• Aucun résultat trouvé

Plasma cells release membrane microparticles in a mouse model of multiple myeloma.

N/A
N/A
Protected

Academic year: 2022

Partager "Plasma cells release membrane microparticles in a mouse model of multiple myeloma."

Copied!
7
0
0

Texte intégral

(1)

ContentslistsavailableatScienceDirect

Micron

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r o n

Plasma cells release membrane microparticles in a mouse model of multiple myeloma

Tarek Benameur

a,b

, Daniel Chappard

a,c

, Elodie Fioleau

a,c

, Ramaroson Andriantsitohaina

a,b

, M. Carmen Martinez

a,b

, Nicolas Clere

a,b

, Hélène Marchand-Libouban

a,c,∗

aL’UNAMUniversité,Angers,France

bINSERMU1063,Angers,France

cGEROMGroupeEtudesRemodelageOsseuxetbioMatériauxLHEA,IRIS-IBSInstitutdeBiologieenSanté,CHUd’Angers,49933AngersCedex,France

a r t i c l e i n f o

Articlehistory:

Received4June2013

Receivedinrevisedform28August2013 Accepted28August2013

Keywords:

Multiplemyeloma Microparticles PlasmaBcells Microenvironment CD138

a b s t r a c t

Microparticles(MPs)releasedfromtheplasmamembraneplayaroleintumorprogression.Involvement ofMPsinmyeloma(MM)hasbeenpoorlyinvestigated.BecauseofthestronginteractionofMMcells withbonemicroenvironment,wehypothesizedanimplicationofMPsinMMusingamurinemodel.

Forty-fourmicewereinjectedwith5THL-MMcellsandcomparedwith14non-injectedmice.Bloodwas collectedattheearlyandendstagesofMMdevelopment(EMMandLMM)tocharacterizethecirculating MPs.AtLMM,MPswereisolatedfrombonemarrow(BM)oflongbonesof22mice,aftercentrifugation.

ElectronmicroscopyimmunohistochemistryandWesternblottingusingCD138wereperformedonBM- derivedMPs.AtEMM,MPscirculatinglevelwassignificantlylowerversuscontrols.InLMM,asignificant increaseofthetotalMPnumberfromplasmawasobservedversuscontrols.Characterizationofcirculating MPsshowedanincreaseofleukocyte-anderythrocyte-derivedMPs.InLMM,serumM-proteinwas correlatedwithcirculatingMPnumber.BM-derivedMPsincreasedinLMMandexpressedCD138.Anti- CD138coupledwithnanobeadslocalizedattheMPsurface.Thereisevidenceofanassociationbetween increaseofMPsandMMdevelopment;theresultsunderscoretheparticipationofplasmacell-derived MPsoriginatingfromBM.

©2013ElsevierLtd.Allrightsreserved.

1. Introduction

Multiplemyeloma(MM)isaBcellmalignancycharacterized bymonoclonalproliferationofplasmacellsinthelocalbonemar- row(BM)environment andthedevelopmentofosteolyticbone lesions.The BM microenvironmenthas a key role in MM as it includesalargespectrumofcellularandmolecularcomponents thatinfluenceplasmacellsgrowthandosteolysis(Asosinghetal., 2003; Lemaire et al., 2011). A true “cious circle” exists where plasmacellsstimulate boneandmedullarcells whichstimulate inreturntheneoplasticgrowth.Demonstrationthatthegrowth ofmalignantplasmacellsisacceleratedwithapre-existinghigh levelofboneremodelinghasbeenshownusingacombinedani- malmodelinwhich ovariectomywasassociated toinjectionof 5T2MMplasmacellsintheC57BL/KaLwRijmouse(Liboubanetal., 2003).Osteoclastsrespondtoavarietyofcytokinesandgrowth

Correspondingauthorat:GEROMLHEA,IRIS-IBS,CHUd’Angers,49933Cedex, France.Tel.:+33244688344.

E-mailaddress:helene.marchand-libouban@univ-angers.fr (H.Marchand-Libouban).

factorsproducedbycellsoftheBMmicroenvironmentandbythe malignantplasmacells.Thesefactorsincludemacrophageinflam- matoryprotein-1-alpha(MIP-1␣),theligandforreceptoractivator ofnucleartranscriptionfactor-␬B(RANKL)andinterleukin-3(IL- 3)(Asosinghetal.,2003;Choietal.,2000;Leeetal.,2004;Pearse etal.,2001).Itisnowwellrecognizedthatadecreaseofboneforma- tionplaysalsoakeyroleinMM.Inhibitionofosteoblastogenesis isduetoosteoblastinhibitorsreleasedbyplasmacellsincluding Wnt-signalinginhibitorsDickkopf-1(DKK1)andSecretedfrizzled- relatedprotein2(Sfrp2),hepatocytegrowthfactor(HGF)andIL-7 (Giulianietal.,2005;Oshimaetal.,2005;Standaletal.,2007;Tian etal.,2003).Cellsinvolvedinthe“viciouscircle”areofhemato- logicaloriginatvarious differentiationstages, BMstromalcells, endothelialandbonecells(osteoblastsandosteoclasts)(Mitsiades et al.,2007).Interaction withendothelialcells mainlysupports tumorgrowthandneoangiogenesisisobservedinMM(Roccaro etal.,2006).Morerecently,interactionswithT-anddendriticcells (Giulianietal.,2006;Kukrejaetal.,2006)wererevealedthusampli- fyingthe“viciouscircle”.Thus,MMappearsasanexcellentmodel forstudyingtumor-microenvironment.Implicationofthemicroen- vironmentinteractionsinthedevelopmentofatumorhasbeen showninothertypeofcancers.

