Selectin ligands are known to be upregulated in metastatic tumor cell lines, and growing evidence suggests selectin/selectin-ligand interactions mediate the adhesion of circulating tumor cells to distant sites. Thus, novel methods of characterizing selectin ligands expressed on human tissue may serve as valuable assays with significant diagnostic and prognostic potential. Because the kinetic and tensile properties of selectin/selectin-ligand bonds are influenced by local physiological conditions, i.e., wall shear stress due to hydrodynamic force, traditional immunohistochemical approaches cannot adequately detect selectin ligands. That is, though immunohistochemistry is very successful in identifying high affinity interactions, selectin/selectin-ligand interactions exhibit high affinity only when the external force applied on the bond is at an optimal value. This affinity is considerably weaker when applied force on the bond is significantly larger than this optimal value, or else is low or zero, as in traditional immunohistochemistry. Therefore, we have developed a method for detecting selectin ligands expressed on human tissue using a dynamic approach that allows for precise control of the force applied to the bonds between probe and target molecules. More specifically, we conjugated recombinant P-selectin to polystyrene microspheres and perfused this probe over tissue sections of colon cancer - which is known to express P-selectin ligands. Selectin/selectin-ligand adhesive interaction in the form of microsphere rolling on tissue was observed, and specific interaction was confirmed using 10 mM EDTA as a negative control. Increased probe surface coverage (i.e., higher density of P-selectin molecules coated on the surface of the microspheres) resulted in increased interaction with colon cancer tissue. In addition, rolling velocities measured between physiological wall shear stresses 0.25-2.0 dyne/cm2 indicated adhesive interaction of the selectin-conjugated probe with ligands in colon cancer tissues was stabilized at 0.75 dyne/cm2. Analysis of distinct regions within colon cancer tissue sections subjected to this dynamic biochemical tissue analysis revealed different levels of microsphere adhesion in discrete areas, evidence of heterogeneous cellular expression of P-selectin ligands. Taken together, these results demonstrate that functional P-selectin ligands are detectable using dynamic biochemical tissue analysis.

Citation Format: Eric W. Martin, Venktesh S. Shirure, Ramiro Malgor, Vicente A. Resto, Douglas J. Goetz, Monica M. Burdick. Dynamic biochemical tissue analysis of P-selectin ligands expressed on colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 45. doi:10.1158/1538-7445.AM2013-45