The use of animal models in multiple myeloma

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GENERAL REVIEW

The use of animal models in multiple myeloma

L’utilisation des modèles animaux dans le myélome multiple H. Libouban

GEROMgroupeétudesremodelageosseuxetbiomatériaux—LHEA,IRIS-IBSinstitutdebiologieensanté, LUNAMuniversité,CHUd’Angers,49933Angerscedex,France

Availableonline17April2015

KEYWORDS Murinemodelof myeloma;

Osteolysis;

BALB/c;

SCID;

SCID-hu;

5T2MM;

5T33MM;

5TGM1;

C57BL/KalwRij;

Xenograft;

Pristane

Summary Inmyeloma,theunderstandingofthetissular,cellularandmolecularmechanisms oftheinteractionsbetweentumorplasmacellsandbonecellshaveprogressedfrominvitro andinvivostudies.Howevernoneoftheknownanimalmodelsofmyelomareproduceexactly thehumanformofthedisease.Therearecurrentlythreetypesofanimalmodels:(1)injection ofpristaneoilinBALB/cmiceleadstointraperitonealplasmacytomasbutwithoutbonemarrow colonizationandosteolysis;(2)injectionofmalignantplasmacelllinesinimmunodeﬁcientmice SCIDorNOD/SCID;theuseoftheSCID-huorSCID-rabmodelallowstheuseoffreshplasmacells obtainedfromMMpatients;(3)injectionofallogeneicmalignantplasmacells(5T2MM,5T33) intheC57BL/KalwRijmouseinducesbonemarrowproliferationandosteolyticlesions.These cellsdidnotgrowinvitroandcanbepropagatedbyinjectionofplasmacellsisolatedfrom bonemarrowofamouseatendstageofthediseaseintoyoungrecipientmice.The5TGM1isa subcloneof5T33MMcellsandcangrowinvitro.Amongthedifferentmodels,the5TMMmodels andSCID-hu/SCID-rabmodelswereextensivelyusedtotestpathophysiologicalhypothesesand toassessanti-osteoclastic,anti-osteoblasticoranti-tumortherapiesinmyeloma.Inthepresent review,wereportthedifferenttypesofanimalmodelsofMManddescribetheirinterestsand limitations.

MOTSCLÉS Modèlemurinde myélome; Ostéolyse; BALB/c; SCID;

Résumé Dans le myélome, la compréhension des mécanismes tissulaires, cellulaires et moléculaires desinteractionsentrelesplasmocytestumorauxetlescellulesosseusesapro- gresséàpartirdesétudesinvitroetinvivo.Cependant,aucundesmodèlesanimauxdemyélome nereproduit exactementlemyélomehumain.Ilexisteactuellementtroistypesdemodèles animaux:(1)l’injectiond’huilepristanechezdessourisBALB/cconduitàdesplasmacytomes intrapéritonéauxmaissansqu’ily’aitcolonisationdelamoelleosseuseetdéveloppementd’une ostéolyse;(2)l’injectiondelignéesdeplasmocytesmalinschezdessourisimmunodéﬁcientes

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SCID-hu; 5T2MM; 5T33MM; 5TGM1; C57BL/KalwRij; Xénogreffe; Pristane

SCIDouNOD/SCID;l’utilisationdumodèledesourisSCID-huouSCID-rabpermetd’injecterdes plasmocytestumorauxfraisobtenusàpartirdepatientsatteintsdemyélome;(3)l’injection decellulesplasmocytairesallogéniques(5T2MM,5T33)chezlasourisC57BL/KalwRijinduitune proliférationtumoraledanslamoelleosseuseetdeslésionsostéolytiques.Cescellulesnese cultiventpasinvitroet peuventêtrepropagéesparinjectiondeplasmocytesisolés,àpar- tirdemoelleosseusedesourisaustadeterminal delamaladie,dansdessourisreceveuses jeunes.Lalignée5TGM1estunsous-clonedescellules5T33MMetpeutsedévelopperinvitro.

Parmilesdifférentsmodèlesdemyélome,lesmodèles5TMMetlesmodèlesSCID-hu/SCID-rab ontétélargementutilisés pourtesterdeshypothèsesphysiopathologiquesetévaluerl’effet de thérapies anti-ostéoclastiques, de thérapies anti-ostéoblastiques ou des thérapies anti- tumoralesdumyélome.Cetterevuedela littératuredécritlesdifférentstypes demodèles existantsavecleursintérêtsetleurslimitations.

