Efficient Isolation and Accurate In Situ Analysis of Circulating Tumor Cells Using Detachable Beads and a High-Pore-Density Filter * *

Circulating tumor cells (CTCs) in the bloodstream of cancer patients may indicate the likelihood and severity of metastatic progression. Identification, enumeration, and characterization of CTCs may provide a minimally invasive method for assessing cancer status of patients and prescribing personalized anticancer therapy. However, examination of CTCs requires isolation of these cells from whole blood of patients, which is difficult owing to their low quantity (around one CTC per 10 non-cancerous hematopoietic cells in patient blood). Many techniques are used to isolate CTCs, including density gradient centrifugation and dielectrophoresis, but methods using either size-based exclusion or affinity-based enrichment are common. Size-based exclusion assumes that CTCs are larger than most hematopoietic cells and removes all cells smaller than a pre-determined size threshold. Affinity-based enrichment relies on the expression of surface proteins specific to cancer cells and absent in hematopoietic cells. These methods generally use antibody-conjugated magnetic beads and enrich for CTCs by magnetic separation, such as the CellSearch system. Owing to their heterogeneous nature, it may be practically impossible to isolate CTCs with high isolation efficiency using the aforementioned methods. Some CTCs are reported to be nearly identical in size or even smaller than leukocytes, making them difficult to discriminate by size. As for protein expression, epithelial markers, such as EpCAM (epithelial cell adhesion molecule), are downregulated during epithelialto-mesenchymal transition (EMT). Furthermore, the type and expression levels of surface proteins may vary greatly depending on cancer histological subtype. Considering these variations, finding the right antibody or combination of antibodies that consistently captures all CTCs may prove to be difficult. To maximize isolation efficiency, we devised a dual-mode isolation strategy that combines affinity-based enrichment and size-based exclusion. By using microbeads conjugated with CTC-specific antibodies, the size of CTCs can be augmented to enable better discrimination against leukocytes. Subsequent size filtration isolates bead-bound CTCs, allowing the recovery of even smaller-sized CTCs. However, all beadbased capture methods have the inherent limitation of prohibiting accurate image analysis, which is due to optical distortion created by the presence of beads attached to cells. The attached beads not only impede observation of cellular morphology but can actually alter fluorescence signal intensities (Figure 1), demonstrating the incompatibility of in situ quantitative analysis with bead-based capture methods (see the Supporting Information). Accurate quantification of protein expression can lead to better clinical management, particularly in regards to personalized therapy. The expression levels of predictive biomarkers, such as HER2 and EGFR, are commonly used to match patients with appropriate treatment strategies and predict the effectiveness of anticancer drugs. Therefore, it is important to accurately characterize CTCs, and removal of beads from the surface of CTCs prior to imaging is necessary. Thus, we have developed a new method to detach beads from beadbound cells: By inserting a photocleavable linker between the bead surface and conjugated antibodies, it is possible to remove the attached beads from cells by light irradiation without affecting cell viability. Herein, we demonstrate a novel approach for isolation and subsequent in situ protein-expression analysis of CTCs using detachable beads termed RIA (reversible bead attachment for cell isolation and analysis). Scheme 1 illustrates the entire RIA process. Detachable beads conjugated to CTC-specific antibodies bind to CTCs in whole blood of patients. After incubation, the entire sample is filtered through a high-pore-density membrane filter chip, which contains a maximal number of uniform-sized (pore diameter 8 mm) evenly spaced circular pores (distance between pores 5 mm). This step eliminates almost all hematopoietic cells, while CTCs remain on the filter surface. The [*] H. J. Lee, Dr. J. H. Oh, J. M. Oh, J. M. Park, Dr. J. G. Lee, Dr. M. S. Kim, Dr. Y. J. Kim, Dr. H. J. Kang, Dr. S. S. Lee, Dr. N. Huh In Vitro Diagnostics Lab, Bio Research Center, Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology Gyeonggi-do 446-712 (Korea) E-mail: soosuk@samsung.com namhuh@samsung.com H. J. Lee, Prof. J. W. Choi Interdisciplinary Program of Integrated Biotechnology, Department of Chemical & Biomolecular Engineering Sogang University, Seoul 121-742, Korea E-mail: jwchoi@sogang.ac. kr