Seahorse-XF售前幻燈完整版(老版本帶注釋)課件

O2O2O2O2O2O2+H+H+H+H+HH+O2O2O2CELLMETABOLISMREVEALED--ANewAspecttoExploreDiseases

MarketingManager,APAC欒曉輝,Ph.D.xhluan@O2O2O2O2O2O2+H+H+H+H+HH+O2O2O21SeahorseBioscience,Inc.FirstXFplatformin2006,~1,000unitsinstalledworldwidetillnowHeadquarterinBoston,regionalofficesinCopenhagen&ShanghaiMission:

Torevolutionizethestudyofmetabolisminlivingcells,providing newopportunitiesforunderstandingandtreatinghumandiseaseSeahorseBioscience,Inc.First2XFeAnalyzersMeasuretheTwoMajorEnergyPathwaysOxygenConsumptionRate(OCR)=Mitochondrial

RespirationExtracellular

AcidificationRate

(ECAR)=

GlycolysisXFeAnalyzersMeasuretheTwo3MetabolicPhenotypesMeasuresthemetabolicstate

ofcells:Metabolicswitchingexamples:WarburgEffectReverseWarburgEffectCrabtreeEffectPasteurEffectMoreMetabolicLessMetabolicGlycolyticAerobicMemoryTCellsNeuronsH460LungCancerCellsPluripotentStemCellsMetabolicPhenotypesMeasurest4SimultaneouslydetectOCR&ECARRealtimedetection,nodamagetosamplesUptofourcompoundscouldbeadded&

monitoredApplyforcells,tissues,modelanimals&

microorganismsTheGoldStandardPlatformforCellMetabolismAssaysXFCellMitoStressTestProfileSpareCapacityXFGlycolysisStressTestProfileGlycolyticReserveSimultaneouslydetectOCR&EC5XFe96CellCultureMicroplateXFAssayMediumSolidStateSensorCellMonolayerSensorProbesFiberOpticsWell1Well2MeasuringMitochondrialRespiration

andGlycolysisinaMicroplateXFe96CellCultureMicroplate6Thetransientmicrochamberenablesrapidmeasurementofmitochondrialrespirationandglycolysisinminutes

Thebio-cartridgeisraised,bringingthesystembacktobaselineThesensorcartridgeisraised,bringingthesystembacktobaselineAtransient

microchamberisformedWell1Well2MeasuringMitochondrialRespiration

andGlycolysisinaMicroplateThetransientmicrochamberena7AutomatedDrugInjectionPorts

EnableKinetic,FunctionalData

4druginjectionportsperwell

Addinhibitors,stimulants,substrates,andcompounds

InjectionsdefinedbytheuserInjectsupto4compounds[NOTE:VideoRequiresQuickTime]AutomatedDrugInjectionPorts8IsBasalMetabolismEnough?200620072008200920102011David

NichollsPhDJNeuroscience

Jul4;(2007)27(27):7310-7

NagendraYadava,

DavidNicholls,etalVictor

Darley-UsmarPhDFreeRadicalBiology&Medicine

51(2011)1621–1635

BrianDranka,Victor

Darley-Usmar,etal“Cellshaveanenergyreserveto

handleacutestressdemands”IsBasalMetabolismEnough?2009MitochondrialFunction“Profiles”inDiseasesDatacourtesyofDr.PinarCoskun,

DeptofNeurobiologyandBehavioratUCIrvineParkinson’sDisease

Lowersparecapacityinpatient

HIV*

LowersparecapacityinpatientsAutism*

Highersparecapacityinpatient

Multipleacyl-CoAdehydrogenasedeficiency(MADD)**

Highersparecapacityinpatient

PLoSOne.2009Dec17;4(12):e8329.,SongYetalLymphocytesFibroblastsFibroblastsPatientPatientPatientPatientDatacourtesyofJamesBennetLab,UniversityofVirginiaDatacourtesyofSherHendrickson,NCI/NIHFrederickFibroblastsMitochondrialFunction“Profil10MaximalRespirationandSpareCapacityBasalRespirationATP

ProductionProtonLeakXFCellMitoStressTestProfileSpare

CapacityMaximalRespirationNon-mitochondrialRespirationMeasurethekeyparameters

ofmitochondrialfunction:BasalRespirationATPProductionProtonLeakMitochondriahaveanenergyreservetohandleacutestressdemandsXFCellMitoStressTestMaximalRespirationandSpare11XFGlycolysisStressTestProfileGlycolytic

