Soluble Rank Ligand Produced by Myeloma Cells Causes Generalised Bone Loss in Multiple Myeloma

Patients with multiple myeloma commonly develop focal osteolytic bone disease, as well as generalised osteoporosis. The mechanisms underlying the development of osteoporosis in patients with myeloma are poorly understood. Although disruption of the RANKL/OPG pathway has been shown to underlie formation of focal osteolytic lesions, its role in the development of osteoporosis in myeloma remains unclear. Increased soluble RANKL in serum from patients with myeloma raises the possibility that this molecule plays a key role. The aim of the present study was to establish whether sRANKL produced by myeloma cells contributes directly to osteoporosis. C57BL/KaLwRij mice were injected with either 5T2MM or 5T33MM murine myeloma cells. 5T2MM-bearing mice developed osteolytic bone lesions (p<0.05) with increased osteoclast surface (p<0.01) and reduced trabecular bone volume (p<0.05). Bone volume was also reduced at sites where 5T2MM cells were not present (p<0.05). In 5T2MM-bearing mice soluble mRANKL was increased (p<0.05), whereas OPG was not altered. In contrast, 5T33MM-bearing mice had no changes in osteoclast surface or trabecular bone volume and did not develop osteolytic lesions. Soluble mRANKL was undetectable in serum from 5T33MM-bearing mice. In separate experiments, RPMI-8226 human myeloma cells were transduced with an human RANKL/eGFP construct, or eGFP alone. RPMI-8226/hRANKL/eGFP cells, but not RPMI-8226/eGFP cells, stimulated osteoclastic bone resorption (p<0.05) in vitro. Sub-cutaneous injection of NOD/SCID mice with RPMI-8226/hRANKL/eGFP or RPMI-8226/eGFP cells resulted in tumour development in all mice. RPMI-8226/hRANKL/eGFP-bearing mice exhibited increased serum soluble hRANKL (p<0.05) and a three-fold increase in osteoclast number (p<0.05) compared to RPMI-8226/eGFP-bearing mice. This was associated with reduced trabecular bone volume (27%, p<0.05), decreased trabecular number (29%, p<0.05) and increased trabecular thickness (8%, p<0.05). Our findings demonstrate that soluble RANKL produced by myeloma cells causes generalised bone loss, suggesting that targeting RANKL may prevent osteoporosis in patients with myeloma

Serum concentrations of sRANKL and OPG are abnormally regulated in mice bearing 5T2MM and 5T33MM cells, respectively.

<p>Serum concentrations of sRANKL and OPG are abnormally regulated in mice bearing 5T2MM and 5T33MM cells, respectively. A. Serum concentrations of sRANKL in naive mice and mice bearing 5T2MM or 5T33MM cells. B. Serum concentrations of OPG in naive animals and animals bearing 5T2MM or 5T33MM cells. Statistical analysis by Mann-Whitney U test. Data = mean± S.E.M.</p

5T2MM bearing mice have increased bone loss in the lumbar vertebrae and calvariae.

<p>A. Photomicrographs of the vertebrae from naïve and 5T2MM bearing mice. Tumour infiltration is identified with black arrows. B. The proportion of trabecular bone area in the vertebrae from naïve and 5T2MM-bearing mice. C. Photomicrographs of the calvariae of naïve and 5T2MM bearing mice. Medullary spaces are identified with black arrows, and OC with yellow arrows. Non tumour-containing spaces are shown. D. The proportion of medullary area as a percentage of total tissue area in naïve and 5T2MM-bearing mice. E. Distribution of medullary spaces in naïve and 5T2MM-bearing mice, showing significant differences in distribution (KS test, p<0.02). Chi-square analysis demonstrated that 5T2MM-bearing mice had significantly fewer, smaller spaces and more, larger spaces than naïve mice (p<0.05). Data = mean± S.E.M. (B and D).</p

5T2MM, but not 5T33MM murine myeloma cells promote osteoclast formation in C57BL/KaLwRij mice.

<p>A. Photomicrographs of sections of the tibia reacted for TRAP activity from naive, 5T2MM and 5T33MM bearing mice showing the cortico-endosteal bone surface. TRAP-positive osteoclasts are arrowed. Original magnification ×40. B. The proportion of the cortical-endosteal bone surface occupied by osteoclasts, expressed as the percentage of the total bone surface, from naive mice and mice bearing 5T2MM or 5T33MM myeloma cells. C. Photomicrographs of sections of the tibia stained for TRAP activity from naive and 5T33MM bearing mice showing areas of trabecular bone. TRAP-positive osteoclasts are arrowed. Original magnification ×40. D. The proportion of trabecular bone covered by osteoclasts in naïve and 5T33MM bearing mice. Statistical analysis by Mann-Whitney U test. Data = mean± S.E.M.</p

5T2MM, but not 5T33MM cells cause osteolytic bone disease and tumour-induced bone loss.

