Cervicalcancercontinuestobeanimportantworldwidehealthproblemforwomen.Upto35%ofpatientswhoarediagnosedwithandappropriatelytreatedforcervicalcancerwillrecurandtreatmentresultsarepoorforrecurrentdisease.Giventhesesoberingstatistics,developmentofnoveltherapiesforcervicalcancerremainsahighpriority.WeevaluatedtheexpressionofTissueFactor(TF)incervicalcancerandthepotentialofhI-con1,anantibody-like-moleculetargetedagainstTF,asanovelformofimmunotherapyagainstmultipleprimarycervicalcarcinomacelllineswithsquamous-andadenocarcinomahistology.
BecauseTFisatransmembranereceptorforcoagulationfactorVII/VIIa(fVII),inthisstudyweevaluatedtheinvitroexpressionofTFincervicalcarcinomacelllinesbyimmunohistochemistry(IHC),realtime-PCR(qRT-PCR)andflowcytometry.SensitivitytohI-con1-dependentcell-mediated-cytotoxicity(IDCC)wasevaluatedin5-hrs-51chromium-release-assaysagainstcervicalcancercelllinesinvitro.
Cytoplasmicand/ormembraneTFexpressionwasobservedin8outof8(100%)ofthetumortissuestestedbyIHCandin100%(11outof11)ofthecervicalcarcinomacelllinestestedbyreal-time-PCRandflowcytometrybutnotinnormalcervicalkeratinocytes(p=0.0023qRT-PCR;p=0.0042flowcytometry).AllprimarycervicalcancercelllinestestedoverexpressingTF,regardlessoftheirhistology,werehighlysensitivetoIDCC(meankilling±SD,56.2%±15.9%,range,32.4%-76.9%,p<0.001),whilenegligiblecytotoxicitywasseenintheabsenceofhI-con1orinthepresenceofrituximab-control-antibody.Lowdosesofinterleukin-2furtherincreasedthecytotoxiceffectinducedbyhI-con1(p=0.025)whilehumanserumdidnotsignificantlydecreaseIDCCagainstcervicalcancercelllines(p=0.597).
TFishighlyexpressedinsquamousandadenocarcinomaoftheuterinecervix.hI-con1inducesstrongcytotoxicityagainstprimarycervicalcancercelllinesoverexpressingTFandmayrepresentanoveltherapeuticagentforthetreatmentofcervicalcancerrefractorytostandardtreatmentmodalities.
Toourknowledge,nostudieshavespecificallydescribedtheexpressionoftissuefactorincervicalcancer.Therefore,inthisreportweinvestigatedtheexpressionofTFatmRNAandproteinlevelsinmultipleprimarycervicaltumorcelllinesandevaluatedforthefirsttimetheinvitropotentialofhI-con1asanovelimmunotherapeuticagentagainstbiologicallyaggressivecervicalcancercelllinesoverexpressingTF.
Toinvestigatetheeffectofinterleukin-2(IL-2)onIDCC,effectorPBLwereincubatedfor5hoursat37°CatafinalconcentrationofIL-2(Aldesleukin;ChironTherapeutics,Emeryville,CA)rangingfrom50to100IU/mlin96-wellmicrotiterplates.TargetcellswereprimarycervicalcancercelllinesexposedtohI-con1(concentrationsrangingfrom1to80μg/ml),whereascontrolsincludedtheincubationoftargetcellsaloneorwithPBLinthepresenceorabsenceofIL-2ormAb,respectively.RituximabwasusedasacontrolmAb.IDCCwascalculatedasthepercentageofkillingoftargetcellsobservedwithmAbpluseffectorPBL,ascomparedwithtargetcellsincubatedalone.EachexperimentwasperformedwithPBLcollectedfrommultiplenormaldonors,withresultsfromarepresentativedonorpresented.
Astandardfive-hourchromium(51Cr)releaseassayidenticaltothepreviousIDCCassayswasused,exceptthathumanseruminadilutionof1:2wasaddedtotheeffectorcells.Thishumanserumwasusedasasourceofcomplementtotestforcomplement-mediatedtargetcelllysis.TotestforthepossibleinhibitionofIDCCagainstachemoresistantsquamouscervicalcancercelllinebyphysiologicalhumanserumconcentrationsofγ-globulin,humanserumwasdiluted1:2beforebeingaddedinthepresenceorabsenceofeffectorPBL.Experimentswerealsoperformedusingheat-inactivatedhumanserum(56°Cfor60minutes;diluted1:2)inthepresenceofeffectorPBL.ControlsincludedtheincubationoftargetcellsaloneorwitheitherPBLormAbseparately.RituxanwasusedasacontrolmAb.
ForqRT-PCRdata,theright-skewingwasremovedbytakingcopy-numberratiosrelativetothelowest-expressingnormalcervicalkeratinocytesample("relativecopynumbers"),log2-transformingthemtoΔCTs,andcomparingtheresultsviaunequal-variancet-testfordifferencesbetweencervicalcancercellsversusnormalcervicalkeratinocytes.Groupmeanswith95%confidenceintervals(CI)werecalculatedbycomputingthemontheΔCTsandthenreverse-transformingtheresultstoobtainmeans(95%CI)ofrelativecopynumbers.DifferencesinTFexpressionbyIHCandflowcytometrywereanalyzedbytheunpairedt-test.Kruskal-Wallistestandchi-squareanalysiswereusedtoevaluatedifferencesinhI-con1-dependentcell-mediatedcytotoxicitylevelsinprimarytumorcelllines.StatisticalanalysiswasperformedusingSPSSversion17(SPSS,Chicago,IL).Ap-valueof<0.05wasconsideredstatisticallysignificant.
