Cell line preparation
A human ovarian adenocarcinoma cell line, SKOV3 (ATCC® HTB-77TM, Manassas, VA, US), was used for the cell spiking test. The SKOV3 cells were cultured in McCoy’s 5A medium (BioConcept, Allschwil, Switzerland), supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin (Gibco, Grand Island, NY, USA). Peripheral blood mononuclear cells (PBMCs) were separated from the whole blood of healthy volunteers by a density gradient centrifugation method.
Two different enrichment strategies were used for spiking test, including biotinylated anti-EpCAM antibody only, and an antibody cocktail combined with biotinylated anti-EpCAM antibody and biotinylated anti-N-cadherin antibody (Combi). Prior to their mixture, both SKOV3 cells and PBMCs were incubated with anti-EpCAM antibody or Combi cocktail at 37oC for 45 minutes in a 15 mL centrifuge tube. Then 3mL DPBS was added to each tube and the tubes were centrifuged at 300g for 10 minutes to collect the cell pallets and remove free antibodies. The cell mixture for spiking test was prepared by spiking 5*103 SKOV3 cells into PBMCs of 2 mL whole blood origin in 200 μL of DPBS. The SKOV3 cell number was counted using CountessTM II FL Automated Cell Counter (ThermoFisher, Waltham, MA, USA).
The V-BioChip (CytoAurora Inc., HsinChu, Taiwan) is a silicon-based chip with nano-pillar arrays on the inside of microfluidic chambers (Fig 1). The structure and production process of the predecessor of V-BioChip, Cral Chip, has been described in detail in Ma's report11. To improve the chip's capture efficiency, we modified original design of Coral Chip to adjust the distance between the nano-pillars on the microfluidic chip and the shape of the nano-pillars. In brief, metal-assisted chemical etching (MACE) technology was used to form matrix-arranged nano-pillars on the chip surface. The tip of the pillars is modified into a shape of volcanic cone to increase the contact surface between the target cells and the nano-pillars, in attempt to reduce cell damage. Thereafter, the polyethylene glycol-biotin (PEG-biotin) layer was modified on the surface of the wafer by vapor deposition method. Streptavidin, a tetrameric protein with high binding affinity to biotin34, was then attached to the biotin end of the PEG-biotin using liquid deposition technology. The streptavidin-biotin interaction is one of the strongest non-covalent biological interactions currently known and can markedly increase the capture efficiency to the target cells35, 36. When the mixed cell suspension flows over the chip, the target cells will be captured by the V-BioChip, and most other cells will be washed away.
Cell spiking test
The Cell RevealTM machine (CytoAurora Inc., HsinChu, Taiwan) was used for the enrichment and staining of the CTCs. Before the test, the V-Biochip was set up inside the machine. The mixed cell suspension of SKOV3 and PBMCs was injected into the Cell RevealTM system, and the system automatically processed the CTC enrichment and staining procedures. The inputted cell mixture was then fixed in 4% paraformaldehyde. Subsequently, 0.1% of Triton X-100 (ThermoFisher, Waltham, MA, USA) and 2% BSA (Bovine serum albumin) were added to increase the cellular permeability. The cell mixture passes through the V-BioChip at a flow rate of 0.6 mL/h, allowing the target cells to fully contact the chip to achieve an optimal capture rate. As the cell-rich centrate flows through the microfluidic chips, the streptavidin on the chips captures the target cells bound with biotinylated anti-EpCAM antibodies. The process of CTC enrichment and staining were done overnight to achieve the best staining. But this process can be completed within four hours.
In order to distinguish CTCs from white blood cells, microfluidic chips were incubated with anti-EpCAM antibody (R&D Systems, Minneapolis, MN, USA) conjugated with FITC (for the detection of CTCs), as well as anti-CD45 (Agilent, Santa Clara, CA, USA) antibody conjugated with TRITC (for the detection of white blood cells) and 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) (for the detection of nucleated cells). The CTC was defined as an EpCAM+/CD45-/DAPI+ intact cell.
