Senologie - Zeitschrift für Mammadiagnostik und -therapie 2015; 12 - A107
DOI: 10.1055/s-0035-1550548

Isolation of circulating tumor cells using CellCelector post CellSearch enables characterization of single cells

M Neumann 1, S Schömer 1, Y Decker 1, H Schneck 1, M Fleisch 2, R Neves 3, N Stoecklein 3, T Fehm 2, F Meier-Stiegen 1, H Neubauer 1, D Niederacher 1
  • 1Universitätsklinik Düsseldorf, Forschungslabore der Frauenklinik, Düsseldorf, Deutschland
  • 2Universitätsklinik Düsseldorf, Frauenklinik, Düsseldorf, Deutschland
  • 3Universitätsklinik Düsseldorf, Experimentelle Chirurgie, Düsseldorf, Deutschland

Background:

Circulating tumor cells (CTC) are rare cells and dissociated successfully from the primary tumor into the blood stream. Only a small proportion is capable to extravasate and form metastases. The presence of CTCs is associated with increased risk to develop metastases. Understanding the heterologous CTC biology enables the choice of the optimal therapy and improves the prognosis. Therefore, detection and isolation of CTCs is necessary. Here we show a workflow to characterize CTCs by combining the advantages of both the CellSearch®- and CellCelector™-System.

Material and Methods:

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-System. CTCs were isolated from CellSearch-Cartridge using the CellCelector-system and deposit for a second immunofluorescence staining of the migration marker CapG (Cy5). CTCs were again isolated using the CellCelector and placed in PCR-tubes for quality control (QC) after whole genome amplification (WGA).

Results:

We found 97% of CellSearch control cells (SkBr3) and 95% of CellSearch proven CTCs by CellCelector scan. Isolation/Deposit-ratio was tested using different cell lines and revealed a ratio of 0.95. CellSearch cartridge content was transferred to a special magnet adapter and 87% cells could be detected after CellCelector scan. We isolated 97% of classified CTCs using the CellCelector-system. CTCs were deposit and stained for the migration marker CapG revealing strong CapG positivity. CTCs were isolated again using the CellCelector and processed for WGA and checked by QC. Furthermore, CTCs were analyzed for PI3KCA mutation.

Conclusion:

Quantification of CTCs by the CellCelector-System is similar to CellSearch. Moreover, the isolation of CTCs after CellSearch using the CellCelector-System enables enrichment of CTCs with elimination of contaminants and improves subsequent analysis regarding protein expression and genomic analyses.