Cancer cell tethering and rolling on the vascular wall is facilitated by various selectin: glycoprotein interactions which lead to eventual extravasation and metastases. The aberrantly underglycosylated mucin MUC1 has been shown to both abundantly express selectin binding moieties (sialyl Lewis x and a) and to consistently expose its core epitope. Flow cytometry was used to determine MUC1 expression on ZR-75-1 and MCF7 cells, while immunofluorescence microscopy was used to confirm the aberrant form of MUC1 and MUC1:ICAM-1 interactions. Each cell line was then perfused through combined E-selectin and ICAM-1 coated microtubes, as a model of the microvascular endothelium. ZR-75-1 and MCF7 were found to express abundant and low levels of underglycosylated MUC1, respectively. The rolling/adhesion profiles showed that ZR-75-1 cells, when compared to MCF7 cells, interact with E-selectin more efficiently resulting in sufficiently slow rolling velocities to form MUC1:ICAM-1 interactions thereby facilitating firm adhesion. The purpose and novelty of this work is the demonstration of the synergistic adhesion capabilities of MUC1 in the metastatic adhesion cascade, where the observed differential adhesion is consistent with the relative metastatic potential of the ZR-75-1 (highly metastatic) and MCF7 (weakly metastatic) cell lines.

Selectins mediate circulatory leukocyte trafficking to sites of inflammation and trauma, and the extracellular microenvironments at these sites often become acidic. In this study, we investigated the influence of slightly acidic pH on the binding dynamics of selectins (P-, L-, and E-selectin) to P-selectin glycoprotein ligand-1 (PSGL-1) via computational modeling (molecular dynamics) and experimental rolling assays under shear in vitro. The P-selectin/PSGL-1 binding is strengthened at acidic pH, as evidenced by the formation of a new hydrogen bond (seen computationally) and the observed decrease in the rolling velocities of model cells. In the case of L-selectin/PSGL-1 binding dynamics, the binding strength and frequency increase at acidic pH, as indicated by the greater cell-rolling flux of neutrophils and slower rolling velocities of L-selectin-coated microspheres, respectively. The cell flux is most likely due to an increased population of L-selectin in the high-affinity conformation as pH decreases, whereas the velocities are due to increased L-selectin/PSGL-1 contacts. In contrast to P- and L-selectin, the E-selectin/PSGL-1 binding does not exhibit significant changes at acidic pH levels, as shown both experimentally and computationally.

Abstract-The underglycosylated form of the MUC1 glycoprotein, uMUC1, has been identified as a ligand for both Eselectin 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 (sLe x ). 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 Lselectin 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 Eselectin/SM3 surface strategy offers a viable method to selectively capture cancer cells from whole blood samples.