Ph.D. Thesis Defense - Quantitative Analysis of Cell-Surface Interactions and Cell Adhesion Process in Real-time
Date: April 9, 2008
Time: 9:00 AM
Location: Bossone Research Enterprise Center, Room: 709
Advisor: Kenneth A. Barbee, Ph.D.
The cell adhesion process and cellular interactions with extracellular matrix (ECM) proteins were quantitatively evaluated using a thickness shear mode (TSM) sensor. The sensor can sensitively detect changes in mechanical properties on the sensor surface, which is contributed to the progression of cell adhesion. For understanding cell-surface interactions, specific effects of receptor-mediated adhesion, the glycocalyx, and surface charge on initial cell-surface attachment and steady state adhesion of bovine aortic endothelial cells (BAECs) were investigated. The strong correlation between resistance changes (∆R) and the development of cell adhesion strength by comparing the sensor readings with independently assessed cell adhesion was demonstrated. The result showed that integrin binding determines the kinetics of initial cell attachment while heparan sulfate proteoglycan (HSPG) modulates steady state adhesion strength. Coating the sensor surface with the positively charged poly-D-lysine (PDL) enhanced the initial interaction with substratum. In order to relate sensor data to the adhesion of the cells, we performed independent measures of cell spreading and adhesion strength. The adhesive interactions of human mammary epithelial cells, MCF-10A, with a fibronectin coated sensor were altered by overexpressing Her2/Neu oncogene and by blocking integrin function with soluble GRGDS. Correlation of ΔR with cell spreading and adhesion measurements revealed three phases: adhesion, spreading and structural reorganization. In the initial adhesion phase, ΔR increases rapidly as the cells bind to the substratum and began to flatten. The rate of change of ΔR in this phase is proportional to the adhesion strength as determined by a spinning disk assay. As the cells spread beyond their initial projected area, ΔR increased in proportion to the change in area. In the final phase, as the cells approach their final spread area, further increases in ΔR reflect structural changes, possibly indicating maturation of cytoskeleton and focal adhesion formation. In order to see the effect of cell deformability on the initial adhesion, the elastic modulus of spherical cells were evaluated. Normal and oncogene expressing cells were compressed with the flat surface of the hemisphere which was attached to the AFM cantilever. The calculation of Young’s moduli showed that oncogene expressing cells were more compliant than normal cells (1.09kPa±0.36 vs. 2.95kPa±0.34), which were reflected to the initial increase of ∆R. The real time monitoring capability of this technique with high temporal resolution provides more detailed information on the kinetics of the different stages of the adhesion process. The analysis of adhesion properties of normal versus transformed mammary epithelia correlates with changes in integrin expression, demonstrating the usefulness of TSM sensor measurement system for understanding adhesion characteristics in real time as it relates to phenotypic variations.
The Bossone Research Enterprise Center is located at the corner of 32nd and Market Streets.