VDCOURTENAYETALatmospherecontaining5025C02and90nitrogenLiquidmediumwasadded5dayslaterandchangedasnecessaryColoniesofmorethan50cellswerecountedunderadissectingmicroscopex40at20to35daysPlatingefficienci ID: 891200
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1 Br.J.Cancer(1978)38,77GROWTHOFHUMANTUMOU
Br.J.Cancer(1978)38,77GROWTHOFHUMANTUMOURCELLCOLONIESFROMBIOPSIESUSINGTWOSOFT-AGARTECHNIQUESV.D.COURTENAY*,P.J.SELBY*,I.E.SMITHt,J.MILLS*ANDM.J.PECKHAMtFromtheDivisionsof*Biophysics,tMedicineandIRadiotherapy,InstituteofCancerResearch,Sutton,SurreyReceived6March1978Accepted24April1978Summary.-Twotechniquesforgrowingcoloniesofhumantumourcellsinsoftagarhavebeenappliedtocellsuspensionsderivedfromfreshtumourtissuefrom48patients.Colonieswereobtainedin31cases,withplatingefficienciesbetween0.01and15%.In11casestheplatingefficiencieswere1%orabove.Therewasevidencethatsomecategoriesoftumourgrewmorereadilythanothersundertheseconditions.Thepotentialapplicationsofthemethodstoclinicalandexperimentaloncologyarediscussed.RECENTLY,inthislaboratory,twomethodshavebeendevelopedforgrowingcoloniesfromcellsuspensionsobtainedfromhumantumoursgrowninimmune-suppressedmice.Oneinvolvesinvitrocultureinsoftagarwithareplenishableliquidphase,addedredbloodcellsandalow02concentration(Courtenayetal.,1976;CourtenayandMills,1978)whiletheotherusesagardiffusionchambers(ADC)implantedi.p.intopre-irradiatedmice(Smithetal.,1976).Colonyassaysarewidelyusedtomea-suretheresponseofestablishedlinesofanimalandhumantumourcellstreatedwithcytotoxicagents,andcolonyforma-tionprobablyprovidesthemostreliablemeasureofcellsurvival(RoperandDrewinko,1976)sinceonlydividingcellsbelievedtobecapableofrepopulatingthetumouraremeasured.Thetechniquesusedinthesestudieshavealreadybeenappliedtomeasuringcellsurvivalincertainxeno-graftedhumantumourstreatedwithradia-tionandothercytotoxicagents.Theirapplicationtohumanbiopsyspecimenscouldleadtodirectsensitivitytestingofchemotherapeuticagentsforthetreat-mentofindividualpatients'tumours.Thegrowthofcoloniesdirectlyfromasolidhumantumourbytheinvitrotech-niquehasbeenreportedbyCourtenayandMills(1978).ThepresentpaperdescribestheresultsfromaseriesoftumourstakendirectlyfrompatientsandsetupinagarusingtheinvitroandADCtechniques.METHODSThetwoassaytechniqueshavebeendes-cribedelsewhereindetail(CourtenayandMills,1978;Smithetal,1976)andarepre-sentedhereonlyinoutline.Thesolidtumourswerefinelychoppedusingcrossedscalpelsandsingle-cellsuspensionswerepreparedbytreat-mentwithtrypsinandcollagenase,orbymechanicaldispersion,andfilteredthrougha30jtmpolyestermeshtoexcludeclumps.Cellswereexaminedunderphasecontrastusingahaemocytometer,andbrightcellsthatexcludedlissaminegreenwerecountedasviable.Insometumoursitwasdifficulttodistinguishbetweentumourandstromalcells,andcellcountsthereforeincludedsomestro-malcells.Forinvitrogrowth,washedredbloodcellsfromtheratweresuspendedtotheoriginalbloodvolumeinHam'sF12medium+l15%foetalcalfserum.1
2 vol.ofa1/8dilutionwasaddedto1vol.oftumou
