Tissue culture and animal studies with an oncolytic bovine enterovirus (bovine enterovirus 1). (1973)

SUMMARYTreatmentwithbovineenterovirusl(BEV-!)inmicro-testplatesofmousecellsderivedfromSarcoma 180,Ehrlichascites,A755,methylcholunthrcne-induced epithelialcarcinoma,andanL-celllinecauseshighlysignificantdecreases(18to98%)inviablecells,whereascellsderivedfromspleen,lung,thymus,liver,andkidneyofnormalmiceshowednosignificantchangeafterexposuretovirus.Applicationofthemethodtocellsderivedfromhumantumorsrevealeddecreasesof0to51%whereasavarietyofnormaltissuesrespondedbydecreasesfrom0to39%.Howeveroverone-halfthehumantumorpreparationsrespondedwithdecreasesgreaterthan17%whereasonly4of22normalpreparationsshoweddeclinesofthatmagnitude.ThesusceptibilityofrabbitandmousekidneycellstoBEVdidnotchangeover3(mouse)to12(rabbit)passagesinculture.HoweverthenormalcellsofthecontinuouslinesVero,BSC-1.andLLC-RK,weresusceptibletothevirusinboththemicrotestplateandplaqueassays.Treatmentofnormalmice,rabbits,anddogswithBEV-1byintracranial injectionledtonoclinicalabnormalities,norcouldvirusberecoveredfromthebrain.Someinconstantchangeswerenotedonpathological examination.MicebearingSarcoma180andtreatedbyi.p.injectionofBEV-Ishowedonlymildsplenichyperplasiaearlyinthecourseoftreatment.INTRODUCTIONPeriodically,virusesofonetypeoranotherareproposedastherapeutic tools.Mostvirusestestedhavefailedtomeasureuptotheexpectationsoftheirproponentsandoncologistsareproperlyskepticalofclaimsmadeonbehalfoftheseagents(7,11).Whilethereisneedforcaution,proposedoncolyticagentsshouldnotbesummarilydismissed,asanumberofvirusesproduceusefulregressions(1,4,8,9,14,15).BEV-13hasbeeninvestigatedbyTayloretal.(IO,12,1ThisworkwassupportedbygrantsfromtheJamesWhitcombRileyMemorialAssociation(Project71-20),theLittleRedDoor,theClintonCountyCancerSociety,andtheIndianaElks.'Towhomrequestsforreprintsshouldbesent,atthisaddress.3Theabbreviationsusedare:BEV-1,bovineenterovirus1;MEM,Eagle'sminimumessentialmedium;PCS,fetalcalfserum;20PCS,20%PCS;PFU,plaque-formingunit.ReceivedMay10,1973;acceptedJune19,1973.13)andbyCasto etal.(2,3,5,16).Taylor (12,foundtheviruslyticforbothsolidandascitestumorsofthemouse.Thevirusproducedacytopathiceffectinanastrocytoma andseveralglioblastomas derivedfromhumansbutdidnotaffecteitheranormalbrainoramengi-omacellline(10).Wehave,utilizinganewtechnique,extendedtheobservationsofTaylor'sgrouptoothertypesoftumorsandtoavariety cellsderivedfromnormaltissues.TheeffectofBEVonintactanimalshasalsobeenexplored.MATERIALSANDMETHODSVirusBEV-1wasobtainedfromDr.M.TaylorandpropagatedinL-cellsasdescribed(13).Clarifiedlysates,producedbyfreezing,thawing,andcentrifugationofinfectedcells,werestoredat-20Â°.TissueCultureSystemsCellsweremaintainedinAuto-PowMEM(FlowLaboratories,Rockville,Md.):5%inactivatedFCS.Themediumwassupplemented with20m\iglutamine,30m\i/V-2-hydroxyethylpiperazine-yV-2-ethanesulfonic acid(pH7.2),0.13Msodiumbicarbonate,100unitspenicillinperml,and100unitsstreptomycinperml.BSC-1andLLC-RK!cellswerefromstocksmaintainedinthelaboratory.L-cellswereobtainedfromDr.TaylorandVerocellswerefromtheAmericanTypeCultureCollection(No.CCL81).MicrotestPlateSystem.Cellsuspensionsweredilutedto1xIO4cellspermA¬inMEM:20FCS.UninfectedL-celllysateorvirussuspensionat20to100PFU/cellwasaddedtothecellsuspensionand200cells(20/ul)weretransferredtoeachof7wellsofamicrotestplate(FalconPlastics,Cockeysville,Md.).After24hrthemediawerecarefullyaspiratedfromthewellsandreplacedbya0.05%solutionoftrypanblue.Thenumberofunstained(living)cellswasrecorded.Inmostexperiments2additionalrowsof6wells,eachcontainingL-cellspluslysateorL-cellsplusBEV-1wereincludedascontrols.Themeandifferenceinsurvivalbetweenvirus-treatedandcontrolcellswascomputedandthesignificanceofthedifferenceswasdeterminedwiththe/test.2408 CANCERRESEARCHVOL.33