0968-4328/$seefrontmatter©2013ElsevierLtd.Allrightsreserved.

http://dx.doi.org/10.1016/j.micron.2013.08.010

(2)

76 T.Benameuretal./Micron54–55(2013)75–81

Recently,anothermechanismhasbeenhighlightedininducing afavorablelocalmicroenvironmentfortumorgrowth:membrane- derivedvesicles(MPs)shedfromdifferentcelltypes(Peinadoetal., 2011).MPsaremembrane-derivedvesicles(diameter≤1␮m)that arereleasedduringcellactivationorapoptosis.Ontheirsurface, MPsbear antigenscharacteristicof thecellof origin,and carry othermembrane andcytoplasmicconstituents.MPs arepresent inbloodfromhealthyandnon-healthyindividualsleadingtothe hypothesisthattheymayplayphysiologicaland/orpathophysio- logicalroles.PreviousstudieshaverevealedvariousrolesofMPsin cancer.IthasbeenrecentlyshownthatMPscaninduceinvitro neo-angiogenesis which might contribute to the generation of avascular network inmalignant diseaseassociatedwithtumor growth(Soletietal.,2009).Inaddition,MPsreleasedfromtumor cellshaveapro-angiogenicactivity(Kimetal.,2002).Othermem- branecompoundslinkedtoMPs,suchasurokinase,canincrease theinvasive capacity of prostate cancer cells (Angelucci et al., 2000). MPs are able to carry intercellular signals allowing the tumorsurvivalandprogression.Consideringthatenhancedlevels ofcirculatingMPshavebeendetectedinpatientswithdifferent typesofcancers,onecanadvancedthehypothesisthatMPsmight playanimportantroleinthetumordevelopment(Martinezand Andriantsitohaina,2011;Mostefaietal.,2008;Tual-Chalotetal., 2011).

Because of the various cell types involved in MM and the stronginteractionof thebone andmedullarmicroenvironment, wehypothesizedanimplicationofMPsinthedevelopmentofMM.

Inthepresentstudy,quantificationandcharacterizationofboth circulating-MPsandBM-derivedMPswereinvestigatedinthe5THL murinemodelofMM.

2. Materialsandmethods 2.1. Mice

C57BL/KaLwRijfemalemice(6–8-week-old)wereusedforthe study(Harlan,Gannat,France).Theywereacclimatedfor1weekto thelocalvivariumconditions(24Cand12h/12hlight/darkcycle) wheretheyweregivenstandardlaboratoryfood(UAR,Villemoi- sonsurOrge,France)andwateradlibitum.TheAnimalCareand Usecommittee attheUniversity of Angersapprovedall proce- dures.

2.2. Culturecellline

Wehave usedthe5THLcelllineaspreviouslycharacterized (Liboubanetal.,2004).Briefly,the5THLcelllineisanaggressive sublineof5T2MMcelllineoriginatingfromelderlyC57BL/KaLwRij micethatspontaneouslydevelopedMM(Croeseetal.,1987).5THL cellscanbepropagatedintoyoungsyngeneicmicebyintravenous transferofthediseasedBM.Progressionofthediseaseinseven recipientmicewasassessedbymeasuringtheserumM-protein (IgG2a␬)levelusingagarelectrophoresis(HydragelProtein,SEBIA, Issy les Moulineaux, France). Around 6 week post-injection of 5THL,micehad a detectableserum M-proteinandwereeutha- nizedafter10–12weeksbycervicaldislocation.Femursandtibias weredissected,cleanedofsurroundingtissuesandBMwasflushed inDulbecco’smodifiedessentialmedium(DMEM.mod,GIBCO,Life Technologies,France)supplementedwithpenicillin–streptomycin, amphotericin–fungizoneandpyruvate.BMcellswerewashedonce inDMEM.mod.MononuclearcellswereisolatedbyaLympholyte- Mcentrifugationgradient(Cedarlane,Hornby,Ontario,Canada)at 1250×gfor20min.Mononuclearcellswerethenwashedtwicein DMEM.modandcounted.

2.3. Experimentaldesign

Forty-fourmice(6–8weeksold)wereinjectedwith1.5×106 5THLcellsinthetailveinand14non-injectedmicewereusedas control(CTL).Theinjectedmiceweredividedin2groupsaccord- ingtothetimeofsacrifice.At6weeks,correspondingtotheearly stageofMMdevelopment,14micewerebledbeforebeingsacri- ficedbycervicaldislocation.Thesemiceconstitutedtheearlystage MMgroup(EMM)andwereusedtoquantifyandcharacterizethe phenotypeofcirculatingMPs.Attheendstageofthedisease(10–12 weeks),whenosteolysiscanbeevidenced onX-rayimages,the remaining30micewerebledbeforebeingsacrificedbycervical dislocation.ThesemiceconstitutedthelatestageMMgroup(LMM) andwereusedtoquantifyandcharacterizethephenotypeofcir- culatingMPs(seebelow).Twenty-twomicefromtheLMMgroup werealsousedtoisolateMPsfromtheBMoffemursandtibias.

The14non-injectedmice(16–18-week-old)werebledasprevi- ouslydescribedtoquantifyandphenotypecirculatingMPs.Eight ofthe14micewerealsousedtoisolateMPsfromBM.

Twoadditionalmiceinjectedwith5THLcellsweresacrificedat theendstageofthediseaseandwereusedfortransmissionelectron microscopy(TEM)observationsandimmunohistochemistryofBM- derivedMPsandMMcells.

