High purity microfluidic sorting and analysis of circulating tumor cells: towards routine mutation detection

Abstract

A new generation of the Ephesia cell capture technology optimized for CTC capture and genetic analysis is presented, characterized in depth and compared with the CellSearch system as a reference. This technology uses magnetic particles bearing tumour-cell specific EpCAM antibodies, self-assembled in a regular array in a microfluidic flow cell. 48,000 high aspect-ratio columns are generated using a magnetic field in a high throughput (>3 ml h(-1)) device and act as sieves to specifically capture the cells of interest through antibody-antigen interactions. Using this device optimized for CTC capture and analysis, we demonstrated the capture of epithelial cells with capture efficiency above 90% for concentrations as low as a few cells per ml. We showed the high specificity of capture with only 0.26% of non-epithelial cells captured for concentrations above 10 million cells per ml. We investigated the capture behavior of cells in the device, and correlated the cell attachment rate with the EpCAM expression on the cell membranes for six different cell lines. We developed and characterized a two-step blood processing method to allow for rapid processing of 10 ml blood tubes in less than 4 hours, and showed a capture rate of 70% for as low as 25 cells spiked in 10 ml blood tubes, with less than 100 contaminating hematopoietic cells. Using this device and procedure, we validated our system on patient samples using an automated cell immunostaining procedure and a semi-automated cell counting method. Our device captured CTCs in 75% of metastatic prostate cancer patients and 80% of metastatic breast cancer patients, and showed similar or better results than the CellSearch device in 10 out of 13 samples. Finally, we demonstrated the possibility of detecting cancer-related PIK3CA gene mutation in 20 cells captured in the chip with a good correlation between the cell count and the quantitation value Cq of the post-capture qPCR.

Highly-sensitive capture of circulating tumor cells using micro-ellipse filters

“…From Gaussian theory, probability density function is defined as , where n is stage number starting from 0 to 13. With μ  = 7.3 and σ  = 3.544, percentage at various stages could be calculated theoretically 35,36 (See Supplementary). Cumulative percentage of CTCs remained at various stages is shown in Fig.…”

Signatures of breast cancer metastasis at a glance

“…As such, those studies mostly pursued only small numbers of gene candidates that were already known to be involved in breast cancer progression, metastasis and therapeutic resistance, rather than global transcriptional profiles (Aktas et al, 2011;Bolke et al, 2009;Gervasoni et al, 2008;Gradilone et al, 2011;Theodoropoulos et al, 2010). Recent technological advancements allow for immunomagnetic-bead-or microfluidicbased isolation of CTCs (Autebert et al, 2015;Magbanua and Park, 2013). When coupled to high-throughput microarray or RNAsequencing technologies and unbiased bioinformatics analyses, the aforementioned obstacles are significantly surmounted (Curtis, 2015;Magbanua and Park, 2014).…”

Circulating tumor cell technologies

“…One example of a technology that has implemented an immunomagnetic strategy on a micro-scale is the Ephesia chip. The Ephesia chip uses microcontact printing to create magnetic traps that promote the self-assembly of functionalized magnetic beads into an array of 48,000 columns within a microfluidic channel (Autebert et al, 2015;Saliba et al, 2010). The Ephesia design combines aspects of immunomagnetic sorting and micropost-based enrichment designs, while overcoming some of the limitations of previous micropost technologies.…”