Abstract 3065: Single cell isolation and DNA analysis from circulating tumor cells using a self sorting nanowell plate
Authors: Swennenhuis Joost F. , Tibbe Arjan G.J. , Stevens Michiel , Tong Hien Duy , Rijn Cees J.M. van , Terstappen Leon W.M.M. 
Published: May 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. As biopsies are difficult if not impossible to obtain, 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
A cell suspension is applied to a self-sorting nanowell plate made out of Silicon and containing 10,000 wells with a diameter of 65 um and a height of 380 um. The bottom of each well is made of a Silicon Nitride membrane with a thickness of 1 µm that contains a center pore with a diameter of 5µm. The fluid flows through the pore, hereby forcing a cell to enter the well and to collect on the pore at the bottom of the well. Once the cell has landed onto the pore, the flow inside the well is stopped and no more cells will enter the well. This enables a perfect distribution of individual cells over the nanowells. The efficiency of this system was investigated by sorting CellTracker Orange (CTO) stained PC-3 and CellTracker Green (CTG) stained LNCaP cells into a nanowell plate. A computer controlled fluorescence microscope was used to verify the presence of cells and to select the wells of interest. Next, a computer controlled stage directs a needle with a diameter of 50 µm to the designated well and pushes the bottom containing the cell into a 384 PCR well plate. A real time monitored Whole Genome Amplification (WGA) reaction was performed on the PCR plate wells that contained a cell. As a control, bottoms without a cell were used. The quality of the amplified DNA was tested by a 10 gene qPCR panel.
Results
Cell suspension containing 950 PC3 and 1577 LNCaP cells were passed through the self-sorting nanowell plate. After fluorescence scanning, 707 (74%) PC3 cells and 1415 LNCap cells (90%) were found as single cells at the bottom of the nanowell plates. After punching of the bottoms of 150 nanowells containing single cells, 131 cells (87%) were present in separate wells of a 384 well PCR plate. WGA was applied to the PCR plate and 61% of the cells were successfully amplified. 79% of these amplified products contained at least 7 out of the 10 PCR targets. This makes it suitable as a template for genetic analysis. In the PCR plate wells that contained only bottoms no DNA was amplified.
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 with an efficiency greater than 75%.
Citation Format: Joost F. Swennenhuis, Arjan G.J. Tibbe, Michiel Stevens, Hien Duy Tong, Cees J.M. van Rijn, Leon W.M.M. Terstappen. Single cell isolation and DNA analysis from circulating tumor cells using a self sorting nanowell plate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3065. doi:10.1158/1538-7445.AM2014-3065