Introduction

Myeloma(MM)isahematologicalmalignancywhosepreva- lence is 1 in 10,000people; the median survival is approximately three years. MM is due to the prolifera- tion of malignant plasma cells (PC) in the bone marrow.

The overtdisease is clinicallycharacterized bybone pain andasthenia.Biologically,amonoclonalprotein(M-protein) is found in blood and/or urine in 98% of cases. Skeletal abnormalities characterizedby osteolytic fociand diffuse osteolysisisobservedonradiographsin90%ofpatientsdur- ing the time course of the disease. Osteolysis is due to anincreasedosteoclastogenesisinduced bythetumorand nottothemalignantPCs,whichdonothavethecytologic machineryforresorbing thecalciﬁedbone matrix. Hyper- calcemia, which reﬂects bone lysis, is often associated.

Osteolysiscausesseriousclinicalproblems(fractures,spinal cordcompression,bonepain...).Pathophysiologyandetiol- ogyofosteolysisinmyelomaarestillimperfectlyknown.It wasshownthatthedestructionofthebonetissuewasnot associatedwith tumor PCs themselves, but by the stimu- lationofosteoclastactivitywhich occursinthevicinityof tumornodules.

Osteolyticcavitiesaregeneratedin responsetoa vari- ety of local factors produced by the malignant PCs and by the bone marrow microenvironment. These factors favoringosteolysisincludetumornecrosisfactoralpha(TNF-

␣),interleukin-1␤(IL-1␤),interleukin-6(IL-6),macrophage inﬂammatoryprotein-1-alphaandbeta(MIP-1␣andMIP-1␤) and ligand for receptor activator of nuclear transcription factor-␬B (RANKL) [1]. However, none of these factors appears to be solely responsible for bone destruction. In addition,the implicationofseveralof thesefactors(IL-6, IL-1,TNF-␣)inosteolysishasbeenshowninvitrobuttheir implicationinvivoisuncertain[2].InMM,interactionwith bonemarrowmicroenvironmentisessentialforMMprogres- sion.TheinteractionofstromalcellswithtumorPCsthrough theadhesionmoleculesVCAM-1/␣4-ß1increasesosteoclast- ogenesis and resorption activity [3]. The VCAM-1/␣4-ß1 interactionisalsoinvolvedinRANK-L/OPGandIL-6released by stromal cells [4,5]. IL-6 association with its soluble receptorpromotes theproliferation andsurvivalof tumor cells. Additional growth factors are also released by the

microenvironmentthatcanpromotethetumorgrowthsuch asIGF-1[6].

In overt MM, a decrease in bone formation is also associated, leading toan uncoupling in bone remodeling.

Inhibitionofosteoblastogenesisisduetoinhibitorsreleased by PCs and depressing the Wnt-signaling pathway: DKK1 (Dickkopf-1)andSfrp2(secretedfrizzled-relatedprotein2) [7,8]or acting ontheosteoblastic precursors:hepatocyte growthfactor(HGF),IL-3andIL-7[9—11].

InMM,thereisaviciouscircleinwhichtumorPCsstim- ulatebonecellswhichinturnstimulatethetumorgrowth.

Understandingofthetissular,cellularandmolecularmechanisms of interactions between tumor PCs andbone cells suffers fromthe low numberof available animal models.

Modelsareinteresting:

• toevaluatepathophysiologyofthediseaseanditseffect onboneremodeling;

• totesttheeffectoftherapeuticwithananti-osteoblastic, anti-osteoblasticoranti-tumoractivity.

Atpresent,onlymousemodelsareavailableandreﬂect more or less perfectly the human disease. Inthe present review,we reportthedifferenttypesofanimal modelsof MManddescribetheirinterestsandlimitations.

Induction of plasmacytomas in BALB/c mice with pristine oil

Theinjectionofthemineraloilpristaneintotheperitoneum of BALB/c mice induces plasmacytomas with a high fre- quency [12]. This model hasbeen discoveredin 1969 and has constituted at that time the ﬁrst one to study MM.

