthatVEGFcouldbeinvolved in CRT-regulated neuronal differentiation and might predict a favorable tumor behavior in NB. Although VEGF-drivenangiogenesishasbeenshowntoplayacriticalroleinthepathogenesisofNBformationandmetas- tasis14, 15, various studies demonstrate conicting results regarding the role of VEGF in the tumor behavior of NB11,12,16¡V19. To better understand the role of VEGF expression in the angiogenesis, neuronal dierentiation, as well as tumorbehaviorinNB,weinvestigatedtheexpressionofVEGFinhumanNBtumors,mousexenogras,andNB cells.eresultswerecomparedtoangiogenesisandneuronaldierentiationmarkersaswellastheclinicopatho- logicalcharacteristicsofNB. Results ??????????????????????????????????????????????????????????????????????- ???????????????????????????????????????????????? Our previous studies have demon- stratedthatCRTmayupregulateVEGFexpressioninNBcells.Inaddition,constitutiveover-expressionofCRT could lead to NB cell dierentiation with suppressed cell proliferation10. To further clarify the role of CRT and VEGFexpressioninhumanNB,themRNAexpressionlevelsofCRTandVEGFin56primaryNBtumorswere evaluated by real-time PCR. e results revealed a signicantly positive correlation between CRT and VEGF expressioninNBtumortissues(Fig.1A, Spearman?s 0.648,P0.001).Furthermore,NBwithdierentiated histologyexhibitedhighermRNAexpressionlevelsofCRTandVEGFthanNBwithundierentiatedhistology (Fig.1B¡VD). e relationship between CRT, VEGF and dierentiation of NB was further examined in inducible-CRT stNBV1 cells. Aer 24h of tetracycline treatment to induce CRT expression in inducible-CRT stNBV1 cells, the expressions of CRT, VEGF, and GAP43 (a neuronal dierentiation marker) were all signicantly increased (Fig.2A). To further determine the eect of CRT and VEGF on tumor growth, a mouse xenogra model of NB using inducible-CRT stNB-V1 cells was established. NB cells inoculated mice were treated with doxycycline in their daily drinking water (2g/L) to induce CRT expression. After doxycycline treatment, the xenograft tumors revealed signicantly suppressed growth as compared to the control group (Fig.3A and B). In addition, the mRNA expression levels of CRT, VEGF and GAP43 were all signicantly increased in xenogra tumors with doxycyclinetreatment(Fig.2B).isresultwasalsoconrmedbyimmunouorescencemicroscopy(Fig.2C). ?????????????????????????????????????????????????????????????????????????? ??????? ToinvestigateifVEGFexpressionwasassociatedwithneuronaldierentiationandangiogenesis invivo,theexpressionsofCRT,VEGF,andbloodvesselmarkerCD34in69humanNBtumortissueswereexam- inedbyimmunohistochemicalstaining.NBtumorswereclassiedintofourcategoriesbasedontheintensityof VEGFimmunostaining:negative,weak,moderate,andstrongVEGFsignals(Fig.4A).eresultsdemonstrated thatVEGFwasmorecommonlyfoundinNBtumorswithmoredierentiatedhistology(DNB,54.3%)thanthose withundierentiatedhistology(UNB,23.5%)(P0.013)(Fig.4B&Table1).However,therewasnocorrelation between VEGF expression and microvessel density as depicted by CD34 staining (P0.808). Besides, CD34 immunostainingrevealednosignicantdierenceofmicrovesseldensitiesbetweenhumanNBtumorswithvar- ioushistology(Fig.5).Takentogether,thestudyofhumanNBtumorsshowedthatVEGFexpressioncorrelated with neuronal dierentiation rather than angiogenesis, suggesting a possible non-angiogenic role