TISSUE-SPECIFIC STEM CELLS

Bone Marrow-Derived Mesenchymal Stromal Cells Are Attracted by

Multiple Myeloma Cell-Produced Chemokine CCL25 and Favor

Myeloma Cell Growth In Vitro and In Vivo

SONG XU,a,b,c ELINE MENU,b ANN DE BECKER,a,b BEN VAN CAMP,b KARIN VANDERKERKEN,b IVAN VAN RIETa,b

aStem Cell Laboratory, Division of Clinical Hematology, Universitair Ziekenhuis Brussel (UZ Brussel), Brussels,

Belgium; bDepartment of Hematology and Immunology, Myeloma Center, Vrije Universiteit Brussel (VUB),

Brussels, Belgium; cDepartment of Lung Cancer Surgery, Lung Cancer Institute, Tianjin Medical University

General Hospital, Tianjin, China

Key Words. Mesenchymal stromal cells • Multiple myeloma • Microenvironment • Chemokine

ABSTRACT

Multiple myeloma (MM) is a malignancy of terminally dif-ferentiated plasma cells that are predominantly localized

in the bone marrow (BM). Mesenchymal stromal cells(MSCs) give rise to most BM stromal cells that interactwith MM cells. However, the direct involvement of MSCs

in the pathophysiology of MM has not been welladdressed. In this study, in vitro and in vivo migration

assays revealed that MSCs have tropism toward MM cells,and CCL25 was identified as a major MM cell-producedchemoattractant for MSCs. By coculture experiments, we

found that MSCs favor the proliferation of stroma-de-pendent MM cells through soluble factors and cell to cell

contact, which was confirmed by intrafemoral coengraft-ment experiments. We also demonstrated that MSCs pro-tected MM cells against spontaneous and Bortezomib-

induced apoptosis. The tumor-promoting effect of MSCs

correlated with their capacity to enhance AKT and ERKactivities in MM cells, accompanied with increased expres-

sion of CyclinD2, CDK4, and Bcl-XL and decreasedcleaved caspase-3 and poly(ADP-ribose) polymeraseexpression. In turn, MM cells upregulated interleukin-6

(IL-6), IL-10, insulin growth factor-1, vascular endothelialgrowth factor, and dickkopf homolog 1 expression in

MSCs. Finally, infusion of in vitro-expanded murineMSCs in 5T33MM mice resulted in a significantly shortersurvival. MSC infusion is a promising way to support he-

matopoietic recovery and to control graft versus host dis-ease in patients after allogeneic hematopoietic stem cell

transplantation. However, our data suggest that MSC-based cytotherapy has a potential risk for MM diseaseprogression or relapse and should be considered with cau-

tion in MM patients. STEM CELLS 2012;30:266–279

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Mesenchymal stromal cells (MSCs) are self-renewing andmultipotent progenitors that can differentiate into a variety ofcell types, such as adipocytes, osteoblasts, and chondrocytes.Several other terms for MSCs have been used interchange-ably, including mesenchymal stem cells, marrow stromalcells, and multipotent stromal cells but at this moment noneis used uniformly. Bone marrow (BM) is the most commonsource of MSCs but these stem cells have also been isolatedfrom various other sources, such as placenta, amniotic fluid,cord blood, retina, inner ear, gastric epithelium, tendons, sy-novial membrane, hair follicle, teeth, fetal liver, and adiposetissue [1–8]. In addition to their multilineage differentiationpotential, MSCs also possess immunomodulary properties andhave the capacity to home to injury/tumor sites as well as to

produce a variety of cytokines/chemokines. All these featuresoffer these stem cells the potential to be used for variouspreclinical and clinical applications, including support ofhematopoietic stem cell engraftment, tissue engineering,gene therapy, and control of immune reactions in graft versushost disease, organ transplantation, and autoimmune diseases[9–14].

Recently, evidence was provided that MSCs have prefer-ential tropism for tumor sites [15–18]. However, the exacteffect of MSCs on tumor growth and development is still indebate [19]. Khakoo et al. [20] reported in experimentalmodels of Kaposi’s sarcoma (KS) that the coinjection ofhuman MSCs (hMSCs) with KS cells inhibited primary tumorgrowth and they identified Akt inhibition in the KS cellsthrough direct contact with MSCs as a possible mechanismfor inhibition of tumor progression. An MSC-mediated tumorinhibition effect has also been observed in experimental

Author contributions: S.X.: conception and design, assembly of data, data analysis and interpretation, and manuscript writing, E.M.:provision of study material, data analysis and interpretation, and manuscript writing, A.D.B.: provision of study material and dataanalysis and interpretation, B.V.C.: financial support and final approval of manuscript, K.V.: conception and design, provision of studymaterial, and final approval of manuscript; I.V.R.: conception and design, financial support, and final approval of manuscript.

Correspondence: Ivan Van Riet, Ph.D., Stem Cell Laboratory, Division of Clinical Hematology, UZ Brussel, Laarbeeklaan 101, B-1090Brussels, Belgium. Telephone: þ32-02-477-6701; Fax: þ32-02-477-6728; e-mail: ivan.vanriet@uzbrussel.be Received March 23, 2011;accepted for publication November 2, 2011; first published online in STEM CELLS EXPRESS November 18, 2011. VC AlphaMed Press1066-5099/2012/$30.00/0 doi: 10.1002/stem.787

STEM CELLS 2012;30:266–279 www.StemCells.com

models of lymphoma, melanoma, and hepatoma [21–23].Meanwhile, many studies have demonstrated the capacity ofMSCs to initiate and/or to support survival, progression, andmetastasis of certain tumor types. Karnoub et al. [24] demon-strated that MSCs could increase the metastasis rate of breastcancer cells through secretion of CCL5 by MSCs. Severalother groups have also reported that MSCs injected togetherwith tumor cells can favor tumor growth in vivo, as observedfor osteosarcoma, large-cell lung cancer, and colon cancer[25–27]. While a lot of studies indicate that nonmodifiedMSCs have no significant influence on the progression oftumors [28, 29], there are two reports showing that MSCs ex-hibit opposite effects on the same tumor model in vitro and invivo [30, 31], indicating that effects of MSCs on cancer cellgrowth are difficult to interpret. Klopp et al. [19] recentlyreviewed the differences in the methodology of these reportedstudies and proposed that the time of MSCs introduction intotumors might be a critical factor for the contradicting results.Other possible explanations for these conflicting outcomesmight include the ratio of MSC numbers to cancer cell num-bers that were used in the different studies as well as the na-ture of the tumor type itself [32].

