Targeting Underglycosylated MUC1 for the Selective Capture of Highly Metastatic Breast Cancer Cells Under Flow

摘要

The underglycosylated form of the MUC1 glycoprotein, uMUCl, 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 uMUCl, 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-selectincoated microtubes resulted in fast rolling velocities and low numbers of interacting cells and blocking uMUCl with the SM3 antibody had no effect. ZR-75-1 cells, on the other hand, were highly dependent on the E-selectin:uMUCl interaction as exemplifiedby significant increases in cell rolling velocities and decreases in the number of interacting cells when blocking with SM3 or when uMUCl expression was knocked down via siRNA transfection. Whereas uMUCl interactions with E-selectin supported cell rolling, P-selectin: uMUCl interactions exclusively facilitated cell tethering, while L-selectin surfaces supported no cell adhesive interactions. These experimental observations are consistent with molecular dynamics simulations of uMUCl bound to E-, P-, andL-selectin where the degree of residue contact correlated with the differential adhesion of uMUCl 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.