Adenosinesignalingplaysanimportantroleininflammationandtheimmuneresponse.ManycellsinthetumormicroenvironmentexpressectopicCD39andCD73,leADIngtothebuildupofextracellularadenosine.EngagementofadenosinewiththehighaffinityA2areceptor(A2aR)onthesurfaceofTcells,macrophages,NKcells,neutrophils,anddendriticcellscausesdownregulationoftheimmuneresponse.Therefore,A2aRisanovelimmunecheckpointprotein,andblockadeofA2aRisbeingactivelyinvestigatedasapotentialimmunotherapy.
SeveralA2aRantagoNISTshaveprogressedtoclinicaltrialsforthetreatmentofParkinson’sdisease,andpreclinicalstudieshaveconfirmedthatblockadeofA2areceptoractivationhastheABIlitytomarkedlyenhanceanti-tumorimmunity.MicetreatedwithA2aRantagonists,suchasZM241385(seedatabelow)orcaffeine,showsignificantlydelayedtumorgrowth,andA2aRknockoutmicedemonstrateincreasedtumorrejection.Mostpromising,A2aRblockadecanbeusedinsynergywiththeinhibitionofotherimmunecheckpointpathways.StudiesshowthatthecombinationofA2aRblockadeandPD-1inhibitionismoreeffectivethaneithertreatmentseparately,andA2aRblockadeincreasestheactivityofCTLA-4andTIM-3inhibitionincontrollingthegrowthofCD73+melanoma.
AdenosineA2areceptor(A2aRorADORA2A)stablyexpressedinHEK-293reportercellswithacAMP-responsiveluciferaseconstruct(CRE/CREBLuc,BPSBioscience,#60515).A2aRisamemberoftheseventransmembraneGprotein-coupledreceptor(GPCR)family.TheactivityofA2aRismediatedbyGsproteinwhichactivatesadenylylcyclase,resultinginthesynthesisofintracellularcAMP.
ThelevelofcAMPcorrelateswiththerespectiveadenosine(agonist)level,andthecelllinecanbeusedtomeasuretheEC50andIC50valuesofA2aRagonistsorantagonistsinaquantitativemanner.BindingofextracellularadenosineligandoritsstableanalogNECA(5′-(N-Ethylcarboxamido)adenosine)totheA2aRexpressedonthesurfaceofthiscelllinecanalsoproduceluciferasereportersignalforqualitativemeasurement.
•AssayMedium:ThawMedium1(BPSBioscience,#60187)
•NECA(Sigma,#79262)
•ZM241385(Sigma,#76263)
•96-welltissueculture-treatedwhiteclear-bottomassayplate
•cAMPGlokit(Promega,#V1501)
•One-Stepluciferaseassaysystem(BPSBioscience,#60690)formeasuringfireflyluciferaseactivity
•Luminometer
ThawMedium1(BPSBioscience,#60187):MEMmedium(Hyclone,#SH30024.01)+10%FBS(LifeTechnologies,#26140-079)+1%non-essentialaminoacids(Hyclone,#SH30238.01)+1mMNapyruvate(Hyclone,#SH30239.01)+1%Penicillin/Streptomycin(Hyclone,SV30010.01)
CompleteGrowthMedium:ThawMedium1(BPSBioscience,#60187) plus0.4mg/mlG418(ThermoFisher,#11811031)and50μg/mlofHygromycinB(Hyclone,#SV30070.01)
Cellsshouldbegrownat37°Cwith5%CO2usingcompletegrowthmedium(ThawMedium1withG418andHygromycinB).
Immediatelyuponreceipt,storeinliquidnitrogen.
•ScreenforagonistsorantagonistsofA2aRincell-basedcAMPassays.
•StudyPD-1andCTLA-4combinationtherapy.
•Screenco-inhibitorimmunecheckpointmoleculesforcancerimmunotherapy.
1.Leone,R.D.,et.al.(2015).Comp.Struct.Biotechnol.J.13:265-272
2.Ma,S-R.,etal.(2017).Molec.Cancer16:99
3.Ohta,A.,etal.(2016).Front.Immunol.7:109.
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