Multiple myeloma (MM) is a malignant plasma cell disor-der characterized by an accumulation of monoclonal termi-nally differentiated plasma cells in the BM and the presenceof a monoclonal immunoglobulin fraction in the blood and/orurine [33]. The involvement of the BM microenvironment inthe pathophysiology of the MM disease is nowadays well-documented. The crosstalk between BM stromal cells andMM cells supports the proliferation, survival, migration, anddrug resistance of MM cells as well as osteoclastogenesis andangiogenesis [34, 35]. Recent insight into the functionalimportance of the BM stroma and its interaction with MMcells lead to the identification of many new molecular targetsand derived treatment regimens [36]. Although MSCs arethe precursors of BM stromal cells, their direct involvementin the progression of MM is not clearly defined. In this study,we demonstrated that MM cells induce in vitro as well asin vivo chemotaxis of MSCs. Through chemokine profileanalysis, we identified CCL25 as a major MM cell-producedchemokine that is involved in MSC chemotaxis. By conduct-ing coculture experiments in vitro as well as in vivoexperiments, we also studied the effect of MSCs on MMcell growth and apoptosis. Despite the fact that MSCs consti-tute only a small population in the BM cell population, evenafter exogenous transplantation, our findings indicate that thesupportive role of MSC in MM cell growth cannot beignored.

MATERIALS AND METHODS

5T33MM Murine Model

The 5T33MM mice originated spontaneously from elderly C57Bl/KaLwRij mice, and this MM model is maintained by i.v. transferof diseased BM cells into young syngeneic C57Bl/KaLwRij mice(Harlan CPB, Horst, The Netherlands, www.harlan.com) at 6–8weeks of age [37, 38]. The development of myeloma wasassessed by the level of monoclonal antibody present in the se-rum (paraprotein) by protein electrophoresis. Mice were housedand maintained following the conditions approved by the EthicalCommittee for Animal Experiments, Vrije Universiteit Brussels(license no. LA1230281).

Myeloma Cell Lines

The murine 5T33MMvv and 5T33MMvt MM cells were used inthis study. The 5T33MMvv cells grow in vitro stroma depend-

ently with a survival of only a few days. The 5T33MMvt is aclonally identical but in vitro stroma-independent growing variantof the 5T33MMvv cells. Stroma-independent human MM celllines RPMI8226, LP-1, Karpas, U266, MM5.2, MM S1 and onestroma-dependent human MM cell line MM5.1 were alsoused in this study. All cell lines were kept in culture as described[39, 40].

Primary Human MM Cells

BM samples of MM patients were collected for routine diagnosticpurposes after obtaining informed consent. Each MM patient wasdiagnosed and staged according to the criteria of Durie andSalmon [41]. The study was approved by the local ethical com-mittee. Primary MM cells were immunomagnetically separatedusing the magnetic cell sorting system (MACS; Miltenyi Biotech,Leiden, The Netherlands, www.miltenyibiotec.com) with CD138microbeads according to the manufacturer’s instruction. CD138(þ) cells were recovered, and viability was assessed with trypanblue staining. MACS purification revealed a pure primary MMcell population (>98% plasma cells) as determined by May-Grun-wald Giemsa-stained cytospin preparations.

Primary Culture of MSCs

hMSCs. Human BM samples, aspirated from the sternum, wereobtained from healthy donors with informed consent. hMSCswere isolated and cultured according to the methods describedpreviously [42]. hMSCs were used at passages 3–5 in this study.

Murine MSCs. Murine MSCs (mMSCs) are more difficult toculture due to the low mMSC numbers in BM and a considerablelevel of contaminated hematopoietic cells in the long-term invitro culture [43]. We established a modified isolation and culturemethod based on the combination of mechanical crushing andcollagenase digestion at the moment of harvest, followed by animmunodepletion step using microbeads coated with CD11b,CD45, and CD34 antibodies [44]. mMSCs were used at passages3–5 for all experiments.

Flow Cytometry Analysis

A two-step staining method was used for immunophenotyping asdescribed [42]. The following antibodies were used: CD14,CD34, CD105, Sca-1, CD45, CD90, vascular cell adhesion mole-cule 1 (VCAM-1), anti-mouse CCR9 (all purchased from eBio-science, San Diego, CA), CD73, integrin a4 (both from BD Bio-sciences, San Diego, CA, www.bdbiosciences.com), integrin b1(Acris Antibodies, Herford, Germany, www.acris-antibodies.com),and anti-human CCR9 (R&D, Minneapolis, MN, www.rndsys-tems.com). Cells were analyzed with a FACSCanto flow cytome-ter (Becton Dickinson, San Jose, CA, www.bdbiosciences.com).WinMDI 2.8 software was used to create the overlap histograms.

Preparation of Conditioned Medium

A total of 2.5 � 105 MSCs were cultured for 2 days in 5 mlserum-free RPMI-1640 medium and culture supernatant washarvested as a source of conditioned medium (CM). CM wascentrifuged at 2000 rpm to remove cell debris and frozen at�20�C until use.

For the collection of MM cell CM, MM cell lines or primaryhuman MM cells (CD138 positive population) were incubated inserum-free RPMI-1640 medium for, respectively, 72 and 24 hours,at a fixed cell concentration of 1 � 106 per milliliter.

In Vitro Migration Assays

The migratory ability of MSCs was determined as described [42].MM cell CM was added as chemoattractant source. Serum-freemedium and 10% fetal calf serum containing medium were usedas negative and positive controls, respectively. Migration experi-ments were also performed using hMSCs with knockdown ofCCR9 via RNA interference.

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In Vivo Migration Assays

5T33MM mice were diseased as described above. After 2 weeksof inoculation, 5T33MM mice and naive mice were injectedi.v. with DiI (1,10dioctadecyl-3,3,30,30tetramethylindocarbocyanineperchlorate) (Molecular Probes, Invitrogen, Merelbeke, Belgium,www.invitrogen.com)–labeled 3 � 105 mMSCs suspended in 200ll of 0.9% NaCl (n ¼ 20 per group). At days 1, 3, 5, and 7 post-MSC injection, five 5T33MM mice and five naive mice were sac-rificed. Lung, heart, liver, spleen, kidney, and tibia were removed,and 5-lm paraffin sections were made. After deparaffin treatment,DiI-labeled mMSCs were observed and counted under fluorescentmicroscope OLYMPUS IX81. To quantify MSCs distributionamong different organs, MSCs were counted in eight randomfields on each section. Five uncontinuous sections were scored tocalculate the mean value. Adjacent sections were analyzed by he-matoxylin and eosin staining. Blood was also taken to determineserum paraprotein concentration at the indicated time.

Chemokine Expression in MM Cells

To detect the presence of chemokines potentially mediating MSCsmigration, CM from the human MM cell line RPMI8226 was col-lected as described above and tested by DiscoveryMAP v1.0 analy-sis (Rules Based Medicine, Austin, TX, www.rulesbasedmedicine.com) using antibodies targeting 22 different chemokines.

