Characterizing Melanoma Adhesion Dynamics: Thrombin Mediated Interactions and Polymorphic Neutrophil Deformability

Open Access
Author:
Lee, Deborah Snyder
Graduate Program:
Bioengineering
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
March 31, 2016
Committee Members:
  • Cheng Dong, Thesis Advisor
Keywords:
  • cancer
  • melanoma
  • thrombin
  • fibrinogen
  • fibrin
  • polymorphic neutrophils
  • cellular adhesions
  • flow assays
Abstract:
In order for cancer metastasis to occur, tumor cells must separate from the primary tumor and enter the circulatory system, and then extravasate through the vascular endothelial cells in order to form a secondary tumor at a distant site. This work focuses on the adhesion of melanoma cells to the endothelium in response to various conditions. Soluble fibrin serves as a cross-linker between endothelial cells and melanoma cells. We used an aptamer, a small oligonucleic acid sequence with a distinct tertiary structure, to inhibit thrombin activity, and therefore formation of soluble fibrin. The apparatus used, a parallel-plate flow chamber, allows for the perfusion of cells over a confluent monolayer, therefore modeling physiological flow conditions over the endothelium in vitro. The results indicated that the aptamer was able to effectively inhibit thrombin activity and subsequent melanoma adhesions decreased after addition of the aptamer. Melanoma interactions involving the effects of activation and sequestration of polymorphic neutrophils (PMNs) were also investigated. PMN activation and sequestration has been associated with inflammation of the pulmonary microcirculation. It was found that activation, which triggers changes in PMN deformability, has a significant effect on the aggregation of melanoma cells on the endothelium whereas environments with high concentrations of PMNs did not yield significant changes. The results suggested that activation plays a key role in aiding metastatic melanoma cells in adhering to the endothelium due to a decrease in neutrophil deformability. This effect was reversed by the addition of cytochalasin B, a mycotoxin that disrupts actin filaments and increases deformability of PMNs, and a decrease in melanoma aggregations on the endothelium was observed. Thus, these results suggest that deformability plays a larger role than accumulation of PMNs in aiding melanoma cells to adhere to the endothelium.