Pristane oil induces a chronic inﬂammatory tissue where plasmacytomasdevelopwitha60%frequencyafter16weeks [12].Plasmacytomascanthenbetransferredtoothermice pretreated with pristane. Plasmacytomas predominantly secreteamonoclonalIgA in60%of cases.Thisis alimita- tionofthemodelbecauseIgGMMisthemostfrequentform in humans[13].The maindisadvantages ofthe modelare that PCsarerestrictivelylocalized inthe peritoneumand donotextendtothebonemarrow;sobonelesionsarenot observed.Thismodeldoes notfaithfullyreproduce human

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MMandthus cannotbeusedtostudythe pathophysiology ofMM-inducedbonelesions.However,itiswelladaptedto studythegeneexpressionproﬁleinPCsduringtumorpro- gression[14,15].Anotherinterestofthepristanemodelis thepossibilitytostudyoftheoncogenicrearrangementsof c-myc[16,17].Besidetocytogeneticstudies,theroleofIL- 6hasalsobeeninvestigatedinthismodel.IL-6isnowwell knowntobeinvolvedinsurvivalandproliferationofPCsin MM[18].IthasbeenshownthatBALB/cmicedeﬁcientfor theIL-6genefailedtodevelopplasmacytomaafterpristane injection[19,20].

Experimentaltherapiesontheplasmacytomamodelhave been tested but theywere focused only on the effectof tumor growth [12]. One interesting study was the use of zoledronic acid (ZOL, the most potent bisphosphonate of the3rdgeneration)currentlyusedtopreventbonelesionsin humanMM.ItiswellknownthatZOLiseffectiveinreducing anddelayingskeletaleventsinMMpatients[21].Thedirect effectofZOLonPCswaslessevidentinvivo.Areductionof plasmacytomadevelopmentandanincreaseinsurvivalhave beenobservedinBALB/cmice,whichhavereceivedZOLon the1stdayafterpristaneinjection[22].

Immuno-deﬁcient mice: the SCID model

ItconsistsintheinjectionofhumanPCsinseverecombined immunodeﬁciency(SCID)mice,soitrepresentsaxenograft model. However, the choice of the plasma human tumor line appears criticalbecause of theycan beof myeloma- tous or lymphoblastoidorigin and their Epstein-Barr virus statusmaydiffer(EBV-orEBV+).Themalignanthumancells colonizethebonemarrowmicroenvironmentanddevelopin themarrowcavities.Osteolyticlesionsoccurandaresimi- lartothosedescribedinhumans;inaddition,aM-proteinis secretedandcanbedosedintheserum[23—27].

Some of these myelomalines, asthe KPMM2, arewell characterized [28]. KPMM2 is EBV- originated from a MM patient. These cells are strongly dependent to IL-6 asin humanMM.Inaddition,theirproliferationisinducedbyIL- 6viaanautocrinemechanism.Intravenousinjectionofthe cellsinSCIDmiceinducesamajordevelopmentwithinthe hematopoietic bone marrow and lymph nodes [27]. Oste- olyticlesionsareobserved30—40daysafterinoculationand mice exhibithind limb paralysis at the end course of the disease.IntheKPMM2model,injectionofanti-humanIL-6 receptorsignificantlyreducesthedevelopmentofthedis- ease.OtherstudieshavebeendonewithhumanPCsfroma leukemialinetransformedbytheEpstein-Barrvirus(ARH77) [23].SCIDmicedevelop paralysisofthelowerlimbs28to 35daysafterinoculationofARH77cells.Hypercalcemiais observedwithin5daysaftertheonsetofparalysis.Histolog- icalexaminationofvariousorgansshowsaPCinfiltrationof theliver,spleen,spineandlongbones.Asignificantincrease isobservedinMIP-1␣levelinthebonemarrowenvironment.

Incontrast,serumormedullarleveloflocalfactorssuchas IL-6,IL-1,TNFTGF-␣,TNF-␣,lymphotoxinandPTHrpwere notsigniﬁcantlyincreased,althoughARH77areabletopro- ducedIL-6invitro.CelllinesJJN3orKPMM2havenotbeen oftenusedasananimalmodelforMM.Incontrast,ARH77, despitethedisadvantageoftheEBV+,statusremainedthe mostwidelyusedcelllineintheSCIDmodel.Severaltypesof

treatmentweretestedintheARH77modellikeibandronate, anantisenseconstructagainstMIP-1␣andtherecombinant RANKantagonistmoleculeRANK-Fc[29—31].

The route of inoculation has also an inﬂuence on the tumor development in the SCID model [32]. The intra- venous route is preferred and is associated with typical osteolyticlesions.Intraperitonealinjectionisaccompanied bylocations in the pancreas,spleen, liver,intestine, kid- neybut bone marrow is rarely invaded.The intra-cardiac or intra-osseous combines injection direct intramedullary tumor growth associated with osteolytic lesions but also extramedullaryproliferations.