CCR9 Knockdown by RNA Interference

To knockdown CCR9 expression, hMSCs were transfected withFlexiTube GeneSolution for CCR9 (GS10803, Qiagen, Hilden,Germany, www.qiagen.com). The FlexiTube GeneSolution forCCR9 provides four nonoverlapping CCR9 RNAi duplexes forthis gene to obtain high knockdown efficiency. These duplexeswere transfected into hMSC with Lipofectamine RNAiMAX(Invitrogen) according to the manufacturer’s protocol. AllStarsNegative Control small interfering RNA (siRNA; SI03650318,Qiagen) was used as negative control. The efficiency of CCR9knockdown was evaluated by real time polymerase chain reaction(PCR) and fluorescence-activated cell sorting (FACS).

The knockdown for CCR9 expression in mMSCs was per-formed using the same strategy but cells were transfected withmouse CCR9 RNAi duplexes (GS12769, Qiagen).

In Vitro Proliferation Assay

A total of 5 � 104 MM cells were plated in 96-well plates for24 hours by direct coculturing with increasing numbers of MSCs(previously irradiated at 1,500 rad) in serum-free RPMI-1640medium or by MSC CM. The proliferation of MM cells was thendetermined by thymidine incorporation assay, described as previ-ously [45]. Results are expressed as the relative DNA synthesiscompared with MM cells alone.

In Vivo Proliferation Assay

Mice were anesthetized, and a small incision was made over theright knee to gain access to the kneecap. A 27-gauge needle wasused to drill a hole in the femur, and 20 ll of RPMI-1640 con-taining 5 � 106 freshly isolated 5T33MMvv cells alone or in thepresence of 0.5 � 106 mMSCs was slowly injected into thecavity with a 26-gauge microliter syringe (n ¼ 5 per group).After 2 weeks, femur with injection in situ, opposite femur andspleen were removed, and the number of 5T33MMvv cells inthese organs was assessed by immunostaining with anti-idiotypeantibodies. The development of the anti-idiotype antibody against5T33MM cells, called 3H2, was described previously [38].

Assessment of Apoptosis

Apoptosis of MM cells was induced by nutritional starvation orBortezomib activity (Janssen Pharmaceutica N.V., J&J PRD).MM cells were washed twice with phosphate-buffered saline(PBS) and stained with 3 ll of 7-amino-actinomycin D (7-AAD;BD Pharmingen, Franklin Lakes, NJ, www.bdbiosciences.com)

and 4 ll of Annexin V–fluorescein isothiocyanate in 100 ll ofbinding buffer for 15 minutes in the dark at room temperature.Then, cells were resuspended in 400 ll of binding buffer and im-mediately analyzed using a FACSCanto flow cytometer. The cellsundergoing early apoptosis are in the lower right quadrant beingAnnexin V positive and 7-AAD negative; late apoptotic or deadcells are in the upper right quadrant being 7-AAD positive andAnnexin V positive; live cells are in the lower left quadrant beingnegative for both fluorescent probes.

Immunofluorescence Staining

To assess MM cell proliferation and apoptosis in situ after intrafe-moral injection, immunofluorescence staining using antiprolifera-tion cell nuclear antigen (PCNA) antibody or TUNEL (terminal de-oxynucleotidyl transferase dUTP nick end labeling) assay wereperformed. The femurs were removed and fixed in decalcificationsolution. All fixed samples were embedded in paraffin and 5-lmsections were cut. After being deparaffinized and rehydrated, slideswere incubated in citrate buffer (pH 6.0), and antigen retrieval wasperformed in a microwave two times for 5 minutes. After coolingdown at room temperature, slides were washed twice for 5 minuteswith PBS and incubated with 10% normal goat serum for 30minutes at room temperature. To determine MM cell proliferation,anti-PCNA antibody (sc-7907, 1/100, rabbit-anti-mouse, SantaCruz Biotechnology, Santa Cruz, CA, www.scbt.com) in PBS wereadded, and the slides were incubated at 4�C overnight. The nextday, after three washes in PBS for 5 minutes, secondary antibodyAlexa Fluor 488 conjugated goat-anti-rabbit IgG (1/250, Invitro-gen) was added and allowed to incubate for 1 hour in the dark atroom temperature. After three washes in PBS for 5 minutes, theslides were counterstained, mounted with SlowFade Gold antifadereagent with 40,6-diamidino-2-phenylindole (DAPI) (Invitrogen),and left for 10 minutes in the dark at room temperature before ex-amination by fluorescence microscopy (Zeiss Axioplan 2, CarlZeiss, Gottingen, Germany, www.zeiss.com). For testing MM cellapoptosis in situ, a TUNEL assay was performed according to themanufacturer’s protocol. In brief, after antigen retrieval, the slideswere washed in PBS three times and incubated with 50 ll ofTUNEL reaction mixture (11684817910, In situ Cell Death Detec-tion Kit, Roche, Mannheim, Germany, www.roche-applied-science.com) for 1 hour at 37�C. After three washes in PBS for 5minutes, the slides were also counterstained by DAPI (Invitrogen)and evaluated by fluorescence microscopy. For both PCNA andTUNEL staining, adjacent sections were analyzed to determineMM cells location. The proliferation index or apoptosis index wasquantified by the average percentage of PCNA(þ)/DAPI(þ) orTUNEL(þ)/DAPI(þ) per field of �200 using Image-Pro Plus 6.0software. Three random fields with more than 90% MM cells werecounted per femur. n ¼ 5 mice per group.

Real Time PCR

Total RNA was isolated using Trizol (Invitrogen) and RNeasyMini Kit (Qiagen, Germany), following the manufacturer’sinstructions. The concentration and purity of RNA was deter-mined by Quant-iT RNA BR Assay kit (Invitrogen) with Qubitfluorometer (Invitrogen). cDNA was synthesized using the Ther-moscript reverse-transcription PCR system (Invitrogen) with ran-dom hexamers as primers. Quantitative real-time PCR analysiswas done using the iCycler (Bio-Rad Laboratories, Hercules, CA,www.bio-rad.com) using the SYBR GreenER qPCR SuperMixfor iCycler (Invitrogen) according to manufacturer’s instructions.The primer sequences used are listed in Table 1. Transcript levelswere normalized to the housekeeping gene b-actin and analyzedby the relative quantification 2�DDCt method.

Western Blot Analysis

mMSCs and 5T33MMvv cells were cocultured for 24 hours at aratio of 1:10. 5T33MMvv cells were then harvested by gentlypipetting to separate them from adherent mMSCs. Preparation ofwhole cell lysates and immunoblotting were performed as

268 Tumor-Promoting Properties of MSCs in MM

previously described [45], using the following antibodies: CylinD2, CDK4, Bcl2, Bcl-XL, Caspase-3, poly(ADP-ribose) polymer-ase (PARP), AKT, Phospho-Akt, p44/42 mitogen-activated pro-tein kinase (MAPK). Phospho-p44/42 MAPK, and b-actin (allfrom Cell Signaling, Danvers, MA, www.cellsignal.com).

