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Abstract
The underglycosylated form of the MUC1 glycoprotein, uMUC1, has been identified as a ligand for both E-selectin and ICAM-1 and can play multiple potential roles during rolling and firm adhesion events in the metastatic cascade. Using flow cytometry and confocal microscopy, the T47D and ZR-75-1 cell lines were verified to highly express uMUC1, however it was found that only ZR-75-1 cells expressed the E-selectin binding moiety sialyl Lewis x (sLex). Furthermore, perfusing T47D cells through E-selectin coated microtubes resulted in fast rolling velocities and low numbers of interacting cells and blocking uMUC1 with the SM3 antibody had no effect. ZR-75-1 cells, on the other hand, were highly dependent on the E-selectin:uMUC1 interaction as exemplified by significant increases in cell rolling velocities and decreases in the number of interacting cells when blocking with SM3 or when uMUC1 expression was knocked down via siRNA transfection. Whereas uMUC1 interactions with E-selectin supported cell rolling, P-selectin: uMUC1 interactions exclusively facilitated cell tethering, while L-selectin surfaces supported no cell adhesive interactions. These experimental observations are consistent with molecular dynamics simulations of uMUC1 bound to E-, P-, and L-selectin where the degree of residue contact correlated with the differential adhesion of uMUC1 to each selectin. Finally, an E-selectin and SM3 combined surface coating captured approximately 30% of the total number of interacting cancer cells comparable to the number of adhered cells when utilizing E-selectin and ICAM-1 combined surfaces. The E-selectin/SM3 surface strategy offers a viable method to selectively capture cancer cells from whole blood samples.
Highlights
In our previous study of MUC1, we examined the adhesion profiles of ZR-75-1 and MCF7 cells on combined surfaces of E-selectin and ICAM-1.14 We found that whereas both cell lines exhibited high levels of MUC1, only ZR-75-1 cells exhibited underglycosylated form of MUC1 (uMUC1) which leads to slower rolling velocities and the existence of firm adhesion events which strongly suggested that the uMUC1 glycoprotein significantly participates in the adhesion of metastatic cancer cells to the inflamed endothelium
We examined two cell lines (MCF7 and ZR-75-1) that exhibited variants of the MUC1 glycoprotein where both cell lines expressed MUC1, only ZR-75-1 expressed the underglycosylated form uMUC1
We had previously found that uMUC1 has the ability to interact with E-selectin and ICAM-1 and, for these two cell lines, the metastatic potential corresponded with uMUC1 expression since ZR-75-1 cells are highly metastatic and MCF7 cells are weakly metastatic.[14]
Summary
Leukocyte recruitment to the inflamed endothelium and cancer metastasis through the bloodstream via circulating tumor cells (CTCs) have been proposed to share a similar stepwise mechanism that allows for cell adhesion and extravasation.[12,28,31,33,36] Referred to as adhesion cascades (leukocyte and CTC), cells first tether and roll on the blood vessel wall via transient interactions between P- and E-selectin present on the inflamed endothelium[4,36] and carbohydrate moieties, such sialyl Lewis x (sLex) or sialyl Lewis a (sLea) found on leukocytes and CTCs.[34,35] Sufficiently slow cell rolling permits firm cell adhesion events mediated by endothelial intercellular adhesion molecule-1 (ICAM1) at locations of transendothelial migration.[2,11] Work by our group has capitalized on these selectin:carbohydrate based interactions to capture CTCs as well as hematopoietic stem and progenitor cells with the ability to maintain cell viability.[18,19,29,30] Further differentiation between CTCs and contaminating leukocytes will allow isolation processes to be further optimized with respect to both yield and purity.Three cell adhesion molecules constitute the members of the selectin family. Leukocyte recruitment to the inflamed endothelium and cancer metastasis through the bloodstream via circulating tumor cells (CTCs) have been proposed to share a similar stepwise mechanism that allows for cell adhesion and extravasation.[12,28,31,33,36] Referred to as adhesion cascades (leukocyte and CTC), cells first tether and roll on the blood vessel wall via transient interactions between P- and E-selectin present on the inflamed endothelium[4,36] and carbohydrate moieties, such sialyl Lewis x (sLex) or sialyl Lewis a (sLea) found on leukocytes and CTCs.[34,35]. These carbohydrate moieties are attached to O-glycosylated proteins on the cell surface, referred to as selectin ligands, and in the
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