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@SeahorseBioscience,Inc.FirstXFplatformin2006,~1,000unitsinstalledworldwidetillnowHeadquarterinBoston,regionalofficesinCopenhagen&ShanghaiMission:

Torevolutionizethestudyofmetabolisminlivingcells,providing newopportunitiesforunderstandingandtreatinghumandiseaseXFeAnalyzersMeasuretheTwoMajorEnergyPathwaysOxygenConsumptionRate(OCR)=Mitochondrial

RespirationExtracellular

AcidificationRate

(ECAR)=

GlycolysisMetabolicPhenotypesMeasuresthemetabolicstate

ofcells:Metabolicswitchingexamples:WarburgEffectReverseWarburgEffectCrabtreeEffectPasteurEffectMoreMetabolicLessMetabolicGlycolyticAerobicMemoryTCellsNeuronsH460LungCancerCellsPluripotentStemCellsSimultaneouslydetectOCR&ECARRealtimedetection,nodamagetosamplesUptofourcompoundscouldbeadded&

monitoredApplyforcells,tissues,modelanimals&

microorganismsTheGoldStandardPlatformforCellMetabolismAssaysXFCellMitoStressTestProfileSpareCapacityXFGlycolysisStressTestProfileGlycolyticReserveXFe96CellCultureMicroplateXFAssayMediumSolidStateSensorCellMonolayerSensorProbesFiberOpticsWell1Well2MeasuringMitochondrialRespiration

andGlycolysisinaMicroplateThetransientmicrochamberenablesrapidmeasurementofmitochondrialrespirationandglycolysisinminutes

Thebio-cartridgeisraised,bringingthesystembacktobaselineThesensorcartridgeisraised,bringingthesystembacktobaselineAtransient

microchamberisformedWell1Well2MeasuringMitochondrialRespiration

andGlycolysisinaMicroplateAutomatedDrugInjectionPorts

EnableKinetic,FunctionalData

4druginjectionportsperwell

Addinhibitors,stimulants,substrates,andcompounds

InjectionsdefinedbytheuserInjectsupto4compounds[NOTE:VideoRequiresQuickTime]IsBasalMetabolismEnough?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”MitochondrialFunction“Profiles”inDiseasesDatacourtesyofDr.PinarCoskun,

DeptofNeurobiologyandBehavioratUCIrvineParkinson’sDisease

Lowersparecapacityinpatient

HIV*

LowersparecapacityinpatientsAutism*

Highersparecapacityinpatient

Multipleacyl-CoAdehydrogenasedeficiency(MADD)**

Highersparecapacityinpatient

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

ProductionProtonLeakXFCellMitoStressTestProfileSpare

CapacityMaximalRespirationNon-mitochondrialRespirationMeasurethekeyparameters

ofmitochondrialfunction:BasalRespirationATPProductionProtonLeakMitochondriahaveanenergyreservetohandleacutestressdemandsXFCellMitoStressTestXFGlycolysisStressTestProfileGlycolytic

CapacityNon-glycolyticAcidificationGlycolyticReserve

CapacityMeasurethekeyparameters

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

Cellshaveanenergyreservetohandleacutestressdemands

XFGlycolysisStressTestSinglePlatform,NumerousApplicationsMitochondrialDiseasesScreeningCancerCardiovascularTranslationalMedicineAgingModelOrganismsObesity,Diabetes,&MetabolicDisordersNeurodegenerationToxicology&HepatobiologyImmunologyApplicationSupportforNumerousDirectionsMetabolismData=SeahorseData169256213723342320062007200820092010201120122013>5002014>1,200Publications!PublicationsbyChinaResearchers

TotalNumber>60,andIncreasingbyatLeast3~4/MonthNature.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.SeahorseImpactsCellularBioenergeticsResearchMetabolismSwitchRecognizedasNewHallmarkofCancerGlycolyticfuelinghasbeenshowntobeassociatedwithactivatedoncogenes

