Abstract
BackgroundCirculating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers. There is great interest in using CTCs to monitor response to therapies, to identify clinically actionable biomarkers, and to provide a non-invasive window on the molecular state of a tumor. Here we characterize the performance of the AccuCyte® – CyteFinder® system, a comprehensive, reproducible and highly sensitive platform for collecting, identifying and retrieving individual CTCs from microscopic slides for molecular analysis after automated immunofluorescence staining for epithelial markers.MethodsAll experiments employed a density-based cell separation apparatus (AccuCyte) to separate nucleated cells from the blood and transfer them to microscopic slides. After staining, the slides were imaged using a digital scanning microscope (CyteFinder). Precisely counted model CTCs (mCTCs) from four cancer cell lines were spiked into whole blood to determine recovery rates. Individual mCTCs were removed from slides using a single-cell retrieval device (CytePicker™) for whole genome amplification and subsequent analysis by PCR and Sanger sequencing, whole exome sequencing, or array-based comparative genomic hybridization. Clinical CTCs were evaluated in blood samples from patients with different cancers in comparison with the CellSearch® system.ResultsAccuCyte – CyteFinder presented high-resolution images that allowed identification of mCTCs by morphologic and phenotypic features. Spike-in mCTC recoveries were between 90 and 91%. More than 80% of single-digit spike-in mCTCs were identified and even a single cell in 7.5 mL could be found. Analysis of single SKBR3 mCTCs identified presence of a known TP53 mutation by both PCR and whole exome sequencing, and confirmed the reported karyotype of this cell line. Patient sample CTC counts matched or exceeded CellSearch CTC counts in a small feasibility cohort.ConclusionThe AccuCyte – CyteFinder system is a comprehensive and sensitive platform for identification and characterization of CTCs that has been applied to the assessment of CTCs in cancer patient samples as well as the isolation of single cells for genomic analysis. It thus enables accurate non-invasive monitoring of CTCs and evolving cancer biology for personalized, molecularly-guided cancer treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1383-x) contains supplementary material, which is available to authorized users.
Highlights
Circulating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers
Tissue culture cells and model CTC spike-in experiments LNCaP and PC3 (Prostate), A549, and MCF7 and SKBR3 cancer cell lines used as model CTCs were all obtained from American Type Culture Collection (ATCC, Manassas, VA, USA)
Recovery of spiked-in model CTCs (mCTCs) from whole blood Four cancer cell lines representing breast, prostate and lung cancer were used for mCTC recovery experiments
Summary
Circulating tumor cells (CTCs) are malignant cells that have migrated from solid cancers into the blood, where they are typically present in rare numbers. The only FDAcleared platform for CTC enumeration is the CellSearch® system (Veridex, Raritan, NJ, USA), and is used for monitoring CTC in patients with colorectal, breast, and prostate cancer. This system is based on automated immunomagnetic capture of EpCAM expressing cells, followed by staining for DNA and cytokeratin to verify that captured cells are nucleated and epithelial in origin. Other technologies for CTC analysis currently under development include other immunomagnetic positive or negative selection methods, microfluidic chips, filters, isolation based on cell deformability or cell density, and dielectrophoretic separation. The ability to robustly retrieve individually identified cells within a practical workflow remains elusive
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