Abstract 335: Dynamic biochemical tissue analysis of novel P-selectin ligands expressed by colon cancer

Abstract

Abstract Selectin/selectin ligand interactions have been implicated in mediating the adhesion of circulating tumor cells to distant sites during metastasis. Previous work from our lab has demonstrated the limitations of static biochemical tissue analysis (SBTA), using both selectins and antibodies against glycotopes, in detecting functional selectin ligands expressed on tissue. To address these shortcomings, we developed a novel tissue interrogation method, termed dynamic biochemical tissue analysis (DBTA), 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 in conjunction with SBTA using antibodies against P-selectin ligands CD24, CD44, and PSGL-1. Using DBTA, calcium-dependent selectin/selectin ligand adhesive interactions in the form of P-selectin microsphere rolling was observed on four distinct cases of colorectal cancer tissue. Examination of serial sections with SBTA in the same regions of tissue displaying P-selectin microsphere rolling revealed no detectable levels of CD24, CD44, or PSGL-1 epitopes, while only one case displayed CD44 expression that was in agreement with DBTA. However, not all regions displaying specific reactivity with the P-selectin microspheres used in DBTA were recognized with the CD44 antibody in SBTA. These results imply the presence of novel P-selectin ligands in the tissue section. Follow-up CD45 SBTA ruled out the possibility of microsphere interaction with infiltrated leukocytes, in agreement with the lack of PSGL-1 detection. Consistent with our DBTA studies using microspheres coated with E- and L-selectin on other types of cancer tissue, DBTA reveals unequivocal detection of functional P-selectin ligands by generating results distinct from SBTA. To further 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 300 frames per second was assessed using a cross-correlation 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 and/or aggressiveness, furthering our understanding of aberrantly expressed functional selectin ligands as potential cancer biomarkers. Citation Format: Eric Martin, Ramiro Malgor, Douglas Goetz, Monica Burdick. Dynamic biochemical tissue analysis of novel P-selectin ligands expressed by colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 335. doi:10.1158/1538-7445.AM2015-335