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Micron 96 (2017) 77–85
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ltered bone microarchitecture and gene expression profile due toalcium deficiency in a mouse model of myeloma
élène Libouban ∗, Daniel ChappardEROM Groupe Etudes Remodelage Osseux et bioMatériaux – IRIS-IBS Institut de Biologie en Santé, Université d’Angers, CHU d’Angers, 49933 Angers Cedex,rance
r t i c l e i n f o
rticle history:eceived 26 September 2016eceived in revised form 24 January 2017ccepted 30 January 2017vailable online 24 February 2017
eywords:ietary calciumyperperathyroidismultiple myelomasteolysis-ray microtomography
a b s t r a c t
It is not clear why patients with an indolent form of multiple myeloma (MM) develop into an aggressiveform with poor prognostic. We investigated the effect of a dietary calcium deficiency on tumor growth,osteolysis and gene expression in the 5T2MM murine model.
Two groups of C57BL/KaLwRij mice received 5T2MM cells and started a diet with normal (0.8%;“normal-Ca-MM”) or low calcium content (0.05%; “low-Ca-MM”). Two control groups (without 5T2MMcells) received either a normal or low calcium diet (normal-Ca and low-Ca groups). Tumor growth, oste-olysis and marrow gene expression of the Wnt pathway, RANKL and MIP-1� were monitored at 6, 8 and10 weeks (w) after cell injection.
In low-Ca mice, serum level of PTH was higher after 10 w; microCT showed trabecular bone loss anddecrease of cortical thickness at the tibia. A higher M-protein level was evidenced at 10 w and 4 micedeveloped paraplegia at 8/9 w in low-Ca-MM group only. Numerous cortical perforations of the tibia wereobserved in MM groups with a marked decrease in cortical thickness in low-Ca-MM. At 6 w, osteoclastnumber from the endosteum was significantly higher in low-Ca-MM compared to normal-Ca MM. Thisobservation was not found at 8 and 10 w. MicroCT of the lumbar vertebrae showed dramatic bone destruc-
tion in the low-Ca-MM group. qPCR revealed no difference in RANKL expression whereas differences wereobtained in the expression of Lrp5/Lrp6 and MIP-1� from 6 w.A low calcium diet induced higher bone destruction in the tibia and vertebra associated with an earlierdecrease in bone formation level and a higher increase in bone resorption level at early time in the MMdevelopment.
© 2017 Published by Elsevier Ltd.
. Introduction
Multiple myeloma (MM) is a hematological malignancy char-cterized by proliferation of malignant plasma cells in the bonearrow. Osteolysis is one of the main clinical consequences of MM
nd concerns 90% of the patients. Osteolytic foci in MM patientsnduce serious clinical manifestations such as bone pain, fracturesf long bones, vertebra and/or hypercalcemia. Bone fractures occur
n 60% of patients (Roodman, 2009). Moreover, bone fractures have higher incidence in MM than in other cancer (breast, prostate andung cancer) with a high frequency of bones metastases. Patho-
ogical fractures in MM are associated with a 20% increased riskf death (Saad et al., 2007). In the last decade, the median sur-ival of MM patients has been improved due to the apparition∗ Corresponding author.E-mail address: [email protected] (H. Libouban).
ttp://dx.doi.org/10.1016/j.micron.2017.01.009968-4328/© 2017 Published by Elsevier Ltd.
of new treatments. The median survival is now approximately5–8 years and reaches 10 years for patients with low-risk cytoge-netic abnormalities (Avet-Loiseau et al., 2012; Chesi and Bergsagel,2015). Bone resorption, due to an increase in osteoclast number,is increased in MM patients in the vicinity of plasma cell nodules(Bataille et al., 1989). Secretion of numerous cytokines by malig-nant plasma cells and the bone marrow microenvironment areresponsible for an increased osteoclastogenesis in MM (Roodman,2009). Several cytokines have been identified to play a key rolein osteolysis: tumor necrosis factor alpha (TNF�, interleukin-1�(IL-1�)), interleukin-6 (IL-6), macrophage inflammatory protein-1-alpha and beta (MIP-1� and MIP-1�) and ligand for receptoractivator of nuclear transcription factor-�B (RANKL) (Kato et al.,2002; Xi et al., 2016). In overt MM, a decrease in bone formationis associated, leading to an uncoupling in bone remodeling at the
later stages of the disease. Inhibition of osteoblastogenesis is due toinhibitors released by plasma cells that depress the Wnt-signalingpathway: DKK1 (Dickkopf-1) and Sfrp2 (Secreted frizzled-related
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8 H. Libouban, D. Chappa
rotein 2) (Tian et al., 2003) or act on osteoblastic precursors: hep-tocyte growth factor (HGF), interleukin-3 (IL-3) and interleukin-7IL-7) (Ehrlich et al., 2005; Standal et al., 2007).
