Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo

EuropeanJournalofCellBiology91 (2012) 930–937

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EuropeanJournalofCell

Biology

journalhomepage:www.elsevier.com/locate/ejcb

Docancercellshavedistinctadhesionsin3Dcollagenmatricesandinvivo?

SaraGeraldoa,b,AnthonySimona,b,NadiaElkhatiba,b,DanielLouvarda,b,

LucFetlera,c,DanijelaM.Vignjevica,b,∗

aInstitutCurie,Paris75005,FrancebUMR144/CNRS,FrancecUMR168/CNRS,France

articleinfo

Articlehistory:Received9March2012Receivedinrevisedform24July2012Accepted25July2012

Keywords:Focaladhesions3DmatrixVinculinCollagenIntravitalimagingabstract

Duringmetastasis,cancercellsbreachthebasementmembraneandmigratethroughthestromamostlycomposedofanetworkofcollagenIfibers.Cellmigrationon2Disinitiatedbyprotrusionofthecellmembranefollowedbyformationofadhesionsthatlinktheactincytoskeletontotheextracellularmatrix(ECM).Cellsthenmoveforwardsbyexertingtractionforcesontheadhesionsatitsfrontandbydisassem-blingadhesionsattherear.In2D,onlytheventralsurfaceofamigratingcellisincontactwiththeECM,wherecell–matrixadhesionsareassembled.In3Dmatrices,eventhoughthewholesurfaceofamigratingcellisavailableforinteractingwiththeECM,itisunclearwhetherdiscreteadhesionstructuresactuallyexist.Usinghigh-resolutionconfocalmicroscopyweimagedtheendogenousadhesomeproteinsinthreedifferentcancercelltypesembeddedinnon-pepsinizedcollagentypeI,polymerizedataslowrate,toallowtheformationofanetworkthatresemblestheorganizationofEMCobservedinvivo.Vinculinaggregatesweredetectedinthecellularprotrusions,frequentlycolocalizingwithcollagenfibers,imply-ingtheycorrespondtoadhesionstructuresin3D.Asthedistancefromthesubstratebottomincreases,adhesionaggregatesbecomesmallerandalmostundetectableinsomecelllines.Usingintravitalimagingweshowhere,forthefirsttime,theexistenceofadhesomeproteinsaggregatesinvivo.Theseaggregatessharesimilaritieswiththeonesfoundin3Dcollagenmatrices.Itstillremainstobedeterminedifadhe-sionsassembledin3Dandinvivosharefunctionalsimilaritiestothewell-describedadhesionsin2D.Thiswillprovideamajorstepforwardinunderstandingcellmigrationinmorephysiologicalenvironments.© 2012 Elsevier GmbH. All rights reserved.

Introduction

Inordertoescapetheprimarytumorandreachthebloodves-

sels,cancercellsencounterdifferentECMsduringtheircourseof

invasivemigration.Firsttheybreachthebasementmembrane,a

thinanddensesheet-likestructurecomposedofanetworkofcol-

lagenIVandlaminin(Yurchenco,2011).Thentheymigratethrough

thestromacomposedoffibrillarcollagens,proteoglycansandvar-

iousglycoproteins(Boot-HandfordandTuckwell,2003;Egeblad

etal.,2010;Nabaetal.,2012).

Cellmigrationisacontinuousprocess,butforsimplicity,itis

oftendescribedinfourindividualsteps.First,actinpolymerization

drivesprotrusionsatthecellleadingedge;second,thosenewly

extendedprotrusionsbecomestabilizedbyadheringtotheextra-

cellularmatrix(ECM);third,thecellmovesforwardbyexerting

∗Correspondingauthorat:EquipedeMorphogeneseetSignalisationcellulaires,UMR144CNRS/InstitutCurie,InstitutCurie,25rued’Ulm,75248Pariscedex05,France.Tel.:+330142346366;fax:+330142346377.E-mailaddress:danijela.vignjevic@curie.fr(D.M.Vignjevic).tractionforcesattheadhesionpoints;andfourth,adhesionsatthe

cellreararereleasedenablingtranslocationofthecellbody.

