ActaMed.Okayama,2002 Vol.56,No.3,pp.129-134 Copyright ｃ2002byOkayamaUniversityMedicalSchool.
OriginalArticle http://www.lib.okayama-u.ac.jp/www/acta/
ComparisonofChemosensitivityTests: ClonogenicAssayversusMTTAssay
KazuhikoKawada ,ToshiroYonei ,HiroshiUeoka ,KatsuyukiKiura , MasahiroTabata ,NagioTakigawa ,MineHarada ,andMitsuneTanimoto
DepartmentofInternalMedicineII,OkayamaUniversityMedicalSchool,Okayama700-8558,Japan, DepartmentofRespiratoryMedicine,NationalOkayamaMedicalCenter,Okayama701-1192,Japan,and DepartmentofInternalMedicine,KyushuUniversitySchoolofMedicine,Fukuoka812-8582,Japan
Whenthedevelopmentofchemotherapeuticagentsreachestheclinicaltrialstage,itisnecessary toperform drug sensitivitytestsquicklyin ordertoselectthemostpromising agentsforthe treatmentofcancer.Inordertoassessthepossibilityofusingthe3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide(MTT)assayasasubstituteforthehumantumorclonogenicassay (HTCA),weevaluatedthecorrelationbetweentheresultsobtainedbythese2assaysin5humanlung cancercelllines.Thecorrelationcoe cientbetweentheresultsoftheHTCAandtheMTTassaywas 0.673,indicatingarelativelygoodcorrelation.Thecorrelationwasmostprominentinplatinum analogues (r＝0.939)and good in anthracyclines/anthracenedione (r＝0.611). However, no significantcorrelationwasobservedinvincaalkaloids,etoposide,irinotecan,SN-38(anactive metaboliteofirinotecan),andrhizoxin.TheresultsoftheMTTassayshowedahighdegreeof correlationwiththoseoftheHTCA inpredictingthesensitivityofcancercelllinestoplatinum analogues,andanthracyclines/anthracenedione.TheseresultssuggestthattheMTTassaymaybe moreconvenientandquicklyperformedthantheHTCA andcanreplaceHTCA inevaluatingthe e ectsofanticanceragents,especiallytheplatinumanaloguesandanthracyclines/anthracenedione.
Keywords:chemosensitivity test, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenicassay
ecently,therehasbeenremarkableprogressin drugsensitivitytestshavebeenintroducedforthispur- R the developmentofchemotherapeutic agents. pose,theyallpossesscertaindisadvantages［1,2］.The However,anenormousamountoftimeandmoneymust humantumorclonogenicassay(HTCA)hasbeenwidely beinvestedbeforeanagentisapprovedforclinicaluse. employedfortheevaluationofdrugsensitivityintumor Therefore,whenthedevelopmentofchemotherapeutic celllinesandtumortissuesobtainedbybiopsyorsurgery agentsreachestheclinicaltrialstage,rapidtestsofdrug ［3,4］.SinceHTCA hasbeenproventohavecertain sensitivitymaybeusefulforselectionofthemostpromis- degreeofcorrelationwithclinicalresponse,ithasbeen ingagentsinthetreatmentofcancer.Althoughvarious routinelyusedinourlaboratory.However,theassayalso hasanumberofdisadvantages,includinglowe ciency andslowturnaroundtimebeforetheresultsareobtained ReceivedOctober23,2001;acceptedDecember17,2001. Correspondingauthor.Phone:＋81-82-235-7229;Fax:＋81-86-232-8226 ［5,6］.Variousmethodshavethereforebeendeveloped E-mail:hueoka＠md.okayama-u.ac.jp(H.Ueoka) toallowformorerapidevaluationofdrugsensitivity.The 130 Kawadaetal. ActaMed.Okayama Vol.56,No.3