Animal models can help to understand the molecular, cellu-ar and tissue aspects of the pathophysiology of human diseasesnd to develop new therapeutic strategies. Several murine modelsf human MM have been described in the literature (see reviews
n (Libouban, 2015; Paton-Hough et al., 2015)). Among them, theT2MM murine model was described in the C57BL/KaLwRij strainRadl et al., 1988, 1979). It presents the most common aspectsf the human disease with moderate growth, homing restrictedo the bone marrow microenvironment and osteolytic lesions. A
onoclonal protein (M-protein) is detected from 6 weeks (w)fter injection and osteolytic lesions are radiographically visiblet 8 w (Libouban et al., 2004). The 5T2MM model was extensivelysed to evaluate the effect of antineoplastic and antiangiogenicrugs (Deleu et al., 2009a; Deleu et al., 2009b) or inhibitors ofone resorption such as bisphosphonates (Croucher et al., 2003;’Diaye et al., 2015; Radl et al., 1985). The model is also of thetpmost importance to elucidate the pathophysiology of the dis-ase (Buckle et al., 2012; Vanderkerken et al., 2000). It has beenhown that a high bone remodeling level (as induced by ovariec-omy in mice) accelerates plasma cell growth (Libouban et al.,003). Similarly, other factors known to increase bone remodelingave also been found to burst the growth of carcinomatous cellsHirbe et al., 2007; Ooi et al., 2010; Zheng et al., 2008, 2007, 2011).
calcium deficient diet stimulates the growth of MCF-7 humanreast cancer cells in the mouse by inducing a secondary hyper-arathyroidism (Zheng et al., 2007). More recently, it was shownhat a vitamin D deficiency promotes MDA-MB-231 breast cancerells in the mouse by also inducing a secondary hyperparathy-oidism (Ooi et al., 2010). These studies highlight the importance ofalcium intake and bone remodeling on tumor growth and osteoly-is. They have analyzed the pathophysiological mechanisms at theell and tissue levels in animal models of bone metastasis; how-ver, molecular alterations in the bone microenvironment haveeceived little consideration. The aim of the present study waso investigate the effect of a dietary calcium deficiency on MMevelopment at the tissue, cell and molecular levels in the 5TMModel. Microcomputed tomography (microCT), histomorphome-
ry and gene expression profile of the microenvironment weresed.
. Material and methods
.1. The multiple myeloma cell line
We have used a 5T2MM cell line (subclone 5THL) in57BL/KaLwRij mice as previously characterized (Libouban et al.,004). These malignant plasma cells can be propagated into youngyngeneic mice by intravenous transfer of cells from invadedone marrow. Progression of the disease in recipient mice wasssessed by measuring the serum M-protein level. Around 6 wost-injection, mice had a detectable serum M-protein and wereacrificed after 10–11 w by cervical dislocation. Femurs and tib-as were dissected, cleaned of surrounding tissues and bone
arrow was flushed in Dulbecco’s modified essential mediumDMEM.mod., GIBCO, Life Technologies, France) supplementedith penicillin-streptomycin, amphotericin-fungizone and pyru-
ate (GIBCO). Bone marrow cells were washed once in DMEM.mod.
ononuclear cells were isolated by a Lympholyte-M centrifugationradient (Cedarlane, Hornby, Ontario, Canada) at 450 g for 25 min.ononuclear cells were then washed twice and counted. The recip-
ent mice received 1.5 × 106 cells in the tail vein.
icron 96 (2017) 77–85
2.2. Animals and study design
Seventy-six C57BL/KaLwRij female mice 6–8-w-old were usedfor the full study (Harlan, Gannat, France). They were acclimatedfor one w to the local vivarium conditions (24 ◦C and 12 h/12 h lightdark cycle) where they were given standard laboratory food (UAR,Villemoison sur Orge, France) and water ad libitum. The animalswere housed at the animal facilities of Angers University MedicalSchool (SCAHU). All procedures have been approved by the Institu-tional Animal Care and Use Committee at the University of Angers(Agreement number 49028). All procedures were done in accor-dance with the 2013 French animal procedure act and under thesupervision of authorized investigators.
Two experimental procedures were conducted. The first oneexamined the modification of bone remodeling by dietary cal-cium deficiency in control animals and used 20 C57BL/KaLwRijmice. The second experimental procedure combined the calciumdeficiency and the injection of 5T2MM cells in mice and used56 C57BL/KaLwRij mice. The two experimental procedures weredescribed in details below.
2.2.1. Modification of bone remodeling by dietary calciumdeficiency
On day 0, twenty mice were randomized into 2 groups (10 ani-mals per group). The first group was given standard laboratorychow with normal calcium content (0.8%); they constituted theNormal-Ca group. The second group was given a calcium deficientlaboratory chow (0.05% – SAFE AUGY, Augy, France); they consti-tuted the Low-Ca group. At 10 w, blood samples were collectedby intracardiac puncture to measure serum parathyroid hormone(PTH) level and mice were then sacrificed by cervical dislocation.
