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Lung Cancer: Methods and Protocols PDF

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Methods in Molecular Biology 2279 Pedro G. Santiago-Cardona Editor Lung Cancer 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. Lung Cancer Methods and Protocols Edited by Pedro G. Santiago-Cardona Biochemistry and Cancer Biology Divisions, Ponce Health Sciences University-Ponce Research Institute, Ponce, Puerto Rico Editor PedroG.Santiago-Cardona BiochemistryandCancerBiology Divisions PonceHealthSciencesUniversity-Ponce ResearchInstitute Ponce,PuertoRico ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-1277-4 ISBN978-1-0716-1278-1 (eBook) https://doi.org/10.1007/978-1-0716-1278-1 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2021 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, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. CoverIllustrationCaption:Formoreinformation,seeFigure3fromChapter4 ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface Lung cancer is characterized by an aggressive nature and a poor patient survival. This is in spite oftherecentsignificant advancesin theunderstandingofthe genetics,histology, and molecularandcellularbiologyofthedisease,aswellasitsclinicalaspects.Theseknowledge gains have resulted in improved detection, diagnosis, and patient treatment. Treatment in particularhasevolvedtoincludetargetedtherapiesbasedonparticular tumorgeneticsand molecular biology. Yet, in spite of this progress, the prognosis for lung cancer patients remains relatively poor, with still low 5-year survival rates when compared to other cancer types.Therefore,researchaimedatfurtheringourunderstandingofthisfataldiseaseismore thanwarranted,atthebasic,translational,andclinicallevels.Together,thechaptersofthis bookhavetheoverarchinggoaltoserveasalaboratorymanualthatcontainsprotocolsand in-depthdiscussionforcommonlyusedexperimentalapproachesforthecharacterizationof severalaspectsoflungtumorbiology. In the handling of many lung cancers cases, information about diagnosis, tumor grading, staging, and histological sub-classification is obtained from the analyses of tumor biopsy or resection specimens. Characterization and detection of clinically informative biomarkers in such biological specimens is thus a priority for lung cancer management. Analysisofbiomarkersinlungtumortissuesguidesclinicalinterventionsandhelpstoassess thehistologicaloriginofthetumor,probabilityofresponsetotargetedtherapy,metastatic potential, probability of disease recurrence, and probability of acquiring resistance to therapy. Along these lines, Chapters 1–3 of this book describe the protocols for the immunohistochemistry (IHC) detection of TTF-1, P40, and NAPSIN-A, respectively. These three protein biomarkers have proven extremely useful in sub-classifying non–small cell lung carcinomas (NSCLC) into adenocarcinomas or squamous cell carcinomas (SCC) whendetectedbyIHCinlungtumorbiopsysamples.Anotherclinicallyvaluablebiomarker is PD-L1. Assessing the expression of PD-L1 in tumoral tissue may help clinicians to determine which patients can benefit from immunotherapy using immune checkpoint inhibitors. Lung cancer patients with strong tumoral PD-L1 expression may show a better response to immunotherapy, and therefore, assessing PD-L1 expression in tumor biopsies byIHCcanhelpclinicianstostratifypatientsbasedonthelikelihoodoffavorableoutcomes from immunotherapy. Chapter 4 describes a detailed protocol for the IHC detection of PD-L1expressioninclinicalsamples,whileChapter5describesaprotocolthatcanbeused tovalidatethespecificityofanti-PD-L1antibodiesbyimmunoblotanalysis,avalidationthat isextremelyimportanttoassesswhetheraparticularPD-L1antibodyissuitableforclinical applications. These chapters dealing with immunological detection of clinically relevant biomarkersarefollowedbytwochaptersdealingwiththeoptimizationaspectsofimmuno- logical detection of antigens. Chapter 6 describes the optimization of the immunohisto- chemistry procedure using lung cancer cell line–derived tridimensional spheroids with a tumor-like tissue architecture. Using these spheroids for protocol optimization purposes will avoid the use of valuable human lung tumor