In Vivo mMSCs Infusion Study

After inoculation of the 5T33MMvv cells (1 � 105 cells permice) on day 0, 5T33MM diseased mice (n ¼ 10) were treatedwith mMSCs (2 � 105 cells in 200 ll of 0.9% NaCl) or vehicle(0.9% NaCl) intravenously on days 6, 10, and 14. To determinethe safety of mMSCs, naive mice (n ¼ 10) received the sameamount of mMSCs according to the same schedule. Additionalnaive mice (n ¼ 10) without treatment were included as negativecontrols. The survival time of each mouse was determined basedon the occurrence of morbidity, namely hind limb paralysis [46].In parallel, an additional in vivo mMSCs infusion study was per-formed. The difference was that the mice were all killed whenthe first mouse showed the sign of morbidity. Blood sampleswere obtained to determine serum paraprotein concentrations.

Statistical Analysis

Statistical analysis was done using GraphPad Prism 5 software. Alldata represent the mean 6 SD, and results were analyzed using theMann–Whitney U test. Survival curves were plotted using theKaplan-Meier method. p < .05 was considered statistically signifi-cant. All experiments were repeated in at least triplicates.

RESULTS

MSCs Can Migrate Toward Myeloma Cells

Prior to their use in experiments, the true nature of the in vitro-expanded MSCs was confirmed by morphological analysis, im-munostaining with specific surface markers, and their differen-tiation ability toward adipocytes, osteoblasts, and chondrocytesin specific induction media (Supporting Information Fig. 1).

The directed migration of MSCs in response to MM cellswas investigated using an in vitro transwell system. CM of mu-rine MM cells and human MM cells were used as source of che-

moattractants, while serum-free medium and 10% fetal calf se-rum were used as negative and positive controls, respectively.We observed a significant MSC migration in response to bothmurine and human MM cells, although hMSC migration towardMM cells was higher than mMSC migration (Fig. 1A).

MSC migration toward MM cells was investigated in vivousing the 5T33MM mouse model. To track the migration oftransplanted MSC, MSCs were labeled with the cell trackerdye DiI prior to in vivo administration. After 2 weeks of theadministration of 5T33MMvv cells, MM and naive mice wereinjected i.v. with DiI-labeled mMSC. On the 1st, 3rd, 5th, and7th day, several organs (heart, lungs, liver, spleen, kidney,and tibia) were harvested for histological analysis, and bloodsamples were taken to measure serum paraprotein concentra-tion (Fig. 1B). DiI-labeled mMSCs were visualized under thefluorescent microscope (Fig. 1C). Microscopic analysis pro-vided evidence that 7 days after MSCs administration therewere many mMSCs detectable in lungs and liver which are‘‘barrier’’ organs. A decreased MSC migration to lungs and anincreased migration to liver were observed over time (Sup-porting Information Fig. 2). However, more mMSCs weredetected in spleen and tibia in 5T33MM mice compared withnaive mice (Fig. 1D). Spleen and BM are two major MM cellinvaded organs in 5T33MM mice [38]. Moreover, on the indi-cated harvest days, we observed that the number of migratedmMSCs was gradually increased in spleen and tibia of5T33MM mice, which was in accordance with the increasinglevel of M compound, a marker representing disease progres-sion (Fig. 1E). Although MSCs do not exclusively home toMM sites, these data demonstrate that MSCs can preferen-tially home to MM sites.

MM Cell-Produced CCL25 Is a PotentChemokine for Attraction of MSCs

Chemotaxis involves both release of migratory signalsthrough chemokines and expression of specific receptors onthe migrating effector cells. Having found that MSCs couldmigrate in response to MM cells, we started to identifyMM cell-secreted chemokines that could induce recruitmentof MSCs. The CM of RPMI8226 MM cells was used for

Table 1. Primers for quantitative real time PCR

Gene Primer GenBank accession Annealing temperature (�C) Size (bp) Species

IL-6 50-GAGGATACCACTCCCAACAGACC-30 NM_031168 60 141 M50-GCCCTCGCTTCCGTACTCG-30

IGF-1 50-TGCTCTTCAGTTCGTGTG-30 NM_001111274 60 144 M50-ACATCTCCAGTCTCCTCAG-30

VEGF 50-ATCTTCAAGCCGTCCTGTGT-30 NM_001025250 60 177 M50-GCATTCACATCTGCTGTGCT-30

IL-10 50-GCTCTTACTGACTGGCATGAG-30 NM_010548 60 105 M50-CGCAGCTCTAGGAGCATGTG-30

DKK1 50CTGAAGATGAGGAGTGCGGCT-30 NM_010051 60 183 M50-GGCTGTGGTCAGAGGGCATG-30

CCL25 50-TTACCAGCACAGGATCAAATGG-30 NM_009138 58 105 M50-CGGAAGTAGAATCTCACAGCAC-30

b-actin 50-GCATTGTTACCAACTGGGACGA-30 NM_007393 60 174 M50-TGGCTGGGGTGTTGAAGGTC-30

CCR9 50-AGGCCATGAGAGCACATACTT-30 NM_006641 60 130 H50-GATTCCTCCTTGATTTGGCTGT-30

CCL25 50-AAAGCTCCACCACAACACGC-30 NM_005624 63 114 H50-CTGCTGCTGATGGGATTGCT-30

b-Actin 50-ATGTGGCCGAGGACTTTGATT-30 NM_001101 60 107 H50-AGTGGGGTGGCTTTTAGGATG-30

Abbreviations: DKK1, dickkopf homolog 1; IGF-1, insulin growth factor-1; IL-6, interleukin-6; IL-10, interleukin-10; PCR, polymerasechain reaction; VEGF, vascular endothelial growth factor.

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DiscoveryMAP v1.0 analysis, which allows simultaneous detec-tion of a large range of chemokines and cytokines. It was foundthat RPMI8226 cells could secrete a variety of inflammatorychemokines, including eotaxin-3, IP-10, MIG, RANTES, andTECK, among which CCL25 (TECK) showed the highest con-centration (Fig. 2A). To identify whether CCL25 is widelyexpressed in MM cells, 6 other human MM cell lines as wellas primary MM cells from 14 patients were tested by semi-quantitative PCR. It was found that CCL25 was detected in 11out of 14 patients and 5 out of 6 MM cell lines (Fig. 2B).

