Chao Huang

Research Proposal

Capture of Circulating Tumor Cells Using Dielectrophoresis and Immunocapture Keywords: circulating tumor cell, dielectrophoresis, immunocapture, lab-on-a-chip I, Chao Huang, attest to the originality of this research proposal. The enumeration of circulating tumor cells (CTCs), often found in the blood of cancer patients, is used clinically as a prognostic indicator of patient survival.1 Capture of peripheral blood CTCs may enable early cancer assessment as well as genetic and pharmacological evaluation of cancer cells.2 However, the rarity of CTCs (one in 109 blood cells) presents a technical challenge for cell capture. Current commercial and research devices for the immunocapture of CTCs use EpCAM antibody-coated surfaces, which capture large numbers of leukocytes in addition to CTCs, thereby resulting in low yields and purity.2,3 Geometricallyenhanced immunocapture microdevices using staggered post arrays have improved the capture efficiency of CTCs compared to similar existing microdevices; however, these techniques still resulted in a relatively low mean capture purity of 62%, largely due to leukocytes nonspecific interaction with the CTC-targeted antibody-coated posts.4 The inadequacy in sorting CTCs and leukocytes with high specificity presents a limitation on the use of immunocapture microdevices for CTC capture with high purity, which in turn limits the use of CTCs in oncology research. Dielectrophoresis (DEP) recently has been used as a standalone technique for isolation of highly concentrated breast tumor cells from peripheral blood based on their distinct total membrane capacitance; however, this yield decreased dramatically as the sample was diluted with more peripheral blood cells.5 Furthermore, applying large electric field magnitudes required for cell capture can result in current-induced heating,6 which may cause physiological damage to the CTCs and make any subsequent biological assessment unfeasible. Thus, while DEP is capable of sorting CTCs from other blood components (based on cell dielectric properties) in artificial samples, the risk of harming CTCs from current-induced heating and the rarity of CTCs in whole blood makes a CTC enrichment of 109 using DEP alone an extremely difficult task. My proposed work bridges the gap between existing DEP-activated rare cell isolation and surface immunocapture techniques by using the distinct DEP force acting on different components of blood to attract CTCs to and repel leukocytes from the posts in an immunocapture microdevice. This approach combines the robustness of surface immunocapture with the exquisite sensitivity of DEP; the applied electric field is tuned low enough to cause no physiological harm to cells while inducing a strong enough DEP force to cause desired interactions with the immunocapture post array. These synergistic effects will minimize leukocytes nonspecific binding to CTC-targeted posts, and will yield higher performance in CTC capture purity. The overall objective of my proposed project is to develop a novel microfluidic device to isolate CTCs from whole blood samples, which will enable researchers to study biological determinants of cancer. The proposed research has two specific aims: 1. Determine the electric field frequency range at which DEP attracts CTCs toward immunocapture surfaces while repelling leukocytes. The DEP force on a cell is a direct function of total membrane capacitance, and its magnitude and direction are determined by the real part of the Clausius-Mossotti factor, Re[fCM], a frequency-dependent term that describes the relationship between the complex permittivities of the cell and its suspending medium, respectively. Previous research has demonstrated the distinct membrane capacitances of breast tumor cells and various blood components;7 I used this data to calculate Re[fCM] (and by extension, the DEP response) of the different cell types across a frequency spectrum (Figure 1). In a certain frequency range (shaded region), breast tumor cells experience a positive (attractive) DEP force while other blood components experience a negative (repelling) DEP force. Based on the distinct DEP response of breast tumor cells due to membrane dielectric properties, I 1

Chao Huang

Research Proposal

hypothesize that CTCs generally will exhibit a distinct DEP response, and I propose to use experimental methods similar to those described in my previous research for DEP characterization of Mycobacteria to characterize the DEP response of hematological cells and prostate cancer tumor cells (available to me through an ongoing clinical collaboration under IRB approval), which will serve as representative CTCs to test and optimize my system. I expect to find the Figure 1. Plot of Re[fCM] vs. frequency of the minimum voltage required to induce a DEP response applied electric field for different components in these cells and a frequency range in which CTCs of blood, calculated using a spherical model 8 (with a thin outer shell) for cells and specific experience a DEP force that is opposite in direction to membrane capacitance data obtained from the that of other blood cells. literature.7 Shaded region illustrates the 2. Capture CTCs by designing an internal frequency range in which breast tumor cells electrode geometry to selectively attract CTCs to experience a positive DEP force while other immunocapture surfaces while repelling leukocytes. blood cells experience a negative DEP force. After characterizing the DEP response of different blood components, I will design an electrode geometry that integrates with existing immunocapture post array devices (Figure 2). I will optimize the electrode geometry to induce a maximal DEP response through FEM simulations. In a frequency range similar to the one highlighted in Figure 1, I hypothesize that as a blood sample is flowed through the device, CTCs will experience a positive DEP force due to the electric field gradient and be attracted toward the antibody-coated posts, while leukocytes and other blood components will experience a negative DEP force and be repelled from the posts. This approach uses surface immunocapture techniques to capture CTCs efficiently and DEP forces to maximize CTC-post interactions. I expect that these synergistic strategies will minimize leukocytes non-specific binding to the CTCtargeted posts and yield higher performance in CTC capture purity to facilitate oncology research. Successful completion of my proposed project will yield a novel method for CTC capture with high yield and purity. This research will have broad impacts on rare Figure 2. Top and side views of cell isolation techniques as well as biological immunocapture device with integrated understanding of cancer. I expect to have many electrodes. Posts are etched in silicon st opportunities to communicate my research at conferences (light grey). 1 gold layer (black) is (e.g. MicroTAS) as well as through outreach projects that I connected to a positive lead. Lid of device consists of glass slide (white) with am organizing with BMES. Through these activities, my 2nd gold layer (black) connected to a research will be disseminated broadly, enhancing negative lead. Insulating layer (dark technological understanding while promoting teaching and grey) distorts the applied electric field, learning. My leadership in the scientific discourse both will creating a gradient (dotted lines) to advance scientific discovery as well as inspire others to induce DEP response. CTCs are attracted toward posts; leukocytes and other blood make a positive global impact through scientific research. cells are repelled (bolded arrows).
Danila DC, et al. Clin Cancer Res, 2007. | 2 Adams AA, et al. J Am Chem Soc, 2008. | 3 Nagrath S, et al. Nature, 2007. | 4 Gleghorn JP, et al. Lab Chip, 2009. | 5 Gascoyne PRC, et al. Electrophoresis, 2009. | 6 Weaver JC, et al. Biophys J, 1999. | 7 Becker FF, et al. PNAS, 1995. | 8 Jones TB. Electromechanics of Particles, 1995.
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