Analysis of endothelial cell contractility in melanoma induced junction disruption of endothelial monolayers

Open Access
Author:
Pohler, Steven E
Graduate Program:
Chemical Engineering
Degree:
Master of Engineering
Document Type:
Master Thesis
Date of Defense:
July 16, 2014
Committee Members:
  • Esther Winter Gomez, Thesis Advisor
Keywords:
  • endothelial
  • junction disruption
  • VE-cadherin
  • melanoma
  • contractility
  • soluble factors
  • receptor-ligand binding
  • interleukins
Abstract:
Tumor metastasis occurs as a multiple step process culminating in tumor cells leaving the bloodstream through the endothelial layer due to the disruption of cell-cell junctions. Soluble signaling from tumor secreted proteins and receptor-ligand binding facilitate interactions between tumor cells and endothelial cells that are known to mediate junction disruption in endothelial cell layers. Previous work has shown that soluble factors alone, such as interleukins which are known to be pro-inflammatory, can promote junction disruption, making a case for endothelial cell contractility as a regulator of junction disruption and extravasation. In this work we used melanoma as a model of metastatic cancer and sought to elucidate the signaling pathways that are involved in mediating junction disruption in the endothelium, in response to tumor cells. Isolation of endothelial signaling was achieved by incubating endothelial cells with cytoskeletal inhibitors before soluble factor treatment or co-culture experiments. A reduction in gap formation was measured when inhibited monolayers were co-cultured with A2058 cells, as well as with K562 cells which were used as a model to isolate receptor-ligand binding. Inhibitor experiments implicate Rho, Rac and Src pathways in junction disruption mediated by melanoma. Furthermore, we measured the contractility of groups of endothelial cells in response to an inflammatory cytokine secreted by melanoma, and found that endothelial monolayer contractility decreased more rapidly than control treatments. These data show that soluble factors secreted by melanoma are able to affect contractility in endothelial cells, which is borne both by cell-cell forces as well as cell-substrate coupling. These results suggest possible therapeutic targets to mitigate the spread of metastatic cancers, through the regulation of endothelial permeability.