2.2.2. Combined model (dietary calcium deficiency + 5T2MMcells)
Fifty mice were injected with 5T2MM cells in the tail vein andimmediately placed on the normal or low calcium diet as above.They constituted respectively the normal-Ca-MM group (n = 25)and the low-Ca-MM group (n = 25). Each group of mice was dividedinto 3 subgroups to perform a time-dependent analysis of osteoly-sis by histomorphometry and quantitative PCR (qPCR). Mice weresacrificed by cervical dislocation at 6, 8 and 10 w. Mice, which devel-oped paraplegia before the end of each time point, were sacrificedfor ethical reason. Six additional mice were sacrificed at the begin-ning of the study at w0 and were used as control (control-w0 group)for biochemical and histomorphometric analysis.
2.3. Bone tissue collection
Right femurs and tibias from all mice were dissected, cleanedof soft tissue, and conserved in an ethanol based fixative +4 ◦C forfurther analysis. Lumbar vertebrae from L2 to L4 were also dissectedfor mice in experimental procedure 2, slightly cleaned of soft tissueand conserved in an ethanol based fixative for further analysis. Leftfemurs of mice, in the experimental procedure 2, were used forqPCR analysis.
2.4. Measurement of the M-protein level in the serum
Progression of MM was monitored by measuring the serumM-protein (IgG2a�) concentration using agar electrophoresis(Hydragel protéine, SEBIA, Issy les Moulineaux, France). The levelof M-protein was expressed as the percentage of the �-peak using
image analysis (NIH image for Windows) on the electrophoreticdiagram. Blood was collected from the tail vein at 6 w for all 5T2MMgroups to check the presence of the M-protein. For kinetic evolu-tion of the M-protein level, blood was also collected at 8 and 10 w
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Fig. 1. Effect of dietary calcium diet in control mice after 10 w. (A) X-ray microCT
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H. Libouban, D. Chappa
n the two groups maintained up to 10 w. Blood was collected athe time of sacrifice for mice that developed paraplegia and did noteach 10 w.
.5. Follow up of osteolytic lesions
Presence of osteolytic bone lesions was checked on digital radio-raphs of the pelvis and hind legs obtained with a Faxitron machinet 26 kV (Faxitron Edimex, Angers, France). Radiographs were per-ormed at the time of sacrifice in all groups.
.6. X-ray microtomography
MicroCT was performed on the proximal tibia and on the lumbarertebrae (L2 to L4) with a Skyscan 1172 microtomograph (BrukericroCT, Kontich, Belgium). Bone samples were transferred to an
ppendorf tube containing the fixative and polyethylene foam wassed to ensure immobilization of the sample. The tube was affixedn a brass stub with plasticine and analyzed at a resolution of.25 �m per pixel for the tibia and 13.38 �m for the vertebrae. Afteregmentation, the 3D models were constructed from the stack ofD images with a surface-rendering program (Ant, release 2.0.5,kyscan).
3D measurements were obtained with the CtAn softwarerelease 2.5, Skyscan). For each tibia sample, 200 section imagesere obtained and corresponded to the cumulated height of the pri-ary and secondary spongiosa (1.05 mm). Trabecular bone analysisas performed on the L2 vertebral body.
The following 3D parameters were calculated: trabecularolume (BV/TV, in%), trabecular number (Tb.N, in mm−1) and tra-ecular separation (Tb.Sp, in �m). Cortical bone measurementsere performed on 2D sections of the tibia at the metaphyseal
egion (1.05 mm under the growth cartilage) and at the diaphysealhaft (3.14 mm under the growth cartilage). Mean cortical thicknessCt.Th, in �m) was determined with the CtAn software.
.7. Histology
Histology was performed on undecalcified femurs after embed-ing in poly (methyl methacrylate). For each mice, four non serialections (50 �m apart, 7 �m in thickness) were cut dry on aeavy-duty microtome equipped with tungsten carbide knivesLeica Polycut S; Rueil-Malmaison, France, with 50◦ knives) andtained with Goldner’s trichrome or tartrate resistant acid phos-hatase (TRAcP) for the histochemical identification of osteoclastsChappard, 2014). The osteoclast number was determined at the
ndosteal surface (on 5 mm from the growth plate) at a magni-cation of ×200 on the TRAcP-stained sections. The percentagerea of the metaphysis invaded by plasma cells was measured onistological sections of the femur.able 1iochemical analysis in the different groups.
Serum assay Control-w0 Control grou
Normal-Ca
Calcium (mmol/L) 2.40 ± 0.02w10 2.32 ± 0.03*
PTH (pg/mL) 39.21 ± 6.05w6 29.5 ± 2.02w8 46.76 ± 16.89
w10 19.37 ± 3.41*,� ,#
* vs control-w.� vs w6.# vs w8.a vs Normal-Ca.b vs Normal-Ca-MM.
reconstructions of the proximal tibia from normal-Ca (a) and low-Ca (b). (B) 3DmicroCT measurements of BV/TV on proximal tibia. (C) 2D microCT measurementsof cortical thickness in the metaphyseal region and the diaphyseal shaft of the tibia.