CapacityNon-glycolyticAcidificationGlycolyticReserve

CapacityMeasurethekeyparameters

ofglycolyticfunction:Non-glycolyticAcidificationGlycolysisGlycolyticCapacityGlycolyticReserveGlycolysisGlycolysisECAR(mpH/min)ExtracellularAcidificationECAR(mpH/min)10mM

Cellshaveanenergyreservetohandleacutestressdemands

XFGlycolysisStressTestXFGlycolysisStressTestProf12SinglePlatform,NumerousApplicationsMitochondrialDiseasesScreeningCancerCardiovascularTranslationalMedicineAgingModelOrganismsObesity,Diabetes,&MetabolicDisordersNeurodegenerationToxicology&HepatobiologyImmunologySinglePlatform,NumerousAppl13ApplicationSupportforNumerousDirectionsApplicationSupportforNumero14MetabolismData=SeahorseData169256213723342320062007200820092010201120122013>5002014>1,200Publications!MetabolismData=SeahorseDat15PublicationsbyChinaResearchers

TotalNumber>60,andIncreasingbyatLeast3~4/MonthPublicationsbyChinaResearch16Nature.2009May21;459(7245):387-92.Bmi1regulatesmitochondrialfunctionandtheDNAdamageresponsepathwayLiuJ,CaoL,ChenJ,SongS,LeeIH,QuijanoC,LiuH,KeyvanfarK,ChenH,CaoLY,AhnB,KumarN,Rovira,Ii,XuXL,VanLohuizenM,MotoyamaN,DengC,Finkel.Cancer.2011Jun30;IndividualizingantimetabolictreatmentstrategiesforheadandnecksquamouscellcarcinomabasedonTP53mutationalstatusSandulacheVC,SkinnerHD,OwTJ,ZhangA,XiaX,LuchakJM,WongLJ,PickeringCR,ZhouG,MyersJN.CancerCardiovascularBBAMolecularCellResearch,2011Aug4Susceptibilitytosimvastatin-inducedtoxicityispartlydeterminedbymitochondrialrespirationandphosphorylationstateofAkt]MullenPJ,ZahnoA,LindingerP,MaseneniS,FelserA,Kr?henbühlS,BrechtK.NeurodegenerationObesity,Diabetes,MetabolicDisordersInvestOphthalmolVisSci.2011Oct6[Epubaheadofprint]HighGlucoseInducesMitochondrialMorphologyandMetabolicChangesinRetinalPericytesTrudeauK,MolinaAJ,RoyS.AgingNature.2010Dec2;468(7324):659-63.TheLkb1metabolicsensormaintainshaematopoieticstemcellsurvival.GurumurthyS,XieSZ,AlagesanB,KimJ,YusufRZ,SaezB,TzatsosA,OzsolakF,MilosP,FerrariF,ParkPJ,ShirihaiOS,ScaddenDT,BardeesyN.StemCellBiologyJNeurochem.2011Jul;118(1):79-92.Mitochondrialaconitaseknockdownattenuatesparaquat-induceddopaminergiccelldeathviadecreasedcellularmetabolismandreleaseofironandH(2)O(2)CantuD,FultonRE,DrechselDA,PatelM.SeahorseImpactsCellularBioenergeticsResearchNature.2009May21;459(7245)17MetabolismSwitchRecognizedasNewHallmarkofCancerGlycolyticfuelinghasbeenshowntobeassociatedwithactivatedoncogenes

(e.g.,RAS,MYC)andmutanttumorsuppressors

(e.g.,TP53)HallmarksofCancer:TheNextGeneration,Cell144,March4,2011MetabolismSwitchRecognizeda18TypicalGlycolysisIncreaseinCancerCellsPC-3MPCSOligo2-DGProstateTumorCellsPrimaryProstateEpithelialCellsGlucose

