Abstract
Normally, bone sialoprotein (BSP) is an important contributor to bone micro-calcification. However, it is also highly expressed in bone-metastatic malignancies, including prostate, lung, and breast cancer. In these disorders, BSP correlates with poor prognosis. Its expression in triple-negative breast cancer cells is enhanced by the transcription factor RUNX2, and both, BSP and RUNX2 are under control of IGF-1 and TGFβ1. Knockdown of BSP or its inactivation by specific antibodies were found to reduce the metastatic potential of MDA-MB-231 triple-negative breast cancer cells in xenografts. While the role of BSP in bone metastasis was studied using such in vivo models, valid in vitro test systems to investigate BSP biology have been lacking since this protein is expressed at very low levels in classical 2D cell cultures and the frequently used breast cancer cell line MDA-MB-231 is difficult to grow in 3D. Here, we have developed a long-term 3D spheroid culture model using MDA-MB-231 cells in a sandwich approach using cell embedding between a non-adherent surface and basement membrane extracts. This allowed consistent growth of spheroids for more than 21 days. Also, co-culturing of MDA-MB-231 with CCD-1137Sk fibroblasts yielded stably growing spheroids, suggesting the importance of extracellular matrix (ECM) in this process. In addition, we have set up a novel and simple open source analysis tool to characterize protein expression in 2D cultures and spheroids by immunofluorescence. Using this approach in combination with Western blot analysis, the expression profile of BSP was analyzed. BSP was enriched at the rims of spheroids, both in mono- and co-cultures and its abundance in general correlated with that of TGFβ1 under different conditions, including spheroid maturation, cytostatic treatment, and fibroblast co-culture. Conversely, correlation of IGF-1 and BSP was limited to mono-culture time course profiles. In conclusion, we present novel tools to study the regulation of gene expression in combination with cell proliferation and apoptosis in a long-term 3D model of breast cancer and find dynamic abundance profiles of the metastasis-relevant protein BSP and its regulators.
Highlights
Breast cancer is the most frequent neoplastic lesion in women
Cells were grown under four different conditions, i.e., hanging drop (HD), inlay (IM), on top (OM), and sandwich methods (SM)
Cells in Hanging Drop Technique (HD), Inlay Method (IM), and On-Top Method (OM) cultures did not form spheroids (Figure 1A) but remained loose cell aggregates that decreased in size during the observation period of 24 day in vitro (DiV) (Figure 1B)
Summary
Breast cancer is the most frequent neoplastic lesion in women. When associated with distant metastasis, the overall prognosis for breast cancer is poor with a 5-years survival in stage IV of about 27% [1]. Metastasis is frequent in breast cancer and typically affects liver, bone, lung, or a combination of these [3,4,5], with bone being the most frequently targeted organ of breast cancer metastasis [6]. This might be due to bone’s rich depots of nutrients, growth factors (TGFβ, IGF, VEGF, M-CSF, FGF, MCP, BMP2) and fine blood supplies [7]. Bone contains a special type of capillaries called sinusoids, which are characterized by slow blood circulation and porous endothelial walls, that facilitate the extravasation of metastatic cells into the bone marrow [8]. Strategies which could reduce the incidence and morbidity of bone metastases are of great clinical importance
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