Altered bone microarchitecture and gene expression profile due to calcium deficiency in a mouse model of myeloma

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Micron

j o u r n a l h o m e p 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

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Altered bone microarchitecture and gene expression proﬁle due to calcium deﬁciency in a mouse model of myeloma

Hélène Libouban

^∗

, Daniel Chappard

GEROMGroupeEtudesRemodelageOsseuxetbioMatériaux–IRIS-IBSInstitutdeBiologieenSanté,Universitéd’Angers,CHUd’Angers,49933AngersCedex, France

a r t i c l e i n f o

Articlehistory:

Received26September2016

Receivedinrevisedform24January2017 Accepted30January2017

Availableonline24February2017

Keywords:

Dietarycalcium Hyperperathyroidism Multiplemyeloma Osteolysis

X-raymicrotomography

a b s t r a c t

Itisnotclearwhypatientswithanindolentformofmultiplemyeloma(MM)developintoanaggressive formwithpoorprognostic.Weinvestigatedtheeffectofadietarycalciumdeﬁciencyontumorgrowth, osteolysisandgeneexpressioninthe5T2MMmurinemodel.

Twogroups ofC57BL/KaLwRijmicereceived5T2MMcellsand startedadietwithnormal(0.8%;

“normal-Ca-MM”)orlowcalciumcontent(0.05%;“low-Ca-MM”).Twocontrolgroups(without5T2MM cells)receivedeitheranormalorlowcalciumdiet(normal-Caandlow-Cagroups).Tumorgrowth,oste- olysisandmarrowgeneexpressionoftheWntpathway,RANKLandMIP-1␣weremonitoredat6,8and 10weeks(w)aftercellinjection.

Inlow-Camice,serumlevelofPTHwashigherafter10w;microCTshowedtrabecularbonelossand decreaseofcorticalthicknessatthetibia.AhigherM-proteinlevelwasevidencedat10wand4mice developedparaplegiaat8/9winlow-Ca-MMgrouponly.Numerouscorticalperforationsofthetibiawere observedinMMgroupswithamarkeddecreaseincorticalthicknessinlow-Ca-MM.At6w,osteoclast numberfromtheendosteumwassigniﬁcantlyhigherinlow-Ca-MMcomparedtonormal-CaMM.This observationwasnotfoundat8and10w.MicroCTofthelumbarvertebraeshoweddramaticbonedestruc- tioninthelow-Ca-MMgroup.qPCRrevealednodifferenceinRANKLexpressionwhereasdifferenceswere obtainedintheexpressionofLrp5/Lrp6andMIP-1␣from6w.

Alowcalciumdietinducedhigherbonedestructioninthetibiaandvertebraassociatedwithanearlier decreaseinboneformationlevelandahigherincreaseinboneresorptionlevelatearlytimeintheMM development.

1. Introduction

Multiplemyeloma(MM)isahematologicalmalignancychar- acterizedbyproliferationofmalignant plasmacellsinthebone marrow.OsteolysisisoneofthemainclinicalconsequencesofMM andconcerns90%ofthepatients.OsteolyticfociinMMpatients induceseriousclinicalmanifestationssuchasbonepain,fractures oflongbones,vertebraand/orhypercalcemia.Bonefracturesoccur in60%ofpatients(Roodman,2009).Moreover,bonefractureshave ahigherincidenceinMMthaninothercancer(breast,prostateand lungcancer)withahighfrequencyofbonesmetastases.Patho- logicalfracturesinMMareassociatedwitha20%increasedrisk ofdeath(Saadetal.,2007).In thelastdecade,themediansur- vival ofMM patientshasbeenimproveddue tothe apparition

∗Correspondingauthor.

E-mailaddress:[email protected](H.Libouban).

of new treatments. The median survival is now approximately 5–8yearsandreaches10yearsforpatientswithlow-riskcytoge- neticabnormalities(Avet-Loiseauetal.,2012;ChesiandBergsagel, 2015).Boneresorption,duetoanincreaseinosteoclastnumber, isincreasedinMMpatientsinthevicinityofplasmacellnodules (Batailleetal.,1989).Secretionofnumerouscytokinesbymalig- nant plasmacells and the bone marrowmicroenvironmentare responsibleforanincreasedosteoclastogenesisinMM(Roodman, 2009).Severalcytokineshave beenidentiﬁed toplaya keyrole inosteolysis:tumornecrosisfactor alpha(TNF␣,interleukin-1␤ (IL-1␤)),interleukin-6(IL-6),macrophageinﬂammatoryprotein- 1-alphaand beta(MIP-1␣andMIP-1␤)and ligandfor receptor activatorofnucleartranscriptionfactor-␬B(RANKL)(Katoetal., 2002;Xietal.,2016).InovertMM,adecreaseinboneformation isassociated,leadingtoanuncouplinginboneremodelingatthe laterstagesofthedisease.Inhibitionofosteoblastogenesisisdueto inhibitorsreleasedbyplasmacellsthatdepresstheWnt-signaling pathway:DKK1(Dickkopf-1)andSfrp2(Secretedfrizzled-related http://dx.doi.org/10.1016/j.micron.2017.01.009

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protein2)(Tianetal.,2003)oractonosteoblasticprecursors:hep- atocytegrowthfactor(HGF),interleukin-3(IL-3)andinterleukin-7 (IL-7)(Ehrlichetal.,2005;Standaletal.,2007).

