Screening and Molecular Analysis of Single Circulating Tumor Cells Using Micromagnet Array

www.nature.com/scientificreports OPEN Screening and Molecular Analysis of Single Circulating Tumor Cells Using Micromagnet Array received: 01 April 2015 accepted: 21 August 2015 Published: 05 November 2015 Yu-Yen Huang1,*, Peng Chen2,*, Chun-Hsien Wu2, Kazunori Hoshino3, Konstantin Sokolov4, Nancy Lane5, Huaying Liu5, Michael Huebschman5, Eugene Frenkel5 & John X. J. Zhang1 Immunomagnetic assay has been developed to detect rare circulating tumor cells (CTCs), which shows clinical significance in cancer diagnosis and prognosis. The generation and fine-tuning of the magnetic field play essential roles in such assay toward effective single-cell-based analyses of target cells. However, the current assay has a limited range of field gradient, potentially leading to aggregation of cells and nanoparticles. Consequently, quenching of the fluorescence signal and mechanical damage to the cells may occur, which lower the system sensitivity and specificity. We develop a micromagnet-integrated microfluidic system for enhanced CTC detection. The ferromagnetic micromagnets, after being magnetized, generate localized magnetic field up to 8-fold stronger than that without the micromagnets, and strengthen the interactions between CTCs and the magnetic field. The system is demonstrated with four cancer cell lines with over 97% capture rate, as well as with clinical samples from breast, prostate, lung, and colorectal cancer patients. The system captures target CTCs from patient blood samples on a standard glass slide that can be examined using the fluorescence in-situ hybridization method for the single-cell profiling. All cells showed clear hybridization signals, indicating the efficacy of the compact system in providing retrievable cells for molecular studies. Detection and enrichment of target cells, such as stem cells1, disseminated tumor cells (DTCs)2, and circulating tumor cells (CTCs)3 from heterogeneous suspensions play a central role in biomedical research and clinical practice. In particular, circulating tumor cells (CTCs) have been shown to closely relate to cancer metastasis4,5 providing information to assist cancer studies. First, accurate enumeration of CTCs can be used as a key indicator for cancer diagnosis, prognosis, and cancer treatment monitoring6. Beyond enumeration, advanced single cell characterization techniques, such as fluorescence in-situ hybridization (FISH)7, reverse transcription polymerase chain reaction (RT-PCR)8, and quantitative RT-PCR9, can provide insights into the biologic characterization of the CTCs. CTCs have the potential of providing a non-invasive “liquid-biopsy” to study the heterogeneity of cancer cells and eventually aid the development of personalized therapy10–12. A combination of rapid enumeration and molecular profiling are critical to exploit the full potential of CTCs. The challenges associated with CTC detection and analyses begin with the natural scarcity of CTCs (the estimated ratio between CTCs and normal leukocytes is 1:107–109), therefore platforms for CTC detection with high sensitivity, specificity, and reliability are in need4. A great number of separation systems have been developed, such as an antibody mediated immunoassay13, size-based filtration method14, 1 Thayer School of Engineering, Dartmouth College, Hanover, NH 03755. 2Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX 78712-0238. 3Department of Biomedical Engineering, University of Connecticut, Storrs, CT 06269-3247. 4Department of Imaging Physics, Division of Diagnostic Imaging, The University of Texas MD Anderson Cancer Center, Houston, TX 77030. 5Harold C. Simons Comprehensive Center of the University of Texas Southwestern Medical Center, Dallas TX 75390. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.X.J.Z. (email: john.zhang@ dartmouth.edu) Scientific Reports | 5:16047 | DOI: 10.1038/srep16047 1 www.nature.com/scientificreports/ fluorescence-activated cell sorting (FACS)15, immunomagnetic separation16–19, and dielectrophoresis force separation20, and others as summarized in previous reviews21. Among the popular methods, the immunomagnetic cell separation assay, which works by selectively labeling the CTCs with magnetic nanoparticles, and using an external magnetic field to capture target cells, provides an effective solution for the translational clinical applications22–24. The immunomagnetic assay exhibits good sensitivity and specificity that arises from the cancer specific antibody-antigen interactions. In addition, the large effective range of magnetic attraction enables the larger channel size and allows for higher throughput. The immunomagnetic assay can also be integrated with multiple separation mechanisms, such as size filtration and inertial focusing25. The immunomagnetic assay has been widely applied for cell separation from heterogeneous suspensions16. Approaches with engineered functional surface using techniques such as chemically modified three dimensional micro/nano-structures are proposed to enhance the sensitivity of rare cell detection26–29. For immunomagnetic assays, several isolation methods integrated with non-functionalized 3-D structures in the microchannel have been employed for particle sorting and cell detection with large populations30–32. To achieve high detection sensitivity and retain both the physical and biological integrities of the target cells, we propose a patterned thin-film micromagnet design, which can be integrated into a microchip based immunomagnetic assay to improve the detection and analysis of the CTCs. Results Design and fabrication of micromagnet-integrated microfluidic screening system. When placed in an external magnetic field, the micromagnets can be magnetized to generate a localized strong magnetic field that can enhance the attractive interactions between cells and the capture surface in the microchannel. Compared to the conventional magnetic activated cell sorting system, where permanent magnets are used as the only magnetic flux source, the micromagnet approach increases the magnetic trap density throughout the whole microchannel surface and local magnetic field gradient. The micromagnets are designed to yield better capture sensitivity, achieve better capture distribution, and facilitate the downstream analyses. To fulfill these purposes, several design factors need to be considered, including the thickness, the lateral dimension, and the spatial periodicity of the micromagnets. Thickness of a micromagnet determines the magnitude of the magnetic force and the vertical effective range of the micromagnet. To minimize the physical damages to the cells due to collision, we decreased the thickness of the micromagnets compared with previous structures. Lateral dimension determines the lateral magnetic effective range of each micromagnet. Another key design parameter is the spatial periodicity of the (...truncated)