- Open Access
Binary-blend fibber-based capture assay of circulating tumor cells for clinical diagnosis of colorectal cancer
© The Author(s) 2018
Received: 23 July 2017
Accepted: 20 December 2017
Published: 16 January 2018
In addition to conventional approaches, detecting and characterizing CTCs in patient blood allows for early diagnosis of cancer metastasis.
We blended poly(ethylene oxide) (PEO) into nylon-6 through electrospinning to generate a fibrous matbased circulating tumour cells (CTCs) assay. The contents of nylon-6 and PEO in the electrospun blend fibrous mats (EBFMs) were optimized to facilitate high cell-substrate affinity and low leukocyte adsorption.
Compared with the IsoFlux System, a commercial instrument for CTC detection, the CTC assay of EBFMs exhibited lower false positive readings and high sensitivity and selectivity with preclinical specimens. Furthermore, we examined the clinical diagnosis accuracy of colorectal cancer, using the CTC assay and compared the results with those identified through pathological analyses of biopsies from colonoscopies. Our positive expressions of colorectal cancer through CTC detection completely matched those recognized through the pathological analyses for the individuals having stage II, III, and IV colorectal cancer. Nevertheless, two in four individuals having stage I colorectal cancer, recognized through pathological analysis of biopsies from colonoscopies, exhibited positive expression of CTCs. Ten individuals were identified through pathological analysis as having no colorectal tumours. Nevertheless, two of these ten individuals exhibited positive expression of CTCs.
Thus, in this population, the low cost EBFMs exhibited considerable capture efficiency for the non-invasive diagnosis of colorectal cancer.
Metastasis is the most common cause of cancer-related death in patients with solid tumours. A considerable body of evidence indicates that tumour cells are shed from primary and metastatic tumour masses at different stages of malignant progression. These breakaway circulating tumour cells (CTCs)  enter the bloodstream and travel to different tissues of the body as a crucial means of spreading cancer. The current gold standard for diagnosing tumour status requires invasive biopsy and pathological analysis. In addition to conventional approaches, detecting and characterizing CTCs in patient blood allows for early diagnosis of cancer metastasis. To address this unmet need, significant research endeavours, especially in the fields of chemistry, materials science, and bioengineering, have been devoted to developing CTC detection, isolation, and characterization technologies. Identifying CTCs in blood samples has, however, been technically challenging, because of the extremely low abundance (a few to hundreds per millilitre) of CTCs among a large number (109 mL−1) of hematological cells.
A great number of separation systems have been developed, such as an antibody mediated immunoassay , size-based filtration method , fluorescence-activated cell sorting (FACS) , immunomagnetic separation [5, 6], dielectrophoresis force separation , and others, as summarized in previous reviews . Among the popular methods, the immunomagnetic cell separation assay, which works by selectively labelling the CTCs with magnetic nanoparticles and using an external magnetic field to capture target cells, provides an effective solution for the translational clinical applications . The immunomagnetic assay exhibits good sensitivity and specificity that arises from the cancer-specific antibody-antigen interactions. Therefore, some commercial instruments have been well-developed, such as the gold standard CellSearch system and IsoFlux system. These systems have exhibited outstanding cell capture efficiency (40–70%) when employed to isolate viable cancer cells from peripheral blood samples. However, sometimes a few leukocytes contaminate the CTC labelling system, resulting in false positive clinical diagnoses. In addition, positive expression of CTC detection alone is not enough to proceed with a diagnosis and treatment, limiting the clinical use of CTC detection. Most reports of CTC detection are focused on the high selectivity, specificity, and throughput of cell separation. Clinical diagnoses of cancer species by CTC detection are extremely rare .
Polycaprolactam (nylon 6, Polysciences) and PEO (Acros Organics), having average molecular weights (Mw) of 18,000 and 1,000,000 g mol−1, respectively, were used as received. Red blood cell lysing buffer and hybri-maxTM fibrinogen from human plasma (50–70%, Mw: 340 kDa), and sodium phosphate dibasic dihydrate were purchased from Sigma-Aldrich. Formic acid (Acros Organics) and NHS (Acros Organics, 98%) were used without further purification. EDC was purchased from Alfa-Aesar. Biotin anti-human CD326 (anti-EpCAM) and the FITC-streptavidin were purchased from BioLegeng (San Diego, CA). FITC-anti-EpCAM antibody and Cy5-biotin were purchased from Milli-Mark™ (Germany) and Click Chemistry Tools, respectively.