Animalmodelscanhelp tounderstandthemolecular,cellu- larandtissueaspectsofthepathophysiologyofhumandiseases andtodevelopnewtherapeuticstrategies.Severalmurinemodels ofhumanMMhavebeendescribedintheliterature(seereviews in(Libouban,2015;Paton-Houghetal.,2015)).Amongthem,the 5T2MMmurinemodelwasdescribedintheC57BL/KaLwRijstrain (Radl et al.,1988,1979).It presentsthemost commonaspects ofthehumandiseasewithmoderategrowth,homingrestricted tothebonemarrowmicroenvironmentandosteolyticlesions.A monoclonalprotein (M-protein) is detected from 6 weeks (w) afterinjectionandosteolyticlesionsareradiographically visible at8w(Liboubanetal.,2004).The5T2MMmodelwasextensively usedtoevaluatetheeffectof antineoplasticand antiangiogenic drugs(Deleu etal., 2009a;Deleu etal., 2009b)orinhibitors of boneresorptionsuchasbisphosphonates(Croucheretal.,2003;

N’Diayeetal.,2015;Radl etal.,1985).Themodelisalsoofthe utpmostimportancetoelucidatethepathophysiologyofthedis- ease(Buckleetal.,2012;Vanderkerkenetal.,2000).Ithasbeen shownthatahighboneremodelinglevel(asinducedbyovariec- tomy in mice) accelerates plasma cell growth (Libouban et al., 2003).Similarly,otherfactorsknowntoincreaseboneremodeling havealsobeenfoundtoburstthegrowthofcarcinomatouscells (Hirbeetal.,2007;Ooietal.,2010;Zhengetal.,2008,2007,2011).

AcalciumdeficientdietstimulatesthegrowthofMCF-7human breastcancercellsinthemousebyinducingasecondaryhyper- parathyroidism(Zhengetal.,2007).Morerecently,itwasshown thatavitaminDdeficiencypromotesMDA-MB-231breastcancer cells inthemouse by alsoinducinga secondary hyperparathyroidism(Ooietal.,2010).Thesestudieshighlighttheimportanceof calciumintakeandboneremodelingontumorgrowthandosteoly- sis.Theyhaveanalyzedthepathophysiologicalmechanismsatthe cellandtissuelevelsinanimalmodelsofbonemetastasis;however,molecularalterationsin thebone microenvironmenthave receivedlittleconsideration. The aimof thepresent studywas toinvestigatetheeffectof adietarycalciumdeficiency onMM developmentatthetissue,cellandmolecularlevelsinthe5TMM model.Microcomputedtomography(microCT),histomorphome- tryand gene expression profileof the microenvironmentwere used.

2. Materialandmethods 2.1. Themultiplemyelomacellline

We have used a 5T2MM cell line (subclone 5THL) in C57BL/KaLwRijmiceaspreviouslycharacterized(Liboubanetal., 2004).Thesemalignantplasmacellscanbepropagatedintoyoung syngeneic mice by intravenous transfer of cells from invaded bonemarrow. Progressionof thediseasein recipientmicewas assessed by measuringthe serum M-proteinlevel. Around 6w post-injection,micehadadetectableserumM-proteinandwere sacrificedafter10–11wbycervicaldislocation.Femursandtib- ias were dissected, cleaned of surrounding tissues and bone marrow was flushed in Dulbecco’s modified essential medium (DMEM.mod., GIBCO, Life Technologies, France) supplemented withpenicillin-streptomycin,amphotericin-fungizone andpyru- vate(GIBCO).BonemarrowcellswerewashedonceinDMEM.mod.

MononuclearcellswereisolatedbyaLympholyte-Mcentrifugation gradient(Cedarlane,Hornby,Ontario,Canada)at450gfor25min.

Mononuclearcellswerethenwashedtwiceandcounted.Therecip- ientmicereceived1.5×10⁶cellsinthetailvein.

2.2. Animalsandstudydesign

Seventy-sixC57BL/KaLwRijfemalemice6–8-w-oldwereused forthefullstudy(Harlan,Gannat,France).Theywereacclimated foronewtothelocalvivariumconditions(24^◦Cand12h/12hlight darkcycle)wheretheyweregivenstandardlaboratoryfood(UAR, VillemoisonsurOrge,France)andwateradlibitum.Theanimals werehousedattheanimalfacilitiesofAngersUniversityMedical School(SCAHU).AllprocedureshavebeenapprovedbytheInstitu- tionalAnimalCareandUseCommitteeattheUniversityofAngers (Agreementnumber49028).Allproceduresweredoneinaccor- dancewiththe2013Frenchanimalprocedureactandunderthe supervisionofauthorizedinvestigators.

Twoexperimentalprocedureswereconducted.The firstone examinedthe modification of bone remodeling by dietary cal- ciumdeficiency incontrol animalsand used 20 C57BL/KaLwRij mice.Thesecondexperimentalprocedurecombinedthecalcium deficiency and the injection of 5T2MMcells in mice and used 56C57BL/KaLwRijmice.Thetwoexperimentalprocedureswere describedindetailsbelow.

2.2.1. Modiﬁcationofboneremodelingbydietarycalcium deﬁciency

Onday0,twentymicewererandomizedinto2groups(10ani- malsper group).The firstgroupwasgiven standard laboratory chowwithnormal calciumcontent(0.8%);theyconstitutedthe Normal-Cagroup.Thesecondgroupwasgivenacalciumdeficient laboratorychow(0.05%–SAFEAUGY,Augy,France);theyconsti- tutedtheLow-Ca group.At10w,blood sampleswerecollected byintracardiacpuncturetomeasureserumparathyroidhormone (PTH)levelandmicewerethensacrificedbycervicaldislocation.