2.4. CirculatingMPisolationandcharacterization

C57BL/KaLwRij mice at 6 weeks or at 10–12 weeks were anesthetized usingIsoflurane (Baxter,Maurepas, France) before sacrifice,and∼800␮lofbloodwerecollectedbyaspirationfrom theleftventricle.Bloodwasplacedincitratedtubesandcentrifuged at1900×gfor3minforseparationofplatelet-richplasmafrom wholeblood.Then,platelet-richplasmawascentrifugedat5000×g for4mintoobtainplatelet-freeplasma(PFP).Sixtymicrolitersof PFPwerefrozenandstoredat−80Cuntilsubsequentuse.Inorder topelletMPsforinvitrostudies,circulatingMPswereconcentrated fromPFPbythreeseriesofcentrifugationsat21,000×gfor45min andre-suspendedinsalineandstoredat4Cuntilsubsequentuse.

Membrane MP subpopulations were discriminated in PFP accordingtheexpressionofmembrane-specificantigens.Pheno- typeofendothelialMPswasperformedusinganti-CD54labeling;

characterizationofplatelet,leukocyteanderythrocyteMPs was performed using respectively anti-CD61, anti-CD45 and Ter- 119/erythroidcelllabeling.IrrelevantmouseIgGwasusedasan isotype-matchednegativecontrolforeachsample.

Fornumerationstudies,8␮lofPFPwereincubatedwitheither 1␮lofspecificantibody(BioLegend,SanDiego,CA).After45minof incubationatroomtemperature,samplesweredilutedin300␮lof saline.AnnexinV(BioVision,Inc.,MountainView,CA)bindingwas usedtonumeratecirculatingphosphatidylserine-expressingMPs (2␮lofannexinV/5␮lPFP).Then,inordertoenumerateMPs,an equalvolumeofsampleandFlowcountbeadswereaddedandsam- pleswereanalyzedinaflowcytometer500MPLsystem(Beckman Coulter,Roissy,France)aspreviouslydescribed(Agounietal.,2008;

Mostefaietal.,2008).Flow-countfluorospheresconsistof10␮m polystyrenefluorospheresinanaqueoussuspensionmedium.Each fluorospherecontainsadyethathasafluorescenceemissionrange of525–700nmwhenexcitatedat488nm.Theconcentrationof eachlotofFlow-countfluorospheresisderivedfrommultiplerepli- cateanalysesonaCOULTERparticlesizeanalyzerperformedby themanufacturer.EachlotofFlow-countfluorosphereshasaspe- cificconcentrationoffluorospheres.Whenidenticalvolumesofa sampleandFlow-countfluorospheresareused,aratioofMPsin thesampletofluorospheresisestablished.Sincetheconcentration offluorospheresisknown,theabsolutecountoftheMPscanbe automaticallydeterminedbytheMXPsoftware.

(3)

Fig.1.FlowcytometricanalysisofcirculatingMPs.(A)CirculatingMPsfromcontrol(CTL),earlyMMstage(EMM)andlateMMstage(LMM)miceandFlowcountbeads (beadsregion,10␮mdiameter)arevisualizedinasidescatter(SS)/forwardscatter(FS)logarithmicrepresentation.MPsaredefinedaseventswithsize0.1–1␮mgatedin the“MPs”windows.(B)TotalcirculatingMPlevelsinEMMandLMMcomparedwithCTLmiceanddifferentpopulations:(C)endothelial-,(D)platelet-,(E)procoagulant-,(F) erythrocyte-and(G)leukocyte-derivedMPsfrommiceatEMMandLMMstagescomparedwithCTLmice.Resultsareexpressedasevents/␮lofplasmaandgivenasmean SEM.

2.5. Bonemarrow(BM)-derivedMPisolation

BMcellsfrom5THL-MMandcontrolmice(10–12weeks)were flushedfromlongbonesandcentrifuged;supernatantwasused toisolateMPs.Briefly,femursandtibiasweredissected,cleaned ofsurroundingtissuesand BMwasflushedinDMEM.mod sup- plementedwithpenicillin–streptomycin,amphotericin–fungizone andpyruvate.Cellsuspension(∼8ml)wasfilteredandcentrifuged at300×gfor5min.MPswereisolatedfromsupernatants,collected andclearedfromdetachedcells orlargecellfragmentsbycen- trifugationasdescribedabove.Thewashingmediumfromthelast supernatantwasusedascontrol.

2.6. Westernblottingatendstageofdisease

ProteinanalysisbyWesternblottingwasperformedonhomog- enizedMPsobtainedfromBMof2mice.Becauseplasmacellsare asubpopulationofleukocytesexpressingheterogeneouslyCD45 andmorespecificallyCD138(syndecan-1),weusedanantiCD138 monoclonal antibody (Wijdenes et al., 2002). Total protein of

BM-derivedMPsfrom5THL-MMmice(20␮g)wereprobedwith ratanti-CD138(BDBiosciences,SanJosé,CA,Ref.553712).

2.7. Transmissionelectronmicroscopyand immunohistochemistry

Cells and MPs were fixed with a freshly prepared 2%

paraformaldehydein0.1Msodiumcacodylatebuffer(pH7.4)for 1h30minat20C.Theywerethendehydratedingradingconcen- trationofethanolbetween−20Cand−40C.Embeddingwasdone inLowicrylK4M(Polysciences,Paris)andpolymerizedat−40C byUVlight(=360nm)(AFS,Leica).Sectionsof60nminthickness wereperformedusinganUltracutSmicrotome(LeicaUltracutE) andcollectedoncoppergrids.

AfterrinsingwithPBSbuffer,gridswereincubatedin50mM NH4Clfollowedby1%bovinealbumin.Goatpolyclonalanti-mouse CD138antibody(R&DSystems,FranceRef.AF-3190)wasusedas theprimaryantibody(dilution1/50inPBSbuffer,incubation1h 30min).AfterseveralrinsesinPBS,gridswereincubatedfor30min withthesecondaryantibodyIgGanti-goatcoupledwithcolloidal goldparticles(10nmindiameter,SigmaRef.G-5402).Gridswere

(4)

78 T.Benameuretal./Micron54–55(2013)75–81

rinsedwithPBS,contrastedwithuranylacetate(saturatedindis- tilledwater)and observedwitha JEOL2010TEMunder200kV voltage.