for VEGF in promotingneuroblastomadierentiation. ???????????????????????????????????????????? e clinical signicance of VEGF expressioninNBwasfurtherexaminedbycomparingproteinexpressionofVEGFinvariousclinicopathologic andbiologicalvariablesofNB(Table1).eresultsshowedthatpositiveVEGFproteinexpressionwassigni- cantlycorrelatedwithCRTexpression(P0.001),inadditiontopreviouslymentioneddierentiatedhistology (P0.013).Furthermore,VEGFexpressionalsoshowedastronginversecorrelationwithMYCNamplication,a signicantbiologicalmarkerofpoorprognosisofNB(P0.034).Insummary,theseresultsconrmedapositive correlationbetweenVEGFexpressionandfavorablebiomarkersincludingCRTexpression,dierentiatedtumor histologyandunampliedMYCN. Kaplan-MeieranalysisfurtherrevealedthatNBpatientswithpositiveVEGFexpressionintumortissueshada signicantlyhigher5-yearpredictivesurvivalrateascomparedtothosepatientswithnegativeVEGFexpression (Fig.4C;P0.013,Log-ranktest),suggestingthatpositiveVEGFexpressioncouldpredictafavorableoutcome inNBpatients. Discussion ConvergingevidencerevealedthatNBcellsexhibitacapacityofdierentiatingintoneuron-likecellsorregression byapoptosis,andhenceabenigntumorbehavior20,21.Inpreviousstudies,wedemonstratedthatCRTcouldsup- presscellproliferationandenhancecelldierentiation10.HighlevelofCRTexpressionintumortissuescorrelates SCIENTIfICREPORTS? ?? 11212 ?DOI???????????????-???-?????-? 2 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientificreports/ Figure1. VEGFexpressionwaspositivelycorrelatedwithCRTexpressionanddierentiatedhistologyin humanNBtumors.(A)CRTandVEGFmRNAexpressionsin56humanNBtumorsweredeterminedbyreal- timePCRandnormalizedtotheinternalcontrolGAPDH.ecorrelationbetweenexpressionlevelsofCRT (xaxis)andVEGF(yaxis)wasanalyzedbySpearman?scorrelationtest(Spearman?s 0.648,P0.001).(B) CRTandVEGFmRNAlevelsin31NBtumorswereevaluatedbyreal-timePCR.elevelsofCRT(shadedbar) andVEGF(solidbar)arehigherindierentiatedNB(DNB)thaninundierentiatedNB(UNB).(C)eCRT mRNAexpressionwasdeterminedbyreal-timePCRin56humanNBtumorsandpresentedasthemeanSEM ofeachgroup,undierentiatedNB(N27)versusdierentiatedNB(N29).eCRTmRNAexpressionwas signicantlyincreasedindierentiatedNB.(D)eVEGFmRNAexpressionwasdeterminedbyreal-timePCR in56humanNBtumorsandpresentedasthemeanSEMofeachgroup,UNB(N27)versusDNB(N29). eVEGFmRNAexpressionwassignicantlyincreasedindierentiatedNB.**P0.01,***P0.005. histological grades of dierentiation and predicts a favorable prognosis8. In addition, CRT can up-regulate the expressionandsecretionofVEGFproteininvariousNBcelllines8,10.BlockageofVEGFsignalinginturnmay suppressneuronaldierentiationinCRT-overexpressedNBcells.eselinesofevidencesuggestthatthatVEGF isinvolvedinCRT-drivenneuronaldierentiationofNB.HereweclearlydemonstratethatVEGFexpressionis positivelyassociatedwithCRTexpressionaswellasneuronaldierentiationofNBcellsinhumantumors,mouse xenograsandcelllinemodels,andmaypredictafavorablepatientoutcome.However,VEGFexpressionisnot SCUENTIfICREPORTS? ?? 11212 ?DOI???????????????-???-?????-? 3 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.niture.com/scientificreports/ Figura2. VEGFexpressionwaspositivelycorrelatedwithCRTexpressionandneuronaldierentiationinNB cellsandNBxenogras.(A)HumanstNB-V1NBcellswerestimulatedwith1 g/mltetracyclinetoinduce CRTexpression.ExpressionofCRT,VEGFandGAP43wasvisualizedandquantitatedbyimmunouorescence microscopyandImageJ.Dataareshownasmeanuorescenceintensity(SD)fromatleastthreeindependent experiments.