Fluid flow exposure promotes Epithelial-to-Mesenchymal Transition and adhesion of breast cancer cells to endothelial cells

Abstract

Background: Mechanical interactions between tumor cells and microenvironments are frequent phenomena during breast cancer progression, however, it is not well understood how interactions in the magnitude of those encountered in the vascular microenvironment affect expression of genes related to Epithelial-to-Mesenchymal Transition (EMT) and adhesion to endothelial cells. 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. Methods: MDA-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 gene expression were quantified using microarrays and qPCR, gene-gene interactions were elucidated using network analysis, and key modified genes were examined in clinical datasets. Changes in protein expression of key EMT markers between chemically induced EMT and flow-exposed cells were compared in immunocytochemistry assays. Finally, the ability of flow-stimulated and unstimulated cancer cells to adhere to an endothelial monolayer was evaluated in flow and static adhesion experiments. Results: Fluid 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, suggesting progression towards an EMT phenotype. 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 most subtypes of breast cancer. We also observed increased adhesion of flow-stimulated breast cancer cells compared to unstimulated controls, suggesting an increased potential to form secondary tumors at metastatic sites. Conclusions: This 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.