tissue samples in the optimization stage. Some clinically informative biomarkers are phosphorylation of specific proteins, and the immunologic detection of these phospho-proteins presents the additional challenge of v vi Preface ensuring that the used antibody is able to detect specifically the phosphorylated version of the protein. Chapter7 describesa methodfor theimmunoblot validation of thephospho- specificityofantibodiesusinglungcancercelllines. Chapters8–12aredevotedtothetopicofthegeneticandmolecularcharacterizationof lungcancerbiologicalsamples.Thisisacentraltopicinlungcancerbiologydue,first,tothe variety of mutated alleles that have been found to have a strong oncogenic driver effect in lungcancerand,second,totheimportanceoftumorgeneticsindeterminingmanyaspects of tumor biology, as well as many aspects of the clinical interventions and management of lung cancer. Aspects such as response to targeted therapy, acquisition of resistance to anti- cancer treatments,anddiseaseprognosiscanbestronglyinfluencedbytumorgeneticsand molecular biology. The experimental approaches in this group of chapters include the detectionofoncogenicgenefusions,splicevariants,andabnormalgeneexpressionprofiles using NanoString technology (Chapter 8), protocols for PCR-based approaches for the detection of mutations in EGFR, KRAS, and BRAS genes (Chapters 9 and 10), a PCR-basedapproachtodetectMETexonskipping(Chapter11),andanimmunocytochem- ical approach for detecting ALK and ROS1 rearrangements in lung cancer cytological samples(Chapter12). The following group of chapters of this book switch their focus to protocols for the generation of research tools and preclinical lung cancer models that can be extremely valuable to achieve a better understanding of lung tumor biology. Chapter 13 describes a procedure for the generation of patient-derived xenografts by implanting human lung tumor tissue into immunodeficient mice. This mouse-humanized xenograft model can prove extremely valuable as a preclinical model to study various aspects of lung tumor biology.Chapter14describesthegenerationofcarcinogen-inducedmousecelllinesmim- ickingtraitsoflungadenocarcinomas.Beingderivedfrommiceexposedtotobaccosmoke carcinogens,thesecelllinesareextremelyusefulsincetheiroriginrecapitulatestheetiology ofthehumandisease.Manyaspectsoftheaggressivenatureoflungcancer,includingrelapse andresistanceagainsttherapy,havebeenattributedtothepresenceoftumoralcancerstem cells. Chapter 15 describes a procedure for the in vitro enrichment of mouse lung cancer stem cells, together with a protocol for their characterization using a whole transcriptome analysis.Chapter16describesaninvivoimagingproceduretomonitor tumorgrowthand progression in an orthotopic lung cancer model in mice. This chapter demonstrates the power of such a model to monitor tumor response to chemotherapy. The disruption of apoptotic pathways is one among the many traits associated with the cancer state, and understandinghowthisbreakdownoccursincancercellsisstillthetopicofintenseresearch. Chapter 17 describes an annexin V/propidium iodide–based staining protocol to assess apoptosis in lung cancer cells. It is generally accepted that aldehydes and carcinogens in tobaccosmokehavethecapacitytoreactwithDNAbases,creatingmutagenicadducts.Such mutagenic adductsplay animportant etiologicalroleinlungcancerand canbeconsidered biomarkers for aldehyde exposure. Chapter 18 describes a high-performance liquid chromatography-tandem mass spectrometry–based protocol to detect such DNA adducts withspecificityandsensitivity.Lastbutnotleast,thebookcloseswithachapteraddressing theveryimportanttopicoftheeffectivityofanti-cancerdrugdelivery.Chapter19describes amethodforcisplatinencapsulationinliposomes,withtheaimofincreasingdrugdelivery whilereducingtoxicity. Takentogether,wehopethechaptersofthisbookgivethereaderaglobalperspectiveof theresearcheffortsrelatedtolungcancer,whileallowingthemtoexperimentallyprobethe different aspects of lung cancer research in their laboratories, including the experimentally Preface vii relevant tests used in the establishment of the diagnosis and prognosis of lung cancer. It is hopedthatthebookservesasaguidetoassistthemolecularcancerbiologistsintheirsearch for theunderstandingofthemolecularaspectsofthisdisease. Ponce,PuertoRico PedroG.Santiago-Cardona Contents Preface ..................................................................... v Contributors................................................................. xi 1 AutomatedTTF-1ImmunohistochemistryAssayfor the DifferentiationofLungAdenocarcinomaVersusLung SquamousCellCarcinoma ............................................... 