Abstract 3512: Dielectrophoretic capture and genetic analysis of individual disseminated solid tumor cells.
Authors: Carpenter Erica L. , Rader JulieAnn , Krytska Kateryna , Ruden Jacob , Rappaport Eric , Mosse Yael , Fontana Francesca , Banzi Manuela , Medoro Gianni , Manaresi Nicolo , O'Dwyer Peter , Maris John 
Published: August 2014

Abstract
Background: The purpose of this study was to develop a novel approach for isolation and genetic analysis of individual circulating and disseminated tumor cells (CTC, DTC) from patient samples. This work is motivated by our desire to develop non-invasive molecular diagnostics and understand single tumor cell heterogeneity.
Methods: Here we used the DEPArray microelectronics and microfluidics platform for the manipulation and capture of individual tumor cells. A suspension of live or fixed cells, prelabeled with fluorochrome-conjugated antibodies, was loaded into the DEPArray cartridge, and application of non-uniform electrical fields (dielectrophoresis) was employed to trap individual cells into movable micro-cages. Cell images were acquired via the incorporated fluorescence microscope and CCD camera, and cells were selected based on morphology and expression of tumor antigens. To determine sensitivity and specificity of the capture process, cell lines were spiked into normal donor white blood cells (WBC) over a 5-log range. Pre-enrichment by FACSAria was employed when the starting tumor cell concentration was below 1 in 1,000 WBC. The whole genome amplification (WGA) product of single cells was then subjected to targeted sequencing. To apply this approach to patient samples, we obtained blood and bone marrow samples from patients with refractory high-risk neuroblastoma, a cancer with a high rate and quantity of distant metastatic disease.
Results: Single tumor cells were recovered from all spiking experiments, including spiked samples with a tumor cell concentration as low as 1 in 106 WBC. Recovery of EpCAM-negative single cells was accomplished with the neuroblastoma cell lines SY5Y, NB1643, and Kelly labeled with an antibody for the disialoganglioside GD2, the breast cancer cell line MCF-7 with a HER2 antibody, and for the lung cancer cell line H292 with EGFR labeling. Recovery of EpCAM-positive cells was achieved with the colon carcinoma cell line SW480. Captured single cells were WGA'd and specificity was verified using Sanger sequencing. For the 2 neuroblastoma spiking experiments for which sequencing has been completed, the known ALK mutation (F1174L) was detected in 100% of cells. The known KRAS mutation (G12V) was detected in 100% of single SW480 cells sequenced to date. No mutations were detected in isolated WBC. We next analyzed the post-Ficoll fraction of 12 patient bone marrow and 1 patient blood sample, with a median tumor cell concentration of 0.119% (range 0.003 - 80.0%). A minimum of 10 single tumor cells were captured per patient. Genome-wide analysis of DNA and RNA copy number and mutation status from these samples is ongoing.
Conclusion: These data demonstrate the development of a novel workflow for the reliable capture and genetic analysis of rare CTC. Efforts are ongoing to join this approach with a more comprehensive genome-wide analysis of single cells from neuroblastoma and other cancers.
Citation Format: Erica L. Carpenter, JulieAnn Rader, Kateryna Krytska, Jacob Ruden, Eric Rappaport, Yael Mosse, Francesca Fontana, Manuela Banzi, Gianni Medoro, Nicolo Manaresi, Peter O'Dwyer, John Maris. Dielectrophoretic capture and genetic analysis of individual disseminated solid tumor cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3512. doi:10.1158/1538-7445.AM2013-3512