2.8. Statisticalanalysis

Statistical study was done with SYSTAT statistical software (Systat, San José, CA, release 13.0). All data were reported as mean±standarderrorofthemean(SEM).Significantdifferences betweengroupswereanalyzedbyanalysisofvariance(ANOVA) withtheFisher’sleastsignificantdifferenceposthoctest.Inorder toevaluateifcorrelationsexistbetweenthelevelofM-proteinand thelevelofcirculatingMPs,alinearregressionanalysiswasper- formedusing7miceattheendstageofthedisease;thePearson’s coefficientofcorrelationwasdetermined.Resultswereconsidered assignificantwhenP<0.05.

3. Results

3.1. CirculatinglevelsofMPsattheendstageofMMcomparedwiththeearly stage

CirculatinglevelsofMPsweredeterminedattheearly(6weeks)andatthe endstages(10–12weeks)ofMMdevelopment(Fig.1A–G).Foreachgroupsofmice (CTL,EMMandLMM),histograms/bivariateplotsresultingfromMPsenumeration arerepresentedinFig.1A.AtEMM,thetotalnumberofcirculatingMPswassignif- icantlyreducedby∼30.5%comparedtocontrolmice(Fig.1B).Incontrast,atLMM, asignificantincreaseofthetotalnumberofMPsfromplasmawasobservedcom- paredtocontrolmice.CirculatinglevelofMPswasfoundsignificantlyhigherat LMMcomparedtoEMM.

PhenotypiccharacterizationofthecellularoriginofMPsshowedsignificant increasesofthecirculatinglevelsofMPswithaprocoagulantpotential(Annexin V+)-,andthoseofplatelet(CD61+)-,leukocyte(CD45+)-,endothelial(CD54+)-and erythrocyte-derivedMPsinMMmiceatLMMcomparedwithEMM(Fig.1C–G).We foundasignificantcorrelationbetweenthelevelofserumM-proteinandthelevel ofcirculatingMPs(r=0.77;P<0.05)(Fig.2).

3.2. BonemarrowlevelsofMPsatLMM

Flowcytometeranalysisrevealedasignificantincreased(∼3.7-fold,P<0.05) amountofMPsfromBMinmiceatLMM(Fig.3)probablyduetotheincreased numberofcellsintoBM.Indeed,theenhancedproductionofMPsfromBMwas positivelycorrelatedwiththenumberofBM-derivedcellsatLMM(Fig.4)suggesting thatelevatednumberofMPsresultsfromenhancedcellcountinBMandnotfrom anincreasedabilityofcellstovesiculate.ThepresenceofMPswithintheBMwas clearlyevidencedbyTEMobservationofisolatedMPs;theyareheterogeneousin sizewithamaximum750nmdiameter(Fig.5A).

3.3. ExpressionofplasmacellbiomarkerCD138onBM-derivedMPs

TEMimmunochemistryshowedthepresenceofseveralgoldnanobeadsaround MPs;somebeingclearlylocalizedatthesurfaceoftheMPs(Fig.5B).Westernblot revealedalsotheexpressionoftheplasmacellmarkerCD138(syndecan-1)byBM- derivedMPsfrom2miceatLMM(Fig.5C).Goldnanobeadscoupledwithanti-CD138

Fig.2. CorrelationbetweenthelevelofserumM-proteinandthelevelofcirculating MPs.

Fig.3. Flowcytometryanalysisofbonemarrow-derivedMPs.Bonemarrow-derived MPlevelsweresignificantlyincreasedatthelatestageofMM(LMM)comparedwith control(CTL)mice.

Fig.4.PositivecorrelationbetweenthetotalMPsandthenumberofcellsderived fromBMof5THL-MMmice.

werealsoidentifiedatthesurfaceof5THLcells(Fig.5D)isolatedfromthesamemice attheendstageofMM(Fig.4E).

4. Discussion

The allograft of 5TMM cells in C57BL/KaLwRij mice is one of the main models to study the pathophysiology of MM, the influenceofBMmicroenvironmentontumorprogressionandto evaluatedrugeffectsonosteolysisand tumorgrowth(Asosingh etal.,2000).The5T2MMrepresentsamodelsituationofthemost commonforms ofhumanMMdisease,withamoderategrowth andosteolyticlesions.Inthepresentstudy,weused5THLcells inwhichM-proteininwasdetectedafter6weeksandosteolysis detected after 8 weeks post-injection, as previously described (Liboubanetal.,2004).Itiswellestablishedthatinteractionsexist betweenmalignantplasmacells,BMstromalcells,osteoclastpre- cursorsandendothelialcells.Stromalcellsplayakeyroleintumor progressionasmostofthefactorsinvolvedinMMarereleasedin themicroenvironmentduetomolecularinteractionswithstromal cells (Abeet al.,2009; Michigami et al.,2000).The role ofthe microenvironmentwasprovenbyvariousstudiesinwhichafactor implicatedintheMMviciouscirclewasinhibited:asanexample, it wasshown that inhibition of cell–cellcontact (between MM andstromalcells)completelypreventedtheincreaseofresorption (Abeetal.,2004).Otherstudiesshowedthatinhibitionofosteo- clasticactivityconsiderablyreducedtumorprogressionwithouta completeabolishingofthetumorgrowth(Croucheretal.,2003).