1 RosaVe´lezCintr(cid:1)on,Andre´sJ.Martı´nez,JoAnnJusino, Marı´aConte-Miller,andAdalbertoMendoza 2 ImmunohistochemicalDetectionofp40ExpressioninLung CancerClinicalSamples.................................................. 13 ArunaNambirajanandDeepaliJain 3 AutomatedImmunohistochemistryAssayfor theDetectionofNapsin-A inFormalin-FixedParaffin-EmbeddedTissuesfromLungTumors............ 23 RosaVe´lezCintr(cid:1)on,JoAnnJusino,Marı´aConte-Miller, Andre´sJ.Martı´nez,andAdalbertoMendoza 4 DetectionofProgrammedCellDeathLigand1ExpressioninLung CancerClinicalSamplesbyanAutomatedImmunohistochemistrySystem..... 35 EdwinRogerParraandShariaHerna´ndezRuiz 5 WesternBlotasaSupportTechniqueforImmunohistochemistry toDetectProgrammedCellDeathLigand1Expression..................... 49 EdwinRogerParraandShariaHerna´ndezRuiz 6 CreationofFormalin-Fixed,Paraffin-Embedded3DLungCancer CellularSpheroidsfor theOptimizationofImmunohistochemistry StainingProcedures..................................................... 59 JenniferCaba´n-Rivera,CamilleChard(cid:1)on-Col(cid:1)on, AlbertoPedraza-Torres,YoanE.Rodrı´guez, RaymondQuin˜ones-Alvarado, andPedroG.Santiago-Cardona 7 ImmunoblotValidationofPhospho-SpecificAntibodiesUsing LungCancerCellLines.................................................. 75 WilfredoM.Pedreira-Garcı´a,JaileenePe´rez-Morales, CamilleChard(cid:1)on-Col(cid:1)on,JenniferCaba´n-Rivera, andPedroG.Santiago-Cardona 8 DetectionofNon-SmallLungCellCarcinoma-Associated GeneticAlterationsUsingaNanoStringGeneExpression PlatformApproach...................................................... 91 JohanStaaf,MatsJ¨onsson,andAnnaF.Karlsson 9 APCR-BasedApproachforDriverMutationAnalysisof EGFR,KRAS,andBRAFGenesinLungCancerTissueSections............. 109 RodrigodeOliveiraCavagna,LeticiaFerroLeal, Fla´viaEscremimdePaula,GustavoNorizBernardinelli, andRuiManuelReis ix x Contents 10 6-ColorCrystalDigitalPCRTMfor theHigh-PlexDetection ofEGFRMutationsinNon-SmallCellLungCancer........................ 127 JordanMadic,Ce´cileJovelet,ImaneDehri,andAllisonC.Mallory 11 DetectionofMETExon14SkippingAlterationsinLung CancerClinicalSamplesUsingaPCR-BasedApproach...................... 145 JaneS.Y.Sui,StephenP.Finn,andStevenG.Gray 12 ImmunocytochemicalDetectionofALKandROS1Rearrangements inLungCancerCytologicalSamples ...................................... 157 DianeFrankel,EliseKaspi,andPatriceRoll 13 AMethodfor theEstablishmentofHumanLungAdenocarcinoma Patient-DerivedXenograftsinMice....................................... 165 JoanneLundy,BrendanJ.Jenkins,andMohamedI.Saad 14 AMethodfor theEstablishmentandCharacterizationofMouse LungAdenocarcinomaCellLinesthatMimicTraitsofHuman Adenocarcinomas....................................................... 175 MagdaSpella,IoannisLilis,andGeorgiosT.Stathopoulos 15 WholeTranscriptomeSequencingAnalysisofCancerStem/ ProgenitorCellsObtainedfromMouseLungAdenocarcinomas.............. 187 AnsamSinjab,ReemDaouk,WassimAbou-Kheir, andHumamKadara 16 InVivoImagingofOrthotopicLungCancerModelsinMice................ 199 PengLiu,LiweiZhao,LauraSenovilla,OliverKepp, andGuidoKroemer 17 AnAnnexinV-FITC—PropidiumIodide-BasedMethodfor DetectingApoptosisinaNon-SmallCellLungCancerCellLine............. 213 RobinKumar,AnkitSaneja,andAmulyaK.Panda 18 DetectionofDNAAdductFormationinRatLungsbya Micro-HPLC/MS/MSApproach......................................... 225 Ange´licaB.Sanchez,CamilaC.M.Garcia,PaoloDiMascio, andMarisaH.G.Medeiros 19 AMethodforLiposomeCo-encapsulationofPhenethylIsothiocyanate andCisplatinandDeterminingItsToxicityAgainstLungand LungCancerCells ...................................................... 241 MengweiSunandAnthonyJ.DiPasqua Index ...................................................................... 255

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