Human Placenta-Derived Adherent Cells Delivered Intralesionally Inhibit Myeloma Bone Disease and Tumor Growth, While Intravenously Are Capable of Trafficking to Myelomatous Bone.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3717-3717
Author(s):  
Xin Li ◽  
Wen Ling ◽  
Angela Pennisi ◽  
Yuping Wang ◽  
Sharmin Khan ◽  
...  
Keyword(s):  
Bone Formation ◽  
Tumor Growth ◽  
Human Placenta ◽  
Bone Disease ◽  
Adherent Cells ◽  
X Rays ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
Cellular Therapeutics

Abstract Abstract 3717 Human placenta has emerged as a valuable, uncontroversial source of transplantable cells for many cytotherapeutic purposes, including modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDAC) are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Multiple myeloma (MM) is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. The aim of the study was to evaluate the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDAC in the SCID-rab model of MM-associated bone disease. SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which primary human myeloma cells were directly injected (Yatta et al., Leukemia 2004; Yaccoby et al., Blood 2007). Bone disease was evaluated by measurements of bone mineral density (BMD) and X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and histologically. For in vivo tracking PDAC were transduced with a luciferase/GFP reporter in a lentiviral vector. SCID-rab mice engrafted with primary myeloma cells from 2 patients. Upon establishment of MM growth, PDAC (1×106 cells/bone) or vehicle were injected into the implanted myelomatous bone (Patient's 1, 5 mice/group; Patient's 2, 7 mice/group). While BMD of the implanted bones was significantly reduced in control hosts, intralesional PDAC cytotherapy significantly increased BMD of the implanted bones from pretreatment levels by >37% (p<0.01 versus control) and inhibited MM growth in the 2 sets of experiments (p<0.04). The bone anabolic effect of PDAC was associated with increased number of osteoblasts (p<0.003) and reduced number of osteoclasts (p<0.004). Intralesional PDAC cytotherapy also promoted bone formation in nonmyelomatous SCID-rab mice. Intralesional but not subcutaneous engraftment of PDAC inhibited bone disease and tumor growth in SCID-rab mice. In contrast to intra-bone injection in SCID-rab mice, intra-tumor injection of PDAC had no effect on subcutaneous growth of the H929 myeloma cell line in SCID mice (8 mice/group). Live-animal imaging revealed that the majority of PDAC disappeared from the injected bones within 4 weeks. To test their systemic behavior, PDAC were intravenously injected into 18 SCID-rab mice engrafted with H929 myeloma cells. The presences of PDAC in various organs were evaluated 1, 2 and 7 days after injection. Ex vivo bioluminescence analysis of the implanted myelomatous bones detected PDAC in two of five bones on day 1, in four of four bones on day 2, and in four of nine bones on day 7. Intravenously injected PDAC were also detected in lungs not in any other murine tissues. Immunohistochemical staining for GFP in myelomatous bone sections detected GFP-expressing PDAC in rabbit marrow areas infiltrated with myeloma cells, supporting bioluminescence analysis. Our study suggest that PDAC stimulate bone formation by acting as bystander cells that increase endogenous osteoblastogenesis and inhibit osteoclastogenesis, and that alteration of the bone marrow microenvironment by PDAC attenuates growth of MM. PDAC cytotherapy is a promising therapeutic approach for myeloma bone disease. Disclosures: Khan: Celgene Cellular Therapeutics: Research Funding. Heidaran:Celgene Cellular Therapeutics: Employment. Pal:Celgene Cellular Therapeutics: Employment. Zhang:Celgene Cellular Therapeutics: Employment. He:Celgene Cellular Therapeutics: Employment. Zeitlin:Celgene Cellular Therapeutics: Employment. Abbot:Celgene Cellular Therapeutics: Employment. Faleck:Celgene Cellular Therapeutics: Employment. Hariri:Celgene Cellular Therapeutics: Employment.

