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Methods in Molecular Biology 2054 Jyotsna Batra Srilakshmi Srinivasan Editors Theranostics Methods and Protocols 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. Theranostics Methods and Protocols Edited by Jyotsna Batra and Srilakshmi Srinivasan School of Biomedical Sciences, Institute of Health and Biomedical Innovation (IHBI), Translational Research Institute, Queensland University of Technology (QUT), Brisbane, QLD, Australia Editors JyotsnaBatra SrilakshmiSrinivasan SchoolofBiomedicalSciences SchoolofBiomedicalSciences InstituteofHealthandBiomedical InstituteofHealthandBiomedical Innovation(IHBI),Translational Innovation(IHBI),Translational ResearchInstitute ResearchInstitute QueenslandUniversity QueenslandUniversity ofTechnology(QUT) ofTechnology(QUT) Brisbane,QLD,Australia Brisbane,QLD,Australia ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-4939-9768-8 ISBN978-1-4939-9769-5 (eBook) https://doi.org/10.1007/978-1-4939-9769-5 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2019 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeenmade. Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:233SpringStreet,NewYork,NY10013,U.S.A. Preface Theterm “theranostics”implies toa combinationoftherapyand diagnosticsand hasbeen used by scientific community in a variety of contexts. Over the past decade, the field of theranosticshasadvancedconsiderablywithrespecttoadvancesinbiomarkeridentification, newmolecularimagingprobes,geneticsandhigh-throughputtechniques,imaging-guided molecular therapy, and new nanotheranostics. The field is appropriate to explain the advancementinsciencetoestablishaccurateandpersonalizedtherapiesfor variousdiseases andtocombinediagnosticandtherapeuticapplicationsintoasingleagentfor thedevelop- mentofapromisingparadigminvolvingdiagnosis,drugdelivery,andmonitoringofdisease responsetotreatment. Newclinicians,researchers,andstudentsworkinginthismultidisciplinaryfieldoftenask howtoobtainwell-optimizedandwell-detailedprotocolstoachievetheirresearchoutcome inthisfield.Thisbookisdesignedspecificallytomeetthatdemandandispublishedintime to meet theneeds ofmedical researchersin acomprehensive mannerencompassingbioen- gineering,diagnostics,invivoimaginganditsuseforimage-guidedtherapy,andavarietyof othermiscellaneoussubjects.Toaccomplishthisdauntingtask,wehadthegoodfortuneto recruitnearly52leadersinthefieldworldwidecontributingtothe18chapters. Given the multidisciplinary nature of the field, the book is broken into five different sections. PartI(Bioengineering)consistsofthreechapters(Chapters1–3).Manytheranostic approacheshasbeenlimitedduetothelackofrepresentativelaboratorymodelsthatmimic thebiologicalprocessesoccurringinvivoandtherestrictiveaccessandlimitedliveimaging capabilitiesthatinvivomodelsprovide.Syntheticscaffoldmaterialsallowtoengineerbone- likemicroenvironmentsderivedfromprimaryhumancells,whichcanfurtherbeco-cultured with metastatic cell lines. Chapter 1 reviews the several easy-to-follow methods for the characterizationofcellsgrowninhydrogelsandalsodescribesanenzymaticapproachforan improved cell recovery for subsequent analysis. Chapter 2 reviews the application of melt electrowriting technology (MEW) to provide 3D microfiber scaffolds suitable for this purpose. Using primary human cells, MEW scaffolds support the reproducible formation of human bone-like 3D microenvironments. Chapter 3 describes imaging techniques to obtain multidimensional real-time data of cancer cells in the microenvironment context. Thisnovelpreclinicalplatformwillcontributetothebetter understandingofthenatureof thedynamicinteractionsbetweenboneandcancer,aswellasservetomeasuretheefficiency ofanticancerdrugs. Part II (Molecular Diagnostics) consists of Chapters 4–9. The current diagnostic methods,suchasbiopsysampling,tumortissuestaining,andimagingtechniques,whichare invasive,painful,time-consuming,andexpensiverequireskilledpersonnel.Therefore,there isanunmetclinicalneedtodevelopnoninvasive,easy-to-usetoolstoidentifypatientsbefore clinical manifestation. Furthermore, the quality of the samples for analysis remains an undeniable challenge which was also discussed in these chapters. Chapter 4 describes exosome-based therapeutics that represent a most promising next-generation approach and are considered valid diagnostic biomarkers and potential therapeutic tools. Exosomes are widely disseminated, heterogenous entities, and the