Furthermore, to examine whether MM cell-secretedCCL25 is functionally involved in MSC migration, theCCL25 unique receptor, CCR9, was knocked-down in hMSCsby RNA interference. FACS analysis showed the efficiency ofCCR9 knockdown (Fig. 2C). hMSCs with CCR9 knockdownexhibited a significant decrease in migration (32%–43%) to-ward MM cells using an in vitro transwell system (Fig. 2D).In addition, we confirmed that 5T33MM murine MM cellswere positive for CCL25 (Supporting Information Fig. 3) andknocked down CCR9 expression in mMSCs by RNA

Figure 1. MM cells stimulate migration of MSCs in vitro and in vivo. (A): Quantification of MSC migration in vitro in response to factorssecreted by MM cells (5T33MMvt and 5TMMvv MM cells for mMSCs, RPMI8226, and LP-1 MM cells for humans MSCs). Data are the mean6 SD of results from three independent experiments. (B): Schematic chart of in vivo MSCs migration assay. mMSCs were labeled with the celltracker DiI beforehand and injected i.v. into 5T33MM mice on 4th day. Naıve mice were used as control. On the indicated days post-mMSCadministration, mice were sacrificed, organs were harvested, and paraffin sections were made. (C): DiI-labeled mMSCs were visualized under flu-orescence microscope. Representative photographs are shown for lung, heart, liver, spleen, kidney, and tibia of 5T33MM mice on 7th day aftermMSCs injection. Scale bar ¼ 50 lm. (D): In vivo mMSC distribution in these organs was compared between naive and 5T33MM mice on day7 post-mMSC administration. *, p < .05. (E): Quantification of the number of MSC that migrated to MM-infected organs (spleen and tibia) post-mMSC administration is consistent with the MM disease progression as determined by M-compound level. *, p < .05; **, p < .01; ***, p <.001 compared to mMSCs number on day 1 after i.v. injection. Abbreviations: DiI, 1,10dioctadecyl-3,3,30,30tetramethylindocarbocyanine perchlo-rate; FCS, fetal calf serum; mMSC, murine mesenchymal stromal cells; MM, multiple myeloma.

270 Tumor-Promoting Properties of MSCs in MM

Figure 2. Involvement of CCL25 in MM cell-induced migration of mMSCs. (A): Chemokine analysis of serum-free conditioned medium fromthe MM cell line RPMI8226. Results are expressed as concentration of secreted chemokines (pg/ml). (B): CCL25 mRNA expression in isolatedbone marrow (BM) plasma cells from 14 patient samples (MM1 to MM14) and 7 human MM cell lines. b-Actin mRNA expression was used aspositive control. (C): One representative FACS analysis of hMSCs after CCR9 knockdown from three independent experiments. (D): CCR9knocked-down hMSCs exhibited a decreased in vitro migration ability toward primary human MM cells and three MM cell lines. Data are themean 6 SD of results from three experiments. *, p < .05 compared to hMSCs transfected with control siRNA. (E): One representative fluores-cence-activated cell sorting (FACS) analysis of mMSCs after CCR9 knockdown from three independent experiments. (F): After 7 days of i.v.injection into 5T33MM mice, CCR9 knocked-down mMSCs exhibited a significantly decreased in vivo migration toward the MM-invaded spleen,as well as a decreased migration toward the tibia. n ¼ 5 per group; *, p < .05 compared to mMSCs transfected with control siRNA. Abbrevia-tions: DiI, 1,10dioctadecyl-3,3,30,30tetramethylindocarbocyanine perchlorate; hMSCs, human mesenchymal stromal cells; mMSCs, murine mesen-chymal stromal cells; MM, multiple myeloma; PE, phycoerythrin; siRNA, small interfering RNA.

interference (Fig. 2E). Then, an in vivo migration assay wasperformed using CCR9 knockdown or control siRNA trans-fected mMSCs into 5T33MM mice. After 7 days of injection,we could observe that there were significantly less mMSCs inthe spleen of 5T33MM mice with CCR9 knockdown mMSCsinjection. There were less mMSCs in the tibia as well,although this was not significant (Fig. 2F).

MSCs Stimulate Myeloma Cell ProliferationIn Vitro and In Vivo

To test the effect of MSCs on the proliferation of MM cells,we first cocultured the human MM cell lines MM5.1 (stroma-

dependent) and RPMI8226 (stroma-independent) as wellas the murine MM cell lines 5T33MMvv (stroma-dependent)and 5T33MMvt (stroma-independent), with hMSCs ormMSCs, respectively, at different ratios for 24 hours in se-rum-free medium. MSCs exhibited a dose-dependent effect onthe proliferation of human and murine stroma-dependent MMcells but had no apparent effect on stroma-independent MMcells (Fig. 3A). To analyze whether soluble factors fromMSCs are involved in the stimulation of MM proliferation,MSC CM was used to culture stroma-dependent MM cells,MM5.1 and 5T33MMvv. A stimulatory effect was alsodetected but less pronounced when compared with the effect

Figure 3. MSCs favor stroma-dependent MM cell growth in vitro and in vivo. (A): The proliferation of MM cells was assessed after coculturewith pre-irradiated MSCs. Human MSCs and mMSCs contribute to the proliferation of the stroma-dependent MM cell line MM5.1 and5T33MMvv, in a dosage-dependent manner but have no remarkable effect on the stroma-independent MM cell line RPMI8226 and 5T33MMvt.*, p < .05; **, p < .01; ***, p < .001 compared to tumor cells alone. (B): Stroma-dependent MM cells were cultured with MSCs (MM cells/MSCs ratio ¼ 10:1) or in the presence of MSCs conditioned medium. A growth promoting effect was detected in both conditions but less pro-nounced with MSC conditioned medium as compared to the direct cell–cell contact condition. (C): Schematic chart of the in vivo proliferationassay. 5T33MMvv cells were injected with or without mMSCs into the right femur. After 2 weeks, cell suspensions from bone marrow of twohind legs and the spleen were evaluated for the presence of MM cells using idiotype-antibody FACS analysis and in situ immunostaining. (D):The level of MM cells in both femurs and spleen was monitored by anti-idiotype FACS analysis at 2 weeks after injection. n ¼ 5 per group. *, p< .05 compared to tumor cell injection alone. (E): 5T33MMvv cells (5 � 106) were injected intrafemorally with or without mMSCs (5 � 105).After 1 week, more MM cells were in a proliferative state as shown by positive PCNA expression in the femur where mMSCs were coinjectedcompared to tumor cell injection alone. The data represented the percentage of PCNA (þ) cells in all DAPI (þ) cells per field. n ¼ 5 mice pergroup. *, p < .05 compared to tumor cell injection alone. Abbreviations: DAPI, 40,6-diamidino-2-phenylindole; FACS, fluorescence-activated cellsorting; mMSCs, murine mesenchymal stromal cells; MM, multiple myeloma; PCNA, proliferation cell nuclear antigen.

272 Tumor-Promoting Properties of MSCs in MM

observed in the cultures with direct cell–cell contact(Fig. 3B).

