Cholesterol Loading and Ultrastable Protein Interactions Determine the Level of Tumor Marker Required for Optimal Isolation of Cancer Cells

Cell isolation via antibody-targeted magnetic beads is a powerful tool for research and clinical applications, most recently for isolating circulating tumor cells (CTC). Nonetheless fundamental features of the cell-bead interface are still unknown. Here we apply a clinically relevant antibody against the cancer target HER2 (ErbB2) for magnetic cell isolation. We investigate how many target proteins per cell are sufficient for a cell to be isolated. To understand the importance of primary antibody affinity, we compared a series of point mutants with known affinities and show that even starting with subnanomolar affinity, improving antibody affinity improved cell isolation. To test the importance of the connection between the primary antibody and the magnetic bead, we compared bridging the antibody to the beads with Protein L, secondary antibody, or streptavidin: the high-stability streptavidin-biotin linkage improved sensitivity by an order of magnitude. Cytoskeletal polymerization did not have a major effect on cell isolation, but isolation was inhibited by cholesterol depletion and enhanced by cholesterol loading of cells. Analyzing a panel of human cancer cell lines spanning a wide range of expression showed that the standard approach could only isolate the highest expressing cells. However, our optimization of cholesterol level, primary antibody affinity, and antibody-bead linkage allowed efficient and specific isolation of cells expressing low levels of HER2 or epithelial cell adhesion molecule. These insights should guide future approaches to cell isolation, either magnetically or using other means, and extend the range of cellular antigens and biomarkers that can be targeted for CTC isolation in cancer research and diagnosis. Cancer Res; 73(7); 2310-21. (C)2013 AACR.