Abstract 1606: Single cell isolation and DNA analysis from circulating tumor cells using self-sorting nanowell plates
Authors: Swennenhuis Joost F. , Tibbe Arjan GJ , Stevens Michiel , Stoecklein Nikolas H. , Neves Rui , Tong Hien D. , van Rijn Cees J.M. , Terstappen Leon W.M.M 
Published: August 2015

Abstract
Introduction The heterogeneity of tumor cells dictates the need for analysis at the single cell level. In addition, gene expression can be altered during the course of the disease and is accelerated under the influence of therapy. This imposes the need for a tumor biopsy each time therapeutic intervention is required.
Circulating Tumor Cells (CTC) represent a unique opportunity for a “real time liquid biopsy”. Current available CTC counting technologies are hampered by inefficiency to isolate individual CTC for further molecular characterization to unveil the best treatment strategy. Here we introduce a simple solution to obtain and analyze the genetic make-up of individual CTC from a CTC enriched cell suspension.
Methods Healthy donor blood is drawn into CellSave tubes and spiked with cells of the prostate cancer cell line LNCaP. Samples are processed between 24 and 96 hours using the CellSearch system. The full content of the CellSearch cartridge is transferred to a self-sorting nanowell plate. After identification on a fluorescence microscope single and multiple LNCaP's and WBC are punched out of the plate using a needle and deposited into tubes by using an automated X,Y stage. The DNA is amplified using the Ampli-1 kit from Silicon Biosystems (SB).
Whole Genome Amplification (WGA) products are labeled with Cy-3 and Cy-5 and hybridized to an Agilent sureprint microarray slide. Data was analyzed with the Agilent Genomic workbench software. To show the compatibility with different WGA kits, Self Sorting Nanowell plates with mixed and sorted, differentially stained LNCaP, PC-3 and SKBR3 cells were prepared. Single cells were punched into wells and amplified with three WGA kits: New England Biolabs (NEB) Single Cell WGA kit, the GE Genomiphi kit and the SB Ampli-1 Kit. Evagreen was added to follow the reactions in realtime. Specificity of the system was shown by Sanger sequencing of each cell line for two specific mutations in the ROBO-1 and Pten genes.
Results
Cells that are captured, stained and quantified by the CellSearch system have been isolated using self-sorting nanowell Plates. Array CGH using the Ampli-1 WGA products of these cells shows the identification of the LNCaP cell line for amplified single cell and multi cell samples. Whole Genome Amplified DNA was found to be produced in 60% of the wells for the NEB kit, 77% for the GE kit and 67% for the SB kit. The punched single cells are correctly identified by Sanger sequencing and detection of the mutations in the Robo-1 and the PTEN genes.
Conclusions
We introduced a self-sorting nanowell plate in combination with an efficient method for the isolation of single cells from a CTC enriched cell suspension. 68% of the single isolated cells can be amplified with different WGA kits and cells isolated with CellSearch can subsequently be used for downstream applications such as sequencing and arrayCGH
Citation Format: Joost F. Swennenhuis, Arjan GJ Tibbe, Michiel Stevens, Nikolas H. Stoecklein, Rui Neves, Hien D. Tong, Cees J.M. van Rijn, Leon W.M.M Terstappen. Single cell isolation and DNA analysis from circulating tumor cells using self-sorting nanowell plates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1606. doi:10.1158/1538-7445.AM2015-1606