osteoprotegerin and Bone-Marrow-Neoplasms

The aim of our study was to investigate whether the defective function of osteogenic cells induced by neuroblastoma might play a role in the development of skeletal metastases. This mechanism has been extensively demonstrated for multiple myeloma, in which the blockage of osteoblast differentiation has been ascribed to the inhibitors of canonical Wingless pathway (Wnt), namely Dickkopf 1 (Dkk1). Our purpose was to verify if neuroblastoma cells derived from bone marrow metastases (SH-SY5Y, LAN1) or primaries (NB100, CHP212) hamper the differentiation of mesenchymal stem cells (hMSCs) into osteoblasts in a paracrine manner, and to test whether this ability depends on Dkk1 activity. We found that all neuroblastoma cells increased the proliferation of hMSCs collected from pediatric-aged donors, with a corresponding decrease in osteoblast differentiation markers, including alkaline phosphatase (ALP), analyzed as gene expression, enzymatic activity and number of ALP-positive colony forming units, osteoprotegerin (OPG) release, OPG and osteocalcin gene-expression. Dkk1 mRNA and protein were detectable in all cell lines, and the use of neutralizing anti-Dkk1 antibody reversed the effects induced by SH-SY5Y cells. Taken together, our results confirm that neuroblastoma hinders osteoblastogenesis, and that Dkk1 release seems to play a crucial role in blocking the differentiation of osteoprogenitor cells, though the ability to promote osteoclast activation remains an essential requirement for the development of skeletal metastases. Finally, our findings suggest that strategies regulating Wnt signaling and Dkk1 activity could be considered for adjuvant therapies in neuroblastoma metastasizing to the skeleton.

Topics: Animals; Base Sequence; Bone Marrow Neoplasms; Cell Differentiation; Cell Proliferation; Culture Media, Conditioned; DNA Primers; Gene Expression Profiling; Intercellular Signaling Peptides and Proteins; Neuroblastoma; Osteoblasts; Osteocalcin; Osteoprotegerin; Primates; RNA, Messenger

The skeletal system is the most frequent metastatic site of hematogenous spread of urologic carcinomas. Osseus metastases are classified as osteoneutral, osteolytic, osteoblastic and combinations thereof. Osteolytic metastases lead to bone resorption by activating osteoclasts, while osteoblastic metastases stimulate osteoblasts by paracrine mechanisms. The local osteoblastic effect is associated with secondary systemic bone resorption. The use of bisphosphonates is now an established supportive therapy and newer treatment strategies including targeted intervention in the pathophysiology of bone metastases and radioimmunotherapy are being applied or will be coming soon.

Topics: Bone and Bones; Bone Marrow Neoplasms; Bone Neoplasms; Bone Resorption; Cytokines; Diphosphonates; Humans; Osteoblasts; Osteoclasts; Osteolysis; Osteoprotegerin; Parathyroid Hormone-Related Protein; Radioimmunotherapy; RANK Ligand; Urologic Neoplasms

We have been using the B9/BM1 murine bone marrow metastasis model to study the function of adhesion molecules in the cell-cell interactions and transendothelial migration, necessary for tumor metastasis. The cell surface phenotype of these cells, which colonize vertebral and femoral marrow after intravenous injection, shows great similarity to that of human myeloma cells. In the present study, we investigated the interaction between B9/BM1 cells and osteoclasts, which likely support tumor metastasis in bone marrow. We found that co-culturing B9/BM1 cells and bone marrow-derived endothelial cells (BMECs) in the presence of vitamin D3 and M-CSF promoted differentiation of primary osteoclast progenitors to osteoclasts (detected by TRAP staining), and that this effect was blocked when BMECs were separated from the other cells by a porous polycarbonate membrane. Flow cytometry analysis showed that BMECs expressed RANKL (receptor activator of NF-kappaB ligand) protein on their surface, and that this expression was up-regulated by co-culture with B9/BM1 cells. Accordingly, RT-PCR showed expression of RANKL mRNA also to be up-regulated in BMECs co-cultured with B9/BM1 cells. Addition of OPG (osteoprotegerin, a decoy RANKL receptor) to the co-culture system completely blocked osteoclast induction, as did addition of anti-CD44 antibody. Furthermore, intravenous injection of B9/BM1 cells substantially increased the numbers of TRAP-positive osteoclasts detected in mice in vivo. Taken together, these findings suggest that B9/BM1 myeloma cells act via CD44 to stimulate RANKL expression on BMECs, which in turn physically interact with osteoclast progenitors to promote their differentiation to osteoclasts and metastasis in bone marrow.

Topics: Animals; Bone Marrow Cells; Bone Marrow Neoplasms; Cell Communication; Cell Differentiation; Coculture Techniques; Endothelial Cells; Female; Glycoproteins; Interleukin-6; Mice; Multiple Myeloma; Osteoclasts; Osteoprotegerin; Receptors, Cytoplasmic and Nuclear; Receptors, Tumor Necrosis Factor; Tumor Cells, Cultured