FreeBaseLysineIncreasesSurvivalandReducesMetastasisinProstateCancerModel

Ibrahim-Hashim,etal.,JCancerSciTher2011,S1

TypicalGlycolysisIncreasein19Hedgehog-InducedSwitchtoaGlycolyticPhenotypeSmoothenedknock-outs[Smo-sh]losetheabilitytoutilizeglucoseSmoothenedagonistHedgehogsignalingviaSAG[smoothenedagonist]shiftsadipocytestoaglycolyticphenotypeAerobicMoreMetabolicLessMetabolicGlycolyticHedgehog-InducedSwitchtoaG20Howp53GetInvolvedinCancer?MolCell.2014Jun19;54(6):960-74.Howp53GetInvolvedinCancer21Parks2013NatureReviewsCancerUnderstandingtheTumorMicroenvironmentParks2013NatureReviewsCanc22SpheroidPlatefor3DCulturedCellsA.TheuniquegeometryoftheXFe96SpheroidMicroplate.B.Thecorrectpositionofthespheroidatthebottomofthewell.Designedtoholdspheroidsbetween200and500μmindiameter.C.Theventingsystemthatallowsforoptimalmediamixingaroundeachspheroid.SpheroidPlatefor3DCultured23XFTechnologyunderHypoxiaConditionMCF-7cells(humanmetastaticbreastcancercellline)exhibitacharacteristicWarburgshiftfollowingexposureto5%O2asdepictedintheXFPhenogramDecreasedmitochondrialfunctionintumorcelllinesunder5%O2XFTechnologyunderHypoxiaCo24DynamicMetabolismSwitchReflectingDrugResponse“TheynoticedthattumorsdrivenbyBRAFgeneshadparticularlyhighuseofglucose,whichwasturnedoffveryrapidlyafterdrugtherapy–thisindicatedthemedicationwasworking.”“Theresearchersalsonoticedthatbeforethetumorsstartedtogrowagain,themelanoma’sglucosesupplywasturnedbackonandbecameresistanttotheeffectsofthedrug.”DynamicMetabolismSwitchRefl25MitochondriaBecomesCancerTherapyTargetMitochondriaBecomesCancerTh26MetabolismShiftinCancer/focus/cancermetabolism/index.htmlMetabolismShiftinCancerhttp27CancerImmunotherapyBreakthrough……CancerImmunotherapyBreakthro28……byEnhancingTCellEnergeticPerformance2DGContMelanomaVehicle2DGAdoptivetransfer(spleen)CD62LKLRG-12DGContControl2DG……byEnhancingTCellEnergeti29PublicationinImmunity2012Reveals……

MitochondrialrespiratorycapacityisacriticalregulatorofCD8+TcellmemorydevelopmentvanderWindt,JW,…andPearce,EL,WashingtonSchoolofMedicine.Immunity2011December28PublicationinImmunity2012R30IL-15MemoryTcellIL-2EffectorTcellEffectorTCellsDependonGlycolysistoMeetTheirNeeds

MemoryTcellsDependonFAOtoPersistforYearsWaitingfortheEnemyIL-15MemoryTcellIL-2Effect31

MitochondrialrespiratorycapacityisacriticalregulatorofCD8+Tcellmemorydevelopment

vanderWindt,JW,…andPearce,EL,WashingtonSchoolofMedicine,Immunity2011December28MouseLymphocytes

FattyacidsourceisFBSSpareCapacityinCD8+TCellsisDependenton

MitochondrialFattyAcidOxidation

Mitochondrialrespiratorycap32MemoryCD8+TcellsFunctionRequiresEarlyGlycolyticSwitchRapideffectorfunctionofmemoryCD8+Tcellsrequiresanimmediate-earlyglycolyticswitch,PatrickMGubser,etal.,DepartmentofBiomedicine,Immunobiology,UniversityofBasel.NatureImmunology,13Aug,2013Effectormemory(EM)CD8+Tcells,activatedthroughstimulationarelinkedtoincreasedglycolyticflux,exhibitmoreGAPDHactivityatearlytimepoints,beforeproliferationcommences,thannaivecellsactivatedundersimilarconditionsMemoryCD8+TcellsFunctionR33Mitoresp.GlycoysisMetabolicSwitchingisDeterministicforTCell’sFateMitoresp.GlycoysisMetabolicS34EtomoxirMemoryTcellsIncreasedsurvivalMemoryTCellDevelopmentisDependenton

IncreasedSpareRespiratoryCapacityFueledbyFattyAcidOxidationEtomoxirMemoryTcellsIncrease35The3carbonsourcesthatfuelmitochondriaThe3carbonsourcesthatfuel36UK5099AnneMurphy/MartinJastroch