AmericanNationalInstituteofHealth(NIH)employeda adensityof5×10 cells/mlbytreatmentwith0.25 sulforhodamineB (SRB)assaytoscreenfore ective trypsin＋0.05 EDTA.Afterseveralconcentrationsof chemotherapeuticagentsusinghumantumorcelllinesin drugspreparedbyserialdilution,wereaddedtothe adisease-orientedapproach［7,8］.TheMTTassayhas cultures,thecellswereincubatedfor1hat37°C.After alsobeenwidelyusedinmanylaboratories［9］.We theywerewashedinordertoremovethedrug,thecells employedtheMTT assayandestablishedapanelof weresuspendedin15 FBS＋RPMI-1640＋0.3 humanlungcancercelllinestoscreenfore ectiveagents agarose(TakaraShuzoCo.,Ltd.Kyoto,Japan)and againstlungcancer［10］.TheMTTassayisbasedon platedontoafeederlayer(15 FBS＋RPMI-1640＋ theabilityofviabletumorcellstoreduceatetrazolium 0.5 agarose),followedbyculturewith5 CO2inair basecompoundtoablueformazanproduct［11］.The (NAPCO)at37°Cfor2weeks.Thenumberofcolonies MTT assayisgenerallycarriedoutina96-wellplate ateachdrugconcentrationwascountedusingaparticle format,andtheMTTformazanproductisanalyzedwith counter(ShiraimatsuInstrumentCompany,CP-3000). ascanningmultiplespectrophotometersuchthatanumber TheMTT assaywasperformed ofsamplescanbeanalyzedquicklyandsimply［13-15］. accordingtothemethod ofMosmann［11］. Serial However,itisnecessarytocomparetheMTTassaywith dilutionsofchemotherapeuticagentspreparedbythe otherassaymethodsalreadyconfirmedtocorrelatewith multiplicationmethodwereplacedina96-wellmicroplate. clinicalactivity,becausetheMTTassaydoesnotdirectly Sincethecelllinesemployedinthepresentstudyhad evaluatethecytocidalactivityofcancercells. di erentproliferationrates,thenumberofculturedcells Theaimofthepresentstudyistoassesstheuseful- wasadjustedtoadensitythatallowedtheuntreated nessoftheMTTassayincomparisonwiththeHTCAin controlcellstogrowexponentially.Thatis,SBC-3was thescreeningvariousanticancerdrugsoflungcancercell inoculatedintothemicroplatewellsatadensityof2,000 lines. cells/wellandtheothercelllineswereinoculatedata densityof5,000cells/well.Afterexposureofthecellsto MaterialsandMethods thetestdrug for96h at37°C, 10mlof3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Fivehuman (MTT;SigmaChemicalCo.,St.Louis,MO,USA) lungcancercelllines,SBC-1(JCRB 0816),SBC-2 solutioninPBS(5mg/ml)wasadded,andthecellswere (JCRB 0817),SBC-3(JCRB 0818),ABC-1(JCRB incubatedforanother4h.Then125mlofisopropanol＋ 0815),andEBC-1(JCRB 0820),whichhavebeen 0.04N HClwasadded,andtheabsorbancewasdeter- establishedandmaintainedinourlaboratory,wereem- minedatawavelengthof560nmusinganELISAreader. ployedinthepresentstudy［5,6,10,12］.SBC-1, Theagentsused SBC-2,andSBC-3arehumansmall-celllungcancer inthepresentstudywereasfollows:Anthracyclines: (SCLC)celllines,ABC-1isanadenocarcinomacellline, doxorubicin (ADM), daunorubicin (DNR), epirubicin andEBC-1isasquamouscellcarcinomacellline.SBC-3 (EPI),pirarubicin(THP),aclarubicin(ACR),amrubicin wasestablishedfromapreviouslyuntreatedpatientwith (SM-5887),ME2303,andKRN8602(MX-2);anthra- SCLC,whileSBC-1andSBC-2wereestablishedfrom cenedione:mitoxantrone (MXT);platinum analogues: patientswithSCLCwhohadbeenclinicallyresistantto cisplatin (CDDP), carboplatin (CBDCA), nedaplatin chemotherapy.ABC-1andEBC-1werealsoestablished (254-S), NK121, and DWA2114R;vinca alkaloids: frompatientsreceivinganticanceragents.Thecellswere vincristine(VCR),vindesine(VDS),vinblastine(VLB), culturedinRPMI-1640(GIBCO)supplemented with and vinorelbine (VNR);a podophyllotoxin;etoposide 15 fetalbovineserum(FBS)at37°Cwith5 CO2in (VP-16);topoisomeraseIinhibitors:irinotecan(CPT- humidifiedair.Cellconcentrationsintheculturewere 11)anditsactivemetabolite(SN-38);andanewmacro- adjustedtoallowforexponentialgrowth. lide:rhizoxin(RZX).ADM,EPI,andVNR were providedbyKyowaHakkoKogyoCo.,Ltd.,Tokyo, HTCA was performed according to the Salmon- Japan,CDDP,CBDCA,andVP-16byBristol-Myers Hamburgermethod［3,4］.Forthesuspensionculture, SquibbK.K.,Tokyo,Japan,NK121byNipponKayaku cellswereseparatedbypipetting.Forthemonolayer Co.,Ltd.,Tokyo,Japan,CPT-11andSN-38 by cultureinflasks,asinglecellsuspensionwaspreparedat YakultHonshaCo.,Ltd.,Tokyo,Japan,SM-5887by June2002 ClonogenicAssayversusMTTAssay 131
SumitomoPharmaceuticalsCo.,Ltd.,Osaka,Japan, ofthedrugconcentration.Fromtheregressionlinethus DWA2114R by ChugaiPharmaceuticalCo., Ltd., obtained,the70 lethaldrugconcentration(LD70)was Tokyo,Japan,DNR,THPandME2303byMeijiSeika determinedasanindexofthecytocidale ectofthedrug. kaisha,Ltd.,Tokyo,Japan,ACR byYamanouchi TheMTTassaywasperformedatleast3timesforeach PharmaceuticalCo.,Ltd.,Tokyo,Japan,KRN8602 celllineinquadruplicatecultures.Theratioofabsorbance (MX-2)byKirinBreweryCo.,Ltd.,Tokyo,Japan, oftreatedculturestothatofuntreatedcontrolcultures MXTbyLederleJapan,Ltd.,Tokyo,Japan,VCRand wasobtainedforallconcentrationsofeverydrug.From VLBbyEliLillyJapanK.K.,Kobe,Japan,254-Sand thedose-responsecurvethusobtained,a50 inhibitory VDSbyShionogi& Co.,Ltd.,Osaka,Japan,and concentration(IC50)wasdeterminedasanindexof RZX byFujisawaPharmaceuticalCo.,Ltd.,Osaka, antitumoractivity.Thedegreeofcorrelationofthedata Japan.Thedrugsweredilutedwithphysiologicalsaline, obtainedwiththese2assaymethodswasevaluatedusing mannitol,ordimethylsulfoxide(DMSO).Eachdrugwas Pearson’stest. thenexaminedatvariousdoselevels,includingatadose of1/10peakplasmaconcentration,asreportedinthe Results clinicalphaseIstudy. Usingduplicateculturesofeach TheIC50valuesofeachdrugobtainedwiththeMTT cellline,theHTCAwasperformed3times.Considering assayforthe5humanlungcancercelllinesareshownon thecolonycountofuntreatedtumorcellsasacontrol,the therightsideofTable1andtheLD70valuesobtained percentageofviablecellswasplottedagainstthelogarithm withHTCAareshownontheleftside.Fig.1showsthe
Table1 TheLD70valuesobtainedbythehumantumorclonogenicassay(HTCA)andIC50valuesobtainedbytheMTTassay
TheLD70valuesofeachdrugobtainedbytheHTCA(nM)TheIC50valuesofeachdrugobtainedbytheMTTassay(nM)