2.2.2. Combinedmodel(dietarycalciumdeﬁciency+5T2MM cells)

Fiftymicewereinjectedwith5T2MMcellsinthetailveinand immediatelyplacedonthenormalorlowcalciumdietasabove.

Theyconstitutedrespectively thenormal-Ca-MMgroup(n=25) andthelow-Ca-MMgroup(n=25).Eachgroupofmicewasdivided into3subgroupstoperformatime-dependentanalysisofosteoly- sisbyhistomorphometryandquantitativePCR(qPCR).Micewere sacrificedbycervicaldislocationat6,8and10w.Mice,whichdevel- opedparaplegiabeforetheendofeachtimepoint,weresacrificed forethicalreason.Sixadditionalmiceweresacrificedatthebegin- ningofthestudyatw0andwereusedascontrol(control-w0group) forbiochemicalandhistomorphometricanalysis.

2.3. Bonetissuecollection

Rightfemursandtibiasfromallmiceweredissected,cleaned ofsofttissue,andconservedinanethanolbasedﬁxative+4^◦Cfor furtheranalysis.LumbarvertebraefromL2toL4werealsodissected formiceinexperimentalprocedure2,slightlycleanedofsofttissue andconservedinanethanolbasedﬁxativeforfurtheranalysis.Left femursofmice,intheexperimentalprocedure2,wereusedfor qPCRanalysis.

2.4. MeasurementoftheM-proteinlevelintheserum

Progressionof MM wasmonitored bymeasuring theserum M-protein (IgG2a␬) concentration using agar electrophoresis (Hydragelprotéine,SEBIA,IssylesMoulineaux,France).Thelevel ofM-proteinwasexpressedasthepercentageofthe␥-peakusing imageanalysis(NIHimageforWindows)ontheelectrophoretic diagram.Bloodwascollectedfromthetailveinat6wforall5T2MM groupstocheckthepresenceoftheM-protein.Forkineticevolu- tionoftheM-proteinlevel,bloodwasalsocollectedat8and10w

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inthetwogroupsmaintainedupto10w.Bloodwascollectedat thetimeofsacriﬁceformicethatdevelopedparaplegiaanddidnot reach10w.

2.5. Followupofosteolyticlesions

Presenceofosteolyticbonelesionswascheckedondigitalradio- graphsofthepelvisandhindlegsobtainedwithaFaxitronmachine at26kV(FaxitronEdimex,Angers,France).Radiographswereper- formedatthetimeofsacriﬁceinallgroups.

2.6. X-raymicrotomography

MicroCTwasperformedontheproximaltibiaandonthelumbar vertebrae(L2toL4)withaSkyscan1172microtomograph(Bruker microCT,Kontich,Belgium).Bonesamplesweretransferredtoan Eppendorftubecontainingtheﬁxativeandpolyethylenefoamwas usedtoensureimmobilizationofthesample.Thetubewasafﬁxed ona brassstubwithplasticine and analyzed ata resolution of 5.25␮mperpixelforthetibiaand13.38␮mforthevertebrae.After segmentation,the3Dmodelswereconstructedfromthestackof 2Dimageswithasurface-renderingprogram(Ant,release2.0.5, Skyscan).

3D measurements were obtained with the CtAn software (release2.5,Skyscan).Foreachtibiasample,200sectionimages wereobtainedandcorrespondedtothecumulatedheightofthepri- maryandsecondaryspongiosa(1.05mm).Trabecularboneanalysis wasperformedontheL2vertebralbody.

The following 3D parameters were calculated: trabecular volume(BV/TV,in%),trabecularnumber(Tb.N,inmm⁻¹)andtra- becular separation (Tb.Sp,in ␮m). Corticalbone measurements wereperformedon2Dsectionsofthetibiaatthemetaphyseal region(1.05mmunderthegrowthcartilage)andatthediaphyseal shaft(3.14mmunderthegrowthcartilage).Meancorticalthickness (Ct.Th,in␮m)wasdeterminedwiththeCtAnsoftware.

2.7. Histology

Histologywasperformedonundecalcifiedfemursafterembed- dinginpoly(methylmethacrylate).Foreachmice,fournonserial sections (50␮m apart, 7␮m in thickness) were cut dry on a heavy-duty microtome equipped with tungsten carbide knives (LeicaPolycutS;Rueil-Malmaison,France,with50^◦ knives)and stainedwithGoldner’strichromeortartrateresistantacidphos- phatase(TRAcP)forthehistochemicalidentificationofosteoclasts (Chappard,2014).Theosteoclastnumberwasdeterminedatthe endostealsurface(on5mmfromthegrowthplate) atamagni- ficationof ×200ontheTRAcP-stainedsections.The percentage areaofthemetaphysisinvadedbyplasmacellswasmeasuredon histologicalsectionsofthefemur.

Fig.1.Effectofdietarycalciumdietincontrolmiceafter10w.(A)X-raymicroCT reconstructionsoftheproximaltibiafromnormal-Ca(a)andlow-Ca(b).(B)3D microCTmeasurementsofBV/TVonproximaltibia.(C)2DmicroCTmeasurements ofcorticalthicknessinthemetaphysealregionandthediaphysealshaftofthetibia.

2.8. PTHandcalciumassays

Blood calciumwas determinedat 0 and ateuthanasia after exsanguinationinallgroupsona TechniconSMA analyzer.PTH wasmeasuredat0andat6,8and10winthetwocontrolgroups (normal-Ca;low-Ca)andinthetwoMMgroups(normal-Ca-MM;

low-Ca-MM).IntactPTHwasdosedusingtheMouseIntactPTH ELISAKit(#60–2300,ImmunotopicsInternational,SanClemente, CA),asrecommendedbythesupplier.