vol.ofa1/8dilutionwasaddedto1vol.oftumourcellsinculturemedium.Afteradding3vol.of0.5%agarmedium,1mlaliquotsofthemixturewerepipettedintotesttubes.Whentheagarhadset,thecultureswereincubatedat37°Cinan V.D.COURTENAYETAL.atmospherecontaining5%02,5%C02and90%nitrogen.Liquidmediumwasadded5dayslaterandchangedasnecessary.Coloniesofmorethan50cellswerecountedunderadissectingmicroscope(x40)at20to35days.Platingefficiencies(PEs)werecalculatedasapercentageofthenumberofcellsplatedout.IntheADCmethod,cellsinsimilarme-diumandagarwereintroducedintoMilliporediffusionchamberswhichwerethenimplantedintotheperitonealcavityofC57BLmice.Themicewerepretreatedwith200mg/kgofcytosinearabinoside,followed2dayslaterby900radofwhole-bodyirradiation,anon-lethalcombination(Millaretal.,1978).Thechamberswereimplantedwithin24hofirra-diation.Afterremovalfromthemouseatabout21days,thecoloniesintheADCwerescoredasfortheinvitromethod.RESULTSArangeofdifferenttumourtypes,in-cludingprimaryandmetastatictumours,wasassayedbyoneorbothofthetech-niques.Inpreparingcellsuspensions,therewereconsiderablevariationsinthenum-bersofsuspendedcellsobtainablefromdifferentsolidtumours.Fibroustumoursgenerallygavelowcellyieldsand,inpar-ticular,6/7selerousbreastcarcinomastreatedwithcollagenaseandtrypsinyieldedlessthan2x104singlecellsfromabout1goftissue,andwerethereforenotsetupinagar.Oftheremainingsolidtumoursexamined,80%gavesufficientnumbersofcellsfortestinginagar.Mela-nomaswerefoundtobemostreadilydis-aggregatedand,fromsubcutaneousde-positsandlymphnodes,suspensionswereobtainedsimplybyshakingthecut-uptumourpiecesinculturemediumandfil-teringtoremovetheclumps.Othertu-mours,includingallthecolorectaltumours,yieldedsuspensionslessreadily,evenwithenzymetreatment.Ascitestumourswereeasiertohandle,butsometimescontainedlargenumbersofnon-tumourcells.SolidandascitictumoursweretestedforgrowthinagarandtheresultsareshownintheTable.Of48differenttumours,31gavecoloniesinagar.Thebestresultswereob-tainedfromthemelanomasandovariantumoursandcoloniesweregrownfrom12/14and10/10ofthesetumoursrespec-tively.Nocolonieswereobtainedfrom6breasttumours.ThePEscoveredawiderangefrom0-01to15%,indicatingacon-siderablevariationbetweenindividualtumoursofthesametype.TwoofthemelanomasgavePEsabove10%byoneorotherofthegrowthtechniques.Colonymorphologyvariedwiththetumouroforigin,andthereweredifferencesbetweentumoursofdifferentcategoriesandbetweenindividualtumoursofthesamecategory.Variationoccurredinsize,closenessofpackingandregularityofcolonyoutline(Fig.).Occasionallydiffusecolonieswereobservedwhichweremor-phologicallydistinctfromthepredominantcolonytype,andthesewerenotscoredinthequotedPE
3 s.Smearpreparationsofcellspickedoutfromt
s.Smearpreparationsofcellspickedoutfromthecoloniesshowedamorphologycompatiblewiththetumouroforigin,andmelanoticmelanomasgaveblackcoloniesofcellscontainingmelaningranules.Someofthetumoursexaminedwerealsoimplanteddirectlyintoimmune-sup-pressedmiceandgrownasxenografts.Theappearanceofcoloniesobtainedfor5xeno-graftedmelanomas,anovariantumour,auterinetumourandacoloniccarcinoma,whencomparedwithcorrespondingcolo-niesgrowndirectlyfromtheoriginalbiopsies,showedacloseresemblance.TheirPEswerealsocomparable,andtumourswhichgavehighPEfromtheoriginalbiopsyusuallygavegoodPEwhengrownfromthexenografts.DISCUSSIONTherehavebeennumerouspreviousattemptsatculturingcellsdirectlyfromhumantumours,withtheaimofdevelop-ingmethodsoftestingthechemosensiti-vityofindividualtumours(Hall,1977;Dendyetal.,1970)butthesehavebeenhamperedbythedifficultyofculturingcellstakendirectlyfromtumourspecimens,andofdeterminingwhetherthecellswhichdo78 GROWTHOFCOLONIESINAGARFROMHUMANTUMOURBIOPSIES79TABLE.