Abstract
Bone is the most common site of metastasis for breast cancer, however the reasons for this remain unclear. We hypothesise that under certain conditions mammary cells possess osteomimetic capabilities that may allow them to adapt to, and flourish within, the bone microenvironment. Mammary cells are known to calcify within breast tissue and we have recently reported a novel in vitro model of mammary mineralization using murine mammary adenocarcinoma 4T1 cells. In this study, the osteomimetic properties of the mammary adenocarcinoma cell line and the conditions required to induce mineralization were characterized extensively. It was found that exogenous organic phosphate and inorganic phosphate induce mineralization in a dose dependent manner in 4T1 cells. Ascorbic acid and dexamethasone alone have no effect. 4T1 cells also show enhanced mineralization in response to bone morphogenetic protein 2 in the presence of phosphate supplemented media. The expression of several bone matrix proteins were monitored throughout the process of mineralization and increased expression of collagen type 1 and bone sialoprotein were detected, as determined by real-time RT-PCR. In addition, we have shown for the first time that 3D collagen glycosaminoglycan scaffolds, bioengineered to represent the bone microenvironment, are capable of supporting the growth and mineralization of 4T1 adenocarcinoma cells. These 3D scaffolds represent a novel model system for the study of mammary mineralization and bone metastasis. This work demonstrates that mammary cells are capable of osteomimicry, which may ultimately contribute to their ability to preferentially metastasize to, survive within and colonize the bone microenvironment.
Highlights
Bone is one of the most preferential metastatic target sites for breast cancers [1], the precise molecular mechanisms underlying this preference have yet to be elucidated
We have demonstrated that hydroxyapatite upregulates the production of matrix metalloproteinases (MMPs) in mammary cell lines [9]
In this study we identify the components within the osteogenic cocktail essential for mineralization and we investigate whether mammary cells, which are capable of depositing hydroxyapatite, do so in a manner similar to osteoblasts
Summary
Bone is one of the most preferential metastatic target sites for breast cancers [1], the precise molecular mechanisms underlying this preference have yet to be elucidated. Positive staining for both alizarin red S and von Kossa was detected in the OC&dex group, this was to a much lesser extent than that observed for the OC group Using this established model of mammary mineralization, the expression of several bone markers were investigated using realtime RT-PCR from days 0–28. Positive H&E staining of 4T1 cells was observed in the center of the scaffolds by day 14 at 4006magnification (Figure 6A) confirming cell infiltration of the scaffold At this time point, partial positive staining for calcium (red) and calcium phosphate (black/brown, counterstained with toluidine blue) were detected using alizarin red S and von Kossa staining respectively (Figure 6A).
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