2.9. QuantitativeRT-PCRanalysis

Theleftfemurof eachmousewasdissected, cleanedofsur- rounding tissues and bone marrow was ﬂushed in Dulbecco’s modiﬁed essential medium.Bone marrowcells werelysed and proceededforRNAextractionusingtheRNeasyminikit(Qiagen,

Table1

Biochemicalanalysisinthedifferentgroups.

Serumassay Control-w0 Controlgroups MMgroups

Normal-Ca Low-Ca Normal-Ca-MM Low-Ca-MM

Calcium(mmol/L) 2.40±0.02

w10 2.32±0.03^* 2.34±0.02^* 2.38±0.02 2.31±0.03^*,b

PTH(pg/mL) 39.21±6.05

w6 29.5±2.02 106.13±23.17^*,a 26.09±9.70 92.44±28.75^*,b

w8 46.76±16.89 112.69±21.4^*,a 36.85±4.57 197.43±42.45^*,b

w10 19.37±3.41^*,,# 94.65±27.67^*,^a 42.87±6.26 76.16±21.02^*,#

*vscontrol-w.

vsw6.

#vsw8.

avsNormal-Ca.

bvsNormal-Ca-MM.

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France)followingthemanufacturerprocedure.Afterelution,total RNAwasaliquotedandstoredat−80^◦Cuntiluse.Qualityofthe RNAsampleswasexaminedonadenaturingagarosegelandRNA concentrationsweredeterminedbyspectrometry.One␮goftotal RNA sample was reverse transcripted using random hexamer primersandSuperScriptIIreversetranscriptase(SSII)(Invitrogen, France).Primerswere designedusing Primer3software (http://

www.ncbi.nlm.nih.gov/tools/primer-blast/).Thefollowingprimers were used: MIP-1␣ (forward 5-CCTTGCTGTTCTTCTCTGTACC- 3 and reverse 5-AAATGACACCTGGCTGGG-3);

RANKL (forward 5-TGTACTTTCGAGCGCAGATG- 3 and reverse 5-CCCACAATGTGTTGCAGTTC-3);

Lrp5 (forward 5-GGTCACCTGGACTTCGTCAT-3 and reverse 5-TCCAGCGTGTAGTGTGAAGC-3); Lrp6 (forward 5-TCAACACTTCCCTTGGTTGG-3 and reverse 5-TGTGAGGGATCTTGTCTTCC-3); Dkk1 (forward 5-CTCTGCTAGGAGCCAGTGC-3 and reverse 5- CGCACTCCTCATCTTCAGC-3). Ampliﬁcation was performed in duplicateswith iQSYBR Supermix (Bio-Rad, France) and using a Chromo4^TM real time system (Bio-Rad).Thermal cycling was initiatedwith10minincubationat95^◦Cfollowedby40cyclesof 15sat95^◦C,11sat55^◦Cand22sat72^◦C.Geneexpressionwas calculatedusingthecomparativeC_Tmethodwiththenormaliza- tionofthetargetgenesto6housekeepinggenes(G3PDH,HPRT1, B2M,ACTB,RPS18andHSPCB).

2.10. Statisticalanalysis

StatisticalanalysiswasdonewithSYSTATstatisticalsoftware, release13.00.05(Systat,SanJosé,CA).Alldatawerereportedas mean±standarderrorofthemean(SEM).Signiﬁcantdifferences betweengroupswereanalyzedbyanon-parametricKruskal-Wallis analysisofvariancefollowedbytheConover-Inmantestforallpair- wisecomparisons.Thecomparisonbetweennormal-Cavslow-Ca (incontrolor MMgroups)wasanalyzedbythenon-parametric Mann-WhitneyUtestateachstudytimepoint.Differenceswere consideredassigniﬁcantwhenp<0.05.

3. Results

3.1. Dietarycalciumdietinducedbonelossduetosecondary hyperparathyroidismincontrolmice

Tenweeksof low calciumdietinduced trabecularbone loss andthinningofthecorticesasevidencedon3DmicroCTmodels (Fig.1A).Inthelow-Cagroup,morphometricanalysisrevealeda significantdecreaseinBV/TV(−14.5%,p<0.05)andcorticalthick- ness at the metaphysis (−28.2%, p<0.0001) and the diaphysis (−27.4%,p<0.00001)(Fig.1B–C).Biochemicalresultsforthecontrol groupsappearonTable1.Trabecularandcorticalbonelosseswere associatedwithanincreasedlevelofPTHreflectingthesecondary hyperparathyroidism. No significant differences were observed betweenthetwogroupsafter10w.

3.2. EffectoflowcalciumdietonMMprogression

Duringthecourseofthedisease,detectionoftheM-proteinwas evidencedat6winboth5T2MMgroupswithnodifferenceinthe amountoftheprotein.Inthelow-Ca-MMgroupthekineticbecame differentafter8wwhen an increasedlevel wasobtainedmore rapidlythaninthenormal-Ca-MMgroup.At10w,thedifference betweenthe2groupswassigniﬁcant(p<0.05).Forthelow-Ca- MMgroup,anadditionaltimepointwasaddedonthecurveat9w becauseseveralmicedevelopedparaplegiaandweresacriﬁcedfor ethicalconsiderations.Table2showstheincidenceofparaplegia inthe2groupsof5T2MMmice.Inthetwogroups,noparaplegia

Table2

IncidenceofhindlimbparalysisinnormalandlowcalciumdietMMgroupsat6,8 and10weeks.

