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

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Plasma cells release membrane microparticles in a

mouse model of multiple myeloma.

Tarek Benameur, Daniel Chappard, Elodie Fioleau, Ramaroson

Andriantsitohaina, Maria Carmen Martinez, Nicolas Clere, Hélène

Marchand-Libouban

To cite this version:

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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,∗

a_L’UNAM_Université,_Angers,_France b_INSERM_U1063,_Angers,_France

c_GEROM_Groupe_Etudes_Remodelage_Osseux_et_{bioMatériaux}_–_LHEA,_IRIS-IBS_Institut_de_Biologie_en_Santé,_CHU_d’Angers,₄₉₉₃₃_Angers_Cedex,_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. ElectronmicroscopyimmunohistochemistryandWesternblottingusingCD138wereperformedon BM-derivedMPs.AtEMM,MPscirculatinglevelwassigniﬁcantlylowerversuscontrols.InLMM,asigniﬁcant increaseofthetotalMPnumberfromplasmawasobservedversuscontrols.Characterizationofcirculating MPsshowedanincreaseofleukocyte-anderythrocyte-derivedMPs.InLMM,serumM-proteinwas correlatedwithcirculatingMPnumber.BM-derivedMPsincreasedinLMMandexpressedCD138. Anti-CD138coupledwithnanobeadslocalizedattheMPsurface.Thereisevidenceofanassociationbetween increaseofMPsandMMdevelopment;theresultsunderscoretheparticipationofplasmacell-derived MPsoriginatingfromBM.

1. Introduction

Multiplemyeloma(MM)isaBcellmalignancycharacterized bymonoclonalproliferationofplasmacellsinthelocalbone mar-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 levelofboneremodelinghasbeenshownusingacombined ani-malmodelinwhich ovariectomywasassociated toinjectionof 5T2MMplasmacellsintheC57BL/KaLwRijmouse(Liboubanetal., 2003).Osteoclastsrespondtoavarietyofcytokinesandgrowth

∗ Correspondingauthorat:GEROM–LHEA,IRIS-IBS,CHUd’Angers,49933Cedex, France.Tel.:+33244688344.

E-mailaddress:helene.marchand-libouban@univ-angers.fr

(H.Marchand-Libouban).

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

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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/or pathophysio-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).Other mem-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(24◦Cand12h/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-proteinandwere eutha-nizedafter10–12weeksbycervicaldislocation.Femursandtibias weredissected,cleanedofsurroundingtissuesandBMwasflushed inDulbecco’smodifiedessentialmedium(DMEM.mod,GIBCO,Life Technologies,France)supplementedwithpenicillin–streptomycin, amphotericin–fungizoneandpyruvate.BMcellswerewashedonce inDMEM.mod.Mononuclearcellswereisolatedbya Lympholyte-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).Theinjectedmiceweredividedin2groups accord-ingtothetimeofsacrifice.At6weeks,correspondingtotheearly stageofMMdevelopment,14micewerebledbeforebeing sacri-ficedbycervicaldislocation.Thesemiceconstitutedtheearlystage MMgroup(EMM)andwereusedtoquantifyandcharacterizethe phenotypeofcirculatingMPs.Attheendstageofthedisease(10–12 weeks),whenosteolysiscanbeevidenced onX-rayimages,the remaining30micewerebledbeforebeingsacrificedbycervical dislocation.ThesemiceconstitutedthelatestageMMgroup(LMM) andwereusedtoquantifyandcharacterizethephenotypeof cir-culatingMPs(seebelow).Twenty-twomicefromtheLMMgroup werealsousedtoisolateMPsfromtheBMoffemursandtibias. The14non-injectedmice(16–18-week-old)werebledas previ-ouslydescribedtoquantifyandphenotypecirculatingMPs.Eight ofthe14micewerealsousedtoisolateMPsfromBM.

Twoadditionalmiceinjectedwith5THLcellsweresacriﬁcedat theendstageofthediseaseandwereusedfortransmissionelectron microscopy(TEM)observationsandimmunohistochemistryof BM-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−80◦_C_until_subsequent_use._In_order

topelletMPsforinvitrostudies,circulatingMPswereconcentrated fromPFPbythreeseriesofcentrifugationsat21,000×gfor45min andre-suspendedinsalineandstoredat4◦Cuntilsubsequentuse. 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.

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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 orlargecellfragmentsby cen-trifugationasdescribedabove.Thewashingmediumfromthelast supernatantwasusedascontrol.

2.6. Westernblottingatendstageofdisease

ProteinanalysisbyWesternblottingwasperformedon homog-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 1h30minat20◦C.Theywerethendehydratedingrading concen-trationofethanolbetween−20◦_C_and₋₄₀◦_C._Embedding_was_done

inLowicrylK4M(Polysciences,Paris)andpolymerizedat−40◦_C

byUVlight(=360nm)(AFS,Leica).Sectionsof60nminthickness wereperformedusinganUltracutSmicrotome(LeicaUltracutE) andcollectedoncoppergrids.