However,mostnormalorcancercellsmigratingintwo-

dimensional(2D)andthree-dimensional(3D)substratesdifferin

theirmorphologyandmodesofmigration(Petrieetal.,2009;

Meyeretal.,2012).Cellsmigratingonplanar2Dsubstrates,either

plainorcoatedwithECMproteinssuchaslaminin,collagenIor

fibronectin,developbroadandflatprotrusionscalledlamellipo-

diaandfinger-likeprotrusionscalledfilopodia(PollardandBorisy,

2003;VignjevicandMontagnac,2008).Thesamecellsgrowing

in3Dmatricesaremoreelongatedandwhilecellularprotrusions

thatresemblelamellipodiaandfilopodiaarestillobserved,they

aresmallerinsizeandinfewernumbers(Wirtzetal.,2011).This

changeinmorphologyispartlyduetodifferencesindimensional-

ityof2Dand3Dsubstrates.Whilein2Donlytheventralsurfaceof

acellmigratingisincontactwiththeECM,in3Dthewholesurface

ofamigratingcelliscontactingECMproteins.Thosedifferencesin

cellmorphologyandcell–matrixinterfaceshaveprobablyamajor

roleindeterminingthemechanismandratesofcellsmigratingin

differentenvironments.

AdhesiontotheECMismostlyachievedbycellsurfacerecep-

torscalledintegrins.Intracellularly,integrinsareconnectedtothe

0171-9335/$–seefrontmatter© 2012 Elsevier GmbH. All rights reserved.http://dx.doi.org/10.1016/j.ejcb.2012.07.005S.Geraldoetal./EuropeanJournalofCellBiology91 (2012) 930–937931

contractileactomyosinbundlesviaanentirecollectionofproteins

namedintegrin“adhesome”thathavemechanosensoryandsig-

nalingfunctions(GeigerandYamada,2011).Efficientcellmigration

dependsonthecoordinationofchangesintheactomyosinintracel-

lularmachineryanddynamicsofadhesionswiththeextracellular

environment(Parsonsetal.,2010).

Cellsmigratingon2Dsubstratesassembleseveraltypesofadhe-

sions.Theydifferinsize,proteincomposition,dynamicsandtheir

locationinthecell(GeigerandYamada,2011).However,thereis

controversyinthefieldwhetherdiscreteadhesionstructuresactu-

allyexistincellsmigratingin3Dmatricesand,moreimportantly,

invivo.

Whilesomeofthereportsdescribedistinctadhesionstructures

containing“adhesome”proteinssuchasvinculin,paxillinandzyxin

incellsmigratingindifferent3Dmatrices,othersshowthatadhe-

someproteinsdonotformdistinctaggregatesbutarefairlydiffuse

inthecytoplasm(Cukiermanetal.,2001;TamarizandGrinnell,

2002;Lietal.,2003;PetrollandMa,2003;Wozniaketal.,2003;

MartinsandKolega,2006;Provenzanoetal.,2009;Fraleyetal.,

2010;DeakinandTurner,2011;Hakkinenetal.,2011;Kubowand

Horwitz,2011).Thosestudiesuseddifferentmatrices(cellderived

matricesorcollagenIofdifferentconcentrationsandmethods

ofextraction),andcellsthatwereeitherfullyembeddedin3D

matrixorjustseededontop(HarunagaandYamada,2011).Since

adetailedanalysisofthiscontroversyhasalreadybeenpublished

byHarunagaandYamada(2011),wewillonlyreferheretopos-

sibleexplanationsforthosediscrepancies.First,goingawayfrom

therigidglasscoverslips,theelasticityof3Dmatricesincreases,

whichcouldaffecttheassemblyofdistinctadhesions(Fraleyetal.,

2011;HarunagaandYamada,2011).Second,theorganizationof

the3Dmatricescaninfluencetheassemblyofadhesionstruc-

tures.In3Dcollagenmatricesmostlyconsistingofshortandthin

fibers,celladhesions,whichareinthemicrometerrange,cannot

physicallyassembleonthincollagenfibersthatarenanometers

indiameter.However,moreextensivecollagenbundlingwould

providesufficientwidthtoallowassemblyofadhesions.Third,

over-expressionoffluorescentlylabeledadhesomeproteinscould

obstructthevisualizationofdistinctaggregates.Indeed,distinct

adhesionsweredetectedwhenfluorescentlylabeledadhesome

proteinsareexpressedatlowlevels(KubowandHorwitz,2011).

Fourth,over-expressionofsomeadhesionproteins,suchaspax-

illin,canevenhaveaneffectonthecellmigrationstrategyin

whichadhesionsarenolongerdetected(DeakinandTurner,2011).

Finally,differencesinresolutionandsensitivityofthemicroscopy

systemsusedindifferentstudiescouldalsoprovideanexplanation

forthesediscrepancies.

Toaddressthiscontroversy,herewecombineimagingof

endogenousadhesomeproteinsinthick3Dinvitromatrices,

usinghigh-resolutionconfocalmicroscopy,withimagingoffluo-

rescentlylabeledadhesomeproteinsinxenograftsoflivinganimals

bytwo-photonlaser-scanningmicroscopy.Aggregatesofendoge-

nousproteinsweredetectableinthreedifferentcancercelltypes

fullyembeddedinmatricesmadeofcollagentypeIfibers.Intravital

imagingofxenograftedcancercellsexpressinglowlevelsofGFP-

PaxillinorGFP-Vinculinallowedforthefirsttimeobservationof

aggregatesofadhesomeproteinsinthelivingmouse.

