Download Elucidation of Cell Fate Transitions in Lung Cancer Stem Cells

ElucidationofCellFateTransitionsinLungCancerStemCells
LeadInvestigator:
Joo-HyeonLee
WellcomeTrust–MRCStemCellInstitute
DepartmentofPhysiology,DevelopmentandNeuroscience
http://www.stemcells.cam.ac.uk/researchers/principalinvestigators/dr-joo-hyeon-lee
Co-Investigator:
BenjaminDSimons
GurdonInstitute,DepartmentofPhysics
http://www.stemcells.cam.ac.uk/researchers/principalinvestigators/pressor-ben-simons
OurgoalinthisproposedprojectintheAerodigestiveProgrammeistoexplorecell-fatetransitionsin
aCSCmodelandtousethedatawegeneratetocreatepredictivemodelsthatcapturekey
interactionsinthesystemandwhichcanhelpdistinguishNSCandCSCbyusingadvancedmethods
includingnewlydeveloped3Dorganoidassaysfromhumanandmouselungs,geneticgene
manipulationswithCRISPRtechnologies,andstatisticalphysics,whichwillprovideunique
opportunitiesforstudentstolearncancerbiologywithmultidisciplinaryapproaches.Inparticular,we
willprovideourexpertiseinestablishingpatient-derivedlungcancerorganoidswhichwillbevaluable
resourcetoprovideinsightsintounderstandinglungtumorigenesisandpatient-centredtherapeutic
development.Webelieveourproposedprojectishighlyattractiveforthegroupofcliniciansand
physicistsnotonlytolearnoncogeniccellularchangesofpatient-derivedtumourcellsinthemost
physiologicallyrelevantcontextbutalsotolearnclonaldistributionsoftumourcellsduringearly
tumorigenesisinvivo.
ProjectDescription
Background
Currentcancerresearchaimsatdiscoveringmoleculartargetsthatcancercellsrelyontosurvive,
andwhichmaythereforebeutilizedtokillcancercells.However,cancercellsintumoursdisplay
considerableheterogeneityasaconsequenceofgeneticchange,environmentaldifferencesand
reversiblechangesincellproperties[1].Cancerstemcells(CSCs)arethoughttodrivethis
phenotypicandfunctionalheterogeneityamongcancercellsbyrepopulatingtumorigeniccells[2].
Therefore,identificationofthosetumourcellsthathavethecapacitytoreconstituteatumour
shouldbeournextsteptofocuson.Furthermore,identifyingthemolecularfactorsthatdrivethe
differentiationandproliferationofCSCsinasettingthatmimicsthelungenvironmentcouldprovide
anattractivedrugtargetsforchemorefractorylungtumours.
Inthelung,multipleepithelialcellshavebeenidentifiedasstemorprogenitorcellscapableofselfrenewalanddifferentiationduringregenerationandinjuryrepair[3].Recently,thesestemcell
populationshavealsobeenimplicatedasthecell(s)-of-origininlungcancer;basalcells,whichisthe
stemcellscapableofself-renewalandgivingrisetosecretorycellsintrachea,havebeensuggested
asthecell-of-originintheoncogenicLKB1/PTENmousemodeloflungsquamouscellcarcinoma
(SCC),andalveolartypeIIcells(AT2cells),whichhavebeenproposedtohavestemcellpropertiesin
thedistallung,havealsobeensuggestedasthecell-of-originintheoncogenicKrasmousemodelsof
lungadenocarcinoma(ADC)[4].However,itremainsunknownwhatarethemolecularmechanisms
underlyingderivationoflineagespecificlungcancersandcellularheterogeneitywithinthetumours.
Extrinsicsignallingfactorsderivedfrommicroenvironmenthavebeenemergingasthecritical
regulatorsthatmaintainstemcellsandestablishacellularhierarchy.Thus,studyingstemcellsand
theinteractionswiththeirnichefromwild-typeandoncogenictumourmodelsaffordsthe
comparisonofnormaltissue-specificstemcellsinitialprocessoftransformationandtumour
heterogeneity,CSCs,andnichesignalsthatimpacttheircellularbehaviour.
Aims
Wehypothesizethattherearedistinctdifferencesintheregulatorynetworksandcellfates
associatedwithCSCsandnormaladultstemcells(NSCs).Bydiscoveringanddissectingthe
regulatorynetworks,wewillfindkeyfactorsthatdrivetumourdevelopmentandcellfatedecisions.
Wehavedevelopedthreedimensional(3D)lungorganoidcultureassayswhichwillbeusedtostudy
thedifferentiationofstemcellsisolatedfrommurinelungsorhumanpatientsamples[5].Stemcells
anddifferentiatedprogenywillbeprofiledforgeneexpressionusingRNAseq.Integrativemethods
willbeappliedtointegratethegeneexpressiondatawithpriorknowledgefromtheliteratureand
buildregulatorynetworks.Wewillusetechniquesfromstatisticalphysicstomapwhichdrivergenes
andgenemodulesareassociatedwitheachcellfate.Nichefactorsthatmaintaintheircellular
heterogeneitywillbeevaluatedinourorganoidassays.Finally,wewillinvestigatetheprinciples
governingchangesincelldynamicsandfateduringearlytumorigenesisusingthequantitative
analysisinthemousemodels.Weaimtoidentifygenemodulesthatdrivecancerstemcells,butnot
normallungstemcells,tohelpintheidentificationofdrugtargetsthatarespecifictothecancer
stemcells.Importantly,wewillperformthesestudiesin3Dorganoidsystemsthatmimicthelung
environmentanduseamultidisciplinaryapproachtoexplorethecellularandmolecularmechanisms
underlyingstemcellbehaviourinthetransitiontolungcarcinogenesis.
