We have developed novel co- and triple-culture systems and employed our established in vivo
SCID-hu model to study the effect of osteoblasts on myeloma growth and the association between bone metabolism and tumor progression in vivo
. Our study revealed that osteoblasts inhibited survival and proliferation of myeloma cells from a subset of patients while they had no impact or even stimulated survival in another subset. The effects of osteoblasts on myeloma cells was also observed in the presence of osteoclasts (triple-cultures), which promoted survival and proliferation of myeloma cells from all patients studied.7
Furthermore, in five of nine experiments injection of patients’ MSC into myelomatous bones in SCID-hu mice resulted in inhibition or retardation of myeloma growth, an effect that was associated with increased bone mass. Our study suggests that increased osteoblast activity in myelomatous bones via direct injection of exogenous osteoblast precursors is feasible and that this procedure may not only improve bone density but also inhibit myeloma growth.
Previous studies in murine models showed a close association between myeloma progression and increased osteoclast activity.5,6,14,15,24
To our knowledge, no studies have demonstrated the impact of osteogenic cells on long-term survival and proliferation of myeloma cells. The ex vivo
experiments confirmed our previous observations of the vital role of osteoclasts on maintaining the disease process.6,7
Osteoblasts, in contrast, had diverse effects on myeloma cells, these effects being dependent on the source of the myeloma cells. Interestingly, the majority of patients whose myeloma cells were suppressed by osteoblasts were in clinical stage IIIa/IIIb and had severe bone disease. This suggests that increased osteoblast activity may help control tumor growth even in patients with advanced myeloma. We speculate that in these patients, myeloma cells reduce osteoblast activity, either via induction of osteoblast apoptosis11
or by inhibition of their differentiation,12,13
as part of mechanisms by which myeloma cells alter the bone marrow microenvironment for their advantage.
The mechanisms by which osteoblasts interfere with myeloma cell growth are still unclear. Osteogenic cells produce great amount of different members of the transforming growth factor-β superfamily including BMPs and activin A, which have been shown to induce growth arrest of B lymphocytes and myeloma cells in vitro
Osteoblasts secrete osteonectin, a matrix cellular protein that inhibits survival and growth of epithelial tumor cells.30
Circulating levels of osteonectin seem to be inversely correlated with myeloma stage.31
Osteoblasts express high level of connexins - transmembrane proteins that regulate osteoblast differentiation and apoptosis and mediate intercellular communications between osteogenic cells.32
Connexins are also tumor suppressor genes and their expression is reduced or lost in many tumors. It has been suggested that Cx43 and Cx26 induce their tumor-suppressing properties by a mechanism that is independent of significant gap junctional intercellular communication and possibly through the down-regulation of key genes involved in tumor growth. For instance, transfection of the MDA-231 breast cell line with Cx43 reduced these cells’ growth potential and down-regulated FGFR3 and CXCR4, both of which are involved in myeloma pathology.33
In addition, osteoblasts produce high levels of osteoprotegerin, a factor that indirectly impedes myeloma growth in vivo
through inhibition of osteoclast differentiation.15
Thus, it is also possible that osteoblasts affect myeloma cells, as shown in triple-culture experiments and in vivo
, indirectly through interference with the interaction of myeloma cells with osteoclasts.
Repair of lytic bone lesions is rarely seen, even in myeloma patients with prolonged complete remissions. We hypothesized that prevention of bone repair is caused, at least in part, by reduced numbers and impaired activity of MSC in focal bone lesions. In vitro
, MSC have the ability to differentiate into various cell lineages, including osteoblasts, chondrocytes, adipocytes, skeletal myoblasts and endothelial cells.18,34,35
The osteogenic potential of MSC has been demonstrated in animal models36,37
and in patients with osteogenesis imperfecta.38
We tested the effect of MSC on bone remodeling and tumor growth in myelomatous SCID-hu mice using EGFP-transduced MSC. Histological and immunohistological examinations revealed that patients’ MSC were capable of engraftment and differentiation into various cells of the mesenchymal lineage, including osteoblasts and osteocytes. The differentiated MSC stimulated bone formation in five of nine experiments, an effect that was associated with inhibition of tumor burden in these mice. In non-responding mice, MSC failed to affect either bone mass or myeloma growth.
In summary, we demonstrated that growth of myeloma cells from a subset of patients was restrained by osteoblasts ex vivo
and by MSC injection in myelomatous SCID-hu mice. Further studies to unravel the molecular mechanisms by which osteoblasts affect myeloma cells are warranted. We conclude that increased osteoblast activity via exogenous cytotherapy and/or endogenous approaches, such as treatment with bone anabolic agents, will benefit patients with myeloma for various reasons. First, it will increase bone formation, and relieve skeletal complications. Secondly, it may help control myeloma progression, particularly when combined with specific inhibitors of osteoclast activity. Third, osteoblasts play an important role in maintaining the hematopoietic stem cell niche,39,40
and thus, an increased osteoblast pool may improve hematopoietic recovery, and the ability to mobilize hematopoietic stem cells. Finally, increased bone formation in patients with monoclonal gammopathy of undetermined significance, smoldering myeloma and patients in remission may prevent transformation into active myeloma.