Most of the SCID models which were described in the literatureusedMM cell lines.The transplantof fresh pri- marycellsinduced growthinSCID miceandproductionof monoclonal immunoglobulin but without colonization into thebonemarrow[33,34].Becauseofthedifficultytogen- eratebonemarrowcolonizationoffreshprimaryhumanMM cellsusingconventionalSCIDmice,xenograftsoffreshMM cellswere tested ina highlyimmunodeficient mouse; the non-obesediabetic SCIDmice(NODSCID). Thesemice are characterizedby the absence of NK cells. Therefore,the majority of injection studies with xenogeneic PCs is per- formed in NOD SCID mice since year 2000 [26]. Injection ofPCsfromblood ofMMpatientsusingtheintracardiacor intra-osseous route, leads to colonization into bone marrow andosteolytic lesions[26].The timedevelopment to observesymptomswasfluctuantbetweenonetofivemonths dependingofthePCsorigin.Boneslesionsareobservedin differentskeletalsites:longbones,vertebraeandalsothe skull.

TheSCIDmodelcanbereﬁnedbygraftingfragmentsof livinghumanfetallongboneintherecipientanimals,where they constitute a ‘‘marrow micro-environment’’ favoring the development of transplanted human PCs. This model (referred as SCID-hu) is associated with a homing of the humanPCswithinthefetalbone graft.PrimaryhumanMM cells can also be directly injected into the grafted bone [35]. Osteolytic activity develops only in the xenogeneic bony graft. Several studies on this model were focused onMM therapy such asanti-angiogenic drug thalidomide, inhibitorofDKK1andtheuseofRANK-FC[36—38].Alltreat- mentsdecreaseosteolysisandMMprogression.However,this modeliscumbersome toimplement andmayraiseethical problems.Theauthorshavealsodevelopedasimilarmodel inwhichSCIDmicearegraftedwithapieceofrabbitfetal bone(referredasSCID-rab)graftedsimilarly[39].

The 5TMM model in the C57BL/KalwRij mouse

TheC57BL/KalwRij mousewasdescribed byRadl etal.in 1978.Withage,miceofthisstrain developseveralmono- clonalB-cellproliferativedisorderswithafrequencyof80%

[40].Whenmiceareolderthantwoyears,0.5%developsa typicalMMoraWaldenström’sdisease.Severalspontaneous MMhavebeenisolatedandarecalledthe5TMMseries.Dif- ferent5TMM lines were obtained after transplantation of bonemarrowcellsintosyngeneicyoungmicebytheintra- venousroute (Fig.1).Eight 5TMMwereisolatedandmost of them were characterized in detail: isotype of the M- protein,bone marrowinvolvement,presenceofosteolytic

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Figure 1 Protocol for generation and amplification of the 5TMMmodels.PCswereisolated frombonemarrowof5TMM miceatendstageofthedisease.Bonemarrowisflushedfrom longbone and PCs areisolated after a centrifugation gradi- ent.IsolatedPCsareintravenouslyinjectedinto thetailvein ofyoung healthymice. DevelopmentoftheMMis monitored byelectrophoresisoftheserum.In MMpositivemice, theM- protein is observed on the electrophoresis gel whereas the electrophoresisprofileofcontrolmicedidnotshowM-protein.

Protocolepour laproduction et l’amplification des modèles 5TMM.Lesplasmocytessontisolésàpartirdelamoelleosseuse desouris5TMMaustadeterminaldelamaladie.Unflushdela moelleosseuseesteffectuéàpartirdesoslongsetlesplasmo- cytessontisolésenréalisantungradientdecentrifugation.Les plasmocytesisoléssontinjectésparvoieintraveineusedansla veinecaudaledesourissainesetjeunes.Ledéveloppementdu MMestcontrôléparélectrophorèsedusérum.Chezlessouris positivespourleMM,laprotéinemonoclonaleestobservéesur legeld’électrophorèsetandis queleprofild’électrophorèse desouriscontrôlesnemontrepasdeM-protéine.

lesionsand growthpattern [41]. Seven cell linesproduce an IgGimmunoglobulinwitha light chain.The circulating monoclonalM-proteinreﬂectstheextentofthetumorload andcanbeeasilymonitoredbyelectrophoresisoftheserum (Fig.1).Twolineshavebeenparticularlystudied:the5T2MM and5T33MM. The 5T2MM representsa model situation of themostcommonformsofthehumanMMwithamoderate

growth and osteolytic lesions.In additionand in contrast tothehumandisease,osteolysisrepresentstheuniquecon- sequenceoftheinoculationof5T2MMcellsbecauseofthe absenceofrenallesionsduetolightchaindeposition[42].