Conversely, when using the 5T2MMmodel, we showedthat a favorable microenvironment can enhance tumor growth and

(5)

Fig.5. Transmissionelectronmicroscopyobservations.(A)MPsisolatedfromthebonemarrowof5THLmice.(B)ImmunogoldlocalizationofCD138with10nmnanobeads (arrows)ontheMPs.(C)WesternblotanalysisoftheplasmacellmarkerCD138expressiononbonemarrow-derivedMPsfrom2mice.(D)A5THLplasmacellisolatedfrom BM.(E)ImmunogoldlocalizationofCD138with10nmnanobeads(arrows)atthemembranesurfaceofa5THLplasmacells.

promotethe developmentof osteolyticlesions(Liboubanetal., 2003). Moreover, it had the capability to select an aggressive plasmacellline(5THL)thatcanreproduceamyelomainashorter periodthantheoriginalcellline5T2MM(Liboubanetal.,2004).

Severalmethodsfor detectionand quantificationof MPsare available.Here,byusingtwomethods,flowcytometryandTEM, we show that samples obtained from blood (Fig. 1A) and BM (Fig.5A)areMP-enrichedwithhomogenoussizeanddifferentpro- teinexpression.

Recently,anumberofstudiesreporttheinvolvementofMPsin tumorgrowth(Tavoosidanaetal.,2011).Itisnowadmittedthat MPsplaya rolein variousaspectsoftumorprogression:tumor microenvironment,angiogenesis,evasionofimmunesurveillance, metastasis,acquisition ofaggressive phenotypeand multidrug resistance(Muralidharan-Charietal.,2010).Inthepresentstudy, wefound,atLMM,anincreaseofcirculatingMPsandMPsfrom BM.AnincreasedlevelofMPsexpressingtissuefactorhasbeen observedinuntreatedMMpatientscomparedtohealthycontrols (Auwerdaetal.,2011).Incontrasttothepresentstudyconductedin mice,theseauthorsmainlyfocusedontheclinicalimpactofahigh MPlevelsinrelationshipwithvenousthromboticeventssinceit iswellestablishedthatthesepatientshaveathigherriskofdevel- opingarterio-venousthrombosis(Kristinsson,2010).Itshouldbe

notedthatthe5TMMmodelmimicsthemarrowenvironmentand bonelesionsasobservedinhumansbutotherclinicalcharacter- isticsofthediseasesuchasthrombosisorrenallesionsarenever observed(Liboubanetal.,2006).Indeed,neitherplatelet-derived MPsnorMPsexpressingphosphatidylserine(i.e.withaprocoag- ulantpotential)wereincreasedinthepresentstudy.Inaddition, ahighlevelofMPsincancerhasbeenshowninvitroandinvivo tobecorrelatedwithtumoraggressiveness(Castellanaetal.,2010;

Ginestraetal.,1999).Theseobservationsareinagreementwithour resultsaswefoundahighlevelofMPsatLMM.Ofimportanceis thefactofthesignificantcorrelationbetweencirculatingMPsand M-protein.Thus,circulatingMPsmaybeusedasgooddiagnostic markersofthediseaseevolution.

InadditiontotheincreasedlevelsoftotalcirculatingMPs,those derived from erythrocytes and leukocytes were also enhanced when compared with control mice. In chronic lymphocytic leukemia, it was shown that MPs have mainly a platelet and leukemicB-cellorigin(Ghoshetal.,2010).Thehighlevelofplatelet- derivedMPshasbeenalsoobservedinvarioustypesofcancerand ithasbeenpostulatedthattheymayplayaroleinsolidtumorpro- gressionandmetastasis(Dashevskyetal.,2009;Kimetal.,2003).

Wedidnotobservesuchthisphenomenoninourstudy.Byana- lyzingtheleveloftotalcirculatingMPsandtheirphenotypicorigin

(6)

80 T.Benameuretal./Micron54–55(2013)75–81

accordingtothestageofthedisease,weexpectedtofindinterme- diaryresultsbetweencontrolsandEMM.Wealsoexpectedtofinda phenotypicswitchbetweenEMMandLMMaspreviouslyreported inanother hematologicmalignancy (Ghosh etal., 2010).In the presentmurinemodel,theEMMwascharacterizedbytheonsetofa verysmalllevelofM-protein(notshown),theabsenceofanyoste- olyticlesiononX-raysbutamassiveinfiltrationoftumorplasma cellsinsomebones(Liboubanetal.,2004).Thefactthatearlystage ofmyelomadecreasedlevelsofcirculatingMPsisprobablyassoci- atedwithbloodcellcountintheseanimals.Thus,alterationson thenumber andthefunctionof bloodcells havebeenreported inmultiplemyelomapatients.Concerningplateletsandredblood cells,thesealterationsareassociatedwithhypercoagulablestate butalsowiththrombocytopeniaandhemorrhagiccomplications.

Theseeffects maybe associatedwithchanges in hematological cellsderivedfromBM(inducinganemiaandthrombocytopenia) (AlbarracinandFonseca,2011)butalsowithchangesinbloodvis- cosityandhemostasis(EliceandRodeghiero,2012).Moreover,it hasbeendescribed that blood cells canbesequestered in thy- musandBMof5T2MMmousemodel(Laronne-Bar-Onetal.,2008) reducingthenumberofcirculatingMPsderivedfromthesecells.

PerhapsthisEMMstagewastooprematuretodetectenoughMPs fromplasmacells,suggestingthatthehighlevelofMPsobserved atLMMcouldbeaveryusefulbiomarkerofaprogressionofthe diseaseandpoorprognostic.

Moreinterestingly,wefoundahighquantityofMPsfromBM whichcorrespondstothelocalenvironmentwheretumorplasma cellsgrow.WemanagedtoobserveMPsbyTEMwithimmuno- golddetection,coupledtowesternblotting.CD138wasstrongly expressedonMPsisolatedfromtheBMmicroenvironment,con- firmingthehypothesisthatpartofthemwereshedfromthesurface ofmalignantplasmacells.CD138isatransmembraneheparinsul- fateproteoglycanwithanexpressionhighlyspecificofplasmacells;

itisnotexpressedonBorTlymphocytes(Wijdenesetal.,1996).