Incidenceofhindlimbparalysis 6weeks 8weeks 10weeks

Normal-Ca-MM 0%(0/8) 0%(0/8) 0%(0/9)

Low-Ca-MM 0%(0/8) 50%(4/8) 56%(5/9)

wasobservedat6w.Fiftypercentofparaplegiawereobservedat 8wand56%within9–10winthelow-Ca-MMgroup.Noparaple- giawasobservedinthenormal-Ca-MMgroup.X-raysperformed atthetimeofsacriﬁceshowedminimal“punched-out”osteolytic lesions(preferentiallylocalizedatthetibialcrest)innormal-Ca- MMandlow-Ca-MMgroupssacriﬁcedat8w(Fig.2B:a,c).At8w, osteolyticlesionswereobservedinthelow-Ca-MMgroupatthe tibiaandfemur;theywereassociatedwithamarkedthinningof thecortices.At10w,thenumberofosteolyticlesionswasincreased inthelow-Ca-MMgroupcomparedtothenormal-Ca-MM(Fig.2B:

b,d).BiochemicalresultsfortheMMgroupsappearonTable1.

Brieﬂy, calcemiawassigniﬁcantly decreased inthelow-Ca-MM groupvsthenormal-Ca-MMgroupafter10w.PTHwasconsider- ablyincreasedat6,8and10winthelow-Ca-MMvsinitialvalues at0w.Inthenormal-Ca-MMgroup,nochangeoccurredduringthe timecourseofthestudy.

3.3. Alowcalciumdietinducedagreaterbonelossatthetibia

ThemicroCTmodelsofthetibiaclearlyevidencedthedifferent progressionofosteolyticlesionsupontimebetweenthenormal- Ca-MM(Fig.3A:a–c)andlow-Ca-MMgroups (Fig.3A:d-f).The quantitativemeasurementsareillustratedonFigs.3B–Dand2D corticalmeasurementsonFig.3E–F.

At6w,thetibiahadaratherwellpreservedtrabecularnetwork inthenormal-Ca-MMgroupwithoutcorticalperforations.BV/TV didnotdifferwhencomparedwiththecontrol-w0group;however, Tb.NdecreasedandTb.Spincreasedsigniﬁcantly,revealinganearly disorganizationofthetrabecularmicroarchitecture.Thetrabecular networkwasmarkedlyalteredinthelow-Ca-MMgroup(Fig.3A:

d);BV/TVwassignificantlyreduced(−62%,p<0.001)comparedto normal-Ca-MMgroupat6w.ThedecreaseofBV/TVat6wwasasso- ciatedwithasignificantdecreaseofTb.Nandasignificantincrease ofTb.Sp.Thinnercorticeswereobservedinthelow-Ca-MMgroup inbothmetaphysealanddiaphysealregionsasevidencedbyasig- nificantreductioninCt.Th(Fig.3E–F)comparedtonormal-Ca-MM groupat6w.On thecontrary,onlysignificantdifferenceswere observedforCt.Thfromthemetaphysisat8and10wbetween normal-Ca-MMandcontrol-w0group.At6w,the3Dmodelfrom thelow-Ca-MMgroupclearlyshowedanon-smoothappearance ofendostealsurfacecomparedtothenormal-Ca-MMgroup.This differenceintheappearanceoftheendostealsurfacereflecteda moreerodedsurfaceinthelow-Ca-MMgroup.Theosteoclastnum- berwassignificantlyhigherat6winlow-ca-MMgroupcompared tonormal-CaMMgroup(resp.22.4±4.2vs10.6±0.8,p<0.05).

Theprogressionofosteoclastnumberispresentedasa3Dgraph combinedwithevolution ofthebone marrowinvasion (Fig.4).

At6w,plasmacellinvasionofthefemoraldiaphysiswashigher inthelow-Ca-MMgroupcomparedtothenormal-Ca-MMgroup (resp.64.5%±17.3vs43.1%±15.1)butthisincreasedidnotreach statisticalsigniﬁcance.

At 8w,BV/TV decreased significantly in thenormal-Ca-MM groupwhencomparedtothecontrol-w0group;Tb.Spwassignifi- cantlyhigherandTb.NwassignificantlylowercomparedtoTb.Sp andTb.Nmeasurementsat6w.Bonedestructionwasevidenced in3Dmodelsofthenormal-Ca-MMgroupwiththepresenceof trabecular destruction and small cortical perforations (Fig. 3A:

b).Osteolyticlesionsweremaximizedinthelow-Ca-MMgroup

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Fig.2. Effectoflow-Calciumdietonmyelomaprogression.(A)EvolutionoftheserumM-proteinlevel(expressedin%)duringthetimecourseofMMdisease.Asigniﬁcant higherM-proteinlevelwasfoundat10winthelow-Ca-MMgroup(*p<0.05).(B)X-rayimagesofthehindlimbofmicefromnormal-Ca-MMgroupat8w(a)and10w(b);

fromlow-Ca-MMgroupat8(c)and10w(d).Osteolyticlacunae(→)areevidencedat8winnormalandlowcalciumdietmice.Notethethinningofthecortices(→)with lowcalciumdietat8w.Greaterbonedestructionisobservedwithlowcalciumdietat10w(d).Lacunaappearedsmallerbutmorenumerousinthetibialcrest(→).