Human Placenta-Derived Adherent Stem Cells Prevent Bone Loss and Stimulate Bone Formation in Myelomatous Bones, and Suppress Growth of Primary Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 646-646
Author(s):  
Xin Li ◽  
Wen Ling ◽  
Angela Pennisi ◽  
Jianmei Chen ◽  
Sharmin Khan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Formation ◽  
Human Placenta ◽  
Bone Disease ◽  
Adherent Cells ◽  
Live Animal ◽  
Prevent Bone Loss ◽  
Animal Imaging ◽  
Live Animal Imaging

Abstract Induction of osteolytic bone disease in multiple myeloma (MM) is caused by activation of osteoclastogenesis and suppression of osteoblastogenesis. Bone formation is reduced mainly through production of inhibitors of osteoblast differentiation by MM cells and by impaired osteogenic differentiation of endogenous mesenchymal stem cells (MSCs). Recently, human placenta has emerged as a potentially valuable source of progenitor cells for multiple therapeutic purposes, including bone repair and cancer (Parolini et al., Stem Cells26:300–311, 2008). The aim of the study was to investigate the effects of human placenta-derived adherent cells (PDAC™) on MM bone disease and tumor growth in the SCID-rab mouse model for MM. PDAC™ are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Bone disease was evaluated by measurements of bone mineral density (BMD) and visualized by X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and live animal imaging. For in vivo tracking PDAC™ or our stroma-dependent BN MM cell line was transduced with a luciferase/GFP reporter in a lentiviral vector. In the first in vivo experiment, 10 SCID-rab mice were engrafted with a patient’s MM cells. Following establishment of MM and detection of bone disease, luciferase-expressing PDAC™ (1×106 cells/bone) or phosphate-buffered saline (PBS) were injected directly into implanted myelomatous bones in SCID-rab mice (5 mice/group). At experiment’s end (5 wk after cytotherapy) PDAC™ could be detected in mice by live animal imaging. Whereas in control mice, BMD of the implanted bone was reduced from pretreatment levels by 8±4%, administration of PDAC™ resulted in increased BMD of the implanted bone in all mice by 132±20% from pretreatment levels (p<0.0006). Levels of hIg in mice sera (tumor burden) at experiment’s end were 202±56 μg/ml and 12±4 μg/ml in PBS- and PDAC™ -treated hosts, respectively (p<0.007). In the second in vivo experiment hosts engrafted with our luciferase-expressing BN MM line (Li et al., BJH 2007) were similarly injected with PDAC™ or PBS (8 mice/group). Six wk following treatment the BMD of the implanted bone in the control PBS group was reduced by 31±33% (p<0.0009) from pretreatment levels while in PDAC™ –treated group it was slightly reduced by 2±6% from pretreatment levels. Treatment with PDAC™ had no effect on in vivo growth of the BN MM cell line, indicating that prevention of bone disease by PDAC™ was not a consequence of reduced MM. In contrast to fetal MSCs, PDAC™ expressed high levels of OPG (>30 fold) and low levels of RANKL (<5 fold) as determined by qRT-PCR. Differentiation of osteoclast precursors in media supplemented with RANKL and M-CSF was reduced in the presence of PDAC™ or their conditioned media by 60±6% (p<0.004), an effect that was partially blocked by OPG neutralizing antibody (p<0.04). PDAC™ also induced apoptosis of osteoclast precursors as determined by annexin V/PI staining. We conclude that PDAC™ prevent bone loss and promote bone formation in myelomatous bone through simultaneous inhibition of osteoclastogenesis and stimulation of osteoblastogenesis, and that engraftment of PDAC™ inhibits growth of primary MM in vivo.

scholarly journals The ephrinB2/EphB4 axis is dysregulated in osteoprogenitors from myeloma patients and its activation affects myeloma bone disease and tumor growth

Blood ◽  
2009 ◽  
Vol 114 (9) ◽  
pp. 1803-1812 ◽  
Author(s):  
Angela Pennisi ◽  
Wen Ling ◽  
Xin Li ◽  
Sharmin Khan ◽  
John D. Shaughnessy ◽  
...  
Keyword(s):  
Bone Formation ◽  
Bone Disease ◽  
Bone Cells ◽  
Myeloma Cell ◽  
Chimeric Proteins ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
Bidirectional Signaling ◽  
Surface Ligand