(B,C)Inducible-CRTstNB-V1cellswereinjectedsubcutaneouslyintonudemice.Tumor inoculatedmiceweretreatedwithdoxycyclineintheirdailydrinkingwater(2g/L)toinduceCRTexpression. Miceweresacricedaer15-daystreatmentandthetumorwasremovedforexperiments.emRNA expressionlevelsofCRT,VEGF,andGAP43wereconrmedbyreal-timePCRandwereincreasedaer doxycyclinetreatment.emRNAexpressionlevelwasnormalizedtotheinternalcontrolHSP60.Eachbar ofthehistogramrepresentsquantiedresultsandisshownasthemeanSD.Statisticaldierenceswere comparedwiththecontrollevel.eexpressionsofCRT,VEGFandGAP43werevisualizedandquantitatedby immunouorescencemicroscopy.*P0.05,**P0.01. SCIUNTIfICREPORTS? ?? 11212 ?DOI???????????????-???-?????-? 4 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientificreperts/ Figure3. CRTexpressionsuppressedNBtumorgrowthinmousexenogramodel.(A)Inducible-CRT stNB-V1cellswereemployedinmousexenogramodel.NBcellsinoculatedmiceweretreatedwith doxycyclineintheirdailydrinkingwater(2g/L)toinduceCRTexpression.egrowthoftumorwasmeasured for15days.(B)Tumorinoculatedmiceweresacricedaer15-daystreatmentandthetumorweredissectedto measurethetumorsize.Statisticaldierenceswerecomparedwiththecontrollevel.**P0.01. correlated with the quantitative markers for endothelial cells, indicating an essential role of VEGF in neuronal dierentiationratherthanangiogenesisofNB. VEGF is a well-recognized pro-angiogenic factor and a key regulator of physiologic and pathologic angi- ogenesis22. In addition, angiogenesis has been proposed to play a crucial role in regulating NB formation and metastasis14,15,23.However,thereareinconsistentconclusionsregardingtheroleofangiogenesisaswellasVEGF expression in the tumor behavior of NB. Some studies showed that high vascular index or over-expression of VEGFwascorrelatedwithadverseprognosisofNBpatients24,25,whereasotherstudiesrevealedthattumorvascu- larityorVEGFexpressionwasnotcorrelatedwithprognosisandtumorstageinNBpatients17,26.Moreover,ithas beenshownthatthereisnosignicantsuppressioneectontumorgrowthinmouseNBxenograstreatedwith angiogenesisinhibitororanti-VEGFantibody18,27.eanti-angiogenesistherapiesinNBpatientsalsorevealed limited and modest success3, 28. In the present study of NB, the microvessel density revealed no signicant dif- ferencebetweenNBtumorswithvarioushistology(Fig.5andTable1),suggestingthatangiogenesisisnotonly relatedtotumoraggressivenessbutalsoplaysancriticalroleforNBdierentiation,whichresemblesthephysio- logiceventsgivingrisetothematurationofthevasculatureinnormalneuronaldevelopment.Besides,wedidnot ndacorrelationbetweenVEGFexpressionandvascularindexineitherthemousexenograsorhumantumor tissues(Table1).PreviousstudiesmainlyevaluatedtheangiogenicroleofVEGFinNBanimalsorsmallsample sizeswithoutlongitudinalfollow-upandgotconictresults.Inthisstudy,wefoundnocorrelationbetweenVEGF and vascular index, but a positive correlation between VEGF and neuronal dierentiation of NB. e present studyfocusestherelationshipbetweenNBdierentiationandVEGFintumorsofNBpatientsandfoundapos- siblenon-angiogenicroleofVEGFinNBapartfromitsangiogenicfunction.Besides,ourlongitudinalinvestiga- tionfromacohortof69patientsfoundthatpositiveVEGFexpressionintumortissueshadasignicantlyhigher 5-year predictive survival rate. VEGF expression did predict a favorable tumor behavior in NB. ese results indicateaparticularroleofVEGFexpressioninNBtumorbehavior. SCIENTIfICREPORTS? ?? 