Microchip-based immunomagnetic detection of circulating tumor cells

Abstract

Screening for circulating tumor cells (CTCs) in blood has been an object of interest for evidence of progressive disease, status of disease activity, recognition of clonal evolution of molecular changes and for possible early diagnosis of cancer. We describe a new method of microchip-based immunomagnetic CTC detection, in which the benefits of both immunomagnetic assay and the microfluidic device are combined. As the blood sample flows through the microchannel closely above arrayed magnets, cancer cells labeled with magnetic nanoparticles are separated from blood flow and deposited at the bottom wall of the glass coverslip, which allows direct observation of captured cells with a fluorescence microscope. A polydimethylsiloxane (PDMS)-based microchannel fixed on a glass coverslip was used to screen blood samples. The thin, flat dimensions of the microchannel, combined with the sharp magnetic field gradient in the vicinity of arrayed magnets with alternate polarities, lead to an effective capture of labeled cells. Comparing to the commercially available CellSearch™ system, less (25%) magnetic particles are required to achieve a comparable capture rate, while the screening speed (at optimal blood flow rate of 10 mL/hour) is more than five times faster than those reported previously with a microchannel-based assay. For the screening experiment, blood drawn from healthy subjects into CellSave™ tubes was spiked with cultured cancer cell lines of COLO205 and SKBR3. The blood was then kept at room temperature for 48 hours before the screening, emulating the actual clinical cases of blood screening. Customized Fe3O4 magnetic nanoparticles (Veridex Ferrofluid™) conjugated to anti-Epithelial cell adhesion molecule (EpCAM) antibodies were introduced into the blood samples to label cancer cells, and the blood was then run through the microchip device to capture the labelled cells. After capture, the cells were stained with fluorescently labelled anti-cytokeratin, DAPI and anti-CD45. Subsequent immunofluorescence images were taken for the captured cells, followed by comprehensive computer aided analysis based on fluorescence intensities and cell morphology. Rare cancer cells (from ~1000 cells down to ~5 cells per mL) with very low tumor cell to blood cell ratios (about 1: 107~109, including red blood cells) were successfully detected. Cancer cell capture rates of 90% and 86% were demonstrated for COLO205 and SKBR3cells, respectively.

Microfluidic device with integrated microfilter of conical-shaped holes for high efficiency and high purity capture of circulating tumor cells

“…Currently, two approaches are in competition, depending on whether or not the capture is affinity-based. The affinity-based capture relies on immunochemical interaction between magnetic beads [11][12][13][14][15][16] or patterned structures [17][18][19][20] and tumor cells that express special surface markers such as EpCAM, an epithelial cell adhesion molecule over expressed by majority of tumor cells. These methods are efficient for capturing CTCs of epithelial phenotypes but not applicable to those with down-regulated or lost epithelial markers.…”

Rational Design of Materials Interface for Efficient Capture of Circulating Tumor Cells

“…By combing the immunomagnetic separation strategy and microfluidic technique, Immunomagnetic‐based microfluidic systems have also been developed for efficient CTCs capture and isolation 69, 70, 71, 72, 73, 74, 75, 76. Hoshino et al developed an immunomagnetic‐based microfluidic device for CTCs capture 69.…”

“…Hoshino et al developed an immunomagnetic‐based microfluidic device for CTCs capture 69. In this work, blood samples were firstly labelled with magnetic nanoparticles functionalized by EpCAM antibodies, and target CTCs were then efficiently captured with high efficiency of 90% when the blood samples flowed through the microfluidic channel closely above arrayed magnets.…”

Continuous-flow ferrohydrodynamic sorting of particles and cells in microfluidic devices

“…Microfluidic particle and cell sorting plays an important role in environmental monitoring (Liu et al 2004;Beyor et al 2008;Dharmasiri et al 2010), disease diagnostics (Nagrath et al 2007;Adams et al 2008;Hoshino et al 2011), and therapeutics (Toner and Irimia 2005;Yung et al 2009). Compared to high-specificity and label-based cell sorting techniques such as 0fluorescence-activated cell sorter (FACS) (Bonner et al 1972) and magnetic-activated cell sorter (MACS) (Miltenyi et al 1990), microfluidic sortings are mostly label-free, relying on cells' intrinsic properties such as size, shape, density, deformability, electric and magnetic properties for manipulation specificity (Pamme 2007;Tsutsui and Ho 2009;Gossett et al 2010;Lenshof and Laurell 2010).…”