-Platingefficiencies(PE)ofcoloniesgrowninvitroorinADCfromcellsuspensionspreparedfromtumourbiopsiesPETumourtypeMelanomaOvarianCaBreastCancerColorectalCancerTeratomatestisSeminomaPancreaticCaUterineCa(body)(cervix)Oat-cellCabronchusHypernephromaOrchioblastomaOsteosarcomaLeiomyosarcomaFormS.c.depositS.c.depositS.c.depositS.c.depositS.c.depositS.c.depositS.c.depositNodedepositNodedepositNodedepositNodedepositPrimaryAscitesPleuraleffusionAscitesAscitesAscitesAscitesAscitesAscitesAscitesPrimarySecondarydep.Peritonealdep.PrimaryPrimaryPrimaryPleuraleffusionPleuraleffusionPleuraleffusionPrimaryPrimaryPrimaryPrimaryPrimarySecondarydep.PrimaryPrimaryPrimaiyPrimaryPrimarySecondarydep.AscitesSecondarydep.Secondarydep.PrimaryLocalrecurrencePrimaryTreatmentofsuspensionmmmmmmmmmmmmuuuuuuuuuttmtttuuut+ct+ctttt+ct+cttmt+cmmttmmTumourbinvitro0150*53-05-600-2000-50-25002-70-250-020-21-01-00-41-34.5000000000-0300-3000-17120-10-040iopsyXenograftADCinvitro*ADC0-230-21-0�2-520-015-000-011-00-9210-011-011*5--0-06620-070-040-600-072-0-130-01--1-7--2-20-553-00--0--0--0-0-25--2-00-03-0-0-10--0--10-1-0--0--0u=untreatedm=mechanicalseparationt=trypsint+c=trypsin+collagenase-=notdone*Xenograftedtumoursderivedfromtheoriginalbiopsyweretestedafterthe1stor2ndpassageinimmune-suppressedmice.6 V.D.COURTENAYETAL.growareoftumourorstromalorigin.Stromalcellsthatattachtoculturedishesandgrowasmonolayersareunabletoformcoloniesinagar,andtheabilitytoproducecoloniesinagar(Macpherson,1973)isoneofthecriteriaofmalignanttransformation.Softagarhasextensivelybeenusedforthegrowthofmarrowcellsandsomeestablishedcelllines,butthereislittleinformationregardingthegrowthofhuman
4 tumourcellsinagarwithoutpreviousgrowthin
tumourcellsinagarwithoutpreviousgrowthinmonolayerculture,al-thoughcolonyformationfromchildhoodsolidtumours(McAllisterandReed,1968;Altmanetal.,1975)hasbeenreported.T%,'I_TT___1_______'Ial_'I__/,Il"It-ecently,Hamburgerand8almon(1977a,b)havegrownagarcoloniesfromhumanmyelomacellsandalsofromothermalig-nantcellsofmarrow.For3tumours,abronchialcarcinoma,aneuroblastomaandanovarianascites,theyobtainedPEsbe-tween0-02and041%.Inthepresentstudieswehaveexamined48differentsolidorascitestumours.Compactcoloniesofover50cellswereobtainedfromoverhalfofthetumourstestedandPEsaveraging2%weremeasured.Evidencethatcolonieswerederivedfromtumourcellswasprovidedbycellmorphology,andwasparticularlyclearinthecaseofthemelanoticmelanomas,butwasalsoshownfromthedistinctivecolonvmorphologyandhistologicalappearanceofcoloniesgrownfromothertumourtypes.Althoughagarinhibitstheformationofcoloniesbystromalcellsthatgrowasmonolayers,thepossibilitythatbloodcellsfromthetumourmightbecapableofpro-ducingdiffusecoloniessimilartothoseseeninmarrowcultureswasconsidered,andoccasionaldiffusecolonieswereexcludedfromthecolonycounts.Thetwoagartechniquesusedinthesestudiesdifferfromstandardagartech-niquesinprovidingforthereplenishmentoftheagarmediumfromaliquidphase,bytheadditionoffreshculturemediumabovetheagarorbygrowingthecellsinADCinFiG.Coloniesgrowninvitroandviewedinthemousesothatnutrientsarerenewedagar:a.Amelanoticmelanomax150;bydiffusionfromthefluid.Itb.Melanoticmelanomax75;c.Ovarianperitonealascitesx150.mightbethoughtthatunderthesequasi(ai.:.......:a..:i...:*:.::(}))A;....................................:..:...................L.:.;::.:..::.......*:.:(c80vv-k7-."wvv..L.w...LILvll.