11212 ?DOI???????????????-???-?????-? 5 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientoficreports/ Figuru4. VEGFexpressioncorrelatedwiththehistologygradesofNBtumorsandpredictedafavorable clinicaloutcomeofNBpatients.(A)ImmunohistochemicalanalysisofVEGFexpressionintumortissuesof NBpatients.TumorswereclassiedintofourcategoriesbasedontheintensityofVEGFimmunostaining: negative,weak,moderate,andstrongsignalsofVEGF.Upperpanelsshowslowmagnicationimages(200, scalebar,5?m)andlowerpanelsindicatestheboxedimageswithhighmagnication(400,scalebar,2?m). UNB,undierentiatedneuroblastoma.DNB,dierentiatedneuroblastoma.(B)ecorrelationbetweenVEGF expressionlevelandthedierentiationhistologyofNBtumorwasanalyzedin69humanNBtumorsamples. Summationofweak,moderateandstrongVEGFimmunostainingwasrecordedas?positive?,thereby23.5%and 54.3%positiveVEGFexpressionwerefoundinUNBandDNB,respectively.(C)Kaplan-Meiersurvivalanalysis accordingtotheexpressionofVEGFdeterminedbyimmunohistochemistryinacohortof69NBpatients.NB patientswithpositiveVEGFexpressionintumortissueshadasignicantlyhigher5-yearpredictivesurvival ratecomparedtothosepatientswithnegativeVEGFexpression. IthasbeenshownthatVEGFcoulddirectlymodulatevariousneuronalfunctions,includingneuronalprolifer- ation,migration,survival,axonguidance,anddierentiationinadditiontoitsroleinangiogenesis29,30.Evidence has also shown that VEGF up-regulation can promote neuronal dierentiation, while VEGF down-regulation inhibitsneuronaldierentiationbothinstrokeandhypoxiamodels31¡V34.OurpresentstudiesrevealthatVEGF is positively correlated with neuronal dierentiation of NB both in vivo and in vitro, which is compatible with a previous study showing upregulation of VEGF and CEACAM1, a pro-angiogenic factor and mediator of VEGF-driven angiogenesis, during dierentiation and maturation of neuroblastic tumors19. ese ndings stronglysupportthenotionthatVEGFmayaectNBtumorbehaviorbyregulatingtumorcelldierentiation. SCIENTIfICREPORTS? ?? 11212 ?DAI???????????????-???-?????-? 6 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.com/scientifecreports/ Casos PositiveVEGF expression(%) PValue* Ageatdiagnosis 1.5year 22 11(50.0) 0.290 1.5year 47 16(34.0) Sex Male 37 13(35.1) 0.621 Female 32 14(31.1) ClinicalStage 1,2,4S 24 13(54.2) 0.075 3,4 45 14(31.1) Histology Undierentiated 34 8(23.5) 0.013 Dierentiated 35 19(54.3) MYCN Amplied 15 2(13.3) 0.034 Non-amplied 54 25(46.3) CRTexpression Positive 31 19(61.3) 0.001 Negative 38 8(21.1) Microvesseldensity High 35 13(37.1) 0.808 Low 34 14(41.2) Table1. VEGFexpressionandclinicopathologicandbiologiccharacteristicsofNB. *Chi-squaretest. Figure5. MicrovesseldensitydoesnotcorrelatewiththehistologygradeofNBtumors.Shownare representativeimagesofmicrovesselsandquantitativedataofmicrovesseldensityinUNB(N21)andDNB (N42)tumorsstainingwithbloodvesselmarkerCD34.DatashowthemeanSD.Scalebar,2 m. Our previous and present studies demonstrated a strong relationship between CRT and VEGF expression duringthedierentiationofNBcells10.VEGFexpressioniscriticalforCRT-drivenneuronaldierentiationofNB cells10.However,howCRTmayregulateVEGFexpressiontoaectNBcelldierentiationremainsunclear.Ithas SCIENTIfICREPORTS? ?? 