VALIDATION OF EPHESIA DEVICE

Ephesia system had been already validated for high capture efficiency with both cell lines and patients samples compared with CellSearch System⁸⁹.

FIGURE 2-2-4 EPHESIA DEVICE A) FINAL DESIGN OF THE CHIP B) CAPTURE PRINCIPLE OF CTC, C) SCHEME OF EXPERIMENTAL SET-UP WITH MAGNETIC COIL AROUND THE CHIP D)

CELLS CAPTURED ON THE MAGNETIC BEADS AND CELLS AFTER IMMUNOSTAINING IN CHIP.

First, for an efficient capture, the sample processing method had been optimized for Ephesia device. Unfortunately, due its high viscosity, the whole blood cannot be directly processed in the device at high flow rate due to collapsing of the columns. Also high concentration of RBCs might affect the capture efficiency. Therefore, different approaches had been investigated to remove RBC: lysing them or using centrifugation separation. Lysis of RBCs had resulted in high non-specific capture of WBC due to fixation steps. However, gradient-based centrifugation method had yielded less non-specific capturing of WBC;

moreover it helped to reduce the sample size dramatically while not changing the capture efficiency of the cells of interest. Yet, considering the viability of the sample, using semi-fixated conservative Cellsave tubes had given high non-specific capture of WBC. Thereafter, this problem had been solved by using RosetteSep method, as blood processing method, in

63

which the cells are depleted by tetrameric antibodies both against WBC and RBC without any significant decrease in capture efficiency.

The system had been first tested with cell lines with various expressions of EpCAM spiked into PBS (phosphate-buffered saline buffer). Average of 90.6% ± 5.84% (n = 12) capture efficiency had been obtained for high EpCAM cell line (MCF7) with a range of 50-250-1000 cells spiked (Figure 2-2-5a). Depending on EpCAM expression, different profile of capture had been observed, lower the EpCAM expression is lower the capture efficiency (Figure 2-2-5b) , however the capture rates remain higher than what was measured with CellSearch⁸⁹. The specificity of the system was determined by non-specific capture rate of Raji (B lymphocyte cell line) on the EpCAM beads. A high number of (10⁶) Raji cells spiked alone into the PBS had resulted in capture rate of only 0.26% ± 0.13% (n = 3) (Figure 2-2-5b). Similarly, mix of 50 MCF7 cell with 5x10⁶ of Raji cells showed capture rate of 90% ± 1.4% (n = 3) for EpCAM-positive cells whereas only 0.2% ± 0.11% (n = 3) for EpCAM-negative cells. However capture efficiency was shown to be reduced with cells spiked into blood samples, ~70%, but with very high specificity having only around 100 contaminating WBC cells with 10 ml of blood processed.

The capture efficiency in blood samples is lower particularly compared to filtration system such as FMSA⁷² and microfiltration system with conical shaped holes⁷¹ that have capture efficiencies respectively around 90% and 95%. But these systems show lower purity levels (14000fold enrichment, 96% WBC clearance) compared to Ephesia system having below 100 contaminating cells in a 7.5 ml blood sample. This could highly affect their ability to perform mutational analysis. However these kinds of system are more advantageous for subsequent culturing of CTCs which is very important for drug response studies. Ephesia system has limitation in this regard at this point in which captured cells cannot be removed from magnetic beads. Recently developed technology CTC-iChip combines both inertial microfluidics and antibody targeted WBC depletion leading to tumor antigen independent CTC sorting approach. As described before, either positive or negative selection method can be performed with this method. Ozkumur et al demonstrated the capture efficiency as around 97% for both epithelial and highly mesenchymal cell lines spiked into blood with negative selection mode which is unbiased by EpCAM expressions of cells. However this system also shows low purification, 2.5 log for negative depletion method but higher >3.5 log for EpCAM positive selection mode which allowed detection of EML4-ALK translocation by RT-PCR.

64

FIGURE 2-2-5 CAPTURE EFFICIENCY A) FOR MCF7 CELL LINE WITH DIFFERENT NUMBER OF CELL SPIKED INTO PBS, B) FOR DIFFERENT CELL LINES HAVING VARIOUS EXPRESSION OF EPCAM, IMAGE COPIED FROM REF. 89

A detailed investigation of capture principle on Ephesia had been already explained by the previous study so readers are referred to read the reference 79 for more information. The following criteria had been considered⁸⁹: the surface density of antibodies on the beads, ii) the thermodynamic affinity constant between the antibody and the antigen in the capture buffer, iii) the duration of the contact, iv) the hydrodynamic force exerted during contact, and v) the antigen expression levels on the cell membrane. The first two points are intrinsically fixed by the commercial EpCAM beads used in the system. The effect of time and the hydrodynamic force had been tested by using different flow velocity where no difference had been observed between 0.5 ml h⁻¹ to 3 ml h⁻¹. Moreover, a particular capture profile had been observed depending on the expression levels of EpCAM, higher the level more cells are captured in the frontier rows and lower the level, the relative ratio of the capture efficiency decreases between the frontier rows and the latter.

Next the device had been tested with clinical samples from metastatic breast (N=5) cancer and metastatic prostate cancer patients (N=8) and had also been compared with CellSearch system in a blind study. The results had shown that the CTCs were detected in 75% of prostate cancer patients and 80% of breast cancer patients. All in all, in 10 out of 13 samples, the results were similar or better than what obtained with CellSearch device.

65

FIGURE 2-2-6 COMPARISON OF CTC COUNTS OBTAINED WITH EPHESIA AND CELLSEARCH, IMAGE COPIED FROM REF. 89

Moreover, Ephesia had been shown to be compatible with further downstream analysis such as mutation detection by conventional quantitative PCR analysis. The captured cells can actually be recovered from the system by simply removing the magnetic field and flushing the cells from the outlet. E545K mutation could be detected in MCF7 cells captured from spiking into the healthy blood samples at a level of as few as 13 cells.

All in all, Ephesia device offers great advantages for isolation of CTCs. First the system uses commercial antibody coated magnetic beads which can be easily produced in batch processing with a rather homogenous quality of beads and by simply changing the beads or mixing them with another antibody coated beads; one can broaden the range of target cells. Another unique feature of Ephesia is to have very high aspect ratio of column arrays creating a dense sieve giving possibility for cells to collide with the beads multiple times, thus increasing the surface area to be captured on a small footprint. Also, the chip can be easily fabricated by soft-lithography without needing of tedious antibody functionalization into the chip. Meanwhile, the system allows imaging at very high resolutions and/or low resolutions (10X) in an automated way. Lastly, the fixed captured cells can be recovered with a high quality to be processed for downstream analysis due to its high purity.

66

2.2.4 MICROFABRICATION AND CTC CAPTURING PROTOCOL OF