Next, we investigated whether MSCs could favorMM cell growth in vivo. Naive mice received 5 � 106

5T33MMvv cells with or without 5 � 105 mMSCs by intrafe-moral injection in the right leg. After 2 weeks, cell suspen-sions from the BM of two hind legs and the spleen were eval-uated for the presence of MM cells using idiotype-antibodyFACS analysis (Fig. 3C). In the right femur where MM cellsand mMSCs were coinjected, 29.1% MM cells were detected,while only 12.4% MM cells were detected in the femur with-out mMSCs injection. After injection of MM cells into theright femur, some MM cells could be detected in the oppositeleg and in the spleen as well. By anti-idiotype FACS analysis,we found a higher proportion of MM cells in the opposite legand spleen of mice that were coinjected with MSCs whencompared with mice injected with 5T33MM cells alone, how-ever, without significant difference (Fig. 3D). To confirm thein vivo data that MSC can promote proliferation of MM cells,we injected 5T33MMvv cells intrafemorally into naive micetogether with or without mMSCs intrafemorally. After 7 days,by an immunofluorescence staining of the proliferation anti-gen PCNA, we observed that there were significantly moreMM cells positive for PCNA expression in the femur wheremMSCs were coinjected (proliferation index: tumor alone vs.MSC coinjection is 12.4 6 2.8% and 19.8 6 5.3%, respec-tively, p < .05; Fig. 3E, Supporting Information Fig. 4).

MSCs Protect MM Cells Against ApoptosisIn Vitro and In Vivo

Human and murine MM cells were cultured in serum-freemedium for 24 or 48 hours to induce spontaneous apoptosis.In the presence of MSCs, the proportion of apoptotic cellswas dramatically decreased, in a dosage-dependent manner(Fig. 4A). Chemoresistance and relapse is the key feature ofthe clinical course in MM, and it is well-known that the BMmicroenvironment protects MM cells against chemotherapy.Here we examined whether MSCs could protect MM cellsagainst chemotherapy-induced apoptosis. Bortezomib is aclinically available proteasome inhibitor that is currentlyamong the most potent chemotherapeutic drugs used in thetreatment of MM. When the human MM cell line RPMI8226and the murine MM cell line 5T33MMvt were cultured incomplete medium with 5 nM Bortezomib for 48 hours,approximately 70% apoptotic cells (early þ late) wereobserved. However, in the presence of MSCs, the proportionof apoptotic cells significantly decreased to 35% and 53%,respectively. Similar findings were observed for the stroma-dependent human MM cell line MM 5.1 and the murine MMcell line 5T33MMvv (Fig. 4B). After separation from hMSCs,we observed less than 2% hMSCs contamination in thehuman MM fraction, excluding significant influence on theapoptotic analyses of the MM cells (Supporting InformationFig. 5). A comparable finding was also observed in the cocul-ture of mMSCs and murine MM cells.

To confirm in vivo that MSC could protect against sponta-neous apoptosis of MM cells, 5T33MMvv cells were intrafe-morally injected into naive mice together with or withoutmMSCs. By the TUNEL apoptotic assay, less MM cells werefound to undergo apoptosis in mMSC coinjected femurs,although no significance was observed (apoptosis index:tumor alone vs. MSC coinjection is 8.9 6 2.8% and 6.2 62.3%, respectively; Fig. 4C, Supporting Information Fig. 6A).In addition, to explore whether MSC could protect MM cellsagainst Bortezomib-induced apoptosis in vivo, mice weregiven Bortezomib after intrafemoral injection of 5T33MMvv

cells with or without mMSCs, and the TUNEL apoptoticassay was performed. We found significantly less apoptoticMM cells in the mMSC coinjected femur indicating that MSCcould protect Bortezomib-induced MM apoptosis in vivo (ap-optosis index: tumor alone vs. MSC coinjection is 81.4 610.0% and 63.7 6 13.3%, respectively, p < .05; Fig. 4D,Supporting Information Fig. 6B).

Crosstalk Between MM Cells and MSCs

Previous studies have shown that a regulatory network ofcytokines and growth factors exist between BM stromal cellsand MM cells within the MM microenvironment [47–49]. Inthis study, we tested the effect of 5T33MMvt cells on theexpression of MM specific growth and survival factors inmMSCs. After transwell coculture with MM cells for 48hours, interleukin-6 (IL-6), insulin growth factor-1 (IGF-1),vascular endothelial growth factor (VEGF), IL-10, and dick-kopf homolog 1 (DKK1) expression in mMSCs were all sig-nificantly upregulated (Fig. 5A). To determine how MSCscould favor the growth of stroma-dependent MM cells andprotect MM cells against apoptosis, we further examined themolecular changes of MM cells in the presence of MSCs. Af-ter coculturing mMSCs and murine MM cells 5T33MMvv for24 hours, MM cells showed enhanced expression of pAKTand pERK. Positive cell cycle protein Cyclin D2 and CDK4were both upregulated. In addition, the antiapoptotic proteinBcl-XL, but not Bcl-2, was increased in MM cells, accompa-nied with the downregulation of the proapoptotic proteins,cleaved Caspase-3 and PARP (Fig. 5B).

MSCs Infusion Decreases the Survivalof 5T33MM Diseased Mice

As MSCs were able to migrate toward MM sites and favor MMcell growth, we examined whether infusion of MSCs couldaffect MM disease progression and survival in vivo. 5T33MMmice were intraveneously injected with mMSCs on days 6, 10,and 14 after tumor cell inoculation. MSCs infusion led to a sig-nificant decrease in survival, to a mean of 18 days in themMSCs treated MM mice compared to 22 days in vehicle-treated MM mice (n ¼ 10 per group; p ¼ .022; Fig. 5C). Noparticular side effects of mMSCs infusion were observed.

To confirm that MSCs accelerate the development of MMin 5T33MM mice, we performed an additional MSC infusionexperiment. 5T33MM mice were injected with mMSC orvehicle at the same time as shown above. When the firstmouse showed signs of morbidity, all mice were killed, andserum paraprotein, the indicator for MM disease progression,was measured (n ¼ 10 per group). As shown in Figure 5D,the MM mice with MSC infusion had significantly higherparaprotein levels compared to the mice with vehicle treat-ment, indicating that MSC infusion did accelerate MM dis-ease progression.

DISCUSSION

A basic feature of MM cells concerns their reciprocal inter-action with surrounding stromal cells which constitutes theso-called ‘‘MM tumor microenvironment.’’ Cellular compo-nents of the MM tumor microenvironment include osteoclasts,endothelial cells, macrophages, fibroblasts, and others. Thesecells play a very essential role in supporting the proliferation,survival, chemoresistance, and migration of MM cells. Here,we demonstrate that MSCs, the precursors of most BM stro-mal cells, can be recruited by MM cells and have a directimpact on tumor expansion and disease progression (Fig. 6).

