Bone Sialoprotein Shows Enhanced Expression in Early, High-Proliferation Stages of Three-Dimensional Spheroid Cell Cultures of Breast Cancer Cell Line MDA-MB-231

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.

Fibroblasts Accelerate Formation and Improve Reproducibility of 3D Cellular Structures Printed with Magnetic Assistance

“…Similar formation times have been reported for nongrowing aggregates [6] or loose aggregates created with rBM in a scaffold-free environment [1]. Otherwise, longer formation times are required to form robust 3D MDA-MB-231 structures [4,51].…”

“…The long-term growth characteristics of 3D cellular structures, maximum projected areas, and circularities [6,21,23,52,53] are measured. Since the maximum projected structures are not perfect circles, it is not appropriate to use diameter as a metric [4]. Instead, the maximum projected area is used.…”

“…These 3D models exploit in vivo cellular phenomena such as cell-cell interactions, cell polarization, increased drug resistance, and diffusion gradients of O 2 , CO 2 , nutrients, and metabolites that lead to proliferative, quiescent and necrotic regions, and similar gene expressions [ 1 – 3 ], which are unattainable and therefore not observed in 2D cell models. MDA-MB-231, a human epithelial triple-negative metastatic breast cancer cell line, is notorious for being difficult to grow in 3D [ 4 ]. Efforts to form 3D cellular structures with MDA-MB-231 often incorporate biologically based extracellular matrix (ECM) constituents, such as recombinant basement membrane (rBM) [ 1 ] or Matrigel [ 5 , 6 ].…”

The MicroRNA-130a-5p/RUNX2/STK32A Network Modulates Tumor Invasive and Metastatic Potential in Non-Small Cell Lung Cancer

“…Samarakkody et al has been recently proposed that the transcription factor RUNX2 in mammals is one of the key players during development and differentiation, and is commonly dysregulated in cancers [26]. Rustamov et al established that bone sialoprotein predicts unsatisfactory prognosis in triple-negative breast cancer, while its expression is elevated by RUNX2 [27]. Besides, it has been suggested by Villanueva et al that enhanced RUNX2 might transcriptionally induce gene expression engaged in tumor development and metastasis [28].…”

<p>Role of αVβ3 in Prostate Cancer: Metastasis Initiator and Important Therapeutic Target</p>

“…BSP and OPN have been more extensively studied than other SIBLING proteins. [24][25][26][27][28][29] These two proteins have been identified as primary inducers in the progression of PCa metastasis, especially bone metastasis. As mentioned previously, BSP and OPN contain an integrin-binding RGD sequence, which can bind to αVβ3 on the cell surface.…”