Tumor-specific resistance in mice detected by inhibition of macrophage migration.

Abstract

JanVaage,2R.DavidJones,3andBarryW.BrownSectionofExperimentalRadiotherapyfJ.V.,R.D.//,andDepartmentofBiomathematics[B.W.B.Âi,UniversityofTexasatHouston,M.D.AndersonHospitalandTumorInstitute,Houston,Texas77025SUMMARYResistanceagainstamethylcholanthrene-induced fibrosarcomaandaspontaneousmammarycarcinomainsyn-geneicC3Hfmicewasdemonstratedinvitrobyspecificinhibitionbytumorantigensofperitonealcellemigrationfromcapillarytubes.Thesolubleantigensofthetwotumorsdidnotcross-react,butstrongnonspecificinhibitionofmigrationwasobservedwhentumorcellsorparticulatematterwaspresentinthereactionmixture.Thedegreeofinhibitionofmacrophagemigrationwasrelatedtothestageofthedevelopmentofantitumorimmunityintheperitonealcelldonor.Depressionoffullydevelopedimmunitybyexcesstumorantigen(desensitization)wasnotreflectedinthemotilityoftheperitonealcellsofthehostinthepresenceoftumorantigen.Hostresistancecouldalsobedetectedbyasimplebutnonquantitative procedureinwhichtheeffectoftumorantigensonmacrophagespreadingwasobservedinordinaryculturedishormicrotestplateculturesofperitonealexAodatecells.INTRODUCTIONTheMIF4testisnowwellestablishedasaspecificandsensitiveindicatorofdelayedhypersensitivitystatesinman(3,10-12,20,23,24,27),inguineapigs(2,4-6,8,9,13,14,16-18),inrats(11,25),andinmice(1,11,15,19).Theabove-mentionedstudiesdetectedreactivitydirectedagainsttuberculin(2,4,5,8,14,20,25,27),othermicrobialantigens(8,9,20,23,24,27),pureproteinantigens(2,14,16),alloantigens (1,10-13, 18,19),andtumor-specifictransplantation antigens(3,6,15,17).Alloftheseinvestigationsexceptone(9)usedthecapillarytubetechniqueintroducedbyGeorgeandVaughan(14)anddevelopedbyDavidetal.(8).Precedingreports(29,30)haveshownthatpreviousexposure to growing implants of ameth-'ThisworkwassupportedinpartbyUSPHSGrantsCA05047,CA6294,CAI1138,andCAI1430.'Presentaddress:DepartmentofRadiationMedicine,MassachusettsGeneralHospital,Boston,Mass.02114.3DoctoralcandidateatTheUniversityofTexasatHoustonGraduateSchoolofBiomedicaiSciences,Houston,Texas77025.4Theabbreviationusedis:MIF,migration-inhibitoryfactor.ReceivedJuly1,1971;acceptedDecember28,1971.ylcholanthrene-induced sarcomaandaspontaneousmammarycarcinomaresultedinresistancetochallengebyreinjectionofthehomologoustumorifthesensitizingtumorimplantwasresectedpriortochallengeandthattheresistancewasimpairedbutnotabrogatedifthesensitizingtumorwasleftinsituuntilafterthechallenge.Inthestudyreportedhere,themacrophagemigrationinhibitiontestwasusedtoinvestigatethepossiblecorrelationbetweenthelevelofspecifichostresistancetotumorgrowtliandthedegreeofspecificinhibitionofmacrophagemotilityinvitro,withtheuseofperitonealexAodatecellsfromsensitizedmiceandkilledcellsorsolubleantigenfromthesensitizingtumor.MATERIALSANDMETHODSMice.Alloftheanimalsusedintheseexperimentswere12-week-oldfemalemiceofinbredstrainC3Hf/Bufromthedefined-flora,5pathogen-freebreedingcolonymaintainedbytheSectionofExperimentalRadiotherapyofTheUniversityofTexasatHouston,M.D.AndersonHospitalandTumorInstitute.Tumors.ThefibrosarcomahadbeeninducedinthefemaleC3Hmousebymethylcholanthrene(26).Thetumorhadbeenkeptinliquidnitrogenandwasreintroducedintosyngeneicmicetobeusedintheseexperimentsinthe3rdand4thtransplantgenerationsforsensitizingimplantationsandforpreparingtumorcellsuspensions.ThemammarycarcinomahaddevelopedspontaneouslyinamultiparousC3H/Hemouse(21).Italsohadbeenkeptinliquidnitrogenandwasreintroducedintosyngeneicmicetobeusedinthe3rdand4thtransplantgenerations.TumorImplantation.Thes.c.implantationof1-x1-mmpiecesoflivingtumortissuewasusedtoinitiatetumorgrowthforthepurposeofimmunization.Anincisionwasmadeintheskinoftherightflank,andatumorpiecewasplacedundertheskinbymeansofatrocar.Theincisionwasclosedwithawoundclip.Challengeimplantationofpresensitizedandcontrolmicewasbys.c.injectionintheleftsideofviable(trypanblue-negative)tumorcellssuspendedinMedium199(DifcoLaboratories, Inc., Detroit, Mich.) and consistingpredominantlyofsinglecellsandafewclumpsofupto10cellseach.Themechanicalpreparationofsingle-cellsuspensionsfromtumortissuehasbeendescribedelsewhere(28).5Themicecarryonlythefollowingentericbacteria:Clostridiumsp.,Bacteroidessp.,Peptostreptococcussp.,andBacillussp.