The enrichment and enumeration of exfoliated epithelial cells, from populations of fixed cells immunomagnetically purified from enriched blood samples, has led to the suggestion that these cells are circulating tumor cells (""""""""CTCs"""""""") and that the abundance of these cells in peripheral blood samples correlates with the progression of cancer, Cristofanilli et al. (2004). The precise identification and characterization of these exfoliated cells is currently an area of intense investigation, Chen et al. (2005). Two questions are critical in these investigations: ? ? 1. Are these exfoliated epithelial cells really CTCs? ? 2. Is there clinical value in analyzing these CTCs? ? ? A microfluidics-based mammalian cell sorter that is capable of sorting viable, low abundance, mammalian cells from very small cell populations, typically 1,000 to 100,000 cells in 5 30 80% purity with 70-90% recovery. Specifically, the project will first recapitulate the isolation of fixed epithelial cells using a model of MCF-7 breast cancer cells in normal donor blood. Second, the project will isolate viable epithelial cells using the same model. Third, the project will isolate and recover viable primary epithelial cells from 20 ml blood samples obtained from breast cancer patients diagnosed with metastatic disease. Fourth, the viable epithelial cells isolated from patient blood samples will be identified as CTCs using clinically established assays for estrogen receptor, progesterone receptor and HER2/neu by immunohistochemistry and for HER2/neu by fluorescence in situ hybridization of the isolated cells compared with the identical assays performed on the subject's primary tumor. Alternate approaches will also be available. Using UCSD's Biogem core facility, comparative single-nucleotide polymorphism (SNP) analysis of primary tumor versus isolated exfoliated epithelial cells can be performed as the definitive experiment. ? ? ? ?