(2014MethodsinEnzymology)BPTESChristianMetallo/AnneMurphy(2014MolecularCell)DavidNicholls(2009J.Neurochemistry)Calithera/MDAnderson(2014MolecularCancerTherapeutics)EtomoxirMetallo/Murphy(2014MolecularCell)NikaDanial(2014CellMetabolism)UsinginhibitorsofindividualsubstratepathwaysUK5099AnneMurphy/MartinJastr37MitochondrialDeficiencyinDeafnessMitochondrialDeficiencyinDe38NeurodegenerationDrugDevelopmentNeurodegenerationDrugDevelop39Ten-foldIncreaseinMitochondrialTherapiesClinicaltrialsPhaseI-IV2000-20052005-20112011tillnowMitochondrialtargets10105Metabolictargets3505,425“MitochondrialdrugsaregoingtorevolutionizeWesternmedicine”DougWallace,Director,CenterforMitochondrialandEpigenomicMedicine,CHOPNeurodegeneration:Parkinson’s,Huntington’s,AlzheimersdiabetesmellitushepaticfailureCancerautoimmunityGVHDRenaldisease:proximaltubulopathyCardiovasculardisease:?Ten-foldIncreaseinMitochond40Seahorse-XF售前幻燈完整版(老版本帶注釋)課件41BioenergeticPhenotypeinalcoholliverdiseaseRats:OligomycinFCCPAntimycinARotenoneHowdoweintegratethisdatafortheclinic?ZelicksonBRetal2011.BiochimBiophysActa.BioenergeticPhenotypeinalco42BasalNon-Mitochondrial

MaximalReserveCapacityATPLinkedH+LeakAsinglevaluethatdefinesbioenergetichealthandcanbeusedasaprognosticanddiagnosticindicatorforpatientssusceptibletometabolicstress.OligoFCCPAntBHI=(ReserveCapacity)x(ATPlinked)(H+Leak)x(Non-mitochondrial)BHI=(ReserveCapacity)x(ATPlinked)(H+Leak)x(Non-mitochondrial)BHI=(ReserveCapacity)x(ATPlinked)(H+Leak)x(Non-mitochondrial)BHI=(ReserveCapacity)x(ATPlinked)(H+Leak)x(Non-mitochondrial)BHI=(ReserveCapacity)x(ATPlinked)(H+Leak)x(Non-mitochondrial)BasalNon-MitochondrialMaxim43BioenergeticHealthIndexinalcoholliverdiseasepatientsBHI=Log()RCaxAlb

NMcxPLd*p=0.019BioenergeticHealthIndexina44BHIasadynamicmeasureofbody’sresponsetostressBHICircadianHealthyDietExerciseGeneticsShiftWorkPoorDietPollutionInactivityVirus,AgingIllnessInflammationToxinsTherapiesBHIasadynamicmeasureofbo45Seahorse-XF售前幻燈完整版(老版本帶注釋)課件46CurrentUsersinChina(Partial)浙江大學(xué)生命科學(xué)院上海藥物研究所西安交通大學(xué)生命科學(xué)院中科院營(yíng)養(yǎng)所天津體育學(xué)院國(guó)家新藥篩選中心湖南湘雅第二醫(yī)院中科院生化細(xì)胞所上海交通大學(xué)仁濟(jì)醫(yī)院第四軍醫(yī)大學(xué)溫州醫(yī)學(xué)院中科院動(dòng)物所計(jì)劃生育與生殖健康中心南京醫(yī)科大學(xué)中山大學(xué)腫瘤防治中心第三軍醫(yī)大學(xué)高原所中國(guó)醫(yī)科大學(xué)內(nèi)分泌研究所北京大學(xué)分子醫(yī)學(xué)研究所中科院有機(jī)所中科院生物物理研究所東北大學(xué)禮來(lái)制藥中國(guó)研發(fā)中心葛蘭素史克中國(guó)研發(fā)中心北京大學(xué)醫(yī)學(xué)部301醫(yī)院老年血管研究所復(fù)旦大學(xué)醫(yī)學(xué)院香港大學(xué)李嘉誠(chéng)醫(yī)學(xué)院上海瑞金醫(yī)院內(nèi)分泌研究所清華大學(xué)生命科學(xué)學(xué)院CurrentUsersinChina(Partia47ThankYou

forYourAttention!Jan,2015ThankYouforYourAttention!J48O2O2O2O2O2O2+H+H+H+H+HH+O2O2O2CELLMETABOLISMREVEALED--ANewAspecttoExploreDiseases