On the other hand, the 5T33MM is a veryaggressive line witharapidtumorgrowthandinvolvementofotherorgans (non-hematopoietic),acondition,whichisrarelyobserved inhumans.Kineticdevelopmentsof thesetwomodelsare strongly different: 5T2MM cells can be detected at nine weeksafter inoculationwhereas 5T33canbe observedas early as two weeks after inoculation [43]. In the 5T2MM model, osteolytic lesions can be observed from the 11th weekandtheend-stageoccursat16-weekpost-injectionof thePCs.Inthe5T33MMmodel,theterminalstageofthedis- easeisobtained4—5weeksafterinoculation.Asubcloneof 5T33MMcells,whichcangrowinvitro,hasbeenestablished andnamedthe5TGM1cells[41,44].When itisinoculated intravenously toyoung syngeneic mice, it reproduces the samepathologicalcharacteristicsthanthoseobservedinthe 5T33MM.Morerecently,both5TGM1and5T33MMcellwere usedtogenerateﬂuorescentcellsallowingvisualizationand localizationofMMcellsinwholebodybyﬂuorescenceimag- ing[45,46].Sincethediscoveryofthe5TGM1subclone,the 5T33MM has been usedless frequently. Moreover, despite thecapacityof5TGM1togrowin vitro,the5TGM1model didnotreplacethe5T2MMmodel,whichisremainingexten- sivelyusedtostudypathophysiologyofMMandtoevaluate newtherapeutics.

Several studies have shown that 5T2MM and 5T33MM models have similar pathophysiological characteristics to those of human MM: the cytokine network involved and theadhesionmoleculesarethesame.5T2MMand5T33MM expressthefollowingadhesionmolecules:CD44,␣4/␤1and

␣5/␤1[43,47,48].Moreoverininvitroexperiments,5TGM1 cells establish direct contacts with stromal cells via the interaction of ␣4/␤1 and VCAM-1. This ﬁnding is particu- larlyinterestingasinteractionofPCswithstromalcells is essentialforthetumorgrowth[2].

Osteolyticlesions have been well characterized in the 5T2MM mice [43,49,50]. X-ray assessment of bone lesions revealed that lesions are preferentially localized in long bones (proximal tibia, distal femur and humerus). In the mouse, these zones have physiologically a high bone turnover, this leads to a preferential localization of the tumorandsecondarilypromotesthetumorgrowthasseveral cytokinesaremitogenicfactorsforPCs.Thedevelopmentof osteolyticlesionsiswell-studiedusingdigitalX-rayormam- mography ﬁlms [49,50]. Bonelesions appearasnumerous smalland roundcavities thatdevelop from theendosteal envelope.Lesionsarepreferentiallylocalizedinthemeta- physesandalsosystematicallyobservedatthetibialcrest (Fig.2).Lesionscanbeobservedintheacetabulumandin theiliaccrestwhenusinganaggressivesubcloneof5T2MM cellscharacterizedbyafasterdevelopmentrate[51].Bone lesionsinthe5T2MMarethereforesimilartothoseobserved inhumanwithoneexception: theabsenceofbonelesions in theskull. However,the tibialcrestof themousehas a bony architecture,which mimics thehuman bones of the skullwherethediploe(akindofcancellousbonewithlarge trabeculae)islimitedbytwothickcortices.

Histological examination of invaded bone shows that the marrowis replacedby tumor nodules at the terminal

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Figure 2 X-ray image of the pelvis and hind legs of a C57BL/KalwRijmouseinjectedwith5T2MMcellsatendstageof thedisease.Notetheexistenceofnumerousosteolyticlesions onbothdistalfemurandproximaltibia.

Radiographie du bassin et des membres postérieurs d’une sourisC57BL/KalwRijinjectéesavecdescellules5T2MMetau stadeterminaldelamaladie.Notezl’existencedenombreuses lésionsostéolytiquesàlafois surlefémurdistal etle tibia proximal.

stageofthedisease.Aroundthetumornodules,thereisa considerable increase in the number of osteoclasts. They areresponsiblefor theresorptionoftrabecularaswellas corticalbone. Attheend stageof thedisease,trabecular bonehascompletelydisappearedandcorticalperforations areevidenced (Fig. 3).Scanning electron microscopywas usedtovisualizeandquantifytheendostealresorptionon

Figure4 Scanning electronmicroscopy micrographs ofthe endostealsurfacefromvertebralbodyofa5T2MMmouse.The wholesurfaceiseroded.Notethetypicalappearanceoferoded surfacesformedofvalleysandpits.

