Abstract
BackgroundMechanical interactions between tumor cells and microenvironments are frequent phenomena during breast cancer progression, however, it is not well understood how these interactions affect Epithelial-to-Mesenchymal Transition (EMT). EMT is associated with the progression of most carcinomas through induction of new transcriptional programs within affected epithelial cells, resulting in cells becoming more motile and adhesive to endothelial cells.MethodsMDA-MB-231, SK-BR-3, BT-474, and MCF-7 cells and normal Human Mammary Epithelial Cells (HMECs) were exposed to fluid flow in a parallel-plate bioreactor system. Changes in expression were quantified using microarrays, qPCR, immunocytochemistry, and western blots. Gene–gene interactions were elucidated using network analysis, and key modified genes were examined in clinical datasets. Potential involvement of Smads was investigated using siRNA knockdown studies. Finally, the ability of flow-stimulated and unstimulated cancer cells to adhere to an endothelial monolayer, migrate and invade membrane pores was evaluated in flow and static adhesion experiments.ResultsFluid flow stimulation resulted in upregulation of EMT inducers and downregulation of repressors. Specifically, Vimentin and Snail were upregulated both at the gene and protein expression levels in flow stimulated HMECs and MDA-MB-231 cells, suggesting progression towards an EMT phenotype. Flow-stimulated SNAI2 was abrogated with Smad3 siRNA. Flow-induced overexpression of a panel of cell adhesion genes was also observed. Network analysis revealed genes involved in cell flow responses including FN1, PLAU, and ALCAM. When evaluated in clinical datasets, overexpression of FN1, PLAU, and ALCAM was observed in patients with different subtypes of breast cancer. We also observed increased adhesion, migration and invasion of flow-stimulated breast cancer cells compared to unstimulated controls.ConclusionsThis study shows that fluid forces on the order of 1 Pa promote EMT and adhesion of breast cancer cells to an endothelial monolayer and identified biomarkers were distinctly expressed in patient populations. A better understanding of how biophysical forces such as shear stress affect cellular processes involved in metastatic progression of breast cancer is important for identifying new molecular markers for disease progression, and for predicting metastatic risk.
Highlights
Breast cancer is a heterogeneous disease that progresses from oncogenic transformation in local epithelial cells to formation of tumors at distant organ sites [1, 2]
Fluid flow affects breast cancer cell line gene expression and enriches cellular processes involved in metastasis To investigate the effect of fluid flow on breast cancer cell line gene expression, BT-474, MCF-7, MDAMB-231, and SK-BR-3 breast cancer cells were exposed to fluid flow at 1 Pa derived from the parallel-plate bioreactor system
Our results show that exposure to fluid forces on the order of 1 Pa promotes Epithelial-to-Mesenchymal Transition (EMT) and adhesion of breast cancer cells to endothelial cells
Summary
Breast cancer is a heterogeneous disease that progresses from oncogenic transformation in local epithelial cells to formation of tumors at distant organ sites [1, 2]. Fluid flow is known to significantly affect cancer cell’s behavior, not much information is available on how forces of the magnitude of those experienced in the vascular microenvironment affect cellular events during cancer progression. Forces of such magnitudes (0.1–1 Pa) have been shown to affect cell phenotypes by activating signaling pathways and inducing transcription factors, some of which are involved in EMT [15, 16]. Mechanical interactions between tumor cells and microenvironments are frequent phenomena during breast cancer progression, it is not well understood how these interactions affect Epithelial-to-Mesenchymal Transition (EMT). EMT is associated with the progression of most carcinomas through induction of new transcriptional programs within affected epithelial cells, resulting in cells becoming more motile and adhesive to endothelial cells
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