Myeloma bone disease is caused by uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Bidirectional signaling between the cell-surface ligand ephrinB2 and its receptor, EphB4, is involved in the coupling of osteoblastogenesis and osteoclastogenesis and in angiogenesis. EphrinB2 and EphB4 expression in mesenchymal stem cells (MSCs) from myeloma patients and in bone cells in myelomatous bones was lower than in healthy counterparts. Wnt3a induced up-regulation of EphB4 in patient MSCs. Myeloma cells reduced expression of these genes in MSCs, whereas in vivo myeloma cell-conditioned media reduced EphB4 expression in bone. In osteoclast precursors, EphB4-Fc induced ephrinB2 phosphorylation with subsequent inhibition of NFATc1 and differentiation. In MSCs, EphB4-Fc did not induce ephrinB2 phosphorylation, whereas ephrinB2-Fc induced EphB4 phosphorylation and osteogenic differentiation. EphB4-Fc treatment of myelomatous SCID-hu mice inhibited myeloma growth, osteoclastosis, and angiogenesis and stimulated osteoblastogenesis and bone formation, whereas ephrinB2-Fc stimulated angiogenesis, osteoblastogenesis, and bone formation but had no effect on osteoclastogenesis and myeloma growth. These chimeric proteins had similar effects on normal bone. Myeloma cells expressed low to undetectable ephrinB2 and EphB4 and did not respond to the chimeric proteins. The ephrinB2/EphB4 axis is dysregulated in MM, and its activation by EphB4-Fc inhibits myeloma growth and bone disease.

Anti-Sclerostin Treatment Prevents Multiple Myeloma Induced Bone Loss and Reduces Tumor Burden

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 119-119 ◽  
Author(s):  
Michaela R. Reagan ◽  
Michelle McDonald ◽  
Rachael Terry ◽  
Jessica Pettitt ◽  
Lawrence Le ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Resorption ◽  
Bone Loss ◽  
Bone Formation ◽  
Tumor Growth ◽  
Bone Disease ◽  
Bone Volume ◽  
Antibody Treatment ◽  
Myeloma Cells ◽  
Myeloma Bone Disease

Abstract Multiple myeloma (MM) is a malignancy of plasma cells and is characterized by unrestricted tumor cell growth in bone marrow (BM). MM causes destructive osteolytic lesions causing bone fracture, bone pain, hypercalcaemia, and nerve-compression, resulting from increased bone resorption and suppressed bone formation. Despite the introduction of agents to inhibit bone resorption, such as bisphosphonates, which prevent further bone loss, approaches to preventing osteoblast suppression and repair bone lesions are limited and there are no agents available clinically. The wnt/β-catenin pathway plays a critical role in the regulation of bone formation. Production of the soluble wnt antagonist dickkopf1 (Dkk1) by MM cells has been implicated in MM inhibition of bone formation. As such, Anti-Dkk1 treatment prevents bone disease in pre-clinical models of MM and is in early clinical development. However, Dkk1 is not expressed by all myeloma cells; hence only a proportion of patients may respond to anti-Dkk1 therapy. Sclerostin (Scl) is a soluble wnt antagonist whose expression, unlike Dkk1, is restricted to osteocytes; therefore Scl targeted agents may have less off target effects. Anti-Sclerostin (Anti-Scl) treatment increased bone formation and bone volume in experimental models of osteoporosis, and increased bone mineral density in phase II osteoporosis clinical trials. However, Anti-Scl treatment effects on myeloma bone disease are unknown. Further, cells of the BM such as osteoblasts have been implicated in the regulation of MM cell survival and growth. Thus, in the present study we explored the potential for Anti-Scl therapy to prevent MM induced bone loss and inhibit MM growth in both murine and human xenograft MM models. Female C57BLKalwRij mice (n=8) were injected i.v, with 5TGM1/eGFP murine MM cells (1×106) and female SCID/beige mice (n=10) were injected i.v. with MM1S/Luc/eGFP human MM cells (4 × 106). 24 hours later, naïve mice (without tumor cells) or mice bearing MM cells were treated with anti-sclerostin antibody (Anti-Scl) (100mg/kg i.v) or control antibody. Mice were sacrificed at day 21 (MM1S) or day 28 (5TGM1) and the effect of Anti-Scl on bone structure in the femora and vertebrae were determined by microCT analysis. The effect of Anti-Scl on MM burden was determined by bioluminescent imaging (BLI) performed twice weekly from week 1 using a Xenogen IVIS system, whereas MM burden in 5TGM1/eGFP bearing mice was examined by FACS analysis. Anti-Scl treatment in naïve C57BLKalwRij mice increased trabecular bone volume fraction (BV/TV, 39%, p<0.01, Fig. 1B) in the femur, which was mediated by increases in trabecular thickness (Tb.Th, 42%, p<0.01). Treatment also increased cortical bone volume (22%, p<0.01) in the femur and increased trabecular BV/TV in the vertebra (32%, p<0.01). This demonstrated the potent bone anabolic effect of Anti-Scl independent of myeloma cells. Injection of 5TGM1 cells resulted in a decrease in femoral trabecular BV/TV (30%, p<0.01) through a 30% reduction in trabecular number (TbN) (p<0.01), but no effect on Tb.Th (Figs. 1A and B), whilst also reducing cortical bone volume (BV) by 6% (p<0.05). Vertebrae were also impacted by 5TGM1 tumor growth with a 29% reduction in Tb BV/TV through a 23% reduction in Tb.Th (p<0.01) and also a 15% reduction in cortical BV (p<0.01). Treatment of 5TGM1-bearing mice with Anti-Scl increased trabecular BV/TV (46%, p<0.01) and Tb.Th (30%, p<0.01) to values equivalent to femora of naïve, non-tumor bearing, control mice. Treatment with Anti-Scl also increased cortical BV by 16% (p<0.01), vertebral Tb BV/TV by 29% and cortical BV by 36% in 5TGM1 burdened mice (p<0.01). Treatment of 5TGM1-bearing mice with Anti-Scl had no effect on the proportion 5TGM1/eGFP cells in the BM or spleen. However Anti-Scl treatment significantly suppressed tumor progression in the MM1S model at 3, 3.5 and 4 weeks post cell injection, as determined by BLI imaging (p=0.02, wk 3; p=0.0019, wk 3.5, and p=0.0068, wk 4, 2-tailed t-tests, Fig. 1C). These data demonstrate that Anti-Scl antibody treatment can prevent development of myeloma bone disease. Furthermore, Anti-Scl treatment also suppressed tumor growth, supporting the possibility that targeting the BM microenvironment with this agent may slow disease progression. Our findings highlight the potential clinical application of Anti-Scl antibody treatment in patients with MM and other bone destructive cancers. Disclosures Kneissel: Novartis Institutes for Biomedical Research, Novartis Pharma AG: Employment. Kramer:Novartis Pharma AG: Employment. Brooks:Spouse works for Boston Biomedical Inc: Employment.

