The molecular characterization of circulating tumor cells (CTCs) is widely used to identify targets for individualized treatment options. However, analyzing the whole bulk of CTCs does not give information whether a co-expression of more than one CTC-marker is derived from CTCs, actually co-expressing these markers on the same cell, or from separate CTCs. Here we present a new device, the QIAscout (QIAGEN, Germany), offering the possibility of microscopic single cell isolation for subsequent molecular analysis.
CTCs were isolated from 2x 5 ml whole blood of 15 metastatic breast cancer (MBC) patients (pts) using positive immunomagnetic selection (AdnaTest EMT2/StemCell Select) followed by QIAscout single cell isolation in one of the two blood samples. Cells added to the QIAscout array settle down and adhere to microrafts which is supported by pre-treating the array with Corning Cell-Tak Adhesive. Using an inverted microscope, single cells can be mechanically isolated by picking out one of the 12000 microrafts. Subsequently, in both samples, mRNA bound to Oligo-dT beads was purified, reverse transcribed (AdnaTest EMT2/StemCell Detect) and the cDNA was pre-amplified and analyzed by a multimarker qPCR Panel (AKT2, ALK, AR, AURKA, BRCA1, KIT, MET, EGFR, ERCC1, ERBB2, ERBB3, KRT5, mTOR, NOTCH1, PARP1, PIK3CA, SRC, GAPDH), normalized by data of healthy donors (n=20) with CD45 as leukocyte control. QIAscout further allowed single cells analysis, amplified by whole transcriptome amplification (WTA – REPLI-g WTA Single Cell Kit) followed by qPCR analysis as performed for the bulk of cells. Establishment of the methods was performed using the breast cancer cell line MCF-7 spiked into blood of healthy donors. All consumables were purchased from QIAGEN (Germany).
“Spike-in” experiments revealed CTC recovery rates of about 70% and subsequent CTC characterization in MCF-7 cells and pts` blood samples resulted in a markedly decreased leukocyte contamination implementing the QIAscout into CTC analysis. However, the positivity rate of overexpressed genes was reduced in MBC pts up to 33% using QIAscout as compared to bulk samples. In five pts, gene expression in the bulk of CTCs was compared with at least 2–4 single cells of the same pts, using QIAscout. A good concordance was achieved for CTC-positivity and all single CTCs showed a broad variety in gene expression. At least one of the single cells expressed more genes than the bulk of CTCs and the expression of target genes that define treatment options, e.g., HER2, was often expressed on only 1/4 analyzed cells whereas resistance markers were detected in nearly every CTC of the same pts.
The implementation of the QIAscout in CTC analysis allows the molecular characterization of single tumor cells to get insights into single cell heterogeneity for further therapeutic strategies.
Janina Levermann (University Hospital of Essen), Norbert Hochstein (QIAGEN) Siegfried Hauch (QIAGEN), Ruth Klaever (QIAGEN), Rainer Kimmig (University Hospital of Essen), Sabine Kasimir-Bauer (University Hospital of Essen)