Imagede microscopie électroniqueà balayagede lasurface endostéed’uncorpsvertébralchezunesouris5T2MM.Toutela surfaceestérodée.Noterl’aspecttypiquedessurfacesérodées (aspectde«coupsdedents»ostéoclastiques).

longbones.Erodedsurfaceoccupiedalmostthewholebone surface (Fig. 4). It has been shown that eroded surfaces occupy 76.2±17.1% in 5T2MM mice cells vs. 19.2±7.1%

in control mice [49]. X-ray microcomputed tomography (microCT)with3D reconstructioncan accuratelyvisualize bone resorption, it conﬁrms radiological ﬁndings, allows quantitativeanalysisof trabecularand corticalbone, and histological analysis is possible because the technique is non-destructiveforthesamples(Fig.5).

Figure3 Goldnertrichromestainedsectionofthefemurfrom5T2MMmiceatearlystage(A)andendstage(B)ofthedisease.

Atearlystage,corticalboneandtrabecularbonearepreservedwhereasattheendstage,trabecularbonehasalmostdisappeared andseveralcorticalperforationswereobserved(arrow).Notetheexistenceofextramedullaryextensionofthetumorunderthe periosteum.

Coupe histologiquedefémur coloréeautrichrome deGoldnereteffectuéechezdessouris 5T2MMàun stadeprécoce(A)et terminal(B)delamaladie.Austadeprécoce,l’oscorticaletl’ostrabéculairesontpréservéstandisqu’austadeterminal,l’os trabéculaireapresqueentièrementdisparuetplusieursperforationscorticalessontobservées(ﬂèche).Noterl’existenced’une proliférationtumoraleextra-médullaire,souslepérioste.

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Figure5 X-raymicrotomographicimagesofthetibiaofacontrolmouse(A)andofa5T2MMmouseatendstageofthedisease.

Notetheexistenceofnumerouscorticalperforationsextendingfromtheendostealtotheperiostealsurfaceoftheboneandthe completedisappearanceofthetrabecularnetwork.

Microtomographie-Xdutibiad’unesouriscontrôle(A)etd’unesouris5T2MMaustadeterminaldelamaladie.Noterl’existencede nombreusesperforationscorticaless’étendantdelasurfaceendostéeverslasurfacepériostéeetladisparitiontotaleduréseau trabéculaire.

Thethreemodels5T2MM,5T33MMand5TGM1havepro- videdveryusefuldataonkeyactorsinthepathogenesisin MM andon the testing of new drugs. In MM, osteolysis is theconsequenceofbothanincreaseinosteoclasticresorp- tion and a decrease in osteoblastic formation. Alteration oftheboneturnoverisduetothereleaseoflocalfactors secreted by PCs directly or indirectly via normal cells of thebonemicroenvironment.Identiﬁcationofthesefactors (termedosteoclastactivatingfactors[OAF]byMundyetal.

[52])camefirstfrominvitrostudiesbuttheirrolesinvivo wereconfirmedbyusingthe5TMMmodels.Thekeyroleof theRANK/RANKL/OPGsysteminosteolysishasbeenshown in5T2MMmicetreatedwitharecombinantosteoprotegerin protein.AdministrationofOPGpreventsosteolysisandpre- servestrabecularbone[53].Morerecentlyithasbeenshown anincreaseofsolubleRANKLintheserumof5T2MMmiceas seeninMMpatients[54,55].Theincreasedlevelofsoluble RANKLisassociatedwithdramaticbonedestructionstrongly suggestingakeyroleforsolubleRANKLinthepathogenesis ofosteolysis[54].InadditionsolubleRANKLisnotdetected in5T33MMmice,thusconfirmingthat5T2MMmodelismore closelyrelatedtothehumandisease.Increasedresorptionin MMisalsoduetomacrophageinflammatoryprotein-1␣(MIP- 1␣)producedandreleasedbyPCs.Inthe5TGM1model,the increasein MIP-1␣ stimulates osteoclastic resorption;this increaseis,in part,dependentfromRANKLpathway[56].

InhibitionofMIP-1␣in vivohasaneffectbothonosteoly- sisand onthetumor burdenleadingtoan increaseinthe survivalofmice.