scholarly journals Increasing Wnt signaling in the bone marrow microenvironment inhibits the development of myeloma bone disease and reduces tumor burden in bone in vivo

Blood ◽  
2008 ◽  
Vol 111 (5) ◽  
pp. 2833-2842 ◽  
Author(s):  
Claire M. Edwards ◽  
James R. Edwards ◽  
Seint T. Lwin ◽  
Javier Esparza ◽  
Babatunde O. Oyajobi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Wnt Signaling ◽  
Bone Disease ◽  
Critical Role ◽  
Tumor Burden ◽  
Bone Marrow Microenvironment ◽  
Myeloma Bone Disease ◽  
Osteolytic Bone Disease

There is increasing evidence to suggest that the Wnt signaling pathway plays a critical role in the pathogenesis of myeloma bone disease. In the present study, we determined whether increasing Wnt signaling within the bone marrow microenvironment in myeloma counteracts development of osteolytic bone disease. C57BL/KaLwRij mice were inoculated intravenously with murine 5TGM1 myeloma cells, resulting in tumor growth in bone and development of myeloma bone disease. Lithium chloride (LiCl) treatment activated Wnt signaling in osteoblasts, inhibited myeloma bone disease, and decreased tumor burden in bone, but increased tumor growth when 5TGM1 cells were inoculated subcutaneously. Abrogation of β-catenin activity and disruption of Wnt signaling in 5TGM1 cells by stable overexpression of a dominant-negative TCF4 prevented the LiCl-induced increase in subcutaneous growth but had no effect on LiCl-induced reduction in tumor burden within bone or on osteolysis in myeloma-bearing mice. Together, these data highlight the importance of the local microenvironment in the effect of Wnt signaling on the development of myeloma bone disease and demonstrate that, despite a direct effect to increase tumor growth at extraosseous sites, increasing Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease and inhibit myeloma growth within bone in vivo.

