Isolation of circulating tumor cells enables additional marker staining following panel sequencing

Circulating tumor cells (CTC) are rare cells dissociated successfully from the primary tumor into the blood stream. The presence of CTCs is associated with increased risk to develop metastases and a short survival. Detection and isolation of CTCs is necessary. Here we show a workflow to isolate single CTCs for molecular characterization by combining the CellSearch and CellCelector.

Single cells were classified as CTCs if nucleus (DAPI) and cytokeratin (FITC and/or TRITC) positive while CD45 (Cy5) negative. Detection and quantification of single cells from control samples and patient samples were performed using CellSearch- and CellCelector. CTCs were isolated from CellSearch-Cartridge using the CellCelector and transferred into PCR-tubes for whole genome amplification (WGA).

After standard CellSearch analysis 97% of spiked SKBR3 cells and 95% of patient CTCs were detected by rescanning using the CellCelector system. Isolation/Deposit-ratio was tested using different cell lines and a recovery rate of > 95% was determined. After transfer of the CellSearch cartridge content to chamber slides on a special magnet adapter 87% of CellSearch classified CTCs could be detected and isolated using the CellCelector following transfer to PCR tubes. Next, CTCs were processed for WGA. Quality of WGA products was checked by the Ampli1-QC protocol and genomic analyses (Panel-Sequencing) were performed successfully.

The established work-flow for enrichment of CTCs by CellSearch analysis followed by the isolation of single CTCs using the CellCelector enables the enumeration and molecular characterization of single CTCs to investigate the heterogeneity of CTCs in patient samples.