Circulating tumor cells (CTC) are an important
biomarker for several solid
cancers. Most of the commercially available systems for enumeration of CTC are based on immunomagnetic enrichment of
epithelial cell adhesion molecule (
EpCAM/CD326)-expressing CTC before microscopic cell imaging or reverse-transcription PCR (RT-PCR). The aim of this study was to establish a practical method for enumeration of CTC using a novel flow cytometer that has a disposable microfluidic chip, which is designed to realize absolute cross contamination-free measurements and to collect the analyzed cell sample. Although the process of enumeration and labeling of CTC was optimized for this device, the simplified protocol described here could be applied to other flow cytometers. Cultured
cancer cells spiked into normal blood were enriched using MACS®
EpCAM-
MicroBeads following cell labeling with an
allophycocyanin (APC)-conjugated
EpCAM mAb, instead of by intracellular staining of cytokeratins (CK). The
EpCAM double-positive selection/labeling method allows enumeration of intact CTC, maintenance of cellular integrity, and the concomitant performance of a CTC viability test. The combination of the fine-tuned CTC enrichment process and the cytometric multicolor analysis resulted in a linear relationship between the output cell count and the input cell number from zero to hundreds of cells. In particular, a satisfactory signal/noise ratio was obtained by gate-exclusion of leukocyte signals using an anti-CD45 mAb. The entire process had little influence on the viability of the spiked
lung cancer cell PC-9. Measured PC-9 and
breast cancer MCF-7 cells bearing
EpCAM-
MicroBeads, APC-conjugated
EpCAM mAb, and the
DNA staining
dye SYTO9 grew normally, demonstrating the potential usefulness of the collected samples for further studies. This intact CTC enumeration and analysis procedure (iCeap) would be of great benefit to clinicians by providing them with rapid stratification of antitumor
therapy, and to basic researchers by permitting further molecular and cellular characterization of CTC.