In this investigation, we used histologic evaluation of GCTB and in vitro
cell culture studies to more rigorously characterize the phenotype and lineage of the stromal cell population in GCTB, and to define the cellular and molecular mechanisms that are responsible for their unique osteoclast-inducing function within the tumor. By using freshly harvested, highly enriched GCTB cell populations that were maintained in vitro
for a very limited period, we sought to avoid the altered phenotypic features and loss of osteoclast-inducing capacities typically exhibited by the longstanding, repeatedly passaged cultures of GCTB stromal cells used in studies to date [3
It is conceivable that our strategy of extracting CD14+ monocyte-lineage cells and co-localizing MNGC’s from total dispersed cells did not effectively separate other cells known to be present in GCTB such as macrophage precursors, osteoclasts, endothelial cells, or cells of the adaptive immune system. However, GCTB stromal cells isolated using the CD14 negative selection technique (1) were morphologically consistent with cells of osteoblast-lineage; (2) were enriched in osteoblast markers; (3) were capable of undergoing terminal differentiation in the presence of mineralizing culture conditions; and (4) strongly induced osteoclastogenesis in co-culture with human monocytes. These data indicate that our method of isolation was specific for isolating a highly enriched, functional population of GCTB stromal cells. Further refinement of this isolation method is ongoing, as is an effort to validate these findings beyond the limited number of samples demonstrated herein.
Our experiments demonstrated that negatively selected, uncultured stromal cells can undergo further differentiation and matrix mineralization under pro-osteogenic conditions. Although activation of Wnt and BMP signaling pathways promoted differentiation, the baseline characteristics and responsiveness to Wnt and BMP agonists varied extensively between different tumors. The observed variability in osteoblast-associated gene expression, the effects of the various culture conditions on gene expression profiles, and the functional properties of the cells may have resulted from the heterogeneity of the patients from which specimens were obtained, as well as their varied treatment-related factors (. Furthermore, certain tumors were harvested in limited quantities such that all samples were not included in every experiment. This heterogeneity, evident in the histologic features of the harvested tumor tissue and in the in vitro
studies of the CD14+ cell fractions, may have also contributed to the variable functional properties of the stromal cell isolates. Importantly, our data provide further insights into the mechanisms by which the stromal cells play a pivotal role in the recruitment and induction of myeloid lineage cells to form the characteristic osteoclast-like giant cells of the tumors [6
Previous assertions that GCTB stromal cells are of osteoblast lineage were based on their expression of osteoblast and pre-osteoblast molecular markers [16
]. The expression of multiple osteoblast- and pre-osteoblast-associated genes and gene products in our isolated, unpassaged stromal cells provide additional support for this conclusion. Further, we demonstrated that bone formation could be induced in stromal cells by culture in osteogenic medium. Of note, this characteristic was displayed by the stromal cell populations isolated from all of the harvested tumors, supporting the general concept that cells of osteoblast lineage do, in fact, exist within the tumor stromal cell population. The absence of mineralized tissue in the original tumor and the relatively low levels of expression of osteocalcin, a marker of late-stage osteoblast differentiation, are consistent with an arrest of the cells in an intermediate stage of differentiation with failure to reach terminal stages of osteoblast differentiation. Whether this is related to an intrinsic property of the cells or the unique tumor microenvironment and interaction with other cellular components of the tumor remains unclear. Interestingly, other studies have shown that osteoclasts are a source of products that can either inhibit or enhance osteoblast differentiation [38
Previous studies have provided evidence that GCTB stromal cells exhibit osteoclastogenic activity when co-cultured with monocytic osteoclast precursors [23
]. Such activity has been attributed to stromal cell production of numerous factors involved in recruitment and induction of osteoclast differentiation and activation, including RANKL [3
]. Other potent osteoclastogenic proteins have been reported in GCTB, including M-CSF [6
] and its structural homolog interleukin 34 [48
], as well as Siglecs [49
]. These latter membrane-bound glycoproteins co-activate the immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway responsible for intracellular calcium signaling, a key component of induction of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), the master transcriptional regulator of the osteoclast [49
]. Our freshly harvested and purified stromal cell populations exhibited potent osteoclast-inducing activity when co-cultured with peripheral blood mononuclear cells or with the CD14+ myeloid lineage cells from the original tumors. Consistent with previously published data, a rapid loss of osteoclastogenic capacity occurred in our cell populations with prolonged time in culture or repeated passaging [24
]. We speculate that the decrease in RANKL production and up-regulation of OPG provide a plausible explanation for the loss of osteoclast-inducing activity.