Fig.3. Effectoflow-Calciumdietonbonelossatthetibia.(A)X-raymicroCTreconstructionsoftheproximaltibiafromnormal-Ca-MMgroup(a–c)andlow-Ca-MMgroup (d–f)resp.at6,8and10w.(B–D)3DmicroCTmeasurementsofresp.BV/TV,Tb.SpandTb.Nonproximaltibia.(E–F)2DmicroCTmeasurementsofcorticalthicknessinresp.

themetaphysealregionandthediaphysealshaftofthetibia.

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Fig.4. 3Devolutionoftheosteoclastnumberfromtheendosteumandbonemarrow invasionat6,8and10w.Normal-Ca-MMgroup();Low-Ca-MMgroup(䊉).

(Fig.3A:e).NumerousTRAcPpositiveosteoclastswereobserved inbothgroups.Theosteoclastnumberfrombothgroupswassig- niﬁcantlyincreasedat8wcomparedto6w.Comparisonofboth groupsat8wdidnotshowsigniﬁcantdifferencebetweenlow-Ca andnormal-CaMMgroups(resp.33.1±2.8vs39.2±5.2)(Fig.4).

At10w,severetrabecularandcorticalbonedestructionwere evidencedinbothMMgroupswithanincreaseincorticalperfora- tions.However,lesionsweremorepronouncedinthelow-Ca-MM group(Fig.3A:d,f).Theosteoclastnumberoflow-Caandnormal- CaMMgroupswasnotsigniﬁcantlydifferent(resp.38.4±5.1vs 39.9±2.1) andwassimilartothenumber of osteoclastsat8w (Fig.4).At10w,thebonemarrowwasentirelyinvadedbyplasma cellsinthelow-Ca-MMgroup(100%);inthenormal-Ca-MM,the percentageofbonemarrowinvasionwas82.5±17.5%.

3.4. Alowcalciumdietinducedseverevertebralbonedestruction

3Dmodelsoflumbarvertebrashowedprogressivedisappear- anceoftrabecularbonefrom6to10winthelow-Ca-MMgroup (Fig. 5A:d–f). Lumbarvertebra in thetwo groups showed two anatomicholesattheposteriorsideofthelumbarvertebralbody;

theseholesarenotduetothetumorastheyareobservedincontrol mice(datanotshown)andcorrespondtovascularsinuses.Corti- calperforationswereobservedfrom8winthelow-Ca-MMgroup inducinganenlargementoftheanatomicalholes(Fig.5A:e–f).Sim- ilarcorticalperforationscouldbeobservedinthenormal-Ca-MM groupbutonlyattheterminalstageofthedisease(Fig.5A:c).

Morphometric analysis showed no signiﬁcant difference in BV/TV,Tb.NandTb.Spinthenormal-Cagroupcomparedtocontrol- w0at6,8and10w(Fig.5B–D).Inthelow-Ca-MMgroup,BV/TV decreased signiﬁcantly from 6w (w6: −33.65%, p<0.01; w10:

−57.61%,p<0.01)comparedtothenormal-Ca-MMgroup.Reduc- tionofBV/TVwasassociatedwithasigniﬁcantdisappearanceof trabeculaeasfrom6w(reducedTb.NandincreasedTb.Sp).

3.5. Alowcalciumdietmodiﬁedgeneexpressionproﬁle

AnincreaseinMIP-1␣expressionwasfoundinthebonemar- rowof micefrom thelow-Ca-MM groups at 6 and 8wwith a

ratiorespectivelyof1.6and3.9comparedtotheexpressioninthe normal-Ca-MMgroup(Fig.6A).Ontheopposite,expressionofMIP- 1␣wassignificantlylowerat10winthelow-Cagroupcompared tothenormal-Cagroup.Therewasaninsignificantreductioninthe expressionofRANKL(Fig.6B)at6and8wSignificantdifferences wereobtainedintheexpressionofsomegenesoftheWntpath- way.TheLrp5co-receptorwassignificantlylessexpressedat6w inthelow-Ca-MMgroupcomparedtothenormal-Ca-MMgroup witharatioof−2.6(Fig.6C).ExpressionofDkk1wassignificantly reducedat6and8winthelow-Ca-MMgroupwithratioofrespec- tively−2.5and−4.5(Fig.6D).NosignificantdifferencesofDkk1 expressionwereobservedbetweenthetwogroupsat10w.

4. Discussion

Theuseofadietarycalciumdiettoincreaseboneremodeling wasbasedonpreviousdatainboth animal andclinicalstudies whichshowedanincreaseinboneresorptionduetolowcalcium intake(Eklou-Kalonjietal.,1999;Iwamotoetal.,2004;Lips,2001;

Setoetal.,1999).Inthepresentstudy,along-termcalciumdietdeﬁ- ciencyinducedasustainedsecondaryhyperparathyroidismwhich, inturn,causedamajorbonelossatthetrabecularandcorticallevel.

