title: Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells.
abstract: Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 10(6) white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.
authors.affiliation: Division of Hematology and Oncology, Perelman School of Medicine at the University of Pennsylvania , Philadelphia, PA , USA.
authors.forename: Erica L
authors.initials: EL
authors.lastname: Carpenter
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, Children's Hospital of Philadelphia , Philadelphia, PA , USA.
authors.forename: JulieAnn
authors.initials: J
authors.lastname: Rader
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, Children's Hospital of Philadelphia , Philadelphia, PA , USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Ruden
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, Children's Hospital of Philadelphia , Philadelphia, PA , USA.
authors.forename: Eric F
authors.initials: EF
authors.lastname: Rappaport
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, Children's Hospital of Philadelphia , Philadelphia, PA , USA.
authors.forename: Kristen N
authors.initials: KN
authors.lastname: Hunter
authors.affiliation: Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania , Philadelphia, PA , USA.
authors.forename: Paul L
authors.initials: PL
authors.lastname: Hallberg
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, Children's Hospital of Philadelphia , Philadelphia, PA , USA.
authors.forename: Kate
authors.initials: K
authors.lastname: Krytska
authors.affiliation: Division of Hematology and Oncology, Perelman School of Medicine at the University of Pennsylvania , Philadelphia, PA , USA.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: O'Dwyer
authors.affiliation: Division of Oncology, Center for Childhood Cancer Research, Children's Hospital of Philadelphia , Philadelphia, PA , USA ; Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania , Philadelphia, PA , USA.
authors.forename: Yael P
authors.initials: YP
authors.lastname: Mosse
keywords: circulating tumor cell; disseminated tumor cell; neuroblastoma; single cell capture; targeted sequencing; whole genome amplification