A workflow for the isolation and molecular characterization of Individual Circulating Tumor Cells (CTCs) to enable cell heterogeneity analysis and personalized therapy

Abstract

Study of rare cells has become increasingly attractive in various areas of basic and translational bio-medical research, drug discovery and advanced diagnostics. In oncology, molecular characterization of Circulating Tumor Cells (CTCs) is one of the main application areas so far. CTCs hold the promise to deliver an up-to date picture of the patient mutational profile, which is key for therapy selection in the framework of personalized therapy.
The purification of cells which, like CTCs, have a frequency as low as 1 in a billion (10-9) within heterogeneous suspension of other cells, is the critical issue. Due to the low numbers and the possible heterogeneity of the target cells themselves, a highly automated method to reliably isolate 100% pure cells with single cell resolution would be ideal. While significant progresses have been achieved in the enrichment methods, no platform so far had been able to fill the gap from enriched populations to single pure cells. Another key issue comes downstream of cell isolation, as the low amount of DNA or RNA included in a single cell needs to be amplified in a highly balanced and accurate way to reflect the original content of a single cell in order to allow meaningful molecular profiling.
We show that DEPArray™, an automated, image-based sorting platform, achieves the goal of isolating 100% pure cells with single cell resolution from enriched suspensions. It employs the principle of dielectrophoresis to individually trap and move fluorescently labeled cells on a microelectronic chip with over 300,000 programmable electrodes in a digital, deterministic and highly controlled way.
Furthermore, using Ampli1™ WGA, a method of whole genome amplification from single cells based on ligation mediated PCR, we demonstrate molecular characterization of single CTCs from cancer patients, including mutation detection, genotyping and next generation sequencing analysis. We show that digitizing the sample (i.e. analyzing pure single CTCs collected by DEPArray™) and amplifying the DNA with Ampli1™ WGA it is possible to tap into the power of Next Generation Sequencing (using targeted semiconductor based sequencing with IonTorrent® PGM), overcoming the sensitivity – specificity trade-off which otherwise prevent their use due to the sequencing noise.
Experiments assessing cell viability, when working with live cells will also be presented. The potential impact on translational oncology research will be discussed, as the opportunity to move from simple counting of CTC further deep into CTC or Cancer Stem Cells (CSC) characterization, offers an unprecedented insight into a tumor development and a unique opportunity to develop accurate companion diagnostics and personalized therapies.