MarketingManager,APAC欒曉輝,Ph.D.xhluan@O2O2O2O2O2O2+H+H+H+H+HH+O2O2O249SeahorseBioscience,Inc.FirstXFplatformin2006,~1,000unitsinstalledworldwidetillnowHeadquarterinBoston,regionalofficesinCopenhagen&ShanghaiMission:

Torevolutionizethestudyofmetabolisminlivingcells,providing newopportunitiesforunderstandingandtreatinghumandiseaseSeahorseBioscience,Inc.First50XFeAnalyzersMeasuretheTwoMajorEnergyPathwaysOxygenConsumptionRate(OCR)=Mitochondrial

RespirationExtracellular

AcidificationRate

(ECAR)=

GlycolysisXFeAnalyzersMeasuretheTwo51MetabolicPhenotypesMeasuresthemetabolicstate

ofcells:Metabolicswitchingexamples:WarburgEffectReverseWarburgEffectCrabtreeEffectPasteurEffectMoreMetabolicLessMetabolicGlycolyticAerobicMemoryTCellsNeuronsH460LungCancerCellsPluripotentStemCellsMetabolicPhenotypesMeasurest52SimultaneouslydetectOCR&ECARRealtimedetection,nodamagetosamplesUptofourcompoundscouldbeadded&

monitoredApplyforcells,tissues,modelanimals&

microorganismsTheGoldStandardPlatformforCellMetabolismAssaysXFCellMitoStressTestProfileSpareCapacityXFGlycolysisStressTestProfileGlycolyticReserveSimultaneouslydetectOCR&EC53XFe96CellCultureMicroplateXFAssayMediumSolidStateSensorCellMonolayerSensorProbesFiberOpticsWell1Well2MeasuringMitochondrialRespiration

andGlycolysisinaMicroplateXFe96CellCultureMicroplate54Thetransientmicrochamberenablesrapidmeasurementofmitochondrialrespirationandglycolysisinminutes

Thebio-cartridgeisraised,bringingthesystembacktobaselineThesensorcartridgeisraised,bringingthesystembacktobaselineAtransient

microchamberisformedWell1Well2MeasuringMitochondrialRespiration

andGlycolysisinaMicroplateThetransientmicrochamberena55AutomatedDrugInjectionPorts

EnableKinetic,FunctionalData

4druginjectionportsperwell

Addinhibitors,stimulants,substrates,andcompounds

InjectionsdefinedbytheuserInjectsupto4compounds[NOTE:VideoRequiresQuickTime]AutomatedDrugInjectionPorts56IsBasalMetabolismEnough?200620072008200920102011David

NichollsPhDJNeuroscience

Jul4;(2007)27(27):7310-7

NagendraYadava,

DavidNicholls,etalVictor

Darley-UsmarPhDFreeRadicalBiology&Medicine

51(2011)1621–1635

BrianDranka,Victor

Darley-Usmar,etal“Cellshaveanenergyreserveto

handleacutestressdemands”IsBasalMetabolismEnough?20057MitochondrialFunction“Profiles”inDiseasesDatacourtesyofDr.PinarCoskun,

DeptofNeurobiologyandBehavioratUCIrvineParkinson’sDisease

Lowersparecapacityinpatient

HIV*

LowersparecapacityinpatientsAutism*

Highersparecapacityinpatient

Multipleacyl-CoAdehydrogenasedeficiency(MADD)**

Highersparecapacityinpatient

PLoSOne.2009Dec17;4(12):e8329.,SongYetalLymphocytesFibroblastsFibroblastsPatientPatientPatientPatientDatacourtesyofJamesBennetLab,UniversityofVirginiaDatacourtesyofSherHendrickson,NCI/NIHFrederickFibroblastsMitochondrialFunction“Profil58MaximalRespirationandSpareCapacityBasalRespirationATP

ProductionProtonLeakXFCellMitoStressTestProfileSpare

CapacityMaximalRespirationNon-mitochondrialRespirationMeasurethekeyparameters

ofmitochondrialfunction:BasalRespirationATPProductionProtonLeakMitochondriahaveanenergyreservetohandleacutestressdemandsXFCellMitoStressTestMaximalRespirationandSpare59XFGlycolysisStressTestProfileGlycolytic

