Mammary Stem Cells: Methods and Protocols (Methods in Molecular Biology, 2471) PDF
Preview Mammary Stem Cells: Methods and Protocols (Methods in Molecular Biology, 2471)
Methods in Molecular Biology 2471 Maria dM. Vivanco Editor Mammary Stem Cells Methods and Protocols Second Edition M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Mammary Stem Cells Methods and Protocols Second Edition Edited by Maria dM. Vivanco Cancer Heterogeneity Laboratory, Centre for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Derio, Spain Editor MariadM.Vivanco CancerHeterogeneityLaboratory, CentreforCooperativeResearchin Biosciences(CICbioGUNE) BasqueResearchandTechnologyAlliance(BRTA) Derio,Spain ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2192-9 ISBN978-1-0716-2193-6 (eBook) https://doi.org/10.1007/978-1-0716-2193-6 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2015,2022 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. 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Preface Breastcancerremainsthemostcommonlydiagnosedcancerandtheleadingcauseofcancer deathinwomen.In2015,thefirsteditionofMethodsandProtocolsformammarystemcell researchwaspublishedandsincethenthefieldhascontinuedexpandinganddeepeningour understandingofmammaryglandbiologyandtumorigenesis.Theever-growingmammary stemcellresearchdataillustratetheirhugepotentialtoimpactourknowledgeaboutbreast cancer initiation, progression, development of resistance, and metastasis to other organs. This new edition has revisited some of the main techniques used to study the mammary gland. While some methods from the first edition, such as FACS sorting for isolation and characterizationofstemcells,aswellasthevisualizationofthesidepopulationphenotype, havenotsignificantlychangedandthustheyhavenotbeenrepeatedhere,othershavebeen updatedandquiteafewnewmethodshavebeenincorporated.Together,thiscollectionof chaptersaimstoleadbreastresearchersthroughsomeoftheessentialandlateststate-of-the- artmethodstostudythefullspectrumofmammaryglandbiologyandpathologicalstates. Breastcancerisacomplexdisease,oftenviewedas“caricature”ofnormaldevelopment goneawry.Gaininginsightsintotheseearlyeventsprovidesauniqueopportunitytodevelop innovativestrategiestodefinethemolecularinteractionsandgeneticfoundationsofcancers and identify novel biomarkers and therapeutic targets. Collectively, the approaches described in the first three chapters provide a window into mammary developmental dynamics, and the perturbations underlying tissue dysfunction and disease. Thus, Mikkola andhergroupstartfromthebeginning,focusingonmammaryglanddevelopmentduring embryogenesis.Theycontributetounderstandtheearliestmolecularandcellulareventsby describinganupdatedorgancultureprotocolofthemurineembryonicmammaryglandthat allows monitoring of growth and branching morphogenesis of mammary gland ex vivo by liveimaging.Theyfurtherintroduceanovelmethodthatfacilitatestheidentificationofthe direct stromal cues for branching morphogenesis. The group of Lloyd-Lewis presents a powerfulapproachthatusesthelatestmultidimensionalfluorescenceimagingforvisualizing mammary branching morphogenesis and epithelial cell fate dynamics during postnatal and embryonicmammaryglanddevelopment.TheWahllabcontinueswiththethemeofmouse embryo studies and describes single-cell RNA-seq and single-nucleus ATAC-seq methods and discusses the bioinformatic techniques they use to interpret the vast amount of data generated.DelSolandhisgroupcontinuetopursuethisimportantchallengebyreviewing variouscomputationalapproachesthatrelyonsingle-celland/orbulkRNAsequencingdata forelucidatingthemolecular foundationsofcellsubpopulationidentities,lineagespecifica- tion, and cell fate. Further omics analysis is discussed in the chapter by Elortza and colleagues, who present methods for relative protein quantification. They describe sample preparation prior to mass spectrometry analysis, as well as a standard acquisition method, and some common bioinformatics analyses that help extracting biologically relevant infor- mationoutofthelargeamountofdatagenerated. Severalchaptersarededicatedtodifferentfunctionalexperimentalapproaches.Faraldo and colleagues revisit the classic orthotopic transplantation assay, which