Lung Cancer: Methods and Protocols

261 Pages·2021·7.22 MB·English

by  Pedro G. Santiago-Cardona

Methods in
Molecular Biology 2279
Pedro G. Santiago-Cardona Editor
Lung Cancer
Methods and Protocols
M M B
ETHODS IN OLECULAR IO LO GY
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University ofHertfordshire
Hatfield, Hertfordshire, UK
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For over 35 years, biological scientists have come to rely on the research protocols and
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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
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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