Secondaryhyperpathyroidismisduetoextrinsicchangescausing adrasticdecreaseinplasmaionizedcalciumthusinducingPTH secretiontocorrectplasmacalcium.Iflowcalciumavailabilityis notcorrected,patientsarecharacterizedbyalongtermhighlevel ofparathyroidhormone(Fraser,2009).Thisisinaccordancewith dataobtainedfromthepresentstudyandotherswherePTHlevel ismaintainedhighevenuntil10woflowcalciumdiet(Iwamoto etal.,2004).TheincreaseinPTHduetoalowcalciumdietisrapid andobservedassoonas12hafterdietinitiationinrats(Setoetal., 1999).Shorttermstudyofalowcalciumdietinrats,showedthat therapidincreaseinPTHinducedrapideffectsonboneremodel- ing;thenumberofosteoclastsandosteoblastsalongthetrabecular bonesurfaceswereincreasedafter1and2days,respectively(Seto etal.,1999).Suchahighboneresorptionlevelismaintaineddur- ingtheentireperiodofthelowcalciumintake(Eklou-Kalonjietal., 1999;Iwamotoetal.,2004).Moreover,anincreaseinosteoclast number was shown in both cancellous and endosteal surfaces, in accordance withthebone loss atthese two bone envelopes notedinthepresentstudy(Xiong etal.,2014).Whenconsider- ingboneformation,increasesinosteoblastnumberaremaintained throughtheentiretimeofthestudyinanimalswithalowcalcium dietbut boneformation andmineralizationrates aredecreased (Eklou-Kalonjietal.,1999;Iwamotoetal.,2004;Setoetal.,1999).

Biochemicalmarkersofboneformation(alkalinephosphatase,c- propeptides)arealsoincreasedduringalowcalciumdietinanimals andhumans(Eklou-Kalonjietal.,1999;Harrisetal.,2001;Meunier etal.,2005).Secondaryhyperparathyroidismduetolowcalcium intakeiscommoninmiddle-ageandelderlypopulation.Several studieshaveshownthatnutritionalsupplementationofcalciumin foodordrinkingwaterreducesserumPTHlevelandbonemarkers (Bonjouretal.,2013;Meunieretal.,2005).

WhenMMcellsinjectionwasassociatedwithalowcalciumreg- imen,tumorgrowthwasacceleratedfrom8w.Itwasassociated withahighincidenceofparaplegia(whichwereabsentinnormal- Ca-MMmice);thiscorrespondstoamoresevereformofMM.Mice showedanincreasedosteolysisattheendstageofthediseasedueto anearlierdevelopmentofosteolyticlesionsatthetibiaandlumbar vertebrae.Histomorphometricmeasurementsconﬁrmedthatsec- ondaryhyperparathyroidisminMMmicehasledtoanincreased bonedestruction.Moreover,MMmicewithanormalcalciumdiet didnotshowtrabeculardestructioninthelumbarvertebralbody, although lumbar vertebraehad corticalperforations atthe end stageofthedisease.Thedramaticosteolysisatlumbarvertebrae

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Fig.5. Effectoflow-Calciumdietonbonelossatthelumbarvertebrae.(A)MicroCTreconstructionsoflumbarvertebra(L2–L4)fromnormal-Ca-MMgroup(a–c)and low-Ca-MMgroup(d–f)resp.at6,8and10w.(B–D)3DmicroCTmeasurementsofresp.BV/TV,Tb.SpandTb.NattheL2vertebralbody.

mayexplaintheincidenceofparalysisinthedeﬁcientgroup.His- tologicdatashowedthatcorticalperforationsinthevertebralbody allowedextensionofplasmacellsintheextraduralspaceandcom- pression ofthe spinal cord(Libouban,2015).Our resultsbased onX-rayanalysis,microCTandM-proteindataareinaccordance withourpreviousstudyonacombinedmodelassociatingMMand ovariectomy(Liboubanetal.,2003).Theyarealsoinaccordance withdatareportedinmurinemodelwithbonemetastasisinwhich alowcalciumdietpromotedtumorgrowth(Zhengetal.,2008;

Zhengetal.,2007).Theexperimentaldesignsofthesetwostudies slightlydifferedfromourexperimentbecausethelowcalciumdiet wasinitiatedatthesamedaythaninjectionofMMcells.Excepted thisexperimentaldifference,dataaresimilarintermofincreasein thetumorgrowthandosteolyticlesions(Setoetal.,1999).

It now exists strongevidencesthat a preexisting highbone remodelingpromotestumorgrowth(MMormetastasis)andwould

representariskofconvertingasmolderingformintoanovertone.

We andothers havehypothesizedthat ahighbone remodeling altersthebonemicroenvironmentbyenrichingitwithgrowthfac- torsandcytokines,thusprovidingamore“fertilesoil”onwhich tumorcellsgrowthisenhanced(Guise,2000).Moreover,treatment withtheRANK-Linhibitorosteoprotegerinpreventedthedevelop- mentofosteolyticlesionsandreducedthetumorgrowthdespite acalciumdiet(Zhengetal.,2007).Authorsemphasizedthefact thattheincreaseoftumorgrowthandtheextendofbonelesions aredirectlyduetohighboneresorptioninducedbylowcalcium independentlyofdirecteffectofPTH.(Zhengetal.,2007).Inaddi- tion,theintermittentinjectionofPTH(1–34)hasbeentestedin murinemodels ofmyelomaand theeffect ofPTH delayedMM progression(Pennisietal.,2010).Inourmodel,PTHlevelsarecon- tinuouslyelevatedandthustheeffectsonboneremodelingand tumorgrowthappearoppositetotheaboveresults.In addition,

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Fig.6. Effectoflow-Calciumdietongeneexpressionproﬁleinmedullarmicro- environment.RealtimePCRanalysisof(A)MIP-1␣,(B)RANKL,(C)LRP5and(D) DKK1expressioninbonemarrowfromnormal-Ca-MMandlow-Ca-MMgroupat6, 8and10w.

wedidnotincludeneitheragroupwithnormalcalciumdietand continuousPTHinfusionnortestedaneutralizingantibodytover- ifywhetherthecontinuousincreasedinPTHisresponsibleforthe observedeffects.