11212 ?DOI???????????????-???-?????-? 7 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.nature.cam/scientificreports/ beenshownthatCRTmayaectRNAstabilitybybindingtoAU-richelement(ARE)in3-UTR6,35,36.Bysearch- ingtheAREdatabase,VEGFisconrmedtohaveanAREonthe3-UTRregionofitsmRNA37.Wethenpostulate thatCRTmayregulateVEGFexpressionbyaectingVEGFmRNAstabilitythroughthebindingofAREregion. FurtherinvestigationisneededtoverifythemechanismregardingtheregulationofVEGFexpressionbyCRT. erefore, evaluating VEGF expression in NB could oer complementary prognostic information, which will help clinicians to determine the most proper therapeutic strategies for the NB patients. Studies in the role of VEGF in NB tumorigenesis have unveiled inconsistent results. Both in human and in experimental NB, over-expressionofVEGFhasbeendemonstratedandcorrelatedwithahigh-riskphenotype11,12,38.Incontrast, recentstudieshavefoundthatVEGFisnotrelatedtotumorprogressionandmetastasisinNB26.Moreover,ithas been shown that there is no tumor growth dierence in NB xenogra mice treated with or without anti-VEGF antibody27.Inourstudy,forthersttime,wedemonstratedthatpositiveVEGFexpressionwassignicantlycor- relatedwithdierentiatedhistologyandunampliedMYCN,bothofwhicharefavorableprognosticfactors.Our presentresultsunequivocallyestablishVEGFproteinexpressionasanovelindependentfavorableprognosticfac- torofNB.Ourndingsalsosuggestthatanti-angiogeneticoranti-VEGFagentsmaybeinappropriateapproaches inmanagingpatientswithNB. In conclusion, this study examines the role of VEGF in regulating NB behavior focusing on angiogenesis andneuronaldierentiationbothin vitro and in vivo.eresultsshowthatVEGFexpressionisstronglycorre- latedwithneuronaldierentiationofNBcellshistologyinhumantumors,micexenograsandcelllinemodels. PositiveVEGFexpressionissignicantlycorrelatedwithdierentiatedtumorhistologyandnormalMYCNsta- tus,andhencepredictsafavorablepatientsurvival.Onthecontrary,VEGFasanangiogenesisfactorisnotcor- relatedwithmarkersofangiogenesisinhumantumortissuesofNB.OurndingsdelineateanovelroleofVEGF expression in NB. Instead of enhancing angiogenesis, VEGF might play a critical role in CRT-driven neuronal dierentiation of NB. Further studies to decipher the role ofVEGF onthe regulationof NB dierentiationwill shedlighttothemechanismoftumorigenesisaswellasanoveltherapeuticstrategytoimprovetheoutcomeof NBpatientsinthefuture. ????????????????? ?????????????????????????? AcohortofhistologicallyprovenNBpatientswithcompleteclinicaleval- uationandfollow-upinNationalTaiwanUniversityHospitalwereenrolledinthisstudy.erewere37malesand 32femaleswithmedianageatdiagnosis2.5years(range0¡V11.5).NBtumorspecimenswereobtainedduringsur- geryandimmediatelyfrozeninliquidnitrogen.ehistologicfeaturesofNBwereclassiedintoundierentiated NB(UNB,N34)anddierentiatedNB(DNB,N35)accordingtotheInternationalNeuroblastomaPathology Classication scheme39. e clinical stages were determined according to the International NB Staging System (INSS)40.MYCNamplicationwasdeterminedbychromogenicin situ hybridization41.Patientsweretreatedby surgeryaloneoracombinationofmultimodaltherapyincludingchemotherapy,radiotherapy,autologousstem celltransplantation,and13-cis-retinoicacidaccordingtothepatient?sriskgrouping4.eclinicalevaluationand