(e.g.,RAS,MYC)andmutanttumorsuppressors

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

FreeBaseLysineIncreasesSurvivalandReducesMetastasisinProstateCancerModel

Ibrahim-Hashim,etal.,JCancerSciTher2011,S1

Hedgehog-InducedSwitchtoaGlycolyticPhenotypeSmoothenedknock-outs[Smo-sh]losetheabilitytoutilizeglucoseSmoothenedagonistHedgehogsignalingviaSAG[smoothenedagonist]shiftsadipocytestoaglycolyticphenotypeAerobicMoreMetabolicLessMetabolicGlycolyticHowp53GetInvolvedinCancer?MolCell.2014Jun19;54(6):960-74.Parks2013NatureReviewsCancerUnderstandingtheTumorMicroenvironmentSpheroidPlatefor3DCulturedCellsA.TheuniquegeometryoftheXFe96SpheroidMicroplate.B.Thecorrectpositionofthespheroidatthebottomofthewell.Designedtoholdspheroidsbetween200and500μmindiameter.C.Theventingsystemthatallowsforoptimalmediamixingaroundeachspheroid.XFTechnologyunderHypoxiaConditionMCF-7cells(humanmetastaticbreastcancercellline)exhibitacharacteristicWarburgshiftfollowingexposureto5%O2asdepictedintheXFPhenogramDecreasedmitochondrialfunctionintumorcelllinesunder5%O2DynamicMetabolismSwitchReflectingDrugResponse“TheynoticedthattumorsdrivenbyBRAFgeneshadparticularlyhighuseofglucose,whichwasturnedoffveryrapidlyafterdrugtherapy–thisindicatedthemedicationwasworking.”“Theresearchersalsonoticedthatbeforethetumorsstartedtogrowagain,themelanoma’sglucosesupplywasturnedbackonandbecameresistanttotheeffectsofthedrug.”MitochondriaBecomesCancerTherapyTargetMetabolismShiftinCancer/focus/cancermetabolism/index.htmlCancerImmunotherapyBreakthrough…………byEnhancingTCellEnergeticPerformance2DGContMelanomaVehicle2DGAdoptivetransfer(spleen)CD62LKLRG-12DGContControl2DGPublicationinImmunity2012Reveals……

MitochondrialrespiratorycapacityisacriticalregulatorofCD8+TcellmemorydevelopmentvanderWindt,JW,…andPearce,EL,WashingtonSchoolofMedicine.Immunity2011December28IL-15MemoryTcellIL-2EffectorTcellEffectorTCellsDependonGlycolysistoMeetTheirNeeds

MemoryTcellsDependonFAOtoPersistforYearsWaitingfortheEnemy

MitochondrialrespiratorycapacityisacriticalregulatorofCD8+Tcellmemorydevelopment

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

FattyacidsourceisFBSSpareCapacityinCD8+TCellsisDependenton

MitochondrialFattyAcidOxidationMemoryCD8+TcellsFunctionRequiresEarlyGlycolyticSwitchRapideffectorfunctionofmemoryCD8+Tcellsrequiresanimmediate-earlyglycolyticswitch,PatrickMGubser,etal.,DepartmentofBiomedicine,Immunobiology,UniversityofBasel.NatureImmunology,13Aug,2013Effectormemory(EM)CD8+Tcells,activatedthroughstimulationarelinkedtoincreasedglycolyticflux,exhibitmoreGAPDHactivityatearlytimepoints,beforeproliferationcommences,thannaivecellsactivatedundersimilarconditionsMitoresp.GlycoysisMetabolicSwitchingisDeterministicforTCell’sFateEtomoxirMemoryTcellsIncreasedsurvivalMemoryTCellDevelopmentisDependenton

IncreasedSpareRespiratoryCapacityFueledbyFattyAcidOxidationThe3carbonsourcesthatfuelmitochondriaUK5099AnneMurphy/MartinJastroch

(2014MethodsinEnzymology)BPTESChristianMetallo/AnneMurphy(2014MolecularCell)DavidNicholls(2009J.Neurochemistry)Calithera/MDAnderson(2014MolecularCancerTherapeutics)EtomoxirMetallo/Murphy(2014MolecularCell)NikaDanial(2014CellMetabolism)UsinginhibitorsofindividualsubstratepathwaysMitochondrialDeficiencyinDeafnessNeurodegenerationDrugDevelopmentTen-foldIncreaseinMitochondrialTherapiesClinicaltrialsPhaseI-IV2000-20052005-20112011tillnowMitochondrialtargets10105Metabolictargets3505,425“MitochondrialdrugsaregoingtorevolutionizeWesternmedicine”DougWallace,Director,CenterforMitochondrialandEpigenomicMedicine,CHOPNeurodegeneration:Parkinson’s,Huntington’s,AlzheimersdiabetesmellitushepaticfailureCancerautoimmunityGVHDRenaldisease:proximaltubulopathyCardiovasculardisease:?BioenergeticPhenotypeinalcoholliverdiseaseRats:OligomycinFCCPAntimycinARotenoneHowdoweintegratethisdatafortheclinic?ZelicksonBRetal2011.BiochimBiophysActa.BasalNon-Mitochondrial

MaximalReserveCapacityATPLinkedH+LeakAsinglevaluethatdefinesbioenergetichealthandcanbeusedas