Biophysical and biochemical characterization of selectin-ligand interactions pertinent to cancer metastasis and inflammation

Cellular adhesion mediated by biological macromolecules and their respective ligands plays a fundamental role in a number of diverse biological phenomena, including inflammation and cancer metastasis. These processes occur in the fluid dynamic environment of the vasculature and are regulated by highly specific adhesion molecules, which are specialized to interact under physiological shear conditions. While the participation of E-, P- and L-selectin in these pathophysiological phenomena has clearly been demonstrated, the kinetics and molecular constituents that govern these adhesive interactions remain to be fully characterized. Consequently, this study was undertaken to examine the single-molecule biophysics of selectin-mediated binding to leukocytes and colon carcinoma cells. Furthermore, we used novel biochemical techniques to identify and characterize a previously unrecognized selectin counter-receptor on tumor cells that may facilitate hematogenous metastasis.; Using single-molecule force spectroscopy, we probed in situ the rupture forces, unstressed off-rates and the reactive compliances of single selectin receptors to single ligands on whole human polymorphonuclear leukocytes (PMNs) and colon carcinoma cells under conditions which preserve the proper orientation and post-translational modifications of the selectin ligands. The rupture force spectra and corresponding Bell model parameters of selectin-ligand bonds provide mechanistic insights that help explain the differential avidity of these cells for different selectin substrates.; By performing SDS-PAGE analysis of membrane proteins, metabolic inhibition interventions and blot rolling assays of LS174T colon carcinoma cells, we identified the functional selectin counter-receptor on carcinomas. In particular, our studies show that the variant isoforms of CD44 (CD44v) modified with O-linked glycans function as the high-affinity E-, P- and L-selectin glycoprotein ligands on LS174T cells. This finding provides an explanation for the apparent enhanced metastatic potential associated with CD44v over-expression on many types of tumor cells. By combining our knowledge on the biophysics of receptor-ligand binding and drug delivery via the blood, we aim to provide guidelines to engineer novel anti-carcinogenic therapeutic agents that will selectively recognize novel antigens on tumor cells and impede metastatic spread.