<p>A. Reconstructed 3-dimensional micro-CT images of the tibia of naïve mice, mice bearing 5T2MM cells and mice bearing 5T33MM cells. Lesions in the tibia of 5T2MM bearing animals are arrowed. B. Transverse sections of micro-CT images of tibiae from naïve mice, mice bearing 5T2MM cells and mice bearing 5T33MM cells. Lesions are arrowed. C. Radiographs of the tibia of naïve mice, mice bearing 5T2MM cells and mice bearing 5T33MM cells. Lesions are arrowed. D. Number of lesions in the tibia of naïve mice and 5T2MM or 5T33MM bearing mice. E. Trabecular bone area as a proportion of total tissue area in the tibia of naïve mice, and mice bearing 5T2MM or 5T33MM cells. F. Total bone mineral density of naïve mice, and 5T2MM or 5T33MM bearing mice. Statistical analysis by Mann-Whitney U test. Data = mean± S.E.M.</p

SRC Kinase Inhibition with Saracatinib Limits the Development of Osteolytic Bone Disease in Multiple Myeloma

peer reviewe

The myeloma stem cell concept, revisited: From phenomenology to operational terms

The concept of the myeloma stem cell may have important therapeutic implications, yet its demonstration has been hampered by a lack of consistency in terms and definitions. Here, we summarize the current documentation and propose single-cell in vitro studies for future translational studies. By the classical approach, a CD19–/CD45/CD38/CD138+ malignant plasma cell, but not the CD19/CD38–memory B cell compartment, is enriched for tumorigenic cells that initiate myeloma in xenografted immunodeficient mice, supporting that myeloma stem cells are present in the malignant PC compartment. Using a new approach, analysis of c-DNA libraries from CD19/CD27/CD38–single cells has identified clonotypic memory B cell, suggested to be the cell of origin. This is consistent with multiple myeloma being a multistep hierarchical process before or during clinical presentation. We anticipate that further characterization will require single cell geno-and phenotyping combined with clonogenic assays. To implement such technologies, we propose a revision of the concept of a myeloma stem cell by including operational in vitro assays to describe the cellular components of origin, initiation, maintenance, and evolution of multiple myeloma. These terms are in accordance with recent (2012) consensus statements on the definitions, assays, and nomenclature of cancer stem cells, which is technically precise without completely abolishing established terminology. We expect that this operational model will be useful for future reporting of parameters used to identify and characterize the multiple myeloma stem cells. We strongly recommend that these parameters include validated standard technologies, reproducible assays, and, most importantly, supervised prospective sampling of selected biomaterial which reflects clinical stages, disease spectrum, and therapeutic outcome. This framework is key to the characterization of the cellular architecture of multiple myeloma and its use in precision medicine.This work on MMSC and myeloma pathogenesis and classification was supported by research funding from the EU 6th FP to MSCNET (LSHC-CT-2006-037602), the Danish Research Agency (grants no. 99 00 771, 271-05-0286, 271-05-0537, and 22-00-0314; to CHEPRE, #2101-07-0007), the Danish Cancer Society (grants no. DP 07014, DR 07017, and DP 9810009), Novo Nordic Foundation (Senior Fellowship 2001-4), KE Jensen Foundation (2005-2013), the Multiple Myeloma Research Foundation (senior grant 2003-4, contract no. 14).Peer Reviewe

The myeloma stem cell concept, revisited:from phenomenology to operational terms

The concept of the myeloma stem cell may have important therapeutic implications, yet its demonstration has been hampered by a lack of consistency in terms and definitions. Here, we summarize the current documentation and propose single-cell in vitro studies for future translational studies. By the classical approach, a CD19–/CD45low/–/CD38high/CD138+ malignant plasma cell, but not the CD19+/CD38low/–memory B cell compartment, is enriched for tumorigenic cells that initiate myeloma in xenografted immunodeficient mice, supporting that myeloma stem cells are present in the malignant PC compartment. Using a new approach, analysis of c-DNA libraries from CD19+/CD27+/CD38–single cells has identified clonotypic memory B cell, suggested to be the cell of origin. This is consistent with multiple myeloma being a multistep hierarchical process before or during clinical presentation. We anticipate that further characterization will require single cell geno-and phenotyping combined with clonogenic assays. To implement such technologies, we propose a revision of the concept of a myeloma stem cell by including operational in vitro assays to describe the cellular components of origin, initiation, maintenance, and evolution of multiple myeloma. These terms are in accordance with recent (2012) consensus statements on the definitions, assays, and nomenclature of cancer stem cells, which is technically precise without completely abolishing established terminology. We expect that this operational model will be useful for future reporting of parameters used to identify and characterize the multiple myeloma stem cells. We strongly recommend that these parameters include validated standard technologies, reproducible assays, and, most importantly, supervised prospective sampling of selected biomaterial which reflects clinical stages, disease spectrum, and therapeutic outcome. This framework is key to the characterization of the cellular architecture of multiple myeloma and its use in precision medicine