Another speciﬁc aspect of MM concerns the molecular mechanismsleadingtoareducedbone formation.Several factorshavebeenidentiﬁed:DKK1,sFRP-2,IL-3,Runx2and TGF-␤[2].

Most factors implicated in the pathogenesis of MM are produced and secreted in the bone marrow

microenvironment, which is essential for the growth of PCs.Inadditiontolocalfactors,physicalinteractionofPCs withvariouscellsofthebonemarrow(stromalcells,osteoclasts, endothelialcells) is required. Several studieshave suggestedthatPCsmodifybonemarrowatthecellularand molecular leveltopromote growthand survival.A recent study has shown an increase in stromal cell number and withamirrordecreaseinosteoblastsnumberinMMpatients [57]. This hasbeen conﬁrmed inthe 5TGM1model where an increasein stromal cellsassociatedwithadecrease of osteoblastnumbercorrelateswiththetumorburden[57].

Another way to show the key role played by the microenvironmentinMMprogressionistomodifythebone remodelingprior totheinoculation of PCs.Indeed,it has beenshown thatahighbone remodelinglevel(asinduced byovariectomyinmice)acceleratesPCgrowthinthe5T2MM murine model and considerably increases the number of osteolyticfoci(Fig.6)[58].Moreover,asublineof 5T2MM (the5THLcells)hasbeenselectedbythealteredmicroen- vironment resulting in a more aggressive cell line with preservation of the original clone phenotype [51]. 5THL micearecharacterizedbyaM-proteindetectable6weeks after cellsinjection andosteolyticlesionsstart toappear at eight weeks; end stage of the disease being obtained at10weeks.Anothermethodforincreasingboneremodel- ingistofeedanimalswithalowcalciumdiet.Thisinduces asecondaryhyperparathyroidismandanimalsgraftedwith 5THLPCshaveaconsiderableincreaseintheextendofoste- olyticfocitogetherwithamoreseverediseaseassociated withfrequentparaplegiaduetoinvasionofthespinalcanal byPCs(Fig.7)[59].Themicroenvironmentisalsoenriched inmicroparticles(fragmentsofPCmembranecoveredwith CD138)releasedbythemalignantcellsbuttheroleofthese cellular debris remains unknown [60]. Up to recently, no study onthe 5TMMmodel wasconducted toevaluate the

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Figure 6 X-ray microtomographic image of the tibia of a 5T2MMmouse 10weeks afterinoculation ofPCs. Thismouse wascharacterizedbyanacceleratedMMdevelopment,induced byahighboneremodelinglevelinthebonemicroenvironment.

Notethattheosteolysisismorepronouncedcomparedtowhatis observedatalatertimeinanormal5T2MMmouse(seeFig.5B).

Microtomographie-Xdutibiad’unesouris5T2MM,10semaines après inoculation de plasmocytes tumoraux. Cette souris a présenté un développement accéléré du MM, induit par un niveau élevé de remodelage osseux dans le microenviron- nementosseux.Noterquel’ostéolyseestplusprononcée par rapportàcequiestobservéaprèsunepériodepluslonguedans lemodèlestandard5T2MM(voirFig.5B).

existenceof genealterations.Recentdatahave identiﬁed deletionintheSAMS1tumorgenesuppressorinthe5TGM1 model,whichiscorrelatedwithhumanﬁndings[61].These data open the way of new possible investigations in the pathogenesisofMMusingthe5TMMmodel.

Asthe5TMMmodelconstitutesagoodmodeltoevaluate pathophysiologicalaspectsofMM,itisalsowelladaptedto evaluatevarioustherapeuticstrategiesinMM.Theeffectof treatmentcombininginterferon-␣andmelphalanwastested in5T33MMmice[62].Thiscombinationsigniﬁcantlyreduces tumor growth and leads to an increased animal survival.

TheuseofantibodiesagainsttheIgG2a␬M-proteinreduces tumor growth in most animals [63]. Bisphosphonates are currentlythestandardwaytopreventandtreatosteolytic disease in MM. These compounds have anti-osteoclastic propertiesbyinducingapoptosisofthesecells.Severaltypes ofbisphosphonateshavebeentestedinthe5TMMmodels.

