Recovery of viable exfoliated cells using microfluidics for clinical diagnostics

DESCRIPTION (provided by applicant): 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 ce lls ( 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 sorte r 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 ltl volumes, has recently been demonstrated, Wang et al. (2005). Isolation of viable rare cells from clinically relevant patient samples has recently been demonstrated, leading to current efforts to develop clinical assays for applications outside the cancer arena, Marchand et al. (2006a), Marchand et al. (2006b). To provide a path to answering the critical questions, the proposed research project will demonstrate methods that enrich and isolate viable CTCs from 20 ml of whole blood obtained from patients diagnosed with metastatic disease. Typically the cells are isolated at gt80% 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 usi ng 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 pr imary 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 experimen t.