has provided important insights into mammary development, stem cell function, and tumorigenesis, and they highlight the strengths and limitations of this in vivo grafting assay. Lineage v vi Preface tracing,whichinthepreviouseditionwasarelativelynoveladdition,isnowconsideredthe goldstandardapproachtostudycellularhierarchiesandcellfateinvivo,asdiscussedbyFre andRodilla,whoalsopresentthedownstreamanalysesandread-outassaysmostcommonly used. In Chapter 8, Iggo provides a detailed and informative protocol for lentiviral trans- duction of mammary epithelial cells, which can be used to infect cells simultaneously with multiple vectors, greatly facilitating studies on malignant transformation. In contrast, Jechlinger and colleagues also used lentiviral transduction, but just to mark and track a fewcellswithinorganoidsderivedfromgeneticallymodifiedmice,inorder tostudyevents occurringduringhealthytissuedevelopment,aswellasdiseaseprogression. Theneedforfaithfulmodelsofbreastcancerhasbeenaddressedusingdifferentsystems, allofthemhelpfultoevaluatetheresponseoftumorstopotentialtherapeuticagentsandto contribute to identify the mechanisms of resistance to existing therapies. Dobrolecki and Lewisdescribethewell-establishedpatient-derivedxenograft(PDX)model,whichinvolves the orthotopic transplantation of breast cancer tissue (as well as normal or preneoplastic tissue)intothemammaryfatpadofimmunocompromisedmice.Averypracticalalternative invivoPDXmodelisthechickeneggchorioallantoicmembrane(CAM),whichispresented by Villanueva and Sikora, highlighting its potential as an efficient, scalable precision medi- cineplatform.Anotherrelevantpreclinicalmodelisthemammaryintraductalxenografting technique, which has been established to inject cells or other substances directly into the mammaryductsoffemalemice,presentedbyHowardandcolleagues.Thisrefinedmethod provides the possibility of mimicking the normal microenvironment of preinvasive breast lesions to study breast cancer progression in a more physiologically relevant manner than withotherxenograftingmethods.Iggotakesthismethodologyastepfurtherbydescribing the creation of breast cancer models by transducing tumor cells and normal mammary epithelial cells with lentiviral vectors in organoid culture and injecting them into the mammary ducts of mice. Sumbal and Koledova continue taking advantage of organoid cultures, which have become vastly popular in developmental and cancer biology, and exploiting their potential, by presenting protocols for visualization of their architecture andcompositioninhighresolutioninthreedimensions(3D). ThecomplexityofthehumanbreasttissueisdissectedbyArendt,whopresentsmethods toisolateepithelialcells,stromalcells,adipocytes,andmacrophagestoinvestigatehowrisk factors impact distinct cell populations within breast tissue. Koledova and colleagues focus on the epithelial-stromal interactions, by providing a protocol for an organotypic human breast assay to facilitate research in the roles of human breast fibroblasts in epithelial morphogenesis and early tumorigenesis. All modelshave limitations, such aslow takerate, longduration,lackofimmunesystemoranappropriatestroma,amongothers,andsomeof those can be addressed using patient-derived explants, which preserve the native tissue architectureandmicroenvironment,asdescribedbyVivancoandhergroup.Thepossibility ofmonitoringdiseaseprogressionandresponsestotherapythrougharelativelynon-invasive blood-based biopsy is explored by Maheswaran and colleagues. They describe a versatile negative-selection protocol for enrichment of rare circulating tumor cells (CTCs) through removal of blood components including red blood cells, platelets, and white blood cells, whichalsoincludesthepossibilityofexvivocultureofpatient-derivedCTCs.Thebiochem- ical signaling pathways related to cell structure, proliferation, differentiation, motility, and cell fate are functionally linked to the mechanical properties of cells, and in Chapter 19, Toca-Herreraandhiscolleaguesdescribehowtoperformmechanicalexperimentsonbreast cancercellsusingtheatomicforcemicroscope.Finally,Schneider,inthelastchapter,pullsall Preface vii the knowledge gained through these effective methodologies to revise the