Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS

Abstract

BackgroundTumor cells in the blood of patients with metastatic carcinomas are associated with poor survival. Knowledge of the cells’ genetic make-up can help to guide targeted therapy. We evaluated the efficiency and quality of isolation and amplification of DNA from single circulating tumor cells (CTC).MethodsThe efficiency of the procedure was determined by spiking blood with SKBR-3 cells, enrichment with the CellSearch system, followed by single cell sorting by fluorescence-activated cell sorting (FACS) and whole genome amplification. A selection of single cell DNA from fixed and unfixed SKBR-3 cells was exome sequenced and the DNA quality analyzed. Single CTC from patients with lung cancer were used to demonstrate the potential of single CTC molecular characterization.ResultsThe overall efficiency of the procedure from spiked cell to amplified DNA was approximately 20%. Losses attributed to the CellSearch system were around 20%, transfer to FACS around 25%, sorting around 5% and DNA amplification around 25%. Exome sequencing revealed that the quality of the DNA was affected by the fixation of the cells, amplification, and the low starting quantity of DNA. A single fixed cell had an average coverage at 20× depth of 30% when sequencing to an average of 40× depth, whereas a single unfixed cell had 45% coverage. GenomiPhi-amplified genomic DNA had a coverage of 72% versus a coverage of 87% of genomic DNA. Twenty-one percent of the CTC from patients with lung cancer identified by the CellSearch system could be isolated individually and amplified.ConclusionsCTC enriched by the CellSearch system were sorted by FACS, and DNA retrieved and amplified with an overall efficiency of 20%. Analysis of the sequencing data showed that this DNA could be used for variant calling, but not for quantitative measurements such as copy number detection. Close to 55% of the exome of single SKBR-3 cells were successfully sequenced to 20× depth making it possible to call 72% of the variants. The overall coverage was reduced to 30% at 20× depth, making it possible to call 56% of the variants in CellSave-fixed cells.

Genomic High-Resolution Profiling of Single CKpos/CD45neg Flow-Sorting Purified Circulating Tumor Cells from Patients with Metastatic Breast Cancer

“…A different flow-sorting protocol for isolation of CellSearch CTCs was very recently described by Swennenhuis et al (15 ). For 10 tested clinical CellSearch samples of patients with lung cancer, the authors reported a markedly lower CTC detection rate (mean efficiency, 43% vs 102%; median efficiency, 41% vs 83%).…”

“…The very low concentration of CTCs and the unpredictable number of "contaminating" hematopoietic cells always cocaptured as background by the CellSearch system hamper direct downstream molecular analyses, and hence further purification and isolation of the rare CTCs is required. Initial data indicated that flow-sorting approaches might be suitable for CTC purification (15 ), but efficient protocols have been lacking. Here, we present a protocol to efficiently purify single CTCs captured with the CellSearch circulating tumor cell kit that enables their further comprehensive genetic analysis on a single-cell level.…”

Liquid Biopsies for Monitoring Temporal Genomic Heterogeneity in Breast and Colon Cancers

“…One of the major challenges in CTC research concerns the number of CTCs that have to be analyzed to be able to outline the profile of the clones that drive disease in cancer, with conflicting results being provided by case reports involving both CTC and ctDNA analysis in metastatic breast cancer patients [96,97]. However, in recent years, efforts have been made to develop multiple markers to increase the yield of CTCs [98][99][100], and to improve their amplification [101][102][103] and sequencing techniques [104][105][106].…”

Improving the CellSearch® system

“…An inevitable step therefore is to pick and isolate the single CTCs in individual tubes or as a pure CTC fraction for genomic analysis. Methods used to isolate single cell for individual analysis in combination with CellSearch-enriched CTCs: Micromanipulation [113], ALS Cellselector [114], the Silicon Biosystems DEPArray [115][116][117][118], FACS [28,119,120], and the Punch system using self-seeding Microwell Chips [121]. This step seems a minor intermediate part of the whole workflow but is crucial for the end result.…”