AfterrinsingwithPBSbuffer,gridswereincubatedin50mM NH4Clfollowedby1%bovinealbumin.Goatpolyclonalanti-mouse

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rinsedwithPBS,contrastedwithuranylacetate(saturatedin dis-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,alinearregressionanalysiswas per-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,thetotalnumberofcirculatingMPswas signif-icantlyreducedby∼30.5%comparedtocontrolmice(Fig.1B).Incontrast,atLMM, asigniﬁcantincreaseofthetotalnumberofMPsfromplasmawasobserved com-paredtocontrolmice.CirculatinglevelofMPswasfoundsigniﬁcantlyhigherat LMMcomparedtoEMM.

PhenotypiccharacterizationofthecellularoriginofMPsshowedsigniﬁcant increasesofthecirculatinglevelsofMPswithaprocoagulantpotential(Annexin V+_)-,_and_those_of_platelet_(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)by BM-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

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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-enrichedwithhomogenoussizeanddifferent pro-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 iswellestablishedthatthesepatientshaveathigherriskof devel-opingarterio-venousthrombosis(Kristinsson,2010).Itshouldbe

notedthatthe5TMMmodelmimicsthemarrowenvironmentand bonelesionsasobservedinhumansbutotherclinical character-isticsofthediseasesuchasthrombosisorrenallesionsarenever observed(Liboubanetal.,2006).Indeed,neitherplatelet-derived MPsnorMPsexpressingphosphatidylserine(i.e.witha procoag-ulantpotential)wereincreasedinthepresentstudy.Inaddition, ahighlevelofMPsincancerhasbeenshowninvitroandinvivo tobecorrelatedwithtumoraggressiveness(Castellanaetal.,2010; Ginestraetal.,1999).Theseobservationsareinagreementwithour resultsaswefoundahighlevelofMPsatLMM.Ofimportanceis thefactofthesigniﬁcantcorrelationbetweencirculatingMPsand M-protein.Thus,circulatingMPsmaybeusedasgooddiagnostic markersofthediseaseevolution.

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accordingtothestageofthedisease,weexpectedtofind interme-diaryresultsbetweencontrolsandEMM.Wealsoexpectedtofinda phenotypicswitchbetweenEMMandLMMaspreviouslyreported inanother hematologicmalignancy (Ghosh etal., 2010).In the presentmurinemodel,theEMMwascharacterizedbytheonsetofa verysmalllevelofM-protein(notshown),theabsenceofany oste-olyticlesiononX-raysbutamassiveinfiltrationoftumorplasma cellsinsomebones(Liboubanetal.,2004).Thefactthatearlystage ofmyelomadecreasedlevelsofcirculatingMPsisprobably associ-atedwithbloodcellcountintheseanimals.Thus,alterationson thenumber andthefunctionof bloodcells havebeenreported inmultiplemyelomapatients.Concerningplateletsandredblood cells,thesealterationsareassociatedwithhypercoagulablestate butalsowiththrombocytopeniaandhemorrhagiccomplications. Theseeffects maybe associatedwithchanges in hematological cellsderivedfromBM(inducinganemiaandthrombocytopenia) (AlbarracinandFonseca,2011)butalsowithchangesinblood vis-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.WemanagedtoobserveMPsbyTEMwith immuno-golddetection,coupledtowesternblotting.CD138wasstrongly expressedonMPsisolatedfromtheBMmicroenvironment, con-firmingthehypothesisthatpartofthemwereshedfromthesurface ofmalignantplasmacells.CD138isatransmembraneheparin sul-fateproteoglycanwithanexpressionhighlyspecificofplasmacells; itisnotexpressedonBorTlymphocytes(Wijdenesetal.,1996). Becauseofsuchaspecificexpression,CD138isusedasastandard markerinthediagnosisofMM(Batailleetal.,2006).Freecirculating CD138levelisalsocorrelatedwithtumormassanditisimplicated inthecell/celladhesionprocess,inphysicalinteractionwith sol-ublefactorsandmoleculesformtheextracellularmatrix(Bataille etal.,2006;Dhodapkaretal.,1997).

In the 5T2MM model, differences have been shown in the expressionofseveralmarkerslikeCD45andCD138atthestage levelofthedisease(Asosinghetal.,2003).Atthequiescentstage (correspondingtoourEMM),mostMMcellsarehighlyinvasive andexpressCD45+_and_CD138−_,_whereas_at_the_LMM,_MM_cells

aremoremature,lessinvasivewithaCD45−,CD138+_phenotype.

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

5. Conclusion

Inconclusion,weprovideevidencethat,inLMMstage, circu-latinglevelsofMPsfrommicewereincreasedandthatlevelsof BM-derivedMPsexpressingCD138arecorrelatedwiththe pathol-ogy severity.These MPs may play a critical role as vectors of deleteriousbiologicalmessagesleadingtogrowthtumor.This ﬁnd-ingmaysuggestthatMPscouldbenewactorsoftheviciouscircle involvedinMM.

Acknowledgments

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

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