After the enrichment and staining steps, V-BioChip is sent to the CTC scanning platform for further analysis. This state-of-the-art CTC scanning platform is composed three main parts, including a modified upright fluorescent microscope and two sets of self-developed software (CytoAcqImages system for automatic image scanning and CAT automatic cell identification system). The upright fluorescent microscope (Leica DM6 B, Leica Microsystems GmBH, Wetzlar, Germany) is equipped with Leica HC PL APO 10x /0.45 microscope objective, spectra III light engine (Lumencor, Beaverton, OR, USA) with wavelengh range: 380nm~750nm, Andor Zyla 4.2 sCMOS camera and Marzhauser scanning stage for 4 slides and is controlled by the CytoAcqImages (CAI) system. The CAI system can be used with any brand of microscope, light controller, motorized XYZ stages and sCMOS camera.
Under the control of CAI system, the microscope automatically focuses and scans the V-BioChip, as well as activates the exposure with a fluorescent carousel (Fig 4a). A fully automatic segmented photographing method was adopted to obtain high-resolution full-area images of V-BioChips. Then, the image files were montaged together to form a whole biochip TIFF image file.
V-BioChip is pre-installed with special positioning marks. When the CAI system focuses on these marks, it also records the coordinate information to the full chip image file. When the image interpretation system reads the chip image file, it can use the coordinate information to calculate the specific position of the specific cell on the chip. In addition, this precise positioning function can also help to find specific cells on the chip again, which can assist in re-evaluating specific cells and even further single-cell analysis.
The full chip image files were then transmitted to CAT system (Fig 4b). CAT system can identify target cells according to the immunofluorescence staining on the cells by using pre-set parameters and deep learning AI function. Count-in/filter-out criteria were used to identify the cells while EpCAM+/CD45-/DAPI+ for CTCs and EpCAM-/CD45+/DAPI+ for WBCs (Fig 5a).
Three separate spiking tests were performed for both the single ant-EpCAM antibody and Combi strategy. The results are expressed as the mean recovery rate + standard deviation (SD).
Linearity between the numbers of captured cells and spiked cells
The relationship between the captured cell numbers and the CTC numbers was analyzed by spiking SKOV3 cells into DPBS was analyzed. We incubated 625 SKOV3 cells with 5μL cocktail capture antibodies in 200μL DPBS at 37˚C for 45min and then centrifuged with 400g for 10 min. A serial of 2-fold dilutions was performed to allow for linear regression analysis of the estimated cell numbers being 625, 312, 156, 78 and 39. The diluted cell suspensions were then injected into Cell RevealTM System to capture SKOV3 cells by streptavidin–coated chip with a flow speed of 0.6 μL/min. The captured cells were stained by EpCAM/CD45/DAPI on chip and subsequently were scanned and counted by the automatic CAT system.
Each spiking cell number is tested twice. In addition to linear regression analysis, the recovery rate of each test was also calculated. The results of recovery rate were expressed as mean recovery rate + SD.
In order to examine the clinical feasibility of the test, we recruited participants to join the study, including 10 healthy subjects without any type of ovarian tumor, 5 women with benign ovarian tumor and 8 patients with epithelial ovarian cancer (EOC). FIGO criteria were used for the staging of EOC. The study has been approved by the IRB committee in the Taiwan Adventist Hospital and Changhua Christian Hospital. All methods were carried out in accordance with relevant guidelines and regulations and each participant completed a written consent before they received the test.
In this study, anti-CD13 antibody (Abcam, Cambridge, UK) was also used for ICC staining to identify CTCs that carry CD13 (Aminopeptidase N) surface marker. CD13 is a function marker for cancer stem cell. When an intact cell shows ICC staining of CD13+/EpCAM+/CD45-/DAPI+, it is defined as a CD13+ CTC (Fig 5b).