Becauseofsuchaspecificexpression,CD138isusedasastandard markerinthediagnosisofMM(Batailleetal.,2006).Freecirculating CD138levelisalsocorrelatedwithtumormassanditisimplicated inthecell/celladhesionprocess,inphysicalinteractionwithsol- ublefactorsandmoleculesformtheextracellularmatrix(Bataille etal.,2006;Dhodapkaretal.,1997).

In the 5T2MM model, differences have been shown in the expressionofseveralmarkerslikeCD45andCD138atthestage levelofthedisease(Asosinghetal.,2003).Atthequiescentstage (correspondingtoourEMM),mostMMcellsarehighlyinvasive andexpressCD45+andCD138,whereasattheLMM,MMcells aremoremature,lessinvasivewithaCD45,CD138+phenotype.

In the present study, theCD138 expression was assessed only atthelatestagewhere itwasknownthatmostofthecellsare CD138+.

5. Conclusion

Inconclusion,weprovideevidencethat,inLMMstage,circu- latinglevelsofMPsfrommicewereincreasedandthatlevelsof BM-derivedMPsexpressingCD138arecorrelatedwiththepathol- ogy severity.These MPs may play a critical role as vectors of deleteriousbiologicalmessagesleadingtogrowthtumor.Thisfind- ingmaysuggestthatMPscouldbenewactorsoftheviciouscircle involvedinMM.

Acknowledgments

We thank SCIAM (Service Commun d’Imagerie et Analyses Microscopiques),Universitéd’Angers.Thisworkwassupportedby agrantfromContratRégionPaysdelaLoire(Bioregos2program).

References

Abe,M.,Hiura,K.,Ozaki,S.,Kido,S.,Matsumoto,T.,2009.Viciouscyclebetween myelomacellbindingtobonemarrowstromalcellsviaVLA-4-VCAM-1adhe- sionandmacrophageinflammatoryprotein-1alphaandMIP-1betaproduction.

JournalofBoneandMineralMetabolism27,16–23.

Abe,M.,Hiura,K.,Wilde,J.,Shioyasono,A.,Moriyama,K.,Hashimoto,T.,Kido,S., Oshima,T.,Shibata,H.,Ozaki,S.,Inoue,D.,Matsumoto,T.,2004.Osteoclasts enhancemyelomacellgrowthandsurvivalviacell–cellcontact:aviciouscycle betweenbonedestructionandmyelomaexpansion.Blood104,2484–2491.

Agouni,A.,Lagrue-Lak-Hal,A.H.,Ducluzeau,P.H.,Mostefai,H.A.,Draunet-Busson, C.,Leftheriotis,G.,Heymes,C.,Martinez,M.C.,Andriantsitohaina,R.,2008.

Endothelialdysfunctioncausedbycirculatingmicroparticlesfrompatientswith metabolicsyndrome.AmericanJournalofPathology173,1210–1219.

Albarracin,F.,Fonseca,R.,2011.Plasmacellleukemia.BloodReview25,107–112.

Angelucci,A.,D’Ascenzo,S.,Festuccia,C.,Gravina,G.L.,Bologna,M.,Dolo,V.,Pavan, A.,2000.Vesicle-associatedurokinaseplasminogenactivatorpromotesinva- sioninprostatecancercelllines.ClinicalandExperimentalMetastasis18, 163–170.

Asosingh,K.,DeRaeve,H.,VanRiet,I.,VanCamp,B.,Vanderkerken,K.,2003.Multiple myelomatumorprogressioninthe5T2MMmurinemodelisamultistageand dynamicprocessofdifferentiation,proliferation,invasion,andapoptosis.Blood 101,3136–3141.

Asosingh,K.,Radl,J.,VanRiet,I.,VanCamp,B.,Vanderkerken,K.,2000.The5TMM series:ausefulinvivomousemodelofhumanmultiplemyeloma.Hematology Journal1,351–356.

Auwerda,J.J.,Yuana,Y.,Osanto,S.,deMaat,M.P.,Sonneveld,P.,Bertina,R.M.,Lee- beek,F.W.,2011.Microparticle-associatedtissuefactoractivityandvenous thrombosisinmultiplemyeloma.ThrombosisandHaemostasis105,14–20.

Bataille,R.,Jego,G.,Robillard,N.,Barille-Nion,S.,Harousseau,J.L.,Moreau,P.,Amiot, M.,Pellat-Deceunynck,C.,2006.Thephenotypeofnormal,reactiveandmalig- nantplasmacells.Identificationofmanyandmultiplemyelomasandofnew targetsformyelomatherapy.Haematologica91,1234–1240.

Castellana,D.,Toti,F.,Freyssinet,J.M.,2010.Membranemicrovesicles:macromes- sengersincancerdiseaseandprogression.ThrombosisResearch125(Suppl.2), S84L88.

Choi,S.J.,Cruz,J.C.,Craig,F.,Chung,H.,Devlin,R.D.,Roodman,G.D.,Alsina,M.,2000.

Macrophageinflammatoryprotein1-alphaisapotentialosteoclaststimulatory factorinmultiplemyeloma.Blood96,671–675.

Croese,J.W.,VasNunes,C.M.,Radl,J.,vandenEnden-Vieveen,M.H.,Brondijk, R.J.,Boersma,W.J.,1987.The5T2mousemultiplemyelomamodel:charac- terizationof5T2cellswithinthebonemarrow.BritishJournalofCancer56, 555–560.

Croucher,P.I.,Shipman,C.M.,VanCamp,B.,Vanderkerken,K.,2003.Bisphospho- natesandosteoprotegerinasinhibitorsofmyelomabonedisease.Cancer97, 818–824.