Initially, oral pamidronate (APD) was used in the 5T2MM [64]. Treated animals showed preservation of bone mass andpartialrestorationofosteolyticlesions.However,APD hasnoeffectonthetumormassitself[58,64].Theamino- bisphosphonate, ibandronatehasalsobeen tested inboth models 5T2MM and 5T33MM [65]. It induces a signiﬁcant

Figure7 Hematoxylin-eosinstainedsectionoflumbarverte- braeofa5THLmouse.Notetheseveralcortical perforations (arrows)thatallowproliferationofPCsundertheperiosteum;

PCsarethusproliferatinginthemeninges(m).Thisprolifera- tioninducescompressionofthespinalcord(SC)whichisthe mechanismresponsibleforparaplegia.

Coupehistologiquecoloréeàl’hématoxyline-éosineetobtenue àpartird’unevertèbrelombairedesouris5THL.Noterlesper- forationscorticales(ﬂèches)etl’accumulationdeplasmocytes tumorauxsouslepérioste;lesplasmocytesprolifèrentàtravers lesméninges(m)etcetteproliférationinduitunecompression delamoelle épinière(SC),mécanisme responsablede para- plégie.

reductionintheoccurrenceofosteolyticlesions;however, it has no effect on tumor growth or animal survival. It shouldbenotedthatinthesemodels,thedosesofbispho- sphonatesusedaregenerally100 times higherthanthose requiredin other animal modelsof bone hyper-resorption (suchastheovariectomizedmice).Newgenerationsofbis- phosphonatessuch asZOL or apomine weretested in the 5T2MM model and showed interesting results as they act onseveralaspectofMMdisease:osteolysis,tumorgrowth, survivaland angiogenesis [66,67].In these studies, treat- ment wasstarted at early stageof the disease when the M-proteinisdetectableintheserum.ZOLandapominehave differentmodesofaction:ZOLdecreasesosteoclastogene- siswhereasapominedoesnot,suggestingadirecteffectof apomineonPCsinvivo.Thisdirecteffectisnotobserved foralltheotherbisphosphonatesinvivobuthasbeenshown in vitro whenPCs arecultured in presenceof ZOL added tothemedium [68]. A comparisonbetween ZOL andAPD wasrecentlydoneinthe5THLmodel.Previousstudieshave shownthatAPDpreservedthetrabecularbonemassbuthad noeffectontheamountofcorticalperforations.AmicroCT

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analysisevidencedthatZOLwassuperiortoAPDandconsid- erablyreducedthenumberandsizeofcorticalperforations [69].

BecauseofadvancesinthecomprehensionofMMpatho- physiology,noveltherapeuticapproacheshaveemergedand some were tested in the 5TMM model. The proteasome, whichconstitutesthephysiologicalwaytodegradeproteins in eucaryotic cells, was found to play a key role in MM.

Itisinvolved intheregulationof theRANK/RANKLsystem and proteasome inhibitors (such as bortezomid) are cur- rentlyusedinthetreatmentofhumanMM[70].Proteasome inhibitorshavealsobeenshowntopromoteosteoblastdif- ferentiationandboneformation[71].Treatmentof5T2MM micewithbortezomidreducestheincidenceofbonelesions, tumorgrowthandangiogenesis[72].Theeffectofprotea- someinhibitorsin vivoismediatedbyPTHreceptors[73].

In parallel to thesetreatments, radioimmunotherapy tar- getingsyndecan(CD138antigen)wereconductedin5T3MM miceandithasbeensuggestedtouseincombinedtherapies [74—76].

BecauseMMalwaysinducedamarkedreductioninbone formation,severaltrial havebeen donewithdrugs known tostimulateosteoblastsbyactingontheWntpathway:

• ﬂuoridewasusedinclinicalpracticebutinducedﬂuorosis withouthealingofMMlesions[77,78]however,thedrug wasneverevaluatedinthemouse;

• parathyroidhormonewassuccessfullytestedintheSCID- huandSCID-rabmodelandpromotedboneformation[79], however,clinicaldataarecontradictory[80];

• stimulationoftheWntpathwaybylithiumchloridepro- tects5TGM1miceagainstosteolysisanddecreasestumor growth[81].

The use of anti-DKK1 antibodies in the 5T2MM mice inducessimilareffects[82].

Conclusion

Asusualwithanimalmodels,thedevelopmentofsuchsys- temsis veryinteresting sincetheyofferthe possibilityto evaluatenewhypothesisandtherapeuticsstrategies.How- ever, they do not represent exactly the human disease and differences are noted when trying to predict human response to drugs. Animal models are interesting to test hypothesis and understand the pathophysiological mecha- nismsofthediseaseandexploringnewgeneticorpathways hypotheses.

Disclosure of interest

Theauthordeclaresthathehasnoconﬂictsofinterestcon- cerningthisarticle.

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