isolation of large and high-quality exosomes is daunting. In this chapter, we reveal the protocol and key insights into the v vi Preface isolation, purification,and characterization of exosomes usingultracentrifugation method. MicroRNAs(miRNAs)aresmallendogenousnoncodingRNAmoleculeswhicharepower- ful regulators of the different cellular processes involved in the pathogenesis of various diseases. The use of miRNA-based therapies has been proposed with the onset of early- phaseclinicaltrialstoassessthetherapeuticefficacyofmiRNAs.Thestratificationofpatients based ondifferential expression ofmiRNAs and thetherapeutictargeting ofsuchmiRNAs wouldenablepatient-specifictailoredintervention.Chapter5describesexperimentalmeth- odologyforthedetectionofmiRNAsinplasmasamplesbyRT-qPCR.Chapter6describesa robust and cost-effective protocol to isolate and enrich miRNAs from saliva samples. Similarly,circulatingtumorcells(CTCs)haveshownpromisingpotentialasliquidbiopsies thatfacilitateearlydetection,prognosis,therapeutictargetselection,andmonitoringtreat- ment response. Chapter 7 describes a CTC isolation and analysis method by Tianyu Guo et al. for cancer detection and therapeutic response monitoring. Chapter 8 explains the isolationofCTCsusingthespiralmicrofluidictechnologyfor theefficientsortingofCTCs frompatientbloodsamplesfortargetedtherapy.BoththesetwochaptersarebasedonCTC isolation methods using different technologies. While Chapter 7 is a marker-independent CTCenrichmentmethodbasedonthecellsizeanddeformabilityandtheimmunofluores- cencestainingmethodtodetectCTCs,Chapter8detailsthespiralmicrofluidictechnology which is also a marker-independent technique but utilizes hydrodynamic forces for size- basedefficientsortingofCTCsfrompatientbloodsamples. Pharmacogenetics involves the search for genetic variations that lead to interindividual differencesindrugresponse.Thisgenotypingapproachhavepavedthewaytonewoppor- tunities to deliver a better quality of care through more precise characterization of the individual’s genetic makeup that will, in turn, contribute to the interindividual variations in drugresponse. Chapter 9 provides perspectives from theidentification ofsuch causative geneticvariations—toillustratehowtoanalyzetherawdataobtainedbydifferentsequenc- ing techniques while indicating the potential challenges that may arise at each step. Chapter 10 explains the use of automatic Idylla™ system for the analysis of specific muta- tionsincancerpatients. PartIII(MolecularImaging)consistsofChapters11–13.Numerousadvanceshave been made in recent years in exogenous probes and nanoparticles that allow precise and specific imaging in situ as well as label-free clinical imaging approaches. Chapter 11 sum- marizesasimplemethodofgeneratingfluorescentprobesusingbacterialartificialchromo- somes employed in in situ genetic analysis of cells in response to treatment. Chapter 12 focusesonthelabel-freedigitallivecellimagingtechniqueandhasexpandedfromitsusein the laboratory to the clinical setting, and currently, it is being developed for use in theranostics. Chapter 13 provides a concise review and process of utilizing porous silicon nanoparticlesforanimprovedpermeabilityintothetargettissuesandforprecisedeeptissue imaginganddiagnostics. PartIV(Imaging-GuidedTherapy)is composedofChapters 14–18. Thechapters in this section reveal how advanced imaging techniques now make highly precise clinical interventionpossible,furtherprovidingaccurateandefficientdeliveryofdrugtothetarget deliverybycrossingthebarrierandescapeclearancethroughthereticuloendothelialsystem to ensure sustained presence at effective concentrations. Nanoparticle-based drug delivery systemsareemergingasapromisingdrugdeliveryplatform,duetotheirdistinctadvantages leading to various biological actions in the body. Chapter 14 reviews generalized methods for the use of aptamers in diagnostics using optical and electrochemical platforms of detection and in delivering drug to the cancer cells and details a method for the in vitro Preface vii selection of DNA aptamers against a protein target by SELEX, a combinatorial single- stranded oligonucleotide synthesis technique that specifically bind to a target ligand. Chapter 15 highlights how aptamers can be utilized that has the potential to enhance the precision of molecular medicine and targeted therapeutics. Chapter 16 reviews a simple method of generating fluorescence in situ hybridization probes using bacterial