SBC-1 SBC-2 SBC-3 ABC-1 EBC-1 SBC-1 SBC-2 SBC-3 ABC-1 EBC-1 doxorubicin 190 235 60 520 2,800 29 69 22 62 70 daunorubicih NO NO 72 NO NO NO NO 35 NO NO epirubicin NO NO 130 NO NO NO NO 19 NO NO pirarubicin 98 92 16 120 320 76 74 7 105 156 aclarubicin 92 60 58 85 360 23 14 5 12 14 SM-5887 NO NO 23,600 NO NO NO NO 832 NO NO ME2303 NO NO 2,400 NO NO NO NO 14 NO NO KRN8602 NO NO 1,400 NO NO NO NO 45 NO NO mitoxantrone 210 270 34 180 860 86 140 15 350 440 cisplatin 3,800 6,600 4,830 9,170 84,100 646 2,188 603 1,622 6,310 carboplatin 62,400 51,900 77,100 134,800 606,800 6,500 5,800 4,571 14,000 39,000 254-S 10,200 13,900 14,100 19,500 108,300 1,150 2,000 891 3,000 8,600 NK121 32,400 49,200 23,400 158,000 490,400 2,200 4,000 2,000 5,000 15,600 DWA2114R 95,700 91,300 111,300 186,800 685,800 7,600 17,000 9,800 16,000 80,000 vincristine 673 420 12,400 5,500 NO 8 2 2 2 4 viNOesine 500 NO 5,000 3,450 2,400 2 1 4 2 4 vinblastine 750 420 3,200 2,000 420 4 3 2 2 3 vinorelbine 680 260 1,900 3,280 8,900 53 3 5 4 6 etoposide 315,000 438,000 745,000 820,000 245,000 245 1,202 288 1,549 3,802 irinotecan 55,000 32,000 72,300 200,000 500,000 5,754 2,455 316 665 501 SN-38 20 34 61 81 107 5 3 1 2 2 rhizoxin NO 33 19 92 20 NO 1 1 1 1
NO,notobtained. 132 Kawadaetal. ActaMed.Okayama Vol.56,No.3 correlationofmeanIC50valuesobtainedwiththeMTT maypredicthigheractivityforthesedrugsthanHTCA. assayandthemeanLD70valuesobtainedwiththe HTCA.ThecommonlogarithmsofLD70valueswere Discussion plottedalongthelongitudinalaxisandthecommonloga- rithmsofIC50valueswereplottedalongthehorizontal WecomparedtheusefulnessoftheMTTassaywith axis.Thecorrelationcoe cientwas0.673(P＜0.01), thatoftheHTCAwithrespecttodrugsensitivitytesting. indicatingarelativelygoodcorrelationbetweentheresults Thesensitivityofthehumanlungcancercelllinesevaluat- oftheHTCAassayandthoseoftheMTTassay.The edwiththese2assaymethodsgenerallyshowedrelatively correlationcoe cientsofthedataobtainedfromdi erent celllinesareasfollows:0.794,0.861,0.752,0.691,and 0.861inSBC-1,SBC-2,SBC-3,ABC-1,andEBC-1, respectively.Fig.2showsthestratificationofdata accordingtotheclassofeachdrug(platinumanalogues, anthracyclines/anthracenedione,andvincaalkaloids).In theplatinumanalogues,thedataobtainedwiththeMTT assayshowedthemostprominentcorrelation(r＝0.939, P＜0.01)withthedataobtainedbyHTCA.Inanthra- cyclines/anthracenedione,agoodcorrelation(r＝0.611, P＜0.01)wasalsoconfirmed.However,nosignificant correlationwasobservedinthecaseofthevincaalkaloids. Theplots,withina5 controllimitofvincaalkaloids, weredistributedontheupperleftside,incontrastto thoseoftheplatinum analoguesand anthracyclines/ anthracenedione.Fig.3showsthestratificationdataof Fig.2 Thestratificationofdataaccordingtotheclassofdrugs etoposide, irinotecan, SN-38, and rhizoxin. No (platinum analogues, anthracyclines/anthracenedione, and vinca significantcorrelationwasobservedwiththesedrugs. alkaloids).ThedataobtainedbytheMTTassayshowedasignificant Theplotswithina5 controllimitweresimilarly correlationwiththedataobtainedbytheHTCAwithrespectto distributedattheleft,suggestingthattheMTTassay platinum analoguesandanthracyclines/anthracenedione(r＝0.939, P＜0.01;r＝0.611,P＜0.01,respectively).Nosignificantcorrela- tionwasobservedinthecaseofvincaalkaloids.Theplotswithina 5% controllimitarecircled.