Electrospun nylon-6/PEO fibrous mats
An 80% formic acid solution containing nylon-6 was stirred for 1 h. PBS was used to determine the optimal ratio of nylon-6 in the triple-blend electrospun fibrous mats. Binary mixtures of nylon-6 and PEO were formed in the formic acid solution at PEO-to-nylon-6 weight ratios of 10, 20, 30, 40, 50, and 60 wt%, giving samples denoted as N90/P10, N80/P20, N70/P30, N60/P40, N50/P50, and N40/P60, respectively. Formic acid solutions containing PEO were added dropwise into the nylon-6 solution and then the mixture was stirred for 1 h at room temperature. Concentrations of the mixture solutions for electrospinning were controlled at 22 wt%. For electrospinning, a syringe pump (KDS-100, KD Scientific) was fixed to a support that could be moved left and right at a speed of 7 m/min along a slipway to jet the nylon-6/PEO hybrid solution uniformly in the form of films on a rolled cylinder substrate. The metal needle tips of the syringes were connected to the positive electrode of a high voltage-power supply (YSTC Technology). The feeding rate of the polymer solutions was 0.3 mL/h. The applied voltage was 30 kV; the tip-to-collector distance was 15 cm. The EBFMs were collected through electrospinning onto the surface of a glass slip and dried at room temperature under vacuum for 24 h prior to subsequent characterization.
Anti-EpCAM antibody immobilization
Poly(ethylene oxide) possesses carboxyl groups at the end of the polymer chain. Blending PEO within the nylon-6 matrix facilitates the affinity for inter-molecular hydrogen bonding between the amino group of nylon-6 and oxygen atoms of PEO. In addition, the presence of a hydrophobic domain of the alkyl groups within the blend results in the presentation of the carboxyl groups on the surface to immobilize the biotinylated anti-EpCAM (Fig. 1) . Thereafter, EDC/NHS chemistry was employed for the immobilization of antibodies . Briefly, the EBFMs were incubated in a PBS solution (pH 7.2–7.4) of EDC (2 mg/mL) and NHS (2 mg/mL) for 1 h at room temperature prior to modification. Streptavidin or FITC-streptavidin (127 μg/mL) was added to each sample, which was then stored at 4 °C overnight. Unreacted NHS, EDC, and streptavidin were washed away with the PBS for 10 min. The FITC-streptavidin-adhered EBFMs were subsequently observed using a fluorescence microscope (FL Color Imaging System, AMF 4300, Life Technologies). The streptavidin-modified fibrous mats were incubated in biotinylated anti-EpCAM antibody (5 μg/mL) to form the substrate for the cell-capture experiments. The samples were gently washed three times with PBS to remove any non-immobilized antibodies.
Surface chemical characterization of modified blend fibrous mats
The surface morphologies of the electrospun nanofibres were characterized through field emission scanning electron microscopy (FE-SEM), using a JSM 6500F instrument operated at 15–20 kV. Fibre diameters were determined using Image-J image processing software. For each electrospun mat, at least 100 fibres were considered from three different images to calculate the average diameter. The roughness of the triple-blend fibrous mats was examined using an ultra-precision benchtop 3D optical profiler (UPBOP, Talysurf CCI LITE, Taylor Hobson). The distributions of the biomacromolecules on the fibre surfaces were investigated through laser scanning confocal microscopy (LSCM), using a Leica TCS SP5 confocal spectral microscope imaging system featuring a 100-mW Ar blue laser operated at 494 nm. A red fluorescent antibody (Cy5-Biotin, Click Chemistry Tools) was employed to determine the distribution of the biotinylated anti-EpCAM antibodies on the EBFMs. The streptavidin-modified EBFMs were placed in a Cy5-Biotin solution (5 μg/mL) at room temperature. The Cy5-Biotin-adhered EBFMs were subsequently observed through LSCM at 651 nm. The grafting density of anti-EpCAM could be estimated by the emission of Cy5-biotin at 651 nm of wavelength.
Non-biofouling properties of anti-EpCAM-modified EBFMs
Leukocytes were employed to examine the non-biofouling properties arising from adsorption upon the anti-EpCAM-modified EBFMs as well as fibrinogen [28, 29]. Leukocytes generally generate a significant obstruction for CTC absorption and recognition. A drop of a solution of leukocytes (400 μL) isolated from peripheral blood (10 mL) was placed onto the anti-EpCAM-modified EBFMs, and then the system was incubated for 360 min at room temperature. After the leukocytes had been stained with hematoxylin, the samples were observed by optical microscopy (OM, Olympus, BX 43). Total number of leukocytes was counted to calculate the ratio of the adhesive leukocytes. Each final result was the average of three determinations.
