(386e) Multiparameter Analysis of Circulating Tumor Cells

Breast circulating tumor cells (CTC) are commonly isolated by positive selection enrichment technology (i.e. CellSearch?µ), which targets epithelial surface markers such at the epithelial cell adhesion activating molecule (EpCAM). However, CTC with low or absent epithelial markers, are likely to be missed by this technology (Sieuwerts et al., 2009). In contrast to positive selection for epithelial cells, we developed a negative enrichment technology which removes red blood cells and cells expressing CD45. Since it is hypothesized that only a subset of CTC can establish metastatic deposits and that these may have undergone the epithelial mesenchymal transition (EMT), we investigated whether CTC have mesenchymal and stem cell markers.

Since we obtain unbiased, enriched cell suspension, it is highly desired to analyze the cells for as many distinct markers as possible. Typical fluorescent and/or Confocal microscopy is limited due to spectral overlap, sometime referred to as "bleed-through" of one dye into the next "channel". We developing a labeling an analysis protocol which is targeting as many markers as possible with a combination of specifically chosen antibody-fluorescent labels and the application of a spectral deconvolution camera/software referred to as the Nuance system. In this presentation we will present both our methodology and the latest types of circulating tumor cells that we have found, including a family of markers associated with EMT and cancer stem cells.