2.8. PTH and calcium assays
Blood calcium was determined at 0 and at euthanasia afterexsanguination in all groups on a Technicon SMA analyzer. PTHwas measured at 0 and at 6, 8 and 10 w in the two control groups(normal-Ca; low-Ca) and in the two MM groups (normal-Ca-MM;low-Ca-MM). Intact PTH was dosed using the Mouse Intact PTHELISA Kit (#60–2300, Immunotopics International, San Clemente,CA), as recommended by the supplier.
2.9. Quantitative RT-PCR analysis
The left femur of each mouse was dissected, cleaned of sur-rounding tissues and bone marrow was flushed in Dulbecco’smodified essential medium. Bone marrow cells were lysed andproceeded for RNA extraction using the RNeasy mini kit (Qiagen,
ps MM groups
Low-Ca Normal-Ca-MM Low-Ca-MM
2.34 ± 0.02* 2.38 ± 0.02 2.31 ± 0.03*,b
106.13 ± 23.17*,a 26.09 ± 9.70 92.44 ± 28.75*,b
112.69 ± 21.4*,a 36.85 ± 4.57 197.43 ± 42.45*,b
94.65 ± 27.67*,a 42.87 ± 6.26 76.16 ± 21.02*,#
8 rd / Micron 96 (2017) 77–85
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Table 2Incidence of hind limb paralysis in normal and low calcium diet MM groups at 6, 8and 10 weeks.
Incidence of hind limb paralysis 6 weeks 8 weeks 10 weeks
0 H. Libouban, D. Chappa
rance) following the manufacturer procedure. After elution, totalNA was aliquoted and stored at −80 ◦C until use. Quality of theNA samples was examined on a denaturing agarose gel and RNAoncentrations were determined by spectrometry. One �g of totalNA sample was reverse transcripted using random hexamerrimers and SuperScript II reverse transcriptase (SSII) (Invitrogen,rance). Primers were designed using Primer3 software (http://ww.ncbi.nlm.nih.gov/tools/primer-blast/). The following primersere used: MIP-1� (forward 5′-CCTTGCTGTTCTTCTCTGTACC-
′ and reverse 5′-AAATGACACCTGGCTGGG-3′);ANKL (forward 5′-TGTACTTTCGAGCGCAGATG-′ and reverse 5′-CCCACAATGTGTTGCAGTTC-3′);rp5 (forward 5′-GGTCACCTGGACTTCGTCAT-3′ andeverse 5′-TCCAGCGTGTAGTGTGAAGC-3′); Lrp6 (for-ard 5′-TCAACACTTCCCTTGGTTGG-3′ and reverse
′-TGTGAGGGATCTTGTCTTCC-3′); Dkk1 (forward′-CTCTGCTAGGAGCCAGTGC-3′ and reverse 5′-GCACTCCTCATCTTCAGC-3′). Amplification was performed inuplicates with iQ SYBR Supermix (Bio-Rad, France) and using
Chromo4TM real time system (Bio-Rad). Thermal cycling wasnitiated with 10 min incubation at 95 ◦C followed by 40 cycles of5 s at 95 ◦C, 11 s at 55 ◦C and 22 s at 72 ◦C. Gene expression wasalculated using the comparative CT method with the normaliza-ion of the target genes to 6 housekeeping genes (G3PDH, HPRT1,2 M, ACTB, RPS18 and HSPCB).
.10. Statistical analysis
Statistical analysis was done with SYSTAT statistical software,elease 13.00.05 (Systat, San José, CA). All data were reported as
ean ± standard error of the mean (SEM). Significant differencesetween groups were analyzed by a non-parametric Kruskal-Wallisnalysis of variance followed by the Conover-Inman test for all pair-ise comparisons. The comparison between normal-Ca vs low-Ca
in control or MM groups) was analyzed by the non-parametricann-Whitney U test at each study time point. Differences were
onsidered as significant when p < 0.05.
. Results
.1. Dietary calcium diet induced bone loss due to secondaryyperparathyroidism in control mice
Ten weeks of low calcium diet induced trabecular bone lossnd thinning of the cortices as evidenced on 3D microCT modelsFig. 1A). In the low-Ca group, morphometric analysis revealed aignificant decrease in BV/TV (−14.5%, p < 0.05) and cortical thick-ess at the metaphysis (−28.2%, p < 0.0001) and the diaphysis−27.4%, p < 0.00001) (Fig. 1B–C). Biochemical results for the controlroups appear on Table 1. Trabecular and cortical bone losses weressociated with an increased level of PTH reflecting the secondaryyperparathyroidism. No significant differences were observedetween the two groups after 10 w.
