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The expression of functional selectin ligands on human cancer cell lines is well-documented, but little is known about the in situ expression of functional selectin ligands on human carcinoma tissue. Presented here are data that show a new method of tissue interrogation, known as dynamic biochemical tissue analysis (DBTA), that is able to detect functional selectin ligands expressed on tissue from multiple cancer types at both the primary and metastatic sites. It was found that selectin ligand expression on distant metastasis tumor samples was greater than lymphatic metastasis tumor samples. Functional selectin ligand expression as determined through selectin DBTA probe adhesion also had a positive correlation with tumor stage. Consequently, this document considers how tumors that express functional selectin ligands may trigger the expression of and interact with P- selectin; a necessary requirement for P-selectin/P-selectin ligand interactions and a process that is thought to be involved in oncologic hypercoagulability. This document is also focused on the dynamic, force-based characterization of selectin ligands expressed heterogeneously on tumors using DBTA. For this characterization, a high performance tracking program was developed to abstract, wrangle, and analyze the abundant data collected from the flow assay, DBTA. A model rooted in stochastic kinetics was developed in parallel to assist in the understanding of the design space of the complex assay and relates the events observed in DBTA to the underlying biophysical processes. The semi-empirical model consists of a coupled set of stochastic, 4D partial differential equations that accounts for the transport of DBTA probes to the surface of the tissue and the dynamics of DBTA probe adsorption. The model was numerically solved using an upwind, finite difference scheme written in C++ and displayed solid agreement with the empirical data, successfully bridging the gap between the seemingly random rolling interactions observed in the flow assay to the underlying physical mechanisms.

Selectin/selectin ligand interactions have been implicated in mediating the adhesion of circulating tumor cells to distant sites during metastasis. Previous work from the Burdick lab has demonstrated the limitations of static biochemical tissue analysis (SBTA = immunostaining), using both selectins and antibodies against glycotopes, in detecting functional selectin ligands expressed on tissue. To address these shortcomings, a novel tissue interrogation method, termed dynamic biochemical tissue analysis (DBTA), has been developed in which selectin-coated microspheres are perfused over tissues in a microfluidic device to detect functional selectin ligands in situ. In this work, DBTA using P-selectin microspheres was performed on cancer tissue sections. To characterize the adhesion of P-selectin-coated microspheres to functional selectin ligands that were not identified with SBTA, a more refined method of interaction analysis that discretizes rolling into a sequence of succinct pauses (i.e., brief stationary adhesion) was used. In this method, microsphere rolling recorded at relatively high frame rates (~200 fps) was assessed using a cross-correlation particle tracking algorithm to obtain a robust point estimate that represents the off-rate bond breakage of the ensemble of receptor ligand complexes that mediate microsphere adhesion to the tissue surface. This adhesion parameter lays the framework for potentially correlating the density and type of functional ligand(s) expressed with tumor stage, or aggressiveness, furthering the understanding of aberrantly expressed functional selectin ligands as potential, functional cancer biomarkers.