view of the clinician about the promise held by mammary stem cell research and the future impact in theclinic. Revising the classic methods and identifying the excellent new developments has been anexcitingjourney.Ihopethatthereader willfindtheseprotocolsinspiringandhelpfulto navigatetheintricatebehaviorofmammarystemcellsandtogainfurtherknowledgetotake uscloser tothedesignofinnovativestrategiestopreventandtreatbreastcancer. Derio,Spain MariadM.Vivanco Contents Preface ..................................................................... v Contributors................................................................. xi 1 ProtocolforStudyingEmbryonicMammaryGlandBranching MorphogenesisExVivo ................................................. 1 QiangLan,JyotiSatta,Satu-MarjaMyllyma€ki,EwelinaTrela, RiittaLindstr¨om,BeataKaczyn´ska,JohannaEnglund, andMarjaL.Mikkola 2 MultidimensionalFluorescenceImagingofEmbryonicandPostnatal MammaryGlandDevelopment........................................... 19 ClaudiaCaraban˜aandBethanLloyd-Lewis 3 Single-CellTranscriptomicandEpigeneticAnalysesofMouseMammary DevelopmentStartingwiththeEmbryo ................................... 49 ZhiboMa,NikkiK.Lytle,CynthiaRamos,RaziaF.Naeem, andGeoffrey M.Wahl 4 ComputationalMethodstoIdentifyCell-FateDeterminants,Identity TranscriptionFactors,andNiche-InducedSignalingPathways forStemCellResearch................................................... 83 MuhammadAli,MarianaMessiasRibeiro, andAntoniodelSol 5 DifferentialProteomicAnalysisofComplexMixtures byLabel-FreenLCMS/MS.............................................. 111 IraideEscobe´s,MikelAzkargorta,IbonIloro,andFelixElortza 6 OrthotopicTransplantationofMouseMammaryEpithelialCells ............. 123 MarisaM.Faraldo,MarinaA.Glukhova, andMarie-AngeDeugnier 7 LineageTracingMethodstoStudyMammaryEpithelialHierarchies InVivo ................................................................ 141 Ver(cid:2)onicaRodillaandSilviaFre 8 LentiviralTransductionofMammaryEpithelialCells........................ 159 RichardIggo 9 ModificationofSingleCellsWithinMouseMammaryGlandDerived AciniviaViralTransduction.............................................. 185 LuciaGarciadelValle,MartaG.Montero,andMartinJechlinger 10 SurgicalProcedureforImplantationofHumanTumorTissue intotheEpithelium-FreeMammaryFatPadofImmunocompromised MicetoGeneratePatient-DerivedXenografts(PDX)........................ 195 LaceyE.DobroleckiandMichaelT.Lewis 11 TheChickenEmbryoChorioallantoicMembrane(CAM):AVersatileTool for theStudyofPatient-DerivedXenografts................................ 209 HugoVillanuevaandAndrewG.Sikora 12 IntraductalInjectionsintotheMouseMammaryGland ..................... 221 ErikOliemuller,RichardNewman,andBeatriceA.Howard ix x Contents 13 ModelingBreastCancerinOrganoidandIntraductalModels................ 235 RichardIggo 14 SingleOrganoidsDroplet-BasedStainingMethodforHigh-End 3DImagingofMammaryOrganoids...................................... 259 JakubSumbalandZuzanaKoledova 15 DivideandConquer:IsolatingCellPopulationstoInvestigateHow BreastCancerRiskFactorsAlter theBreastMicroenvironment............... 271 LisaM.Arendt 16 AnOrganotypicAssaytoStudyEpithelial-FibroblastInteractions inHumanBreast........................................................ 283 JakubSumbal,ThorarinnGudjonsson,GunnhildurAstaTraustadottir, andZuzanaKoledova 17 Patient-DerivedExplantCulturesofNormalandTumorHuman BreastTissue........................................................... 301 IsabelGris-Ca´rdenas,MiriamRa´bano,andMariadM.Vivanco 18 Negative-SelectionEnrichmentofCirculatingTumorCellsfromPeripheral BloodUsingtheMicrofluidicCTC-iChip.................................. 309 RisaBurr,JonF.Edd,BrianChirn,AvanishMishra, DanielA.Haber,MehmetToner,andShyamalaMaheswaran 19 MeasuringMechanicalPropertiesofBreastCancerCellswithAtomic ForceMicroscopy....................................................... 323 BarbaraZbiral,AndreasWeber,andJose´L.Toca-Herrera 20 BreastCancerStemCells:AClinician’sView............................... 345 JoseSchneiderandFranciscoJ.Vizoso Index ...................................................................... 351
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