Systemically Injected Mesenchymal Stem Cells Traffic to Myelomatous Bone and Inhibit Bone Disease, and Intralesionally Promotes Bone Formation and Delays Myeloma Progression During the Disease Active Stage or Remission

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 980-980
Author(s):  
Xin Li ◽  
Wen Ling ◽  
Sharmin Khan ◽  
Yuping Wang ◽  
Angela Pennisi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Bone Formation ◽  
Tumor Growth ◽  
Cell Line ◽  
Bone Disease ◽  
Active Stage ◽  
Myeloma Cells ◽  
Intravenous Injections ◽  
Bone Injection

Abstract Abstract 980 Mesenchymal stem cells (MSCs) cytotherapy has been clinically tested in various applications including bone regeneration, autoimmune diseases, and cancer. The aims of the study were to test the effect of MSCs cytotherapy on myeloma (MM) bone disease and tumor growth, and determine their ability to traffic into myelomatous bone during active disease stage and induction of remission by melphalan. We exploited the SCID-rab model for MM and a novel human myeloma cell line, Hg, established through our previously reported procedure (Xin et al., BJH 2007). Hg myeloma cells do not grow in culture but are capable of sequential passaging in SCID-rab mice. Microarray analysis revealed similar gene expression profiling between the Hg cell line and the original patient's myeloma plasma cells, and that these cells are classified in the MMSET subgroup and express DKK1, indicating their authenticity and clinical relevancy. The human fetal MSCs were transduced with a luciferase/GFP reporter in a lentiviral vector for in vivo tracking. In Hg-bearing hosts (5 mice/group), intra-bone injection of MSCs (1×106 cells/mouse) increased bone mineral density (BMD) by 21±4% while in control, diluent injected bones it was reduced by 14±5% (p<0.001). Increased bone formation by MSCs was associated with reduced tumor growth by 50% (p<0.01). Bioluminescence analysis revealed disappearance of the majority of the intralesionally injected MSCs within 4 weeks, suggesting that MSCs exert their effects on bone remodeling as a bystander cells (trophic effect). To test effect on relapse, remission was induced by treating Hg-bearing hosts with a total of 4 subcutaneous injections of melphalan (10 mg/kg/4 days), followed by intra-bone injection of diluent or MSCs (10 mice/group). Three weeks post-cytotherapy BMD was increased by 23±5% in bones injected with MSCs and reduced by 23±3% in bones injected with diluent (p<0.001). Eleven weeks post-cytotherapy, BMD was reduced by 33±6% and 9±7% in bones injected with diluent and MSCs, respectively (p<0.03). Following melphalan treatment circulating immunoglobulin (Ig) level (MM burden) was undetected while at 2 weeks after cytotherapy it was detected in 80% and 30% of hosts injected with diluent and MSCs, respectively. At experiment's end, Ig levels were significantly lower by 6 folds in hosts treated with MSCs than diluent (p<0.01). To further validate clinical relevancy, MSCs or diluent were intravenously injected into Hg-bearing hosts. In contrast to a single injection, 4 weekly, intravenous injections of MSCs (10 mice/group) prevented reduction of the BMD of the myelomatous bone while in control hosts the BMD was reduced by 14±3% (p<0.006 vs. pretreatment). MM growth was not affected by single or multiple intravenous injections of MSCs. Ex vivo imaging of tissues from hosts with active MM or treated with melphalan detected MSCs in implanted bones and murine lungs indicating that myeloma cells or conditions induced by MM or melphalan attract MSCs to myelomatous bones. We conclude that intra-bone injection of MSCs effectively promotes bone formation and delays MM progression during the disease active stage or remission. We also conclude that exogenous MSCs are capable of trafficking to myelomatous bone and that systemic, weekly injections of MSCs inhibit MM bone disease. Disclosures: No relevant conflicts of interest to declare.

The EphrinB2/EphB4 Axis Is Dysregulated in Osteoprogenitors from Myeloma Patients and Its Activation by EphrinB2-Fc or EhpB4-Fc Affects Myeloma Bone Disease and Tumor Growth in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 844-844
Author(s):  
Angela Pennisi ◽  
Wen Ling ◽  
Xin Li ◽  
Jianmei Chen ◽  
Sharmin Khan ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Osteogenic Differentiation ◽  
Bone Disease ◽  
Plasma Cells ◽  
Bone Lesions ◽  
Reverse Signaling ◽  
Myeloma Bone Disease ◽  
Bidirectional Signaling ◽  
Healthy Donors