artificial chromosomes that have unlimited possibilities for the analysis of any genomic regions. Chapter 17 explores the method for developing a library of nanoparticles for the efficient delivery of therapeutic agents across the blood-brain barrier. Chapter 18 introduces the biomimeticsyntheticstrategyandproceduresforpreparinggeneticallyengineerednanove- siclesfordiseasecell-specifictargeting. It is impossible to describe all the areas that encompass theranostics or will have an impactonthescienceandpracticeofpersonalizedmedicine.Ouraimistoofferareasonable solutiontowardsdiseasediagnosisandtherapybygivingthereadersasenseofnewmethods andchallengesassociatedwithdevelopingtheranostics.Ithasbeenagreatpleasureworking withtheauthorsofthisbook.Withouttheirenthusiasm,encouragement,andprofessional deliveryoftheircontributionsinatimelymanner,itwouldnothavebeenpossibletomake thisbookareality.Wehopethatthisbookmayformafoundationforfurtheradvancesinthe fieldoftheranostics. Brisbane,QLD,Australia JyotsnaBatra Woolloongabba,QLD,Australia SrilakshmiSrinivasan Contents Preface ..................................................................... v Contributors................................................................. xi PART I BIOENGINEERING 1 CellRecoveryofHydrogel-EncapsulatedCellsforMolecularAnalysis......... 3 EleonoraPeerani,JulianaB.Candido,andDanielaLoessner 2 BioengineeredMicrotissueModelsoftheHumanBoneMetastatic Microenvironment:ANovelInVitroTheranosticsPlatform forCancerResearch..................................................... 23 NathalieBock 3 Real-Timeand3DQuantificationofCancerCellDynamics: ExploitingaBioengineeredHumanBoneMetastaticMicrotissue............. 59 NathalieBockandJoanRöhl PART II MOLECULAR DIAGNOSTICS 4 ExosomesExtractionandIdentification.................................... 81 XiaoxinWu,SalahAliA.Showiheen,AntoniaRujiaSun, RossCrawford,YinXiao,XinzhanMao,andIndiraPrasadam 5 ProfilingMicroRNAMarkersinPlasma:LookingintoBetter ApproachesandRecommendations ....................................... 93 FarhanaMatinandJyotsnaBatra 6 IsolationandQuantificationofMicroRNAsfromHumanSaliva.............. 105 SriRamArunachalam,KaiDunTang,andChamindiePunyadeera 7 TheIsolationandAnalysisofCirculatingTumorCells....................... 115 TianyuGuo,ElzbietaStankiewicz,XueyingMao,andYong-JieLu 8 TheIsolationandCharacterizationofCirculatingTumor CellsfromHeadandNeckCancerPatientBloodSamples UsingSpiralMicrofluidicTechnology ..................................... 129 AruthaKulasinghe,MajidEbrahimiWarkiani, andChamindiePunyadeera 9 Pharmacogenetics:RoleofSingleNucleotidePolymorphisms................ 137 EmrahYucesanandNurOzten 10 EGFRMutationAnalysisinNon-smallCellLungCarcinoma fromTissueSamplesUsingtheFullyAutomatedIdylla™qPCRSystem....... 147 SimonHeekeandPaulHofman ix x Contents PART III MOLECULAR IMAGING 11 SubcellularLocalizationofMicroRNAsbyMicroRNAInSitu Hybridization(miR-ISH)................................................ 159 HarleyRoseRobinson,MichelleMeiChihHill, andAlexandreSantosCristino 12 DigitalHolographicImagingasaMethodforQuantitative, LiveCellImagingofDrugResponsetoNovelTargetedCancerTherapies..... 171 LauraV.Croft,JaimieA.Mulders,DerekJ.Richard, andKennethO’Byrne 13 LuminescentPorousSiliconNanoparticlesforContinuous WaveandTime-GatedPhotoluminescenceImaging......................... 185 TusharKumeria,ZhiQu,AmiraliPopat,TariqAltalhi, andAbelSantos PART IV IMAGE-GUIDED THERAPY 14 NucleicAcidAptamersasEmergingToolsforDiagnostics andTheranostics........................................................ 201 RuchiMutreja,ShahnawazAhmadBaba,andNaveenKumarNavani 15 AptamerSelectionforDetectingMolecularTargetUsing Cell-SELEX(SystematicEvolutionofLigandsbyExponential Enrichment)Technology ................................................ 223 KimberlyD.Stewart,WeihongTan,andJongY.Park 16 FluorescenceInSituHybridizationandRehybridization UsingBacterialArtificialChromosomeProbes ............................. 243 ElzbietaStankiewicz,TianyuGuo,XueyingMao,andYong-JieLu 17 UpconversionNanoparticle-BasedStrategyforCrossingtheBlood-Brain Barrier toTreattheCentralNervousSystemDisease........................ 263 LibingFu,RogerChung,andBingyangShi 18 GeneticallyEngineeredPlasmaMembraneNanovesicles forCancer-TargetedNanotheranostics .................................... 283 PengfeiZhang,HuChen,JingyiLiu,andGangLiu Index ...................................................................... 295