Dashevsky,O.,Varon,D.,Brill,A.,2009.Platelet-derivedmicroparticlespromote invasivenessofprostatecancercellsviaupregulationofMMP-2production.

InternationalJournalofCancer124,1773–1777.

Dhodapkar,M.V.,Kelly,T.,Theus,A.,Athota,A.B.,Barlogie,B.,Sanderson,R.D.,1997.

Elevatedlevelsofshedsyndecan-1correlatewithtumourmassanddecreased matrixmetalloproteinase-9activityintheserumofpatientswithmultiple myeloma.BritishJournalofHaematology99,368–371.

Elice,F.,Rodeghiero,F.,2012.Hematologicmalignanciesandthrombosis.Thrombo- sisResearch129,360–366.

Ghosh,A.K.,Secreto,C.R.,Knox,T.R.,Ding,W.,Mukhopadhyay,D.,Kay,N.E.,2010.

CirculatingmicrovesiclesinB-cellchroniclymphocyticleukemiacanstimu- latemarrowstromalcells:implicationsfordiseaseprogression.Blood115, 1755–1764.

Ginestra,A.,Miceli,D.,Dolo,V.,Romano,F.M.,Vittorelli,M.L.,1999.Membranevesi- clesinovariancancerfluids:anewpotentialmarker.AnticancerResearch19, 3439–3445.

Giuliani,N.,Colla,S.,Morandi,F.,Lazzaretti,M.,Sala,R.,Bonomini,S.,Grano,M., Colucci,S.,Svaldi,M.,Rizzoli,V.,2005.MyelomacellsblockRUNX2/CBFA1 activityinhumanbonemarrowosteoblastprogenitorsandinhibitosteoblast formationanddifferentiation.Blood106,2472–2483.

Giuliani,N.,Morandi,F.,Tagliaferri,S.,Colla,S.,Bonomini,S.,Sammarelli,G.,Rizzoli, V.,2006.Interleukin-3(IL-3)isoverexpressedbyTlymphocytesinmultiple myelomapatients.Blood107,841–842.

Kim,C.W.,Lee,H.M.,Lee,T.H.,Kang,C.,Kleinman,H.K.,Gho,Y.S.,2002.Extracellular membranevesiclesfromtumorcellspromoteangiogenesisviasphingomyelin.

CancerResearch62,6312–6317.

Kim,H.K.,Song,K.S.,Park,Y.S.,Kang,Y.H.,Lee,Y.J.,Lee,K.R.,Ryu,K.W.,Bae,J.M.,Kim, S.,2003.Elevatedlevelsofcirculatingplateletmicroparticles,VEGF,IL-6and RANTESinpatientswithgastriccancer:possibleroleofametastasispredictor.

EuropeanJournalofCancer39,184–191.

Kristinsson,S.Y.,2010.Thrombosisinmultiplemyeloma.Hematology/theEducation ProgramoftheAmericanSocietyofHematology,437–444.

Kukreja,A.,Hutchinson,A.,Dhodapkar,K.,Mazumder,A.,Vesole,D.,Angitapalli,R., Jagannath,S.,Dhodapkar,M.V.,2006.Enhancementofclonogenicityofhuman multiplemyelomabydendriticcells.JournalofExperimentalMedicine203, 1859–1865.

Laronne-Bar-On,A.,Zipori,D.,Haran-Ghera,N.,2008.Increasedregulatoryversus effectorTcelldevelopmentisassociatedwiththymusatrophyinmousemodels ofmultiplemyeloma.JournalofImmunology181,3714–3724.

(7)

Lee,J.W.,Chung,H.Y.,Ehrlich,L.A.,Jelinek,D.F.,Callander,N.S.,Roodman,G.D.,Choi, S.J.,2004.IL-3expressionbymyelomacellsincreasesbothosteoclastformation andgrowthofmyelomacells.Blood103,2308–2315.

Lemaire,M.,Deleu,S.,DeBruyne,E.,VanValckenborgh,E.,Menu,E.,Vanderkerken, K.,2011.Themicroenvironmentandmolecularbiologyofthemultiplemyeloma tumor.AdvancesinCancerResearch110,19–42.

Libouban,H.,Moreau,M.F.,Basle,M.F.,Bataille,R.,Chappard,D.,2003.Increased boneremodelingduetoovariectomydramaticallyincreasestumoralgrowthin the5T2multiplemyelomamousemodel.Bone33,283–292.

Libouban,H.,Moreau,M.F.,Basle,M.F.,Bataille,R.,Chappard,D.,2004.Selectionofa highlyaggressivemyelomacelllinebyanalteredbonemicroenvironmentinthe C57BL/KaLwRijmouse.BiochemicalandBiophysicalResearchCommunications 316,859–866.

Libouban,H.,Onno,C.,Pascaretti-Grizon,F.,Gallois,Y.,Moreau,M.F.,Baslé,M.F., Chappard,D.,2006. Absenceofrenal lesionsinC57BL/KaLwRij micewith advancedmyelomadueto5T2MMcells.LeukemiaResearch30,1371–1375.

Martinez,M.C.,Andriantsitohaina,R.,2011.Microparticlesinangiogenesis:thera- peuticpotential.CirculationResearch109,110–119.

Michigami, T., Shimizu, N., Williams, P.J., Niewolna, M., Dallas, S.L., Mundy, G.R.,Yoneda,T.,2000.Cell–cellcontactbetweenmarrowstromalcellsand myelomacellsviaVCAM-1andalpha(4)beta(1)-integrinenhancesproduction ofosteoclast-stimulatingactivity.Blood96,1953–1960.