Culture of our CD14-negative stromal cells under osteoblast-inducing conditions resulted in a striking reversal in the OPG/RANKL ratios involving both up-regulation of OPG and downregulation of RANKL. Both the osteogenic media and the activation of the Wnt/β-catenin signal pathway by the GSK-3 inhibitor enhanced OPG expression and increased the OPG/RANKL ratio compared to control. Although the mechanism by which the osteogenic media produced these effects is not known, we speculate that the effects of SB415286 were related to increased expression of dephosphorylated β-catenin (. Others have shown that β-catenin regulates OPG gene expression [50
] by interaction with the transcription factors T-cell factor/Lymphoid Enhancer Factor (TCF/LEF) [33
]. These transcription factors also regulate the expression of osteoblast-associated genes, including Runx2, Osterix [34
], and osteocalcin [35
The loss of osteoclast-inducing capacity after induction of differentiation of the stromal cells into a more definitive osteoblast phenotype has important and potentially promising clinical implications for the therapeutic inhibition of GCTB osteolytic activity. In a recent study of patients with refractory, non-resectable GCTB, treatment with denosumab, a human monoclonal antibody that binds and inhibits RANKL, resulted in depletion of tumoral giant cells, accompanied by inhibition of osteolytic progression in a majority of patients [19
]. In several patients, induction of local regions of bone formation within the tumors was evident, which may have been related to tumor stromal cell differentiation into osteoblastic cells. These findings were recently validated by others [18
]. Although the lytic, destructive behavior of GCTB is attributable to robust osteoclastogenesis from monocyte precursors, sole targeting of the myeloid components of the tumor may be insufficient to suppress stromal cell-mediated disease progression in the long term. Thus, stromal cell-directed therapeutic strategies remain a highly relevant approach for treatment of GCTB.
In contrast to the findings with osteogenic media and Wnt/β-catenin pathway activation, we found that BMP treatment of stromal cells inhibited the effects of long-term culture on the increase in the OPG/RANKL ratio. Importantly, these results were accompanied by maintenance of the osteoclast–inducing capacity of the stromal cells even after prolonged culture. BMP ligands are members of the Transforming Growth Factor Beta superfamily of proteins and initially bind to the Type II receptor resulting in recruitment, dimerization and phosphorylation of the Type I receptor. Receptor dimerization drives formation of R-SMAD/co-SMAD complexes that, upon nuclear translocation, function as transcription factors regulating cell proliferation, apoptosis, and differentiation [36
]. The effects of BMP signal activation on OPG and RANKL expression in mesenchymal-lineage have yet to be fully elucidated [55
]. The suppression of OPG and simultaneous up-regulation of RANKL expression in response to BMP2 was seen in C2C12 cells, which are regarded as pluripotent mesenchymal precursors, and has been suggested as a potential mechanism by which early osteoblast lineage cells stimulate osteoclastogenesis [58
]. Interestingly, BMP/SMAD-pathway activation has been associated with osteolysis in sheep and non-human primate models of spinal fusion and fracture repair [55
]. In these models, osteolysis was noted within days after introducing hyperphysiologic levels of BMP2 into the intramedullary canal, with subsequent robust bone formation up to 8 weeks later. Although we used a significantly lower BMP2 concentration, these in vivo
studies support the concept that BMP/SMAD signal activation can induce osteolysis.
Recent data have suggested that activation of the BMP/SMAD signal pathway also may enhance osteoclastogenesis through direct effects on osteoclast precursors [59
]. In our studies, osteoclast precursors were not exposed to exogenous BMP2, so as to avoid potential direct effects of BMP2. Since BMP signaling can keep stromal cells in their tumoral pro-osteoclastogenic state of arrested differentiation, it is possible that aberrant BMP signaling may represent a key component of GCTB pathophysiology. Our findings were consistent with this possibility, as BMP ligands and receptors were expressed in both stromal and myeloid cell populations, suggesting that BMP pathway modulation may represent an additional potential differentiation therapy for GCTB. Future clinical application of this therapeutic strategy raises other issues. Accurate diagnosis is essential, as cancers that are difficult to distinguish from GCTB, such as giant-cell rich osteosarcoma, may be stimulated and worsen prognosis [61
]. Finally, such treatment does not exclude anti-resorptive therapy.
In summary, our studies confirm that GCTB stromal cells are of osteoblast lineage, as evidenced by their capacity for expression of definitive markers of differentiated osteoblasts and initiation of bone mineralization under osteogenic conditions. We speculate that the arrest of the stromal cells in a state of early osteoblast differentiation is associated with up-regulation of RANKL production and that the production of this potent osteoclast-inducing factor accounts for the unique capacity of the tumors to form multinucleated osteoclasts and to induce osteolysis. Our findings that activation of the Wnt/β-catenin and BMP pathways differentially modulate the phenotype and osteoclast-inducing activity of the stromal cells has potential clinical applications for treating patients with refractory destructive GCTB.