Objective1. WewillanalysedifferentiationofNSCsandCSCsinunique3Dsystemsandbuild
genenetworkthatdistinguishNSCsandCSCsandthedifferentiationprocessesin
whichtheyparticipate.
Objective2. WewilldefinethecellularpropagationdynamicsofNSC-andCSC-derivedcell
clonesusingouruniqueinvitro3Dorganoidcultureassaysandinvivomouse
models.
Objective3. WewillelucidatesignallingpathwaysthatestablishandmaintaintheCSCnichein
earlytumorigenesis.
ResearchPlan
1.
Wewillusethenewinvitroandinvivo3Dassaysdevelopedbyourlabtoexamine
differentiationofCSCs,andcompareandcontrastitwiththedifferentiationofNSCs.NSCs
andCSCswillbeisolatedfrommurinelungsandhumanpatientsamplesfollowing3D
culture/transplantation.LineagedifferentiationofNSCsandCSCswillbeassessedby
measuringtheratiosofairwayandalveolarlineagecells.Inaddition,oncogenicgenesor
tumoursuppressorswillbeintroducedordeletedintheorganoidcultureassaysofNSCs
usingCRISPR/Cas9-mediatedgeneeditingtoevaluatetheinitialcellfatetransitionsinthe
contextofoncogenicchanges.Thisworkwilldirectlycomparethedifferentiationpotential
ofcancerstemcellsandtheirnormalcounterparts.
2.
WewillperformRNAisolationandexpressionprofilinginatimecourseduringthe
differentiationprocessin3Dorganoidcultureassaystocontrastthedifferenttrajectories
andendpointsthatareaccessedbyNSCsandCSCs.RNA-seqdatawillbeanalysedtofindthe
networksandgenesthatdrivecancerstemcelldifferentiation.Thenetworktopologieswill
becomparedtohighlightchangesinconnectivitythatcoulddrivethedifferencesbetween
NSCsandCSCs.Usingknockdownorover-expressiongeneticstrategiesandrecombinant
proteinsorchemicalinhibitors,wewillusethe3DassaystotestcandidateCSCregulators.
Modulationofthefactorswillultimatelybeperformedwithinvivo3Dtransplantation
assaystotesttheimpactonCSCactivity.
3.
Wewillusetheuniquemousemodelswhichenabletodefinethecellularpropagation
dynamicsoftumorigenicclonesusingtheinvivoquantitativelineagetracingatdifferent
timepointsfollowingthelineage-labellingevent(incollaborationwithDrBon-KyoungKoo
andProfessorBenjaminD.Simons).Thisapproachallowsustoassesschangesinclone
characteristicsduringearlycarcinogenesiswithinanestablishedtumour.
4.
Weproposetoelucidatethecellularandmolecularmechanismsthatdeterminetheniche
cellfate.Candidatefactorsofinterestswillbevalidatedin3Dassaysandnewlyestablished
mousemodelsthatallowsforcre-dependentfluorescentlabellingofnichecellsandenables
theirspecificablationbydiphtheriatoxinadministration.Isolationofthenichecell
populationwillenabletheirgeneexpressionprofilingaswellasvalidationofnichefunction
ininvitroandinvivoassays.Elucidationandcomparisonofthemolecularandcellular
compositionoftheNSCandCSCnichewillincreaseourunderstandingofcancerbiology,
whichmayopenupnoveltherapeuticavenues.
References
1.
GreavesMetal.,Clonalevolutionincancer.Nature.2012Jan18;481(7381):306-13.
2.
ValentPetal.,Cancerstemcelldefinitionsandterminology:thedevilisinthedetails.Nat
RevCancer.2012Nov;12(11):767-75.
3.
HoganBLetal.,Repairandregenerationoftherespiratorysystem:complexity,plasticity,
andmechanismsoflungstemcellfunction.CellStemCell.2014Aug7;15(2):123-38.
4.
ChenZetal.,Non-small-celllungcancers:aheterogeneoussetofdiseases.NatRevCancer.
2014Aug;14(8):535-46.
5.
LeeJ-Hetal.,LungstemcelldifferentiationinmicedirectedbyendothelialcellsviaaBMP4NFATc1-thrombospondin-1axis.Cell.2014Jan30;156(3):440-55.
Applications
Toapplyforthisstudentshiphttp://www.cambridgecancercentre.org.uk/studentships
ForgeneralenquiriespleasecontactTinaThorntina.thorn@cruk.cam.ac.uk
Forfurtherinformationorquestionsrelatingtothisprojectpleasecontact:
[email protected]