Fig.1 CorrelationofmeanIC50valuesobtainedbytheMTTassay andmeanLD70valuesobtainedbyHTCA.Thecommonlogarithmsof theLD70valueswereplottedalongthelongitudinalaxisandthe Fig.3 Thestratificationdataobtainedwithetoposide,irinotecan, commonlogarithmsoftheIC50valueswereplottedalongthehorizon- SN-38,andrhizoxin.Nosignificantcorrelationwasobservedwith talaxis.Thecorrelationcoe cientwas0.673(P＜0.01). thesedrugs.Theplotswithina5% controllimitarecircled. June2002 ClonogenicAssayversusMTTAssay 133
goodcorrelationamongvarioustypesofchemotherapeutic e cientassessmentofachemotherapeuticagentoran agents.Inparticular,fortheplatinum analoguesand agentexpectedtohavecarcinostaticactivity［13-15］. anthracyclines/anthracenedione,closecorrelationswere TheMTTassayisalsousefulforperformingsmall-scale confirmedintheresultsevaluatedbyHTCA andMTT experimentsusinganexpensivedrug［7］.However,it assay. However, in vinca alkaloids, etoposide, hassomedrawbacksthatcanleadtomisleadingresults, irinotecan,SN-38,andrhizoxin,thesensitivitiespredict- e.g.,evaluationusingtheMTTassayisbasedonthe edbytheMTTassayweregenerallyhigherthanthose existenceofaproportionalrelationshipbetweentheabsor- obtainedbytheHTCA.Theseresultsmayreflectthe banceandthenumberofviablecellsinaculture［18］. timedependencyoftheanticancere ectinthelatter Thereforeweconfirmedthisproportionalrelationship agents. betweentheabsorbanceandthecellcountbypreparinga Currently,invitrodrugsensitivitytestsincludeacell calibrationcurvepriortothepresentexperiment.Since proliferation assay (HTCA), a di erentialstaining thesizeanddoublingtimeofcancercellsvaryfromcell cytotoxicity(DiSC)assay,andmodifiedassayssuchas linetocellline,wealsoperformedapreliminaryexperi- anadherencematrixassayandathree-dimensionalgel menttofindtheoptimalnumberofcellstobeculturedfor assay［1］.HTCA,developedbySalmonandHam- eachcellline.Inaddition,cellsshouldbeexposedtoa burger,judgescellviabilityasclearlybasedonthe testdrugforanadequateamountoftimeinordertocause presenceorabsenceofcellproliferation,i.e.,basedon maximum celldeathandlossofdehydrogenaseactivity. colony-forming ability［3,4］. Roperand Drewinko Wepreviouslydeterminedtheoptimalnumberofcellsper comparedHTCA withalabelingindexassay,adye wellandtheoptimaldurationofcultureforeachofour exclusionassay,a Crreleaseassay,measurementof cancercelllinessothatthemaximum absorbancewas ［ H］thymidineuptake,andmeasurementofdoubling obtained while exponentialgrowth was maintained time［16］.Theyfound thatHTCA wasthemost［10,12］. definitiveindicatorofcelldeath.Inthepresentexperi- Weperformedthepresentstudytoevaluatethe ment,weemployedthe2-layersoftagarmediummethod correlation between tumor sensitivity evaluated by ofHTCA.Withthismethod,itwaspossibletofreely HTCA,whichhasbeentraditionallyusedatourinstitu- changeexposuretimeandtoexpresscelldeathquantita- tion,andthatbytheMTTassay,whichiswidelyused tively.Thus,ourmethodappearstobethemostuseful totestthesensitivityofvariousanticanceragents.There methodforperformingHTCA［17］.Thedrugconcen- havebeenonlyafewreportscomparingtheMTTassay