CapacityNon-glycolyticAcidificationGlycolyticReserve

CapacityMeasurethekeyparameters

ofglycolyticfunction:Non-glycolyticAcidificationGlycolysisGlycolyticCapacityGlycolyticReserveGlycolysisGlycolysisECAR(mpH/min)ExtracellularAcidificationECAR(mpH/min)10mM

Cellshaveanenergyreservetohandleacutestressdemands

XFGlycolysisStressTestXFGlycolysisStressTestProf60SinglePlatform,NumerousApplicationsMitochondrialDiseasesScreeningCancerCardiovascularTranslationalMedicineAgingModelOrganismsObesity,Diabetes,&MetabolicDisordersNeurodegenerationToxicology&HepatobiologyImmunologySinglePlatform,NumerousAppl61ApplicationSupportforNumerousDirectionsApplicationSupportforNumero62MetabolismData=SeahorseData169256213723342320062007200820092010201120122013>5002014>1,200Publications!MetabolismData=SeahorseDat63PublicationsbyChinaResearchers

TotalNumber>60,andIncreasingbyatLeast3~4/MonthPublicationsbyChinaResearch64Nature.2009May21;459(7245):387-92.Bmi1regulatesmitochondrialfunctionandtheDNAdamageresponsepathwayLiuJ,CaoL,ChenJ,SongS,LeeIH,QuijanoC,LiuH,KeyvanfarK,ChenH,CaoLY,AhnB,KumarN,Rovira,Ii,XuXL,VanLohuizenM,MotoyamaN,DengC,Finkel.Cancer.2011Jun30;IndividualizingantimetabolictreatmentstrategiesforheadandnecksquamouscellcarcinomabasedonTP53mutationalstatusSandulacheVC,SkinnerHD,OwTJ,ZhangA,XiaX,LuchakJM,WongLJ,PickeringCR,ZhouG,MyersJN.CancerCardiovascularBBAMolecularCellResearch,2011Aug4Susceptibilitytosimvastatin-inducedtoxicityispartlydeterminedbymitochondrialrespirationandphosphorylationstateofAkt]MullenPJ,ZahnoA,LindingerP,MaseneniS,FelserA,Kr?henbühlS,BrechtK.NeurodegenerationObesity,Diabetes,MetabolicDisordersInvestOphthalmolVisSci.2011Oct6[Epubaheadofprint]HighGlucoseInducesMitochondrialMorphologyandMetabolicChangesinRetinalPericytesTrudeauK,MolinaAJ,RoyS.AgingNature.2010Dec2;468(7324):659-63.TheLkb1metabolicsensormaintainshaematopoieticstemcellsurvival.GurumurthyS,XieSZ,AlagesanB,KimJ,YusufRZ,SaezB,TzatsosA,OzsolakF,MilosP,FerrariF,ParkPJ,ShirihaiOS,ScaddenDT,BardeesyN.StemCellBiologyJNeurochem.2011Jul;118(1):79-92.Mitochondrialaconitaseknockdownattenuatesparaquat-induceddopaminergiccelldeathviadecreasedcellularmetabolismandreleaseofironandH(2)O(2)CantuD,FultonRE,DrechselDA,PatelM.SeahorseImpactsCellularBioenergeticsResearchNature.2009May21;459(7245)65MetabolismSwitchRecognizedasNewHallmarkofCancerGlycolyticfuelinghasbeenshowntobeassociatedwithactivatedoncogenes

(e.g.,RAS,MYC)andmutanttumorsuppressors

(e.g.,TP53)HallmarksofCancer:TheNextGeneration,Cell144,March4,2011MetabolismSwitchRecognizeda66TypicalGlycolysisIncreaseinCancerCellsPC-3MPCSOligo2-DGProstateTumorCellsPrimaryProstateEpithelialCellsGlucose

