Molecular Mechanisms in Compromised Endothelial Barrier during Breast Cancer Metastasis

Abstract

Abstract : We have successfully devised a 3D tumor invasion assay that integrates 3D tissue engineering and FRET imaging to provide us with a means with which we could directly monitor, in real-time, the endothelial signaling event during tumor intravasation. Using this novel assay, we have demonstrated that breast cancer cells induce endothelial cell myosin light chain kinase (MLCK) activation at the site of tumor invasion, and that this transient MLCK activation correlates with localized myosin II regulatory light chain (RLC) phosphorylation and regional endothelial cell stress fiber shortening. We have also investigated the role of RLC phosphorylation using a high throughput tissue cytometric studies and demonstrated that endothelial RLC phosphorylation played a critical role in determining the routes of tumor intravasation. The majority (~56%) of MDA-MB231 breast cancer intravasation events were transcellular (i.e. through individual endothelial cells). However, when challenged by endothelial cells expressing mutagenized RLC that cannot be phosphorylated by MLCK, cancer cells predominantly (~76%) undergo paracellular invasion, indicating that intraendothelial signaling events can be modulated and play an important role in the tumor intravasation. The development of this novel assay adds significantly to our experimental repertoire to directly study an important yet hitherto under-explored perspective of tumor invasion - that of the underlying endothelium.