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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
cGEROMGroupeEtudesRemodelageOsseuxetbioMatériaux–LHEA,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:GEROM–LHEA,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
76 T.Benameuretal./Micron54–55(2013)75–81
Recently,anothermechanismhasbeenhighlightedininducing afavorablelocalmicroenvironmentfortumorgrowth:membrane- derivedvesicles(MPs)shedfromdifferentcelltypes(Peinadoetal., 2011).MPsaremembrane-derivedvesicles(diameter≤1m)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(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-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∼800lofbloodwerecollectedbyaspirationfrom theleftventricle.Bloodwasplacedincitratedtubesandcentrifuged at1900×gfor3minforseparationofplatelet-richplasmafrom wholeblood.Then,platelet-richplasmawascentrifugedat5000×g for4mintoobtainplatelet-freeplasma(PFP).Sixtymicrolitersof PFPwerefrozenandstoredat−80◦Cuntilsubsequentuse.Inorder 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.
Fornumerationstudies,8lofPFPwereincubatedwitheither 1lofspecificantibody(BioLegend,SanDiego,CA).After45minof incubationatroomtemperature,samplesweredilutedin300lof saline.AnnexinV(BioVision,Inc.,MountainView,CA)bindingwas usedtonumeratecirculatingphosphatidylserine-expressingMPs (2lofannexinV/5lPFP).Then,inordertoenumerateMPs,an equalvolumeofsampleandFlowcountbeadswereaddedandsam- pleswereanalyzedinaflowcytometer500MPLsystem(Beckman Coulter,Roissy,France)aspreviouslydescribed(Agounietal.,2008;
Mostefaietal.,2008).Flow-countfluorospheresconsistof10m polystyrenefluorospheresinanaqueoussuspensionmedium.Each fluorospherecontainsadyethathasafluorescenceemissionrange of525–700nmwhenexcitatedat488nm.Theconcentrationof eachlotofFlow-countfluorospheresisderivedfrommultiplerepli- cateanalysesonaCOULTERparticlesizeanalyzerperformedby themanufacturer.EachlotofFlow-countfluorosphereshasaspe- cificconcentrationoffluorospheres.Whenidenticalvolumesofa sampleandFlow-countfluorospheresareused,aratioofMPsin thesampletofluorospheresisestablished.Sincetheconcentration offluorospheresisknown,theabsolutecountoftheMPscanbe automaticallydeterminedbytheMXPsoftware.
Fig.1.FlowcytometricanalysisofcirculatingMPs.(A)CirculatingMPsfromcontrol(CTL),earlyMMstage(EMM)andlateMMstage(LMM)miceandFlowcountbeads (beadsregion,10mdiameter)arevisualizedinasidescatter(SS)/forwardscatter(FS)logarithmicrepresentation.MPsaredefinedaseventswithsize0.1–1mgatedin 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(20g)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.Theywerethendehydratedingradingconcen- trationofethanolbetween−20◦Cand−40◦C.Embeddingwasdone inLowicrylK4M(Polysciences,Paris)andpolymerizedat−40◦C 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
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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
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
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).
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