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.

Conservation and divergence of methylation patterning in plants and animals.

Cytosine DNA methylation is a heritable epigenetic mark present in many eukaryotic organisms. Although DNA methylation likely has a conserved role in gene silencing, the levels and patterns of DNA methylation appear to vary drastically among different organisms. Here we used shotgun genomic bisulfite sequencing (BS-Seq) to compare DNA methylation in eight diverse plant and animal genomes. We found that patterns of methylation are very similar in flowering plants with methylated cytosines detected in all sequence contexts, whereas CG methylation predominates in animals. Vertebrates have methylation throughout the genome except for CpG islands. Gene body methylation is conserved with clear preference for exons in most organisms. Furthermore, genes appear to be the major target of methylation in Ciona and honey bee. Among the eight organisms, the green alga Chlamydomonas has the most unusual pattern of methylation, having non-CG methylation enriched in exons of genes rather than in repeats and transposons. In addition, the Dnmt1 cofactor Uhrf1 has a conserved function in maintaining CG methylation in both transposons and gene bodies in the mouse, Arabidopsis, and zebrafish genomes.