Melanoma Extravasation Through the Endothelium Affects Focal Adhesion Disassembly

Open Access
Author:
Eswar, Vikram Jayraj
Area of Honors:
Biomedical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Cheng Dong, Thesis Supervisor
  • William O Hancock, Honors Advisor
  • Esther Winter Gomez, Faculty Reader
Keywords:
  • melanoma
  • Src
  • focal adhesion
  • endothelium
  • traction force
  • vinculin
  • contractility
  • extravasation
Abstract:
Melanoma is the most serious type of skin cancer and becomes difficult to treat when it metastasizes to secondary locations in the body. The extravasation of malignant cells through the endothelial barrier is a highly intricate process, involving interactions between soluble cytokines and transmembrane ligand-receptors. Extensive work has been done to characterize the signaling cascade associated with endothelial gap formation during extravasation, specifically the breakdown of cell-cell junctions and cytoskeletal rearrangement mediated by activation of Src, a non-receptor tyrosine kinase. Src has been also implicated in focal adhesion turnover and assembly during cell spreading. This study aims to build on past experiments by developing a better understanding of endothelial gap formation during melanoma transmigration and the role that focal adhesions play in the process. To this end, traction force microscopy was used to measure forces exerted by two to six endothelial cells seeded onto 100 µm circles on 5 kPa polyacrylamide gels. Upon incubation of these micro-patterns with A2058 metastatic melanoma for 10 min, 45 min, and 90 min, measured endothelial forces were found to decrease with time. Using a similar experimental set-up, co-incubation with A2058 for 10 min, 45 min, and 90 min, endothelial micro-patterns were fluorescently stained for vinculin and F-actin. A statistically significant reduction in both focal adhesion (FA) number and total FA area per cell was observed at each time point compared to the negative control (no treatment) suggesting a close relationship between FA expression and traction stresses. Co-incubation experiments with a Src inhibitor, PP1, reversed this decrease in FA number and area after 90 min. Overall, these results propose a relationship between decreased traction forces and Src-mediated disassembly of focal adhesions during extravasation.