-/ GROWTHOFCOLONIESINAGARFROMHUMANTUMOURBIOPSIES81invivoconditions,cellgrowthinADCwouldbebetterthaninvitro.Althoughinaminorityofcasesbettergrowthwasob-tainedwithoneorotherofthesystems,therewasnoevidencethateitherofthemwasgenerallysuperior.ThePEsobtainedfromallthetumourswhichgavecoloniescoveredawiderangeofvalues(001to15%).Thereareprob-ablymanyreasonsforthis.SomeofthelowerPEscouldberelatedtotechnicalproblemsinvolvedinpreparingcellsus-pensions,possiblycausingirreparabledamagetothecells.Also,particularlyinprimarytumourswithapartlydifferenti-atedcellpopulation,aconsiderablepro-portionofthettumourcellsmaylackthecapacityforfurtherproliferation.Inothertumours,theremaybeessentialdifferencesinnutritional,hormonalandotherre-quirementsspecifictothetumouroforigin.Alternatively,inmoreslowlygrow-ingtumourstherateofproliferationmaybetoolowfortheproductionofcolonieswithintheperiodofobservation,sincetoproduceacolonyofover50cellsinaperiodof3weeksrequiresthatthecellsdivi
5 deatleastonceevery3-days.Inspiteofthelim
deatleastonceevery3-days.Inspiteofthelimitationsoutlined,colonieshavebeenobtainedfromoverhalfofthetumourstested,andforanumberofthemthePEslargeenoughtoformthebasisofasurvivalassay.Althoughfurtherexperienceandtechnicaldevelopment,particularlyinthepreparationandsepara-tionoftumour-cellsuspensions,isneeded,theseresults,particularlyfromthemela-nomas,suggestthatitcouldeventuallybepossibletoundertakesensitivitytestingoftumoursfromindividualpatients,atleastforcertaindefinedtumourcategories.Otherpossibleclinicalapplicationsareintheestimationofresponsetochemo-therapy,wheremultiplesamplesareavail-ableandthegrowthofcoloniesinagarcouldalsobeofprognosticvalue,sinceitgivesameasureoftheproliferationrateofthetumourcells.Thesetechniquesal-readyprovideausefulresearchtoolfortheclonalisolationoftumourcelllinesandcouldpotentiallyallowtheanalysisofclonalheterogeneityintumours.OurthanksareduetoDrG.G.Steelforhissup-portandencouragement,toallmembersofthemedicalstaffwhosuppliedthematerial,includingDrsJ.Baker,T.J.McElwain,P.TrottandE.Wilt-shawandtothetissuecollectors,MrsJ.Nicholls,MrsJ.WhittakerandMrsS.Schonfield.REFERENCESALTMAN,A.J.,CRUSSI,F.G.,RIERDEN,W.J.&BAEHNER,R.L.(1975)Growthofrhabdomyosar-comacoloniesfrompleuralfluid.CancerRes.,35,1809.COURTENAY,V.D.,SMITH,I.E.,PECKHAM,M.J.&STEEL,G.G.(1976)Invitroandinvivoradio-sensitivityofhumantumourcellsobtainedfromapancreaticcarcinomaxenograft.Nature,263,771.COURTENAY,V.D.&MILLS,J.(1978)Aninvitrocolonyassayforhumantumoursgrowninimmune-suppressedmiceandtreatedinvivowithcytotoxicagents.Br.J.Cancer,37,261.DENDY,P.P.,BOZMAN,G.&WHEELER,T.K.(1970)Invitroscreeningtestforhumanmalignanttum-oursbeforechemotherapy.Lancet,ii,68.HALL,T.C.(1977)Predictionofresponsestotherapyandmechanismofresistance.Semin.Oncol.,4,193.HAMBURGER,A.W.&SALMON,S.E.(1977a)Primarybioassayofhumantumourstemcells.Science,197,460.HAMBURGER,A.W.&SALMON,S.E.(1977b)Primarybioassayofhumanmyelomastemcells.J.Clin.Invest.,60,846.McALLISTER,R.M.&REED,G.(1968)Colonialgrowthinagarofcellsderivedfromneoplasticandnon-neoplastictissuesofchildren.Ped.Res.,2,356.MACPHERSON,I.A.(1973)Softagartechniques.InTissueCultureMethodsandApplications.Eds.Kruse,P.F.andPatterson,M.K.NewYork:Acad.Press.p.267.MILLAR,J.L.,BLACKETT,N.M.&HUDSPITH,B.H.(1978)Enhancedpost-irradiationrecoveryofthehaemopoieticsysteminanimalspre-treatedwithavarietyofcytotoxicagents.CellTissueKinet.11,inpress.ROPER,P.R.&DREWINKO,B.(1976)Comparisonofinvitromethodstodeterminedrug-inducedcelllethality.CancerRes.,36,2182.SMITH,I.E.,COURTENAY,V.D.&GORDON,M.Y.(1976)Acolony-formingassayforhumantumourxenograftsusingagarindiffusionchambers.Br.J.Cancer,34,476.