usageoftumortissuesforthisstudywereapprovedbytheNationalTaiwanUniversityHospitalResearchEthics Committee.emethodswereperformedinaccordancewiththeapprovedguidelines.Writteninformedconsent wasobtainedfromthepatientsbeforesampleswerecollected. ??????????????????????????? Atotalof69tumorspecimenscollectedbeforechemotherapywerexed and embedded in paran. Tissue sections (5 m) of tumors were deparanized and rehydrated in a routine manner. e expression of CRT, VEGF, and the endothelial cell antigen, CD34, were evaluated using a stand- ard streptavidin¡Vbiotin complex immunoperoxidase staining and experimental procedures were performed as describedpreviously8.OneganglioneuromatumorwithconsistentCRTexpressionbyimmunohistochemistry wasusedasapositivecontrol.Non-immunizedrabbitserumwasusedasanegativecontrol.Tumorswithvarious dierentiatinghistologieswereincludedineachstaining.eimmunoreactivityofCRT,VEGF,andCD34were assessedbyonepathologistwhowasblindedtotheclinicalbackgroundofthepatients.eimmunohistochem- ical analysis of VEGF was classied into negative, weak (more than 10%), moderate, and strong VEGF signal. Summation of the numbers of samples with weak, moderate and strong VEGF signal was dened as ?VEGF positive?.MicrovesselsstainingwithCD34werecountedin400eldimagesofthreeseparateintenseneovascu- larizedareas,andthemeanwascalculatedasdescribedpreviously13. ??????????? Sinceconstitutiveover-expressionofCRTleadstoNBcelldierentiationwithoutproliferation, weutilizedstNB-V1NBcelllineandgeneratedaninducible-CRTstNB-V1celllinebyatetracycline-regulated genesystemasdescribedpreviously10.ToinduceCRTexpression,cellsweretreatedwithtetracycline(1g/mL). CellsweremaintainedinDulbecco?smodiedEagle?smedium(DMEM)/highglucosemediumcontaining10% fetalbovineserum(FBS)and1%penicillin/streptomycin.ecellsweregrowninahumidiedatmospherecon- taining5%CO2 and95%airat37¢XC. ??????????????????? e animal experiments were performed aer approval from the Institutional Animal Care and Use Committee at National Taiwan University. All methods involved in animal experiments were performed in accordance with relevant guidelines and regulations. For animal models of NB to measure tumorigenity, four-week old female athymic nude mice were housed in pathogen-free conditions and acclima- tized for one week. Mice were injected subcutaneously with CRT-inducible stNB-V1 cells. Cells (5106) were suspended in PBS and Matrigel (BD Bioscience) in a 1:1 (v/v) ratio. Tumor-bearing mice were randomized intotwogroupsandweretreatedwithdoxycyclineintheirdailydrinkingwater(2g/L)(N7)orvehiclealone (sucrose)(N10)for15days.egrowthrateofxenogratumorsonanimalswasmeasuredeverydayaccord- ingtothemetricmeasurementoftumorsize.Tumordiametersweremeasuredwithcalipers,andvolumeswere SCIENTIfICREPORTS? ?? 11212 ?DOI???????????????-???-?????-? 8 This text was extracted from a PDF document using an unlicensed copy of PDFTextStream. Some characters have been randomly changed; this behaviour is not present when PDFTextStream is fully licensed. Visit http://www.snowtide.com for more information. www.natire.com/scientifecreports/ calculatedaswidth2length0.5.Miceweresacricedaer15days,andsubcutaneoustumorsweresurgically excisedforfurtheranalysis. ????????????????????????? To study CRT, VEGF and GAP43, a neuron specic marker, expression in NB cells, inducible-CRT stNB-V1 cells were