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Mar 15, 2013 (Vol. 33, No. 6)

Molecular Characterization of CTCs

Microfluidic System Designed to Deliver Optimized Sample for Downstream Analysis

  • Circulating tumor cells (CTCs) are shed from primary and metastatic tumors and disseminate into the blood. CTCs are believed to play a key role in the spread of the disease throughout the body. There has been considerable interest in analyzing these cells as a potential source of clinically actionable information relating to molecular profile of the patient’s disease.

    CTCs can be accessed repeatedly and noninvasively from a simple blood draw. This provides a clinically feasible methodology for tracking longitudinal changes in disease profile that is not readily accomplished with conventional biopsy approaches.

    Numerous approaches have been employed to isolate and utilize CTCs for diagnostic and discovery applications. Platforms have been described in the literature or commercially released using alternative isolation modalities such as size-based separation, affinity capture, and imaging cytometry.

    A key limitation of these technologies is the ability to isolate and recover viable, intact CTCs for downstream molecular analysis. Cells that have been isolated onto antibody-coated microfluidic channels, porous filters, and glass slides often adhere tightly to the substrate, which makes it extremely difficult to remove these cells for further analysis. Other limitations such as low CTC recovery, low purity, and diminished viability have also prevented widespread use of CTCs in the laboratory and clinical environments.

  • Microfluidic Flow Focusing Enhances Cell Isolation

    Click Image To Enlarge +
    Figure 1. The IsoFlux System consists of a benchtop instrument and microfluidic cartridge that utilizes immunomagnetic beads to produce enriched rare cell samples for downstream molecular analysis.

    Fluxion Biosciences developed the IsoFlux™ System to enable automated recovery of CTCs from blood samples in a format optimized for downstream molecular analysis. The system consists of a benchtop instrument, microfluidic cartridge (Figure 1), and reagents for enriching rare cells from peripheral whole blood samples.

    The system utilizes immunomagnetic beads targeted toward surface-expressed antigens on the target cell population. These beads come prefunctionalized for convenience in two formats: anti-EpCAM (a widely used marker for cancer cells of epithelial origin) and anti-Mouse IgG (binds mouse IgGs and allows one or more user-defined antibodies to be used for target cell isolation).

    Clinical blood samples are collected (7–10 mL, BD EDTA tubes) and shipped to the processing site within 24 hours. Density centrifugation (Ficoll-Paque, GE Healthcare) is used to recover the mononuclear cell fraction. The magnetic beads are added to the sample and allowed to couple with target cells on a rotator at 4°C. After coupling, the sample is loaded into the inlet well of the microfluidic cartridge and up to four samples at a time are processed in the automated instrument.

  • Click Image To Enlarge +
    Figure 2. Sample flows into an isolation zone where target cells are captured on the CellSpot disk that forms the roof of the channel. The entire CellSpot is carried with the cap and closed onto a small-volume microfuge tube where the cells can be recovered in less than 20 µL media.

    Inside the microfluidic cartridge, the sample flows into an isolation zone featuring an expanded flow cavity (Figure 2). The flow focusing effect of this cavity brings the laminar flow streams in close and uniform proximity to a permanent magnet that comes down in contact with the cartridge. The magnetic forces attract the target cells labeled with magnetic particles while the remaining blood cells follow the flow and gravity forces away from the isolation zone and into a waste reservoir. This leads to high efficiency rare cell isolation with minimal background cell contamination.

  • High Efficiency, Low Volume Cell Transfer

    The roof of the microfluidic cartridge is constructed from a removable polymer disk called the CellSpot™ that is embedded into a standard microfuge cap (Figure 2). The target cells collect on the CellSpot cap while the sample flows across the isolation zone. Once the sample run is complete, the instrument transfers the entire cap onto a microfuge tube and brings with it the entire enriched cell fraction.

    This overcomes previous limitations of cell recovery seen with microfluidic and filter-based separation technologies. The enriched sample transfers with over 90% efficiency to the downstream assay. Additionally, the enriched sample can be eluted in as little as 10 μL. This is of primary importance to molecular assays, many of which have limited volumetric inputs while still demanding high nucleic acid concentration.

  • CTC Recovery Performance

    The actual number of CTCs present in a blood sample is difficult to know with certainty. The absolute number of CTCs in a sample has been correlated with overall survival in cancer patients. When the downstream analysis looks beyond the enumeration and focuses on biomarker detection, the requisite number of CTCs needed for a successful analysis relies heavily on the sensitivity of the chosen assay. State of the art analysis techniques such as next-generation sequencing (NGS), digital PCR (dPCR), and quantitative PCR (qPCR) have been shown to detect mutations and gene expression levels from as few as five target cells among a background of wild type blood cells.

    The IsoFlux System has been tested with hundreds of clinical blood samples from cancer patients with multiple tumor types. More than 75% of these patient samples tested had greater than 5 CTCs (scored using the immunofluorescence CK+, CD45-, nucleated definition). This type of recovery is important to ensure that the technology can reliably be used in clinical testing without having to recruit an excessive number of patients.

  • Downstream Analysis

    A wide range of analysis options can be used to interrogate CTCs for diagnostic content. Conventional technologies such as immunohistochemistry, immunofluorescence, FISH, and qPCR are frequently used in CTC assays to look at cellular and molecular markers. The past decade has seen a rise in new technologies that increase sensitivity and the level of multiplexing that can be achieved. These technologies are ideal fits for CTC analysis and include NGS, dPCR, and more sensitive qPCR assay chemistries.

    For cellular analysis on a microscope, the IsoFlux samples are typically spotted onto a glass slide or plate for imaging. The low volume format helps contain the sample to a small surface area that minimizes scanning time and data capture. For molecular analysis, the samples typically are lysed to recover DNA or RNA content from the cells. Amplification (whole genome or targeted pre-amplification) is sometimes beneficial depending on the assay format and level of multiplexing desired.

  • Click Image To Enlarge +
    Figure 3. KRAS mutation detection in colorectal cancer patients. qPCR readouts identify a positive mutation when the dCt values (difference between mutant and reference assays) fall below the established cutoff.

    As one example, Fluxion has developed a sensitive KRAS mutation detection panel for use with several cancer types known to harbor this mutation. Samples are collected and processed on the IsoFlux System, and then the DNA is amplified for qPCR analysis. A sensitive allele-specific mutation detection assay (castPCR™, Life Technologies) is used to identify a panel of seven common KRAS point mutations. Initial clinical samples tested on this assay suggest that KRAS mutations can be detected in the CTCs at comparable frequencies as in the primary tumors (30–40%), and can be identified in samples having as few as 7 CTCs (Figure 3).

  • Conclusion

    The IsoFlux System provides a robust method for obtaining CTCs and other rare cells from whole blood samples. It prepares CTCs in an optimal format for the most commonly used downstream analytical methods, including next-generation technologies like NGS and dPCR.

    As an established supplier of analytical instruments to the life science and diagnostic industry, Fluxion Biosciences is rapidly developing complete analytical workflows for CTCs using the IsoFlux System for CTC recovery.

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