.2. Effect of low calcium diet on MM progression
During the course of the disease, detection of the M-protein wasvidenced at 6 w in both 5T2MM groups with no difference in themount of the protein. In the low-Ca-MM group the kinetic becameifferent after 8 w when an increased level was obtained moreapidly than in the normal-Ca-MM group. At 10 w, the differenceetween the 2 groups was significant (p < 0.05). For the low-Ca-
M group, an additional time point was added on the curve at 9 wecause several mice developed paraplegia and were sacrificed forthical considerations. Table 2 shows the incidence of paraplegian the 2 groups of 5T2MM mice. In the two groups, no paraplegia
Normal-Ca-MM 0% (0/8) 0% (0/8) 0% (0/9)Low-Ca-MM 0% (0/8) 50% (4/8) 56% (5/9)
was observed at 6 w. Fifty percent of paraplegia were observed at8 w and 56% within 9–10 w in the low-Ca-MM group. No paraple-gia was observed in the normal-Ca-MM group. X-rays performedat the time of sacrifice showed minimal “punched-out” osteolyticlesions (preferentially localized at the tibial crest) in normal-Ca-MM and low-Ca-MM groups sacrificed at 8 w (Fig. 2B: a, c). At 8 w,osteolytic lesions were observed in the low-Ca-MM group at thetibia and femur; they were associated with a marked thinning ofthe cortices. At 10 w, the number of osteolytic lesions was increasedin the low-Ca-MM group compared to the normal-Ca-MM (Fig. 2B:b, d). Biochemical results for the MM groups appear on Table 1.Briefly, calcemia was significantly decreased in the low-Ca-MMgroup vs the normal-Ca-MM group after 10 w. PTH was consider-ably increased at 6, 8 and 10 w in the low-Ca-MM vs initial valuesat 0 w. In the normal-Ca-MM group, no change occurred during thetime course of the study.
3.3. A low calcium diet induced a greater bone loss at the tibia
The microCT models of the tibia clearly evidenced the differentprogression of osteolytic lesions upon time between the normal-Ca-MM (Fig. 3A: a–c) and low-Ca-MM groups (Fig. 3A: d-f). Thequantitative measurements are illustrated on Figs. 3 B–D and 2 Dcortical measurements on Fig. 3E–F.
At 6 w, the tibia had a rather well preserved trabecular networkin the normal-Ca-MM group without cortical perforations. BV/TVdid not differ when compared with the control-w0 group; however,Tb.N decreased and Tb.Sp increased significantly, revealing an earlydisorganization of the trabecular microarchitecture. The trabecularnetwork was markedly altered in the low-Ca-MM group (Fig. 3A:d); BV/TV was significantly reduced (−62%, p < 0.001) compared tonormal-Ca-MM group at 6 w. The decrease of BV/TV at 6 w was asso-ciated with a significant decrease of Tb.N and a significant increaseof Tb.Sp. Thinner cortices were observed in the low-Ca-MM groupin both metaphyseal and diaphyseal regions as evidenced by a sig-nificant reduction in Ct.Th (Fig. 3E–F) compared to normal-Ca-MMgroup at 6 w. On the contrary, only significant differences wereobserved for Ct.Th from the metaphysis at 8 and 10 w betweennormal-Ca-MM and control-w0 group. At 6 w, the 3D model fromthe low-Ca-MM group clearly showed a non-smooth appearanceof endosteal surface compared to the normal-Ca-MM group. Thisdifference in the appearance of the endosteal surface reflected amore eroded surface in the low-Ca-MM group. The osteoclast num-ber was significantly higher at 6 w in low-ca-MM group comparedto normal-Ca MM group (resp. 22.4 ± 4.2 vs 10.6 ± 0.8, p < 0. 05).The progression of osteoclast number is presented as a 3D graphcombined with evolution of the bone marrow invasion (Fig. 4).At 6 w, plasma cell invasion of the femoral diaphysis was higherin the low-Ca-MM group compared to the normal-Ca-MM group(resp. 64.5% ± 17.3 vs 43.1% ± 15.1) but this increase did not reachstatistical significance.
At 8 w, BV/TV decreased significantly in the normal-Ca-MMgroup when compared to the control-w0 group; Tb.Sp was signifi-cantly higher and Tb.N was significantly lower compared to Tb.Sp
and Tb.N measurements at 6 w. Bone destruction was evidencedin 3D models of the normal-Ca-MM group with the presence oftrabecular destruction and small cortical perforations (Fig. 3A:b). Osteolytic lesions were maximized in the low-Ca-MM group
H. Libouban, D. Chappard / Micron 96 (2017) 77–85 81
Fig. 2. Effect of low-Calcium diet on myeloma progression. (A) Evolution of the serum M-protein level (expressed in%) during the time course of MM disease. A significanthigher M-protein level was found at 10 w in the low-Ca-MM group (* p < 0.05). (B) X-ray images of the hindlimb of mice from normal-Ca-MM group at 8 w (a) and 10 w (b);from low-Ca-MM group at 8 (c) and 10 w (d). Osteolytic lacunae (→) are evidenced at 8 w in normal and low calcium diet mice. Note the thinning of the cortices (→) withlow calcium diet at 8 w. Greater bone destruction is observed with low calcium diet at 10 w (d). Lacuna appeared smaller but more numerous in the tibial crest (→).