cultured on coverslips and stimulated with 1g/L tetracycline for 24h to induce CRT expression, and then xed in 4% paraformaldehyde (PFA) for 10min. Aer blocking with 5% bovine serum albumin (BSA) for 1 hr, cells were stained with anti-human CRT antibody (Millipore), anti-human VEGF antibody (Santa Cruz), and anti-human GAP43 antibody (Abcam), respectively, at 4¢XC overnight. Following the removal of unbound antibodies, cells were stained with Alexa Flour 680-conjugated goat anti-rabbit IgG (Invitrogen) at room temperature for 1 hr. Nuclei were visualized by counterstaining with DAPI.FollowingextensivewasheswithPBS,samplesweremountedwithFluoromount-GTM(Emsdiasum,Fort Washington,PA,USA).FluorescenceimageswereacquiredusingZeissAxioPlan2uorescencemicroscopesys- tem.euorescenceintensityofCRT,VEGF,andGAP43werequantitatedbyImageJfromNIH.Totalcellsfrom eachviewingareaweretakenandtheuorescenceintensitieswerequantied.DatawereshownasthemeanSD oftheaverageuorescencefromatleast6dierentviewingareas. To study CRT, VEGF and GAP43 expression in NB xenografts, frozen section slides of tumor samples were rst treated with 10% citric acid for 10mins at 95¢XC. Aer blocking with 5% BSA for 1h, samples were then incubated with the following primary antibodies, rabbit anti-human CRT antibody (Millipore), rabbit anti-human VEGF antibody (Santa Cruz) or rabbit anti-human GAP43 antibody (Abcam) at 4¢XC overnight. Following the removal of unbound antibodies, samples were the incubated with the following secondary anti- bodies,FITC-conjugateddonkeyanti-mouseIgG,AlexaFlour488-conjugatedgoatanti-ratIgGorAlexaFlour 680-conjugated goat anti-rabbit IgG at room temperature for 1h. Nuclei were visualized by counterstaining with DAPI for 5min. Following extensive washed with PBS, samples were mounted with Fluoromount-GTM (Emsdiasum,FortWashington,PA,USA)andvisualizedwithZeissAxioPlan2uorescencemicroscope. ?????????????????????? TotalRNAfromxenograsandhumanNBtumorswasextractedusing theTRIzolreagent(Invitrogen,Carlsbad,CA,USA)followingthemanufacturer?sinstructions.Complementary DNAwassynthesizedwith1?goftotalRNAusingaToyoboReverseTranscription-polymerasechainreaction (PCR)kit(Toyobo,Osaka,Japan).ereal-timePCRwascarriedoutusingaMini-Opticonreal-timedetection system (Bio-Rad, Hercules, CA, USA) with the mixture reagent SYBR-Green as the uorescent dye (Bio-Rad). Gene-specicprimerswereusedandthespecicitywasconrmedbysinglemelting-curveaerreal-timePCR. Cyclingconditionswere95¢XCfor3min,followedby30cyclesof95¢XCfor30s,60¢XCfor30s,and72¢XCfor30s. Forquantication,thetargetgenewasnormalizedtotheGAPDHtoactasaninternalcontrolforhumanNBand heat shock protein 60 (HSP 60) for xenogras. Primers for the real-time PCR were: GAPDH (F-5-AAG GTG AAGGTCGGAGTC-3andR-5-TGTAGTTGAGGTCAATGAAGG-3);HSP60(F-5-CACCGTAAGCC TTTGGTCAT-3 andR-5-CTTGACTGCCACAACCTGAA-3);CRT(F-5-CCTCCTCTTTGCGTTTC TTG-3 and R-5-CAG ACT CCA AGC CTG AGG AC); VEGF (F-5-GGC ACA CAG GAT GGC TTG AAG- 3 and R-5-GGC ACA CAG GAT GGC TTG AAG-3); GAP43 (F-5-TCC GTC GAC ACA TAA CAA-3 and R-5-CAGTAGTGGTGCCTTCTCC-3). ??????????????????? estatisticalanalyseswerecarriedoutwithSPSS20forWindowssoware.ecorre- lationbetweenCRTandVEGFmRNAexpressionlevelwereanalyzedusingnon-parametricWilcoxonrank-sum test and Spearman?s correlation test. 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