trationemployedforHTCAwasbasedontheblooddrug andtheHTCA.Perezetal.showedthattheMTT concentrationachievedwithclinicaldoses.Theblood assaywascomparabletoaclonogenicassayasregards drugconcentrationatthestartofthebeta-phaseof cisplatinsensitivity;thecorrelationcoe cientwas0.810 elimination,followingintravenousbolusadministrationat (P＝0.0074).However,theydidnotreportthesensitiv- thestandardclinicaldose,isusedasthepivotalconcen- ityofotherdrugs［19］.Weintendedtoassessthe trationforHTCA.Thecellsarejudgedtobesensitiveto possibilityofreplacingtheHTCAwiththeMTTassay ananticanceragentwhenatleast70 inhibitionof fordrugsensitivitytestinginthecaseof22anticancer colony-formingactivityisattainedasaresultofa1-hour agentsusing5lungcancercelllines.Generally,ahigh exposureofcellstothedrugatthatconcentration. degreeofcorrelationwasconfirmedbetweentheresults Therefore,thecelldeathobservedasaresultof1-hour obtainedwiththese2assaymethods.However,the exposuretothedruginHTCAisconsideredtobeuseful MTTassaypredictedahigherlevelofanti-tumoractivity foranassessmentoftherelationshipbetweenHTCAdata ofvincaalkaloidsthanthatwaspredictedwithHTCA. andtheclinicale ect.However,disadvantagesofthe VincaalkaloidsaretypeIIadrugsandtheyhavea HTCAincludethecomplexityoftheprocedure,thetime time-dependent action, according to Shimoyama’s requiredtoobtainresults,andthelimitationsonthe classificationofdrugmechanism［s20］.Sincethecells carcinostaticagentsandthenumberofcelllinesthatcan wereexposedtothedrugforalongerdurationinthe betestedsimultaneously［6］. MTTassay(96h)thanintheHTCA (1h),thismay Ontheotherhand,theMTT assay,whichisin haveledtodi erencesintheresultsobtainedbythese2 widespreadclinicaluseatpresent,allowsfortheuseof typesofassay.Similarresultswerealsoobtainedfor varioustypesofcarcinomacellsandthusfacilitatesmore camptothecinanalogues,etoposide,andrhizoxin,andthe 134 Kawadaetal. ActaMed.Okayama Vol.56,No.3 mechanismoftheseagentsmayalsobetime-dependent. celllines.JNatlCancerInst(1990) ,1113-1118. 8. SkehanP,StorengR,ScudieroD,MonksA,McMahonJ,VisticaD, Inconclusion,HTCA andMTT assaysshoweda WarrenJT,BokeschH,KenneySandBoydMR:Newcolorimetric highdegreeofcorrelationasregardsdrugsensitivity cytotoxicityassayforanticancer-drugscreening.JNatlCancerInst testingofplatinum analoguesandanthracyclines/anthra- (1990) ,1107-1112. cenedione.ThepresentresultssuggestthattheMTT 9. CarmichaelJ,DeGra WG,GazdarAF,MinnaJDandMitchellJB: Evaluationofatetrazolium-basedsemiautomatedcolorimetricassay: assaycanbeemployedinsteadoftheHTCAinorderto Assessmentofchemosensitivitytesting.CancerRes(1987) ,936- estimatetheactivityofsuchagentsagainsttumorcells; 942. theadvantagesoftheMTTassayareitssimplicity,low 10. MatsushitaA,TabataM,UeokaH,KiuraK,ShibayamaT,AoeK, KoharaHandHaradaM:Establishmentofadrugsensitivitypanel cost,andshorterduration.However,whenassessing usinghumanlungcancercelllines.ActaMedOkayama(1999) , time-dependentagentssuchasvincaalkaloids,etoposide, 67-75. camptothecinanalogues,andrhizoxin,itshouldbenoted 11. MosmannT:Rapidcolorimetricassayforcellulargrowthandsurvival: thattheMTTassaypredictshighercytotoxicactivitythan Applicationtoproliferationandcytotoxicityassays.JImmunolMethods (1983) ,55-63. thatpredictedbyHTCA. 