Modeling the unique characteristics of the bone-tumor microenvironment is an important endeavour for our understanding of tumor progression and metastasis as well as the development of bone cancer pain. We have established a coculture model that simulates many of the facets of this microenvironment and affords several advantages. Those advantages include: (1) the maintenance of the complexity and relevance of the bone component of this 'microenvironment'; (2) the distinct separation of the biological components to allow accurate analyses of each component; and (3) the ease of in vitro
manipulation of all biological components. The bone component of this model, the intact femur explant, retains the three-dimensional, multi-cellular architecture consisting of ossified bone, hematopoietic cells, osteoblast and osteoclast precursors, stroma and matrix tissue. Our analyses suggest that the femur is viable in these cultures and is capable of active and regulatable secretion of important paracrine factors. The cells of the marrow cavity encased in the diaphysis of this tissue are important contributors to this model since loss of this compartment via marrow depletion attenuates MCP-1 secretion from bone and abolishes the potentiation due to bone-sarcoma interactions. This suggests that paracrine factors are secreted by these marrow cells, interact within this compartment and diffuse into the culture to modulate the tumor cells. Other in vitro models which have been used to test the influence of secreted bone factors on tumor growth and survival or the paracrine effects of tumor cells on bone resorption often employ single cell cultures, calvarial bone or long bone fragments, all of which have little to no marrow component present [33
]. It is true that the bone marrow contribution to this model would be presumed to be limited to the early interactions of the bone and tumor since its viability declines after 24 h. Even with this limitation, a modulation of a paracrine factor due to component interactions can be observed and that modulation is significantly affected within this time frame by the loss of the bone marrow.
The presence of the femur in cocultures with the sarcoma cells results in a strong up- regulation of MCP-1 expression and secretion. Within 24 h, a greater-than-additive increase in MCP-1 concentrations is observed along with increased MCP-1 mRNA in both femur and sarcoma cells and this potentiation is greater in 48 hr cocultures. These results suggest that paracrine and autocrine regulation of MCP-1 occurred in the cocultures due to the action of factors released by the bone, the tumor cells or both components. Numerous factors, known to be present in the bone-tumor microenvironment, have been shown to induce MCP-1 secretion and gene expression including MCP-1 itself [39
], TGF-β [40
], tumor necrosis factor-alpha (TNF-α) [42
], parathyroid hormone related peptide (PTHrP) [39
] and various interleukins [43
]. The mechanism for this potentiation and the factors responsible in our model are currently under investigation.
It is also interesting to note that comparable concentrations of MCP-1 protein secreted by these sarcoma cells in this model have been observed by our research group in in vivo
mouse models of bone cancer pain. Microperfusates collected in situ from tumors generated by these fibrosarcoma cells contained approximately 4–7 ng/ml [45
] which is a range similar to the 5–8 ng/ml observed in the 48 hr bone-tumor cocultures. In addition, strong MCP-1-like immunoreactivity is detected in tumor biopsy samples [45
]. These results suggest that our model can accurately simulate this microenvironment, recapitulating the regulation of this chemokine and potentially other paracrine factors. One future direction for our research is to expand this coculture model to include neurons dissociated from dorsal root ganglia (DRG). This will allow the analysis of local chemical mediators contributing to the production of bone cancer pain which is difficult to conduct in in vivo
mouse models. Research to date has shown that DRG neurons express receptors (CC chemokine receptor 2 and 4) activated by MCP-1 [46
], and that lack of CCR2 receptor can impair inflammatory and neuropathic pain responses [47
]; all supporting a potential role for MCP-1 for the unique type of pain elicited at the site of bone cancers.
In contrast to the upregulation of MCP-1 in the sarcoma cultures, it was also observed that co-culturing with the breast carcinoma cells results in a robust reduction in TGF-β and MCP-1. This suggests that bidirectional communication between the bone and tumor exists in this model where secretions from the tumor cells can regulate the release or secretion of paracrine factors from the bone tissue. The MDA-MB-231 cell lines did not secrete detectable protein in the single-component cultures and human-specific assays did not detect either cytokine in cocultures. So, it is presumed that both are being actively secreted from cells within the bone tissue or passively released by bone resorption during culturing. Transforming growth factor-β is one of the most abundant growth factors stored within the mineralized bone matrix [6
] and some degradation of the cortical surface of the femur was observed after 48 h of culturing. Passive release of TGF-β is therefore possible in these cocultures and factors released by these breast carcinoma cells may be modulating this process. In contrast, although it can be stored in secretory granules of endothelial cells prior to active secretion [49
], MCP-1 is not known to be stored within the bone matrix. Therefore, paracrine factor(s) released by the breast carcinoma cells may regulate the active secretion of MCP-1 from cells of the femur tissue which could include osteoblasts, marrow endothelial cells or immune cells such as monocytes [18
]. It is interesting that both of these cytokines are reduced in parallel raising the possibility that this effect of the femur-carcinoma interactions could reflect linked regulation. It has been shown that TGF-β can have growth-inhibitory and apoptotic effects on normal and malignant cells, including MDA-MB-231 cells [50
]. In addition, TGF-β can enhance MCP-1 expression and secretion from a variety of cell types including osteoblasts and immune cells [40
]. Therefore, during the early interactions in the cocultures, the MDA cells may counter any potential growth inhibition by suppressing TGF-β secretion. A decrease in TGF-β in this microenvironment may cause a loss of positive regulation of MCP-1 secretion, due to direct effects of TGF-β or through changes in the secretion of other paracrine factors.
Like all experimental approaches, this simulated model of the bone-tumor microenvironment has certain limitations that need to be considered. First, it is most applicable for the investigation of early paracrine interactions between tumor and bone which are not mediated by cell-cell or cell-matrix interactions. The contribution of the hematopoietic compartment to this model are limited to the first 24–48 hr of culture. Also, the tumor and the bone are isolated from each other. The advantage of this is that it allows the dissection of short-term, acute responses to secreted factors in this microenvironment but does not address the role of such factors with chronic exposure. Second, the neonatal femurs utilized have a greater amount of cartilage than would be expected in an adult femur. This age of femur was chosen to improve the ease and efficiency of dissection as well as optimize tissue permeability. Such a difference in bone explants from adult bone, where human primary and metastatic cancers most often reside, is also the case for research conducted on cocultures of neonatal calvaria and breast or prostate carcinomas [33
]. Therefore, the assumptions for extrapolation of such results to the disease process would be similar but our model would afford a more complex and representative microenvironment. Finally, as presented here, the major tissue or cell source of paracrine factors modulated by the bone-tumor interactions can only be presumed primarily due to the complexity of the bone tissue. But, in future experiments, this limitation can be eliminated by the use of tumor cell lines or tissues from transgenic animal models where the expression of these factors has been manipulated.