FreeBaseLysineIncreasesSurvivalandReducesMetastasisinProstateCancerModel

Ibrahim-Hashim,etal.,JCancerSciTher2011,S1

TypicalGlycolysisIncreasein67Hedgehog-InducedSwitchtoaGlycolyticPhenotypeSmoothenedknock-outs[Smo-sh]losetheabilitytoutilizeglucoseSmoothenedagonistHedgehogsignalingviaSAG[smoothenedagonist]shiftsadipocytestoaglycolyticphenotypeAerobicMoreMetabolicLessMetabolicGlycolyticHedgehog-InducedSwitchtoaG68Howp53GetInvolvedinCancer?MolCell.2014Jun19;54(6):960-74.Howp53GetInvolvedinCancer69Parks2013NatureReviewsCancerUnderstandingtheTumorMicroenvironmentParks2013NatureReviewsCanc70SpheroidPlatefor3DCulturedCellsA.TheuniquegeometryoftheXFe96SpheroidMicroplate.B.Thecorrectpositionofthespheroidatthebottomofthewell.Designedtoholdspheroidsbetween200and500μmindiameter.C.Theventingsystemthatallowsforoptimalmediamixingaroundeachspheroid.SpheroidPlatefor3DCultured71XFTechnologyunderHypoxiaConditionMCF-7cells(humanmetastaticbreastcancercellline)exhibitacharacteristicWarburgshiftfollowingexposureto5%O2asdepictedintheXFPhenogramDecreasedmitochondrialfunctionintumorcelllinesunder5%O2XFTechnologyunderHypoxiaCo72DynamicMetabolismSwitchReflectingDrugResponse“TheynoticedthattumorsdrivenbyBRAFgeneshadparticularlyhighuseofglucose,whichwasturnedoffveryrapidlyafterdrugtherapy–thisindicatedthemedicationwasworking.”“Theresearchersalsonoticedthatbeforethetumorsstartedtogrowagain,themelanoma’sglucosesupplywasturnedbackonandbecameresistanttotheeffectsofthedrug.”DynamicMetabolismSwitchRefl73MitochondriaBecomesCancerTherapyTargetMitochondriaBecomesCancerTh74MetabolismShiftinCancer/focus/cancermetabolism/index.htmlMetabolismShiftinCancerhttp75CancerImmunotherapyBreakthrough……CancerImmunotherapyBreakthro76……byEnhancingTCellEnergeticPerformance2DGContMelanomaVehicle2DGAdoptivetransfer(spleen)CD62LKLRG-12DGContControl2DG……byEnhancingTCellEnergeti77PublicationinImmunity2012Reveals……

MitochondrialrespiratorycapacityisacriticalregulatorofCD8+TcellmemorydevelopmentvanderWindt,JW,…andPearce,EL,WashingtonSchoolofMedicine.Immunity2011December28PublicationinImmunity2012R78IL-15MemoryTcellIL-2EffectorTcellEffectorTCellsDependonGlycolysistoMeetTheirNeeds

MemoryTcellsDependonFAOtoPersistforYearsWaitingfortheEnemyIL-15MemoryTcellIL-2Effect79

MitochondrialrespiratorycapacityisacriticalregulatorofCD8+Tcellmemorydevelopment

vanderWindt,JW,…andPearce,EL,WashingtonSchoolofMedicine,Immunity2011December28MouseLymphocytes

FattyacidsourceisFBSSpareCapacityinCD8+TCellsisDependenton

MitochondrialFattyAcidOxidation

Mitochondrialrespiratorycap80MemoryCD8+TcellsFunctionRequiresEarlyGlycolyticSwitchRapideffectorfunctionofmemoryCD8+Tcellsrequiresanimmediate-earlyglycolyticswitch,PatrickMGubser,etal.,DepartmentofBiomedicine,Immunobiology,UniversityofBasel.NatureImmunology,13Aug,2013Effectormemory(EM)CD8+Tcells,activatedthroughstimulationarelinkedtoincreasedglycolyticflux,exhibitmoreGAPDHactivityatearlytimepoints,beforeproliferationcommences,thannaivecellsactivatedundersimilarconditionsMemoryCD8+TcellsFunctionR81Mitoresp.GlycoysisMetabolicSwitchingisDeterministicforTCell’sFateMitoresp.GlycoysisMetabolicS82EtomoxirMemoryTcellsIncreasedsurvivalMemoryTCellDevelopmentisDependenton

IncreasedSpareRespiratoryCapacityFueledbyFattyAcidOxidationEtomoxirMemoryTcellsIncrease83The3carbonsourcesthatfuelmitochondriaThe3carbonsourcesthatfuel84UK5099AnneMurphy/MartinJastroch

(2014MethodsinEnzymology)BPTESChrist