Abstract Induction of osteolytic bone lesions in myeloma (MM) is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Recent studies indicate that in addition to role in cell adhesion, repulsion and neovascularization, bidirectional signaling between the cell surface molecules EphrinB2 and EphB4 also mediates the coupling between osteoblasts and osteoblasts. While mesenchymal stem cells (MSCs) and osteoblasts express the ligand EphrinB2 land its receptor, EphB4, osteoclasts and their precursors mainly express EphrinB2. Forward signaling in MSCs promotes osteogenic differentiation and reverse signaling in osteoclast precursors inhibits their differentiation. The aims of the study were to investigate whether the EphrinB2/Eph4 axis is dysregulated in MM osteoprogenitors and whether activation of this axis in myelomatous bone by EphrinB2-Fc or EphB4-Fc affects MM bone disease, angiogenesis and tumor growth. MSCs were generated from bone marrow of healthy donors (n=5) and patients with MM (n=13). Gene expression was determined by qRT-PCR. MSCs from MM patients had reduced expression of EphrinB2 (EFNB2) by 61±6% (p<0.02) and EphB4 by 60±10% (p<0.02) than expression levels of these molecules in MSCs from healthy donors. Expression of other EFN and EPH B genes were detected and similarly expressed in patients and donors MSCs. Differentiation of MSCs from MM patients into osteoblasts resulted in upregulation of EFNB2 and downregulation of EPHB4. MM cell lines and primary MM plasma cells expressed low to undetectable levels of this family of genes. We exploited our SCID-hu system for primary MM to study the consequences of activation of forward signaling by EphrinB2-Fc or reverse signaling by EphB4-Fc on MM-induced bone disease and MM growth. Twelve SCID-hu mice were engrafted with MM cells from a patient with active MM. Upon detection of MM growth (by human Ig ELISA) and bone disease (radiographically), hosts were locally treated with Fc (control), EphrinB2-Fc or EPHB4 (4 mice/group) for 4 weeks using Alzet pump that continually released 0.11 μg/hour of each compound. While in Fc-treated hosts BMD of the implanted bone was reduced by 8±3% from pretreatment levels, it was increased by EphrinB2-Fc and EPhB4-Fc by 15±8% (p<0.03 vs. Fc) and 2±1% (p<0.02 vs. Fc) from pretreatment levels, respectively. At experiment’s end levels of human Ig in mice sera were increased by 308±99% and 244±86% from pretreatment levels in Fc- and EphrinB2- Fc groups, respectively, while were reduced by 92±1% (p<0.02 vs. Fc) from pretreatment levels in EphB4-Fc group. In myelomatous bones, EphB4-Fc and EphrinB2-Fc increased the numbers of osteoblasts by >3 folds (p<0.004) while EphB4-Fc, but not EphrinB2-Fc, reduced osteoclast numbers by 5 folds (p<0.01 vs. Fc group). The numbers of CD34-reactive neovessels were reduced by 2 folds following treatment with EphB4-Fc (p<0.03) and were increased by 2.5 folds following treatment with EphrinB2-Fc (p<0.05). Our study suggests that downregulation of EphrinB2 and EhpB4 in MSCs from MM patients contributes to their impaired osteogenic differentiation and that treatment with EphrinB2-Fc or EphB4-Fc helps restore coupling of bone remodeling in myelomatous bones. The results also indicate that EphB4-Fc treatment is an effective approach to simultaneously inhibit MM and its associated bone disease.

Myeloma Cells Induce Osteoblast Suppression through Sclerostin Secretion

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2961-2961 ◽  
Author(s):  
Silvia Colucci ◽  
Giacomina Brunetti ◽  
Angela Oranger ◽  
Giorgio Mori ◽  
Francesca Sardone ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Formation ◽  
Bone Disease ◽  
Culture System ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Type I ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
Osteoblast Activity