Materialsandmethods

Antibodies

HumanantibodyagainsttubulinwasfromRecombinantPro-

teinandAntibodyPlatform(InstitutCurie).Mouseantibody

againstvinculinwasakindgiftfromM.Gloukhova.Mouseanti-

bodiesagainst␣-tubulin,paxillinandzyxinwerefromSigma,TransductionLaboratoriesandSynapticSystems,respectively.Cy2-

conjugatedsecondaryanti-humanantibodywasfromJackson.

Alexa-conjugatedsecondaryantibodies,Alexa-andrhodamine-

conjugatedphalloidinwerefromInvitrogen.

Cellculture

HCT116,CT26andMDA-MB-231cells(ATCC)wereculturedin

culturemediumcontainingDMEM(Gibco)supplementedwith10%

(v/v)fetalbovineserum(FBS,Gibco).Cellsweremaintainedat37◦C

in10%CO2humidifiedairduringculture.

CollagenIlabeling

RattailcollagenI(non-pepsinized;BDBiosciences)dissolved

inaceticacid0.2%(v/v)wasdialyzedovernightagainstlabeling

buffer(0.25MNaHCO3,0.4MNaCl,pH9.5)at4◦C.Tetramethyl-

rhodamine(TAMRA,Invitrogen),resuspendedinDMSOaccording

tothemanufacturer’sinstructions,wasdilutedinlabelingbuffer,

mixedwithcollagensolutionandincubatedovernightat4◦Cwith

agitation.Freedyewasremovedbydialyzingthelabeledcollagen

againstlabelingbufferovernightat4◦C.TAMRA-labeledcollagen

wasfinallydialyzedovernightagainstaceticacid0.2%(v/v)at4◦C.

2DcollagenIsubstratum

GlasscoverslipswerecoatedwithrattailcollagentypeITAMRA-

labeledmixedwithunlabeledcollagen(BDBiosciences)ina1:6

ratiodilutedin0.02Naceticacidtoachieve33␮g/cm2.After1hat

roomtemperature,coverslipswerewashed3timeswithPBSand

HCT116andCT26cellsplatedatlowdensity.Cellsweremaintained

at37◦Cin10%CO2humidifiedairfor1or2days.

3DcollagenImatrix

HCT116,CT26andMDA-MB-231cellsembeddedin3Dcollagen

ImatriceswerepreparedbymixingTAMRA-labeledcollagenwith

unlabeledrattailcollagentypeIinaceticacidina1:6ratiowith10×

PBSandcellsresuspendedinDMEM,toachieveafinalcelldensity

of105cells/mlin2mg/mlcollagenmatrix.1NofNaOHwasadded

toincreasepHto7.4.Allreagentswerekeptat4◦C.100␮ldrops

ofcollagenmixcontaining104cellswereaddedtoglassbottom

dishes(WorldPrecisionInstruments)andcollagenwasallowedto

polymerizebyincreasingthetemperaturetoeitherroomtempera-

tureor37◦C.Afterpolymerization,sufficientculturemediumwas

addedtocoverthecollagen/cellsdropsandmaintainedat37◦Cin

10%CO2humidifiedairfor1or2days.

2Dand3Dimmunofluorescencestaining

2D–Cellswerepre-extractedwithextractionbuffer(1%Tri-

tonX-100,4%PEG40000inPEMbuffer(100mMPipes,pH

6.9,1mMMgCl2,1mMEGTA))supplementedwith2␮MPhal-

loidin(Invitrogen)and2␮MTaxol(Invitrogen)for30satroom

temperature.After3quickwasheswithPEMbuffersupple-

mentedwith2␮MPhalloidinand2␮MTaxol,cellswerefixed

in4%PFAfor20minatroomtemperature.After3washes

withPBS,cellswereincubatedwithprimaryantibodiesagainst

tubulin,paxillin,vinculinorzyxinfor1hatroomtemperature.

Cellswerewashed3timeswithPBSfollowedbyincubation

withtheappropriateCy2-orAlexa-conjugatedsecondaryanti-

bodyandAlexa-orrhodamine-conjugatedphalloidinfor1hat

roomtemperature,andfurtherwashed3timeswithPBSbefore

mounting.

3D–Cellswereimmunostainedasdescribed(Hakkinenetal.,

2011)withmodifications.Cellsweresimultaneouslyfixedand