12. TakigawaN,OhnoshiT,UeokaH,KiuraKandKimuraI:Comparison ofantitumoractivity ofnew anthracycline analogues, ME2303, References KRN8602,andSM5887usinghumanlungcancercelllines.ActaMed Okayama(1992) ,249-256. 13. CamplingBG,PymJ,BakerHM,ColeSPandLamYM:Chemosen- 1. CarmichaelJ:Chemosensitivitytestinginlungcancer;inLungCan- sitivitytestingofsmallcelllungcancerusingtheMTTassay.BrJ cer,PassHI,MitchellJB,JohnsonDHandTurrisiATeds,Lippincott- Cancer(1991) ,75-83. RavenPublishers,Philadelphia(1996)pp161-167. 14. BeplerGandO’BriantK:Invitrochemosensitivitytestingofhuman 2. SmitEF,deVriesEG,Timmer-BosschaH,deLeijLF,OosterhuisJW, non-smallcelllungcancercelllines.AnticancerRes(1998) ,3181- ScheperRJ,WeeningJJ,PostmusPE andMulderNH:In vitro 3185. responseofhumansmall-celllung-cancercelllinestochemother- 15. MitsudomiT,KanekoS,TateishiM,YanoT,IshidaT,KohnoeS, apeuticdrugs;nocorrelationwithclinicaldata.IntJCancer(1992) MaeharaYandSugimachiK:Chemosensitivitytestingofhumanlung ,72-78. cancertissuesusingthesuccinatedehydrogenaseinhibitiontest. 3. HamburgerAW andSalmonSE:Primarybioassayofhumantumor AnticancerRes(1990) ,987-990. stemcells.Science(1977) ,461-463. 16. RoperPRandDrewinkoB:Comparisonofinvitromethodstodeter- 4. SalmonSE,HamburgerAW,SoehnlenB,DurieBG,AlbertsDSand minedrug-inducedcelllethality.CancerRes(1976) ,2182-2188. MoonTE:Quantitationofdi erentialsensitivityofhuman-tumorstem 17. HirakiS,OhnoshiT,NumataT,KishimotoN,MoriK,YoneiT, cellstoanticancerdrugs.NEnglJMed(1978) ,1321-1327. YamashitaHandKimuraI:Anticancerdrugsensitivitybyhumantumor 5. HirakiS,OhnoshiT,MiyaiM,NumataT,KawaharaS,SetoT, clonogenicassay.ActaMedOkayama(1986) ,265-269. TamuraT,OzawaS andKimuraI:Tumorstem cellassayfor 18. VisticaDT,SkehanP,ScudieroD,MonksA,PittmanAandBoydMR: detectingmetastasesofhumanlungcancer.ActaMedOkayama Tetrazolium-basedassaysforcellularviability:Acriticalexamination (1983) ,141-146. ofselectedparametersa ectingformazanproduction.CancerRes 6. YoneiT,OhnoshiT,HirakiS,UeokaH,KiuraK,MoritakaT, (1991) ,2515-2520. ShibayamaT,TabataM,SegawaY,TakigawaN andKimuraI: 19. PerezRP,GodwinAK,HandelLMandHamiltonTC:Acomparisonof Antitumoractivityofplatinumanalogsagainsthumanlungcancercell clonogenic,microtetrazoliumandsulforhodamineBassaysfordeter- linesandtumorspecimens.ActaMedOkayama(1993) ,233-241. minationofcisplatincytotoxicityinhumanovariancarcinomacelllines. 7. RubinsteinLV,ShoemakerRH,PaullKD,SimonRM,TosiniS, EurJCancer(1993) ,395-399. SkehanP,ScudieroDA,MonksAandBoydMR:Comparisonofin 20. ShimoyamaM:Cytocidalactionofanticancerantigens:Evaluationof vitro anticancer-drug-screeningdatageneratedwithatetrazolium thesensitivityofculturedanimalandhumancancercells. Bibl assayverousaproteinassayagainstadiversepanelofhumantumor Haematol(1975) ,711-722.