Fig. 3. Effect of low-Calcium diet on bone loss at the tibia. (A) X-ray microCT reconstructions of the proximal tibia from normal-Ca-MM group (a–c) and low-Ca-MM group(d–f) resp. at 6, 8 and 10 w. (B–D) 3D microCT measurements of resp. BV/TV, Tb.Sp and Tb.N on proximal tibia. (E–F) 2D microCT measurements of cortical thickness in resp.the metaphyseal region and the diaphyseal shaft of the tibia.
82 H. Libouban, D. Chappard / M
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ig. 4. 3D evolution of the osteoclast number from the endosteum and bone marrownvasion at 6, 8 and 10 w. Normal-Ca-MM group (©); Low-Ca-MM group (�).
Fig. 3A: e). Numerous TRAcP positive osteoclasts were observedn both groups. The osteoclast number from both groups was sig-ificantly increased at 8 w compared to 6 w. Comparison of bothroups at 8 w did not show significant difference between low-Cand normal-Ca MM groups (resp. 33.1 ± 2.8 vs 39.2 ± 5.2) (Fig. 4).
At 10 w, severe trabecular and cortical bone destruction werevidenced in both MM groups with an increase in cortical perfora-ions. However, lesions were more pronounced in the low-Ca-MMroup (Fig. 3A: d, f). The osteoclast number of low-Ca and normal-a MM groups was not significantly different (resp. 38.4 ± 5.1 vs9.9 ± 2.1) and was similar to the number of osteoclasts at 8 wFig. 4). At 10 w, the bone marrow was entirely invaded by plasmaells in the low-Ca-MM group (100%); in the normal-Ca-MM, theercentage of bone marrow invasion was 82.5 ± 17.5%.
.4. A low calcium diet induced severe vertebral bone destruction
3D models of lumbar vertebra showed progressive disappear-nce of trabecular bone from 6 to 10 w in the low-Ca-MM groupFig. 5A: d–f). Lumbar vertebra in the two groups showed twonatomic holes at the posterior side of the lumbar vertebral body;hese holes are not due to the tumor as they are observed in control
ice (data not shown) and correspond to vascular sinuses. Corti-al perforations were observed from 8 w in the low-Ca-MM groupnducing an enlargement of the anatomical holes (Fig. 5A: e–f). Sim-lar cortical perforations could be observed in the normal-Ca-MMroup but only at the terminal stage of the disease (Fig. 5A: c).
Morphometric analysis showed no significant difference inV/TV, Tb.N and Tb.Sp in the normal-Ca group compared to control-0 at 6, 8 and 10 w (Fig. 5B–D). In the low-Ca-MM group, BV/TV
ecreased significantly from 6 w (w6: −33.65%, p < 0.01; w10:57.61%, p < 0.01) compared to the normal-Ca-MM group. Reduc-
ion of BV/TV was associated with a significant disappearance ofrabeculae as from 6 w (reduced Tb.N and increased Tb.Sp).
.5. A low calcium diet modified gene expression profile
An increase in MIP-1� expression was found in the bone mar-ow of mice from the low-Ca-MM groups at 6 and 8 w with a
icron 96 (2017) 77–85
ratio respectively of 1.6 and 3.9 compared to the expression in thenormal-Ca-MM group (Fig. 6A). On the opposite, expression of MIP-1� was significantly lower at 10 w in the low-Ca group comparedto the normal-Ca group. There was an insignificant reduction in theexpression of RANKL (Fig. 6B) at 6 and 8 w Significant differenceswere obtained in the expression of some genes of the Wnt path-way. The Lrp5 co-receptor was significantly less expressed at 6 win the low-Ca-MM group compared to the normal-Ca-MM groupwith a ratio of −2.6 (Fig. 6C). Expression of Dkk1 was significantlyreduced at 6 and 8 w in the low-Ca-MM group with ratio of respec-tively −2.5 and −4.5 (Fig. 6D). No significant differences of Dkk1expression were observed between the two groups at 10 w.