Abstract Abstract 2961 Reduced osteoblast activity contributes to the development of multiple myeloma-bone disease. Wingless-type (Wnt) signalling pathway is critical in osteoblastogenesis, and it is negatively regulated by molecules such as frizzled-related proteins (sFRPs), Dickkopf proteins (DKKs) and sclerostin. Myeloma cells are known to induce inhibition of osteoblastogenesis through Wnt antagonists such as DKK-1 and sFRP-2 and -3 whereas the role of sclerostin, an osteocyte-expressed negative regulator of bone formation, has not been yet investigated. We provide novel evidence showing sclerostin expression by myeloma cells from patients with multiple myeloma-bone disease and human myeloma cell lines (HMCLs). By means of a co-culture system of bone marrow stromal cells (BMSCs) and HMCLs, we demonstrated that sclerostin expression by myeloma cells and HMCLs is responsible for reduced expression of major osteoblastic specific proteins namely bone-specific alkaline phosphatase, collagen-type I, bone sialoprotein II and osteocalcin as well as decreased mineralized nodule formation and attenuated expression of member of the AP-1 transcription factor family (i.e. Fra-1, Fra-2 and Jun-D). The addition of a neutralizing anti-sclerostin antibody to our co-culture system can restore the above parameters, through the intranuclear accumulation of β-catenin in BMSCs. On the other hand, we demonstrated that sclerostin is also involved in inducing increased receptor activator of nuclear factor-k B ligand (RANKL) and decreased osteoprotegerin (OPG) expression in osteoblasts, contributing to the enhanced osteoclast activity occurring in patients with multiple myeloma-bone disease. Our data suggest that myeloma cells contribute to the suppression of bone formation through sclerostin secretion. Disclosures: No relevant conflicts of interest to declare.

Inhibition of DKK1 Activity Is Associated with Increased Osteoblast Numbers and Bone Formation, Reduced Osteoclast Activity and Inhibition of Tumor Growth in the SCID-rab Model for Primary Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 638-638
Author(s):  
Shmuel Yaccoby ◽  
Wen Ling ◽  
Rinku Saha ◽  
Fenghuang Zhan ◽  
Ron Walker ◽  
...  
Keyword(s):  
Bone Formation ◽  
Tumor Growth ◽  
Wnt Signaling ◽  
Bone Disease ◽  
X Rays ◽  
Mineral Density ◽  
Focal Lesions ◽  
Osteoclast Activity ◽  
Osteolytic Bone Disease ◽  
Pre Treatment

Abstract We have previously demonstrated that myeloma (MM) cells produce the Wnt signaling antagonist, Dickkopf-1 (DKK1), and that DKK1 inhibits differentiation of osteoblasts (Tian et al. NEJM 2003). This is thought to result in an uncoupling process that may lead to induction of lytic bone disease in MM and possibly promote tumor growth. The aim of this study was to investigate the role of DKK1 in our established SCID-rab model for primary MM. Growth of primary MM in this system is restricted to the implanted bone and associated with typical disease manifestations including increased osteoclast activity, reduced osteoblast numbers and induction of osteolytic bone disease (Yata & Yaccoby, Leukemia 2004). In this study, SCID-rab mice were engrafted with MM cells expressing DKK1 (assessed by global gene expression profiling) from 8 patients. The level of DKK1 expression was correlated with numbers of MRI and x-ray focal lesions in these patients. Following establishment of MM growth, as monitored by weekly measurement of human monoclonal immunoglobulins (hIg) in mouse sera and radiographically, mice were injected subcutaneously into the surrounding area of the implanted bone with neutralizing antibody (AB, R&D) against DKK1 (polyclonal AB: n=4, 50 μg/injection/2 days; monoclonal AB: n=4, 100 μg/injection/day) or control IgG AB, for 4–6 weeks. Whereas bone mineral density (BMD) in control mice was reduced by 7.1%±4.6% from pre-treatment levels, BMD in mice treated with anti-DKK1 was increased by 5.6%±6.7% from pre-treatment levels (p&lt;0.03). The bone anabolic effect of DKK1 AB was also visualized on x-rays and was detected in mice transplanted with cells from patients with low and high degree of bone disease. Histological examination revealed that myelomatous bones of DKK1 AB-treated mice had increased numbers of osteocalcin-expressing osteoblasts (45±5 vs. 16±2 per mm bone in control mice, p&lt;0.02) and reduced number of multinucleated TRAP-expressing multinucleated osteoclasts (5±3 vs. 13±2 per mm bone in control mice, p&lt;0.004). These results support recent reports demonstrating the critical involvement of Wnt signaling in osteoblasts in regulation of osteoclastogenesis as well (Glass et al., Dev. Cell 2005). Whereas in control mice myeloma burden increased in all experiments, anti-DKK1 treatment was associated with reduced tumor growth from pre-treatment levels in 4 of 8 experiments. Overall, myeloma tumor burden increased by 325%±122% and 165%±53% from pre-treatment levels in control and DKK1 AB-treated mice, respectively (p&lt;0.03). Furthermore, bone marrow areas with locally high numbers of differentiated osteoblasts were depleted of myeloma cells in DKK1 AB-treated hosts. We conclude that DKK1 is a key player in myeloma bone disease and that blocking DKK1 activity in myelomatous bones reduces osteolytic bone resorption, increases bone formation and helps control myeloma progression.