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Figure 4. MSCs protect MM cells against apoptosis in vitro and in vivo. (A): MSCs were cocultured in serum-free medium with stroma-dependentMM cells (5T33MMvv and MM5.1) for 24 hours or stroma-independent MM cells (5T33MMvt and RPMI8226) for 48 hours, at a ratio of 1:100 or1:10 (MSCs/MM cells). As measured by AnnexinV/7-AAD staining, MSCs could protect both stroma-dependent and stroma-independent MM cellsagainst spontaneous apoptosis induced by serum starvation in a dose-dependent manner. (B): In complete growth medium, MSCs had no apparenteffect on apoptosis of stroma-independent MM cell 5T33MMvt and RPMI8226 but could protect against Bortezomib-induced apoptosis at a ratio of1:10 (MSCs/MM cells). Similarly, MSCs can also protect Bortezomib-induced apoptosis of stroma-dependent MM cells 5T33MMvv and MM 5.1.Representative fluorescence-activated cell sorting profiles are shown. *, p < .05; **, p < .01; ***, p < .001. (C): 5T33MMvv cells (5 � 106) wereinjected intrafemorally with or without mMSCs (5 � 105). After 1 week, there were less MM cells undergoing apoptosis in the femur where mMSCswere coinjected, compared to tumor cell injection alone, although this was not significant. TUNEL immunofluorescence staining was performed, andthe slides were counterstained by DAPI. n ¼ 5 mice per group. (D): After 5T33MMvv injection into naive mice intrafemorally, five doses of Bortezo-mib at 0.6 mg/kg i.p. every other day were given, leading to MM cell apoptosis as measured by TUNEL staining. However, with the coinjection ofmMSCs, significantly less MM cells underwent apoptosis. The data represented the percentage of TUNEL (þ) cells in all DAPI (þ) cells per field. n¼ 5 mice per group. *, p < .05 compared to tumor cell injection alone. Abbreviations: 7-AAD, 7-amino-actinomycin D; DAPI, 40,6-diamidino-2-phe-nylindole; hMSCs, human mesenchymal stromal cells; mMSCs, murine mesenchymal stromal cells; MM, multiple myeloma; TUNEL, terminal deoxy-nucleotidyl transferase dUTP nick end labeling.

274 Tumor-Promoting Properties of MSCs in MM

Many studies in tumor models indicated that MSCs, deliv-ered intravenously or intraarterialy, have preferential tropismtoward tumor sites, primarily as a result of tumor cell-secretedparacrine factors and/or damaged tissue-produced inflamma-tory mediators, and therefore some reports have shown theuse of MSCs as vehicles for cancer gene therapy [50–53]. Toincrease the therapeutic efficacy of MSCs in these cases,some intervention measures, including irradiation, hypoxicpreconditioning, and genetic modification, have been used toenhance MSCs migratory capacity [54–56].

Using an in vitro transwell system, we found in this studythat both mMSCs and hMSCs can migrate in response to MMcells. In the in vivo 5TMM mouse model, we demonstratedthat fluorochrome-labeled mMSCs have the ability to homeinto MM cell invaded sites after systemic administration.Although a major part of mMSCs were found 7 days postad-ministration in lungs and liver, there were remarkably moremMSCs detectable in spleen and tibia of 5TMM diseasedmice as compared to naive mice. Moreover, the number ofmigratory mMSC in spleen and tibia in 5TMM diseased mice

Figure 5. Crosstalk between MM cells and MSCs. (A): MM cells alter cytokine expression in MSCs. After coculture with 5T33MMvt cells ina transwell system for 48 hours, IL-6, IGF-1, VEGF, IL-10, and DKK1 expression were all upregulated in mMSCs as measured by real timequantitative PCR. (B): MSC-induced molecular changes in MM cells. By Western blot analysis, 5T33MMvv MM cells, cocultured with mMSCsfor 24 hours, exhibited upregulated expression of the cell cycle proteins cyclin D2 and CDK4, and the antiapoptotic protein Bcl-XL, as well asdecreased expression of cleaved Caspase-3 and PARP. Phosphorylated AKT and phosphorylated ERK1/2 were also increased. One of three inde-pendent experiments is shown. (C): Kaplan Meier survival curve. Mice were injected i.v. with mMSCs or the vehicle on the indicated days(arrows) after inoculation with 5T33MM cells (n ¼ 10 per group). On average, the onset of morbidity in the vehicle group was 22 days, and themMSCs infusion group lived 4 days shorter. *, p < .05; **, p < .01. (D): In a parallel mMSC transplantation experiment, all mice were sacri-ficed and serum paraprotein was analyzed when the first mice showed signs of morbidity. 5T33MM mice with mMSC infusion had higher para-protein levels than the mice without mMSCs infusion. *, p < .05. n ¼ 10 per group. Abbreviations: DKK1, dickkopf homolog 1; IGF-1, insulingrowth factor-1; IL-6, interleukin-6; mMSCs, murine mesenchymal stromal cells; MM, multiple myeloma; PARP, poly(ADP-ribose) polymerase;PCR, polymerase chain reaction; VEGF, vascular endothelial growth factor.

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was consistent with MM disease progression as determined bythe serum paraprotein level. To elucidate the mechanism ofMSC recruitment by MM cells, a chemokine array of MMcells was performed. Among all the chemokines tested,CCL25, also called TECK, was found to be secreted byRPMI8226 MM cells at the highest level. CCL25, a CC che-mokine expressed predominantly in thymus and epithelium ofthe small intestine, mediates chemotaxis of T cells through itscounter receptor CCR9 [57]. In patients with inflammatorybowel disease, it was found that there were increased numbersof CCR9 (þ) lymphocytes circulating in the peripheral blood[58]. In some tumor models, such as melanoma, prostate can-cer, breast cancer, and ovarian cancer, the expression andactivation of CCR9 affect cancer cell migration and invasion,which can modulate cancer cells metastasis to distal sites thatexpress CCL25 [59–62]. Previous studies have demonstratedthat CCL25 can function as a chemoattractant for MSCs andperiosteal progenitor cells in vitro [63, 64]. However, the roleof tumor cell-produced CCL25 in the attraction of other celltypes has not been reported so far. In this study, PCR analysisrevealed that CCL25 was expressed widely in primary humanMM cells and MM cell lines. A previous study has shownthat both human and murine BM-derived MSCs expressCCR9 [65]. Subsequently, we confirmed this expression andused siRNA to downregulate CCR9 expression on hMSCs.We observed that hMSCs transfected with CCR9 siRNAexhibited a profoundly lower migratory ability in response toCM of MM cells in vitro. In addition, CCR9 knocked-downmMSCs exhibited significantly lower migration ability towardMM-invaded spleen of 5T33MM mice in vivo. Both in vitroand in vivo loss of function studies indicate that CCL25 isinvolved in MM cell-mediated chemotaxis of MSCs. It is

worthy of being noticed that MSCs express chemokine receptorsalso for other chemokines produced by MM cells. These includeCCR1, CCR3, CCR5, and CXCR3, which can interact with MMcell-produced CXCL9 (MIG), CXCL10 (IP-10), CCL5(RANTES), and CCL26 (Eotaxin-3) [65]. Some of these chemo-kines/chemokine receptors might play a role in MSCs recruit-ment by MM as well, but this needs to be further explored.

