Kinetics of proliferation, migration, and death of L1210 ascites cells.

Abstract

DepartmentofRadiology,RadiationTherapyDivision[K.G.H.J,andDepartmentofSurgeryfM.HJ,OhioStateUniversity,Columbus,Ohio43210SUMMARYL1210leukemiacellswerelabeledwithmultipleinjectionsofiododeoxyuridine-125Iortritiatedthymidineandinjectedi.p.intonew,nonradioactivehosts.TheoverallgrowthrateoftheprelabeledL1210cellswasevaluatedintermsofcellcyclelength,growthfraction,andcelllossduetocellmigrationandcelldeath.Followingimplantationintoanewhost,therewasa6-to12-hrlagperiodbeforeperitonealLI210cellsstartedtodivide.Duringthefirst3daysoftumorgrowth,theaveragegenerationtimeremainedconstantat8.5to9.5hr.Thenthelengthofthecellcyclegraduallyincreaseduntilitreached32to36hrshortlybeforethedeathofthemice.Thegrowthfraction remained closetounitythroughout tumordevelopment.ImplantationofL1210cellsi.p.wasfollowedbyrapidmigrationoftumorcellsfromtheinjectionsitetootherorgans.FractionalL1210metastasiswasveryhighduringearlyphasesoftumorgrowth,andmuchslowerinadvancedtumors.L1210cellsinjectedatextraperitonealsitesdidnotinvadetheperitonealcavity.Therateofcelldeathdependedonthelocationoftumorcellswithinthehost.L1210cellslocatedoutsidetheperitonealcavitydiedatthefractionalrateof20%/day.CelldeathamongperitonealL1210cellswasmuchlower(lessthan5%/day)anddidnotsignificantlyinfluencetheoverallgrowthofL1210ascitespopulations.INTRODUCTIONNumerousstudieshaveshownthatthegrowthrateofexperimentaltumorsvariesduringthecourseoftumordevelopment.Asarule,theincreaseincellnumberisrapidduring theearlyphasesoftumorgrowthandthengraduallyslowsasthetumorgetsolder.Recentstudiesontherelationshipbetweentheoverallgrowthrateoftumorsandindividualcellproliferationparametershaveindicatedthatduringlaterphasesoftumordevelopmenteitheranincreasingfractionoftumorcellsstopsdividing(2,18,26)orthedurationofthemitoticcycleincreases(1,3,6,8,19,28,30,31).Anotherfactorcontributingtothegradualdecelerationofgrowthinadvancedtumorscouldbeincreasedcelllossduetoeitheracceleratedcelldeath(11,13,20)orincreasedmigrationofcellsawayfromthesiteoftumorinoculation.Sometimes2ormoreofthesefactorscombinetocauseaprogressivedecreaseinthegrowthrate(4,17,27).TdR2 labeling techniques in combination withautoradiographyhavecontributedgreatlytotheevaluationofgrowthparametersatthecellularlevel.However,manyaspectsoftumorgrowth,particularlytumorcellmigrationandtumorcell death, cannot be directly investigated withautoradiographictechniques(16,25).Thisreportdescribesanalternatemethodforevaluatingthegrowthparametersofexperimentaltumorpopulations.ThismethodinvolvesthelabelingoftumorcellswithIUDR-125!,ananalogofTdR.whichisincorporated exclusivelyintotheDNAofproliferatingcells(5,11,12,14,21).AfterincorporationintotheDNA,IUDR-125!remainsboundwithinthecellsuntiltheydie(5,13,14).Whenalabeledcelldies,rapiddeiodinationandexcretionoftheDNAbreakdownproductslimitthereutilizationofradioactivitybyothercells(7,13,14).Itthusbecomespossibletoestimatedirectlytherateoftumorcelldeathinvivobycontinuouslymonitoringtherateof12SIexcretionfromindividuallivemiceinoculatedwithIUDR-12SI-labeledtumorcells(13).Inaddition,the12SIactivityofvariousorganscanbemeasuredatdifferenttimeintervalsafterinjectionofprelabeledtumorcellsintononradioactivehosts,permittingaquantitativestudyoffractionaltumorcellmetastasis(11,13).Thisshouldproveveryusefulindistinguishingbetweentumorcellmetastasisandtumorcelldeath,the2basicmodesofcelllossfromtheoriginalsiteofinoculation.The'2SI-prelabelingproceduremightalsoproveusefulinevaluatingtherateoftumormultiplication byallowingmonitoringoftherateofdecreaseinDNA-specific125Iactivity.InthecaseofperitonealLI210cellswherethegrowthfractionappearstobecloseto100%throughoutthedevelopmentof tumor(30), declinein12SIactivitypercellshouldprovideadirectmeasureofthemeangenerationtimeofthetumor.Ideally,theIUDR-125!prelabelingprocedure couldthusmakeitpossibletoevaluatesimultaneouslyin1singleexperimenttheratesoftumorcellproliferation,cellmetastasis,andcelldeath,the3basicparametersofpopulationgrowth.1ThisinvestigationwassupportedbytheBremerFoundationFund,TheOhioStateUniversityCollegeofMedicine,Columbus,Ohio.ReceivedAugust24,1970;acceptedNovember12,1970.2Theabbreviationsthymidine.usedare:IUDR,iododeoxyuridine;TdR,402 CANCERRESEARCH VOL.31