Mitsiades,C.S.,McMillin,D.W.,Klippel,S.,Hideshima,T.,Chauhan,D.,Richard- son,P.G.,Munshi,N.C.,Anderson,K.C.,2007.Theroleofthebonemarrow microenvironmentinthepathophysiologyofmyelomaanditssignificancein thedevelopmentofmoreeffectivetherapies.Hematology/OncologyClinicsof NorthAmerica21,1007–1034.

Mostefai, H.A.,Andriantsitohaina, R.,Martinez,M.C., 2008. Plasmamembrane microparticlesinangiogenesis:roleinischemicdiseasesandincancer.Phys- iologicalResearch57,311–320.

Muralidharan-Chari, V., Clancy, J.W., Sedgwick, A., D’Souza-Schorey, C., 2010.

Microvesicles:mediatorsofextracellularcommunicationduringcancerpro- gression.JournalofCellScience123,1603–1611.

Oshima,T.,Abe,M.,Asano,J.,Hara,T.,Kitazoe,K.,Sekimoto,E.,Tanaka,Y.,Shibata,H., Hashimoto,T.,Ozaki,S.,Kido,S.,Inoue,D.,Matsumoto,T.,2005.Myelomacells suppressboneformationbysecretingasolubleWntinhibitor,sFRP-2.Blood 106,3160–3165.

Pearse, R.N., Sordillo, E.M., Yaccoby, S., Wong, B.R., Liau, D.F., Colman, N., Michaeli, J., Epstein, J., Choi, Y., 2001. Multiple myeloma disrupts the TRANCE/osteoprotegerincytokineaxistotriggerbonedestructionandpromote tumorprogression.ProceedingsoftheNationalAcademyofSciencesofthe UnitedStatesofAmerica98,11581–11586.

Peinado,H.,Lavotshkin,S.,Lyden,D.,2011.Thesecretedfactorsresponsibleforpre- metastaticnicheformation:oldsayingsandnewthoughts.SeminarsinCancer Biology21,139–146.

Roccaro,A.M.,Hideshima,T.,Raje,N.,Kumar,S.,Ishitsuka,K.,Yasui,H.,Shiraishi, N.,Ribatti,D.,Nico,B.,Vacca,A.,Dammacco,F.,Richardson,P.G.,Anderson,K.C., 2006.Bortezomibmediatesantiangiogenesisinmultiplemyelomaviadirectand indirecteffectsonendothelialcells.CancerResearch66,184–191.

Soleti,R.,Benameur,T.,Porro,C.,Panaro,M.A.,Andriantsitohaina,R.,Martinez,M.C., 2009.MicroparticlesharboringSonicHedgehogpromoteangiogenesisthrough theupregulationofadhesionproteinsandproangiogenicfactors.Carcinogenesis 30,580–588.

Standal,T.,Abildgaard,N.,Fagerli,U.M.,Stordal,B.,Hjertner,O.,Borset,M.,Sundan, A.,2007.HGFinhibitsBMP-inducedosteoblastogenesis:possibleimplications forthebonediseaseofmultiplemyeloma.Blood109,3024–3030.

Tavoosidana,G.,Ronquist,G.,Darmanis,S.,Yan,J.,Carlsson,L.,Wu,D.,Conze,T., Ek,P.,Semjonow,A.,Eltze,E.,Larsson,A.,Landegren,U.D.,Kamali-Moghaddam, M.,2011.Multiplerecognitionassayrevealsprostasomesaspromisingplasma biomarkersforprostatecancer.ProceedingsoftheNationalAcademyofSciences oftheUnitedStatesofAmerica108,8809–8814.

Tian,E.,Zhan,F.,Walker,R.,Rasmussen,E.,Ma,Y.,Barlogie,B.,ShaughnessyJr.,J.D., 2003.TheroleoftheWnt-signalingantagonistDKK1inthedevelopmentof osteolyticlesionsinmultiplemyeloma.NewEnglandJournalofMedicine349, 2483–2494.

Tual-Chalot,S.,Leonetti,D.,Andriantsitohaina,R.,Martinez,M.C.,2011.Microvesi- cles:intercellularvectorsofbiologicalmessages.MolecularInterventions11, 88–94.

Wijdenes,J.,Dore,J.M.,Clement,C.,Vermot-Desroches,C.,2002.CD138.Journalof BiologicalRegulatorsandHomeostaticAgents16,152–155.

Wijdenes,J.,Vooijs,W.C.,Clement,C.,Post,J.,Morard,F.,Vita,N.,Laurent,P.,Sun,R.X., Klein,B.,Dore,J.M.,1996.Aplasmocyteselectivemonoclonalantibody(B-B4) recognizessyndecan-1.BritishJournalofHaematology94,318–323.

Références

Documents relatifs

In our context each expert is a Kalman filter fed by a subset of sensors, and a gating network serves as a mediator between individual filters, basing its decision on sensor inputs

Organic Light Emitting Diodes (OLEDs) work very much like LEDs, except that the emissive (N-type material) and conductive (P-type material) layers are made from organic

L’accès à ce site Web et l’utilisation de son contenu sont assujettis aux conditions présentées dans le site LISEZ CES CONDITIONS ATTENTIVEMENT AVANT D’UTILISER CE SITE WEB.

[1] Signatures of sulfur mass-independent fractionation (S-MIF) are observed for sulfur minerals in Archean rocks, and for modern stratospheric sulfate aerosols (SSA) deposited in

the pseudoknot, respectively, as the set of arcs whose left (resp. right) extremities are in the left (resp. right) part of the pseudoknot, where left and right parts are defined as

Then, in the spirit of [1] for a reaction diffusion equation with Allee effect and initial datum having heavy tails, we investigate the propagation phenomenon occurring in the

The proportion of negative children becoming positive was significantly different according to nutritional status (severe acute malnutrition 0/86 versus healthy children

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des