4. Discussion
The use of a dietary calcium diet to increase bone remodelingwas based on previous data in both animal and clinical studieswhich showed an increase in bone resorption due to low calciumintake (Eklou-Kalonji et al., 1999; Iwamoto et al., 2004; Lips, 2001;Seto et al., 1999). In the present study, a long-term calcium diet defi-ciency induced a sustained secondary hyperparathyroidism which,in turn, caused a major bone loss at the trabecular and cortical level.Secondary hyperpathyroidism is due to extrinsic changes causinga drastic decrease in plasma ionized calcium thus inducing PTHsecretion to correct plasma calcium. If low calcium availability isnot corrected, patients are characterized by a long term high levelof parathyroid hormone (Fraser, 2009). This is in accordance withdata obtained from the present study and others where PTH levelis maintained high even until 10 w of low calcium diet (Iwamotoet al., 2004). The increase in PTH due to a low calcium diet is rapidand observed as soon as 12 h after diet initiation in rats (Seto et al.,1999). Short term study of a low calcium diet in rats, showed thatthe rapid increase in PTH induced rapid effects on bone remodel-ing; the number of osteoclasts and osteoblasts along the trabecularbone surfaces were increased after 1 and 2 days, respectively (Setoet al., 1999). Such a high bone resorption level is maintained dur-ing the entire period of the low calcium intake (Eklou-Kalonji et al.,1999; Iwamoto et al., 2004). Moreover, an increase in osteoclastnumber was shown in both cancellous and endosteal surfaces,in accordance with the bone loss at these two bone envelopesnoted in the present study (Xiong et al., 2014). When consider-ing bone formation, increases in osteoblast number are maintainedthrough the entire time of the study in animals with a low calciumdiet but bone formation and mineralization rates are decreased(Eklou-Kalonji et al., 1999; Iwamoto et al., 2004; Seto et al., 1999).Biochemical markers of bone formation (alkaline phosphatase, c-propeptides) are also increased during a low calcium diet in animalsand humans (Eklou-Kalonji et al., 1999; Harris et al., 2001; Meunieret al., 2005). Secondary hyperparathyroidism due to low calciumintake is common in middle-age and elderly population. Severalstudies have shown that nutritional supplementation of calcium infood or drinking water reduces serum PTH level and bone markers(Bonjour et al., 2013; Meunier et al., 2005).
When MM cells injection was associated with a low calcium reg-imen, tumor growth was accelerated from 8 w. It was associatedwith a high incidence of paraplegia (which were absent in normal-Ca-MM mice); this corresponds to a more severe form of MM. Miceshowed an increased osteolysis at the end stage of the disease due toan earlier development of osteolytic lesions at the tibia and lumbarvertebrae. Histomorphometric measurements confirmed that sec-ondary hyperparathyroidism in MM mice has led to an increased
bone destruction. Moreover, MM mice with a normal calcium dietdid not show trabecular destruction in the lumbar vertebral body,although lumbar vertebrae had cortical perforations at the endstage of the disease. The dramatic osteolysis at lumbar vertebrae
H. Libouban, D. Chappard / Micron 96 (2017) 77–85 83
F oCT rl resp. B
mtapowowaZswtt
r
ig. 5. Effect of low-Calcium diet on bone loss at the lumbar vertebrae. (A) Microw-Ca-MM group (d–f) resp. at 6, 8 and 10 w. (B–D) 3D microCT measurements of
ay explain the incidence of paralysis in the deficient group. His-ologic data showed that cortical perforations in the vertebral bodyllowed extension of plasma cells in the extradural space and com-ression of the spinal cord (Libouban, 2015). Our results basedn X-ray analysis, microCT and M-protein data are in accordanceith our previous study on a combined model associating MM and
variectomy (Libouban et al., 2003). They are also in accordanceith data reported in murine model with bone metastasis in which
low calcium diet promoted tumor growth (Zheng et al., 2008;heng et al., 2007). The experimental designs of these two studieslightly differed from our experiment because the low calcium dietas initiated at the same day than injection of MM cells. Excepted
his experimental difference, data are similar in term of increase inhe tumor growth and osteolytic lesions (Seto et al., 1999).
It now exists strong evidences that a preexisting high boneemodeling promotes tumor growth (MM or metastasis) and would
econstructions of lumbar vertebra (L2–L4) from normal-Ca-MM group (a–c) andV/TV, Tb.Sp and Tb.N at the L2 vertebral body.
represent a risk of converting a smoldering form into an overt one.We and others have hypothesized that a high bone remodelingalters the bone microenvironment by enriching it with growth fac-tors and cytokines, thus providing a more “fertile soil” on whichtumor cells growth is enhanced (Guise, 2000). Moreover, treatmentwith the RANK-L inhibitor osteoprotegerin prevented the develop-ment of osteolytic lesions and reduced the tumor growth despitea calcium diet (Zheng et al., 2007). Authors emphasized the factthat the increase of tumor growth and the extend of bone lesionsare directly due to high bone resorption induced by low calciumindependently of direct effect of PTH. (Zheng et al., 2007). In addi-tion, the intermittent injection of PTH (1–34) has been tested in
murine models of myeloma and the effect of PTH delayed MMprogression (Pennisi et al., 2010). In our model, PTH levels are con-tinuously elevated and thus the effects on bone remodeling andtumor growth appear opposite to the above results. In addition,
84 H. Libouban, D. Chappard / M
Fig. 6. Effect of low-Calcium diet on gene expression profile in medullar micro-eD8
wcio
hfbhrtbldat
ueImR“2ehfg
nvironment. Real time PCR analysis of (A) MIP-1�, (B) RANKL, (C) LRP5 and (D)KK1 expression in bone marrow from normal-Ca-MM and low-Ca-MM group at 6,
and 10 w.
e did not include neither a group with normal calcium diet andontinuous PTH infusion nor tested a neutralizing antibody to ver-fy whether the continuous increased in PTH is responsible for thebserved effects.