Some studies have shown enhancement of tumor growthby MSCs, potentially through immunomodulatory and/orproangiogenic properties of these stem cells. However, otherstudies have demonstrated that inhibition of tumor growth andextended survival could not show any apparent effect [20–31]. Which effect MSCs might have on the tumor growth anddisease progression in MM is currently unclear. In this study,we found through in vitro coculture assays that MSCs favorthe proliferation of stroma-dependent MM cells by secretingsoluble factors and cell–cell contact. This growth-promotingeffect was also confirmed by coengraftment experiments invivo. After 2 weeks of coengraftment of MSCs and MM cellsintrafemorally, there are more than twice MM cells detectablein the in situ femur compared to MM cells injection alone.Additional PCNA immunofluorescence staining on the in situfemur confirmed that MM cells have a higher proliferationability when MSCs are coinjected intrafemorally

Besides favoring MM cell growth, MSCs were also foundto protect MM cells against apoptosis, induced by serum star-vation or by the potent anti-MM drug Bortezomib, as shownby in vitro Annexin V/7-AAD FACS analysis and by in vivoTUNEL immunofluorescence staining. Recent evidenceshowed that survivin was involved in the antiapoptotic effectof MSCs on myeloma cells [66]. In the coculture system, weobserved that MM cells tended to grow adherently on MSCs,

Figure 6. Schematic model of MSCs effects on MM disease. After systemic infusion, in vitro-expanded MSCs are able to migrate toward MMsites, attracted by MM cell-secreted CCL25 and/or other chemokines. Once MSCs localize in MM sites, by means of direct cell–cell contact and/or a paracrine mechanism, they can favor MM cell growth, protect MM cells against apoptosis, and induce drug resistance. Hence, MM diseaseprogression and relapse can be accelerated. Abbreviations: MSC, mesenchymal stromal cell; MM, multiple myeloma.

276 Tumor-Promoting Properties of MSCs in MM

and direct contact could influence MM cells growth and theirdifferentiation phenotype [67]. It has been shown previouslythat very late antigen 4, or integrin a4/b1 complex, is one of themost important surface molecules for MM cell homing, adhe-sion, growth, survival, and resistance to chemotherapy, medi-ated by its ligand VCAM-1 on BM stromal cells [68–70]. How-ever, blocking VCAM-1 on mMSCs could neither decreaseMM cell adhesion significantly nor impair the supporting effecton proliferation of 5T33MMvv myeloma cells (data notshown), indicating that other adhesion molecules appear to beinvolved in the interaction of MSCs and MM cells.

The functional crosstalk between MM cells and BM stromalcells has been emphasized previously [47–49]. It has been dem-onstrated that MSCs can produce a variety of cytokines andgrowth factors, such as IL6, IL-10, hepatocyte growth factor,stem cell factor, VEGF, IGF-1, and tumor necrosis factor a, allof which have evidence to favor MM cell tumorigenesis [71].We found in this study that after indirect coculture with MMcells in vitro, MSCs could express increased levels of IL-6,IGF-1, VEGF, IL-10, and DKK1. There is evidence showingthat MM patient-derived MSCs (MM-MSCs) have the ability toexpress higher levels of cytokines and growth factors, and theyhave a distinct transcriptional pattern, resulting in impairedimmunomodulary effect and elevated angiogenic activity com-pared with their normal counterparts [72–77]. Our study sug-gests that at least some of these abnormalities of MM-MSCsare ‘‘inducible’’ by the interaction with MM cells. Meanwhile,MSCs, in turn, can favor MM cell growth and protect MM cellsagainst spontaneous and drug induced apoptosis. It is believedthat PI3K/AKT and MAPK signaling pathways are important inantiapoptosis and proliferation of MM cells [78, 79]. After co-culture with MSCs, MM cells exhibit an upregulation of phos-phorylated AKT and ERK activity, accompanied with increasedcyclinD2, CDK4, and Bcl-XL, and decreased cleaved Caspase-3 and PARP.

A very recent report indicates that placenta-derived adher-ent cells, which are mesenchymal-like stem cells isolatedfrom postpartum human placenta, effectively suppress bonedestruction and tumor growth in an in vivo severe combinedimmunodeficiency–rab mouse myeloma model although thesestem cells significantly support MM cell growth in vitro [80].We speculate that some factors like differences in cell source(placenta vs. BM) and the nature of the in vivo MM modelused can explain the discrepancy between these publishedfindings and the data presented in our study.

Given recent findings that infusion of MSCs is a promisingway to support hematopoietic recovery and control of graft versus

host disease after allogeneic hematopoietic stem cell transplanta-tion, we have to consider that this type of cell therapy might benot be safe in MM. Recently, a randomized clinical trial indicatedthat infusion of MSCs results in an increased frequency of diseaserelapse in patients with hematological malignancies (acute mye-loid leukemia, chronic myelogenous leukemia, acute lymphoblas-tic leukemia, myelodysplastic syndrome, and non-Hodgkin lym-phoma) [81]. Although MM patients were not included in thisclinical trial, the decrease in survival we observed in this studyusing an in vivo MM mouse model indicates that systemic infu-sion of MSCs might contribute to MM patient disease progression.Infused MSCs might migrate to the patients’ BM and favor thegrowth of residual MM cells. As MSCs are also immunosuppres-sive, both effects (growth stimulation and immune suppression)might probably lead to MM disease relapse. We therefore suggestthat experimental MSC-based cell therapies in MM patientsshould be considered with extreme caution.

CONCLUSION

We demonstrated that MM cells could induce in vitro as well as invivo chemotaxis of MSCs through CCL25/CCR9 axis, and MSCsplay a supportive role in MM cell growth. Therefore, our data sug-gest that MSC-based cytotherapy might have a potential risk forfavoring MM disease progression or relapse in MM patients.

ACKNOWLEDGMENTS

We thank Nicole Arras, Wim Renmans, Angelo Willems, CarinSeynaeve, Prof. Herman Tournaye, and Dr. Ning Liang for theirexpert technical assistance, and Prof. Zhou Qinghua for hisadministrative support. Our research work is supported by grantsfrom the FWO-Vlaanderen, Vlaamse Liga tegen Kanker (Sticht-ing Emmanuel Van der Shueren), the Vrije Universiteit Brussel(HOA), and the Scientific Foundation Willy Gepts (WFWG) UZBrussel. Xu is supported by CSC-VUB scholarship.

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.

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