RepresentativeIHCofTFinsquamous-andadenocarcinomaspecimensversusnormalcervicalcontroltissues.Upperleftpanel:normalectocervicalsquamousepitheliumnegativeforTF.Lowerleftpanel:cervicalsquamouscellcarcinomashowinghighexpressionofTF.Upperrightpanel:normalcervicalglandsnegativeforTF.Lowerrightpanel:cervicaladenocarcinomashowinghighexpressionofTF.Originalmagnification:200×.
RepresentativeTFexpressionbyflowcytometryofcontrolPBL(negativecontrol)andCVX-1ARK-1,CVX-2ARK-2andADX-1ARK-1primarycervicalcelllines.Isotype(solidblack);hI-con1(dashedline).
RepresentativecytotoxicityexperimentsusinghI-con1againstcontrolPBLandCVX-1ARK-1,CVX-2ARK-2andADX-1ARK-1primarycervicalcelllines.NegligiblecytotoxicitywasdetectedintheabsenceofhI-con1orinthepresenceofrituximabcontrolmAb.
Representativeeffectoflowdosesofinterleukin-2(IL-2)incombinationwithhI-con1(30μg/ml)onADCCagainstCVX-2ARK-2primarycellline(Effectorstotargetratio50:1and25:1).PBLfromhealthydonorswereincubatedfor5hoursinthepresenceof100IU/mlofIL-2.hI-con1-DCCwassignificantlyincreasedinthepresenceoflowdosesofIL-2.Nosignificantincreaseincytotoxicitywasdetectedafter5-hIL-2treatmentintheabsenceofhI-con1orinthepresenceoftherituximabisotypecontrolmAb.
CytotoxicityexperimentsaddinghumanserumtohI-con1(30μg/ml)againstonerepresentativeprimarycervicalcancercelllineoverexpressingTF(i.e.,CVX-7ARK-7:effectorstotargetratio50:1and25:1).Cervicalcancercellswerechallengedbydiluted(1:2)serum(withorwithoutheatinactivation)addedinthepresenceoftheeffectorcellsandhI-con1instandard5-h51Crreleaseassays.Additionoftreatedoruntreatedhumanserum(diluted1:2)toPBLinthepresenceofhI-con1didnotsignificantlychangehI-con1-mediatedcytotoxicityagainstCVX-7ARK-7(p=0.597).
Foreffectivecytotoxicity,theeffectorcellsmustbeabletointeractwiththeimmune-conjugateatthetargetsiteinvivo,eveninthepresenceofhighconcentrationsofhumanIgG.InthisstudyweshowedthathI-con1-dependentcytotoxicityagainstcervicalcancercelllineswasnotinhibitedbyhighconcentrations(upto50%)ofhumanserum.Thesedata,therefore,suggestthatinthepresenceofeffectorPBL,humanserummaynotsignificantlydecreaseIDCCagainstcervicalcancercelllines.Moreover,theseresultsindicatethatthebindingofhI-con1totheFcreceptoronmononucleareffectorcellsislikelytooccurinvivo.
Inconclusion,highexpressionofTFinalargenumberofcervicalcarcinomasmakeshI-con1anattractiveagentforimmunotherapyofrecurrent/refractorycervicalcancer.Inthisstudy,wehavedemonstratedsignificanthI-con1-mediatedkillingofmultipleprimarycervicalcancercelllinesregardlessoftheirsquamousoradenocarcinomahistology.hI-con1mightthereforerepresentanoveltreatmentofthisaggressivediseaseand,potentially,multipleotherhumantumorsoverexpressingTF.
tissuefactor
FactorVII
humanimmuno-conjugatemolecule
hI-con1-dependentcell-mediatedcytotoxicity
fetalbovineserum
immunohistochemistry
monoclonalantibody
naturalkillercells
peripheralbloodlymphocytes
quantitativereal-time-polymerasechainreaction
cervicalsquamouscellcarcinoma
cervicaladenocarcinoma.
TheAuthorsthankDr.WilliamKonigsberg,Dr.AlanGarenandDr.ZhiweiHuforinitiatingthecollaborationwithCLonIconimmunotherapyofhumangynecologicmalignanciesandIconicTherapeuticsInc.forprovidinghI-con1proteinfreeofchargeforourstudies.ThisworkwassupportedinpartbygrantsfromtheAngeloNocivelli,theBerlucchiandtheCamilloGolgiFoundation,Brescia,Italy,NIHR01CA122728-01A2toAS,andgrants501/A3/3and0027557fromtheItalianInstituteofHealth(ISS)toAS.ThisinvestigationwasalsosupportedbyNIHResearchGrantCA-16359fromtheNationalCancerInstitute.
Theauthorsdeclarethattheyhavenocompetinginterests.
EC,JV,SB,MG,MB,PT,andLCcarriedoutthemolecularinvitrostudiesincludingRT-PCR,flowcytometryandIDCCassays,aswellasstatisticalanalysis.NBcarriedouttheIHCstudiesonthetissuesamples.DS,MA,PES,TJR,SP,CJL,andASparticipatedinthedesignofthestudyanddraftedthemanuscript.ASconceivedthestudy.Allauthorsreadandapprovedthefinalmanuscript.
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