(622y) The Relationship Between Oxidative Stress and Breast Cancer Cell Adhesion Using an Ischemia/Reperfusion Injury Model

Authors: 
Schumer, C., University of Kentucky
Anderson, K. W., University of Kentucky
Dziubla, T., University of Kentucky

Circulating tumor cells that metastasize from a primary tumor are the leading cause of cancer death in the United States. These circulating tumor cells adhere to the endothelial lining of the vascular system and extravasate through the lining to form deadly secondary tumors. Many pathways of oxidative stress can be found in pathological diseases, including one specific injury that leads to superoxide anion production.  Ischemia, or the blockage of blood flow, can occur during extensive surgery in the removal of primary tumors. During an ischemic cascade, cells are introduced to a hypoxic environment and depleted of oxygen, which promotes purine nucleotide breakdown and produces xanthine and hypoxanthine. In the presence of xanthine oxidase, superoxide anions can be generated as a side reaction during the formation of uric acid. Superoxide anions can promote cellular dysfunction and even cell death. If there is a restoration of flow to the area subject to ischemia, reperfusion can allow for inflammatory responses to persist, permitting an even greater imbalance in the consumption of reactive oxygen species (ROS) and natural occurring antioxidants. If circulating tumor cells are within the system at the time of ROS generation, cancer cells may adhere to the endothelium more readily. The overall objective of this study is to investigate ROS generation under hypoxic conditions and to determine the role of this ROS generation in cancer cell adhesion to endothelial cells.  Static studies were performed where a Hypoxanthine/Xanthine Oxidase (HXXO) injury model was used to mimic a hypoxic environment in the circulatory system and to determine its effect on the adhesion of MDA-MB-231 invasive breast cancer cells to human umbilical vein endothelial cells (HUVECs). ROS generation was measured using CellROX Green Reagent oxidative stress indicator to indicate superoxide anions and other general forms of oxidative stress present in the HUVECs. Endothelial cell monolayers that were exposed the HXXO injury model showed a 60% increase in ROS generation compared to monolayers not exposed to HXXO, providing confirmation that superoxide formation in endothelial cells can be induced in the presence of HXXO.   In addition, MDA-MB-231 invasive breast cancer cells showed a significant increase in adherence to injured monolayers of endothelial cells correlating cellular dysfunction and increase in response to oxidative stress with the promotion of cancer cell binding. This positive correlation of ROS generation and cancer cell binding supports the use of antioxidant therapeutics to mitigate injuries that provide oxidative stress pathways and ultimately limit the occurrence of secondary tumor formation. Currently, a flow system using Ibidi VI0.4 μ-slides is being used to induce flow over endothelial monolayers.  Flow-regulated studies can be used to mimic the circulatory system physiologically and allow for endothelial cells to produce reactive oxygen species naturally by the physical stopping and starting of flow over endothelial monolayers.  Results of ROS generation and cancer cell binding in this flow system will also be presented.