Targeting Wnt Signaling in Myeloma In Vivo; Differential Effects on Tumor Burden and Myeloma Bone Disease.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 812-812
Author(s):  
Claire M. Edwards ◽  
James R. Edwards ◽  
Seint T. Lwin ◽  
Gregory R. Mundy
Keyword(s):  
Bone Marrow ◽  
Wnt Signaling ◽  
Bone Disease ◽  
Tumor Burden ◽  
Bone Microenvironment ◽  
Myeloma Cells ◽  
Myeloma Bone Disease ◽  
Osteolytic Bone Disease

Abstract Multiple myeloma is characterized by uncontrolled proliferation of myeloma cells within the bone marrow and the development of a severe osteolytic bone disease. In addition to a well characterized increase in osteoclastic bone resorption, myeloma bone disease is associated with a reduction in bone formation. Osteoblast differentiation and bone formation are regulated in vivo by canonical Wnt signaling and activation of β-catenin. Therefore increasing Wnt signaling in the bone microenvironment in multiple myeloma may prevent the development of myeloma bone disease. In support of this, we have previously demonstrated that activation of Wnt signaling with lithium chloride (LiCl) in the 5TGM1 murine model of myeloma reduces tumor burden and osteolytic bone disease. However, we also found that LiCl treatment increased subcutaneous (s.c.) tumor growth. This suggests that the reduction in tumor burden within the bone microenvironment may be an indirect effect mediated through the effects of LiCl to prevent myeloma bone disease. The aim of the current study was to determine the effect of specific molecular blockade of Wnt signaling in myeloma cells in vivo. 5TGM1-GFP myeloma cells were transfected by electroporation with either myc-tagged dominant negative TCF4 (DNTCF4) or pcDNA. Following stable selection by culture in G418, expression of DNTCF4 was confirmed by western blot for myc. No difference was found in the growth rates of 5TGM1-pcDNA or 5TGM1-DNTCF4 in vitro. Treatment with LiCl or Wnt3A had no significant effect on cell viability in vitro, but significantly increased β-catenin activity, as measured by TOPFLASH activity in 5TGM1-pcDNA cells. This increase was not observed in 5TGM1-DNTCF4, confirming that expression of DNTCF4 blocked Wnt signaling induced by LiCl in 5TGM1 myeloma cells. C57Bl/KaLwRij mice were inoculated with 5TGM1-pcDNA or 5TGM1-DNTCF4 cells by either intravenous (i.v.) or s.c. injection. Mice were treated from time of tumor cell inoculation with 200mg/kg/day LiCl or vehicle control (d.H20) by oral gavage for 28 days. I.v. inoculation of myeloma cells resulted in a significant increase in serum IgG2bκ concentrations and the proportion of GFP-positive cells in the bone marrow. A significant reduction in trabecular bone volume was also observed. MicroCT analysis of the tibia demonstrated that LiCl significantly increased trabecular bone volume in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. LiCl significantly decreased serum IgG2bκ concentrations in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice, with a greater effect in 5TGM1-DNTCF4 myeloma-bearing mice. FACS analysis of GFP-positive cells demonstrated that LiCl significantly reduced tumor burden in the bone marrow in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. However, following s.c inoculation, LiCl significantly increased s.c. tumor volume of 5TGM1-pcDNA tumors, but had no effect on 5TGM1-DNTCF4 s.c. tumor volume. Taken together these results demonstrate that the effect of increasing Wnt signaling in myeloma is dependent upon the microenvironment. By specific inhibition of β-catenin activity in myeloma cells combined with systemic stimulation of the Wnt signaling pathway, our results suggest that increasing Wnt signaling in myeloma in vivo has dual effects; firstly to enhance myeloma growth directly, and secondly to enhance osteoblast differentiation and thus indirectly reduce tumor burden in bone, highlighting the importance of the bone marrow microenvironment in regulating myeloma growth and survival.

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