Colorectal tumour cell adhesion in preclinical assay
The human colorectal cancer cell lines DLD-1, HCT-116, and HT-29 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell lines were cultured in RPMI-1640 medium (Gibco-Invitrogen, Carlsbad, CA) with 10% foetal bovine serum (Gibco-Invitrogen), glutamine, penicillin, and streptomycin and maintained in a humidified atmosphere containing 5% CO2 at 37 °C. Anti-EpCAM-modified EBFMs were placed in the wells of a 24-well plate (Corning). Cell lines were incubated in PBS containing FITC-anti-EpCAM antibody (5 μg/mL) in the dark for 1 h. Various counts of tumour cell lines were diluted with PBS (2 mL) prior to use. Peripheral blood samples (5 mL) were collected in tubes containing heparin anticoagulant. The PBS (2 mL) possessing various counts of tumour cell lines was mixed with the peripheral blood samples (5 mL), drawn from healthy donors that no other pathologies or negative for colon cancer, as specimens for CTC capture. The protocol for CTC capture by EBFMs from peripheral blood samples was as follows. The specimens (7 mL) were centrifuged at 3000 rpm for 15 min. The buffy coat was collected and mixed with RBC lysis buffer at 1200 rpm for 6 min to remove most of the erythrocytes. The process was repeated four times to collect the buffy coat, which was then incubated in PBS containing FITC-anti-EpCAM or Alexa594-conjugated anti-Ck8+18 (5 μg/mL) in the dark for 1 h. Droplets of a mixture (300 μL) of PBS and buffy coat were placed on the anti-EpCAM-modified EBFMs (1 × 1 cm2) after incubation for 3 h. Thereafter, the fibrous mats were gently washed three times with PBS to remove any uncaught cells and incubated in 4% paraformaldehyde for 10 min to fix the cells adhering to the surface. After staining with hematoxylin, the samples were imaged using OM (Olympus, BX 43) and the CTCs on the anti-EpCAM-modified EBFMs (1 × 1 cm2) were counted using software (Image-Pro Plus V7) by scanning all areas of the EBFMs.
Clinical diagnosis of colorectal cancer by CTC detection
A 7–10 mL peripheral blood specimen is injected into the instrument.
Magnetic beads are modified by cytokeratin antibodies to mix with the specimen. These marker-modified magnetic beads attach to CTCs specifically in the solution.
The magnetic bead-attached CTCs are picked up from the specimens one by one onto a platform (1 × 1 cm2) for CTC identification.
Fluorescence agencies including cytokeratin, CD45 (leukocyte marker), and nucleus (DAPI) marker are exploited to stain all cells on the platform. The system software is used to record fluorescence intensity and species by scanning all regions of the platform to identify all cells as CTCs or leukocytes and obtain CTC counts. Because of the large amount of leukocytes, some leukocytes inevitably appear on the platform. Seven subjects participated in the program; peripheral blood from each subject was collected and equally divided into specimens for the CTC capture assay and IsoFlux system, respectively.
Results and discussion
Colorectal tumour cell capture from peripheral blood specimens
We have developed a CTC capture platform based on a high-affinity cell enrichment assay, employing electrospun nanofibres deposited on a substrate and coated with a cell-capture agent. A non-invasive diagnosis approach with low cost of nylon-6/PEO fibrous mats was developed to facilitate the diagnosis of colorectal cancer, which could motivate people to participate in colorectal health checks more frequently. For colorectal cancer, the cure rate decreases significantly with cancer stage because of cancer cell metastasis. Although the diagnostic accuracy of stage I colorectal cancer was insufficient, it also indicates lower risk of metastasis in stage I colorectal cancer. In addition, false positive diagnosis of colorectal cancer also indicates the high risk of metastasis in other cancers, providing extra information for the patient. The fibrous assay has been used clinically in circulating cells irrespective of the sort of cancer suffered the subjects. Once the antibody with high selectivity for particular sort of cancer has been developed, it could be further universalized to detect particular sort of cancer. Therefore, this platform based on electrospun fibrous mats has high potential applications in early non-invasive diagnosis and longitudinal monitoring of cancer in clinics.
AWL prepared a clinical protocol entitled “Study of detection circulating tumour cell on micro-nano surface”, which was approved by the Institutional Review Board (IRB) of Taipei Medical University (201311008), and took blood from patients or healthy donors following written informed consent, which was also approved by the IRB committee. FXL prepared and analysed all samples and tested the ability of the capture assay to identify circulating tumour cells. PLW performed examination of invasive colonoscopy biopsies with the clinical diagnosis of colorectal cancer. GJW provided the standard colorectal cancer cells and pathological analysis under the clinical protocol. JKC wrote the main manuscript text. All authors reviewed the manuscript. All authors read and approved the final manuscript.
This work was supported by National Ministry of Science and Technology (MOST106-2320-B-038-038 and 106-2221-E-011-135-MY3) and Taipei Medical University (TMU102-AE1-B33106-2221-E-011-135-MY3) in Taiwan, as well as Taipei Medical University-National Taiwan University of Science and Technology Joint Research Program (TMU-NTUST-106-01). We also thanks for Taipei Medical University Hospital supporting the clinical experiments and IsoFlux system.
The authors declare that they have no competing interests.
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