Ourresultsclearlyshowedthatapreexistinghighboneturnover hada strongimpactonMM developmentat anearlytime,6w followingMMinjection.At6w,theM-proteinlevelwassimilar betweenthedifferentgroupsandthebonemarrowinvasionwas higherinthelow-Ca-MMgroupsalthoughthedifferencedidnot reachedstatisticalsigniﬁcance.However,whenplottingtogether the% of marrow invasion and N.Oc in a 3D graph, differences betweenthetwogroupswasmoreclearlyevidencedduetopsycho- logicalperception(Systat,2009).Inaddition,the3Dpresentationof dataclearlyshowedthatthelowcalciumdietacceleratesthemech- anismofMMprogressionattheearlyphaseofMMdevelopment thatinducethenamoreseverebonedestruction.

Untilnow,nostudywasperformedtocharacterizethemolec- ular enrichment of the microenvironment. In this study, we evaluatedtheexpressionofgenesstronglyinvolvedinMMgrowth.

In MM,considerable attentionshave been paidoncellular and molecularmechanisms responsible for a high bone resorption.

RANK/RANKL and the chemokine MIP-1␣ constitutes the key

“osteoclastactivatingfactors”inMM(Choietal.,2000;Pearseetal., 2001).Atlowcalciumdiet,MIP-1␣washighlyexpressedinthe earlierphaseofMMdevelopment.Thisresultisinaccordancewith histologicaldataofTRAcPpositiveosteoclastsattheendostealsur- faces;osteoclastswereincreasedasearlyas6winthelow-Ca-MM group,conﬁrmingthehigherresorptioninthisgroup.MIP-1␣pro-

motesosteoclastogenesisindependentlyofRANKLandenhances interactionbetweenMMcellsandstromalcells(Obaetal.,2005).

Surprisingly,at10wthelevelofMIP-1␣wasmuchlowercompared tothelevelat6and8wandalsocomparedtonormal-Ca-MMgroup atthesametime.However,asthemarrowinvasionisatalevel of100%inthelow-Ca-MMgroupandbecauseMIP-1␣ismainly secretedby medullarcells, itis notsosurprisingthat thelevel expressionofMIP-1␣waslow.Morerecently,ithasbeenshowna roleforMIP-1␣ininhibitionofosteoblastformationviaosteocal- cindownregulation(Valletetal.,2011).InourstudyMIP-1␣was astrongindicatorofMMaggressiveness;surprisingly,nosigniﬁ- cantdifferenceintheexpressionofRANKLwasobserved.RANKL geneexpressionisincreasedinamousemodelofsecondaryhyper- parathyroidismafter7days(O’Brienetal.,2005).Thiswouldbein favorofanearlyhyper-expressionofRANKL inthelow-Ca-MM groupoccurringbefore6w(theonsetofourkineticstudy).

TheWnt pathwayis involved inthe declineof bone forma- tioninMM(Roodman,2010).StudiesonboneformationinMM havefocusedonDDK1expressionthatisupregulatedinmalignant plasmacells(Roodman,2010;Tianetal.,2003).Atanearlystage ofMM,weobservedadecreaseintheco-receptoroftheWntpath- wayLRP5(whichisakeymoleculetoenhanceboneformation).

ThelackofLRP5inmiceinducesaninhibitionofosteoblastfunc- tionassociatedwithadecreaseinosteoblastnumber(Katoetal., 2002).ThelowLRP5expressioninmicewiththelowcalciumdiet canreﬂectthedecreasein boneformation.We didnotobserve ahighlevelofDKK1expressioninthelow-Ca-MMgroup.There isnodataonDKK1expressioninsecondaryhyperparathyroidism whichcouldexplainourresult.However,inapreviousstudyof geneexpressioninamousemodelofdisuse,DDK1appearedasa lateinhibitorofboneformation(Marchand-Liboubanetal.,2013).

Thisobservationcouldbeinaccordancewithourdataat10wwhen anon-signiﬁcantincreaseofDKK1expressionwasobservedinthe low-Ca-group(comparedtothenormal-Ca-group).

5. Conclusions

ThepresentstudybyusingX-raymicrotomographyandquan- titativePCRanalysisclearlyshowedthatalteredboneremodeling duetocalciumdeﬁciencyincreasetheseverityofMMbonedis- ease with a higher osteolysis and a higher alteration of bone genesexpression.Littleattentionhasbeenplacedoninvestigating themechanismsinvolvedinhumansforthesuddenprogression fromindolentmyeloma(revealedbyamonoclonalgammopathy of undetermined signiﬁcance – MGUS) into an aggressiveMM.

However, evolution to an overt MM, is related toboth intrin- sicchanges ofmalignantplasma cellsand modiﬁcationofbone microenvironment(Bataille,2015;Tagejaetal.,2014).So,calcium deﬁciencywouldrepresentarealcauseforprogressionofsmolder- ingMMtoovertMMbyinducingsecondaryhyperparathyroidism.

MMisadiseasefrequentlyobservedintheelderlywhereasec- ondaryhyperparathyroidism(increasingboneremodeling)isalso frequentlyobserved.

Funding

ThisworkwassupportedbygrantfromtheFrenchMinistryof Health.

Acknowledgments

AuthorsthankP.LegrasandJ.Rouxfortheirhelpwiththeani- malcare(SCAHU)andthetechnicalassistanceofthePlate-forme d’AnalyseCellulaireetMoléculaire(PACEM,AngersUniversity)for theqPCRanalysis.