Our results clearly showed that a preexisting high bone turnoverad a strong impact on MM development at an early time, 6 w
ollowing MM injection. At 6 w, the M-protein level was similaretween the different groups and the bone marrow invasion wasigher in the low-Ca-MM groups although the difference did noteached statistical significance. However, when plotting togetherhe% of marrow invasion and N.Oc in a 3D graph, differencesetween the two groups was more clearly evidenced due to psycho-
ogical perception (Systat, 2009). In addition, the 3D presentation ofata clearly showed that the low calcium diet accelerates the mech-nism of MM progression at the early phase of MM developmenthat induce then a more severe bone destruction.
Until now, no study was performed to characterize the molec-lar enrichment of the microenvironment. In this study, wevaluated the expression of genes strongly involved in MM growth.n MM, considerable attentions have been paid on cellular and
olecular mechanisms responsible for a high bone resorption.ANK/RANKL and the chemokine MIP-1� constitutes the keyosteoclast activating factors” in MM (Choi et al., 2000; Pearse et al.,001). At low calcium diet, MIP-1� was highly expressed in the
arlier phase of MM development. This result is in accordance withistological data of TRAcP positive osteoclasts at the endosteal sur-aces; osteoclasts were increased as early as 6 w in the low-Ca-MMroup, confirming the higher resorption in this group. MIP-1� pro-
icron 96 (2017) 77–85
motes osteoclastogenesis independently of RANKL and enhancesinteraction between MM cells and stromal cells (Oba et al., 2005).Surprisingly, at 10 w the level of MIP-1 � was much lower comparedto the level at 6 and 8 w and also compared to normal-Ca-MM groupat the same time. However, as the marrow invasion is at a levelof 100% in the low-Ca-MM group and because MIP-1 � is mainlysecreted by medullar cells, it is not so surprising that the levelexpression of MIP-1 � was low. More recently, it has been shown arole for MIP-1� in inhibition of osteoblast formation via osteocal-cin down regulation (Vallet et al., 2011). In our study MIP-1� wasa strong indicator of MM aggressiveness; surprisingly, no signifi-cant difference in the expression of RANKL was observed. RANKLgene expression is increased in a mouse model of secondary hyper-parathyroidism after 7 days (O’Brien et al., 2005). This would be infavor of an early hyper-expression of RANKL in the low-Ca-MMgroup occurring before 6 w (the onset of our kinetic study).
The Wnt pathway is involved in the decline of bone forma-tion in MM (Roodman, 2010). Studies on bone formation in MMhave focused on DDK1 expression that is upregulated in malignantplasma cells (Roodman, 2010; Tian et al., 2003). At an early stageof MM, we observed a decrease in the co-receptor of the Wnt path-way LRP5 (which is a key molecule to enhance bone formation).The lack of LRP5 in mice induces an inhibition of osteoblast func-tion associated with a decrease in osteoblast number (Kato et al.,2002). The low LRP5 expression in mice with the low calcium dietcan reflect the decrease in bone formation. We did not observea high level of DKK1expression in the low-Ca-MM group. Thereis no data on DKK1 expression in secondary hyperparathyroidismwhich could explain our result. However, in a previous study ofgene expression in a mouse model of disuse, DDK1 appeared as alate inhibitor of bone formation (Marchand-Libouban et al., 2013).This observation could be in accordance with our data at 10 w whena non-significant increase of DKK1 expression was observed in thelow-Ca-group (compared to the normal-Ca-group).
5. Conclusions
The present study by using X-ray microtomography and quan-titative PCR analysis clearly showed that altered bone remodelingdue to calcium deficiency increase the severity of MM bone dis-ease with a higher osteolysis and a higher alteration of bonegenes expression. Little attention has been placed on investigatingthe mechanisms involved in humans for the sudden progressionfrom indolent myeloma (revealed by a monoclonal gammopathyof undetermined significance – MGUS) into an aggressive MM.However, evolution to an overt MM, is related to both intrin-sic changes of malignant plasma cells and modification of bonemicroenvironment (Bataille, 2015; Tageja et al., 2014). So, calciumdeficiency would represent a real cause for progression of smolder-ing MM to overt MM by inducing secondary hyperparathyroidism.MM is a disease frequently observed in the elderly where a sec-ondary hyperparathyroidism (increasing bone remodeling) is alsofrequently observed.
Funding
This work was supported by grant from the French Ministry ofHealth.
Acknowledgments
Authors thank P. Legras and J. Roux for their help with the ani-mal care (SCAHU) and the technical assistance of the Plate-formed’Analyse Cellulaire et Moléculaire (PACEM, Angers University) forthe qPCR analysis.
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Zheng, Y., Zhou, H., Fong-Yee, C., Modzelewski, J.R., Seibel, M.J., Dunstan, C.R., 2008.Bone resorption increases tumour growth in a mouse model of osteosclerotic
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