Journal: Oncology Letters

Article Title: Detection of circulating colorectal cancer cells by a custom microfluid system before and after endoscopic metallic stent placement

doi: 10.3892/ol.2019.11047

Figure Lengend Snippet: Capture of CTCs using the custom polymeric CTC chip. (A) Protocol for CTC capture with the custom CTC chip. Peripheral blood samples (5 ml) were collected from patients into tubes containing ethylenediaminetetraacetic acid. Samples were centrifuged at 300 × g for 5 min at 4°C. After removing plasma, cellular components including buffy coat were dissolved in 1.5 ml PBS. Processed samples were transferred to the CTC chip using automatic syringes. Micropoles on the chip were coated with EpCAM antibodies to capture CTCs. Captured CTCs were stained with CK19 and CD133 or other antibodies. (B) Representative images of the captured CTCs and their schemes. HCT116 cells were captured (arrows) at the micropoles coated with EpCAM antibodies and the cells were stained with DAPI and CK19 antibodies (upper panels). HCT116 cells mixed with blood from a healthy donor were captured. All cells were stained with DAPI, but only HCT116 cells were stained with anti-CK19 (middle panels). CTCs in patient blood were captured. All cells were stained with DAPI, but only the CTCs were stained with anti-CK19 (lower panels). Scale bar, 50 µm. CTC, circulating tumor cells; WBC, white blood cell.

Article Snippet: After fixation, CTCs were incubated with sheep anti-human CK19 antibodies (#sc-33119; Santa Cruz Biotechnology), which were diluted 1:200 and incubated for 1 h. Then, donkey anti-sheep antibodies conjugated with Alexa Fluor 488 (#1807723; Thermo Fisher Scientific), which were diluted 1:1,000, were incubated for 30 min as secondary antibodies to visualize CTCs.

Techniques: Clinical Proteomics, Staining

Journal: Oncology Letters

Article Title: Detection of circulating colorectal cancer cells by a custom microfluid system before and after endoscopic metallic stent placement

doi: 10.3892/ol.2019.11047

Figure Lengend Snippet: Compatibility of antibodies in the custom circulating tumor cell chip for the detection of epithelial cancer cell lines (HCT116; CK19 positive) and WBCs (CD45 positive).

Article Snippet: After fixation, CTCs were incubated with sheep anti-human CK19 antibodies (#sc-33119; Santa Cruz Biotechnology), which were diluted 1:200 and incubated for 1 h. Then, donkey anti-sheep antibodies conjugated with Alexa Fluor 488 (#1807723; Thermo Fisher Scientific), which were diluted 1:1,000, were incubated for 30 min as secondary antibodies to visualize CTCs.

Techniques:

Journal: Oncology Letters

Article Title: Detection of circulating colorectal cancer cells by a custom microfluid system before and after endoscopic metallic stent placement

doi: 10.3892/ol.2019.11047

Figure Lengend Snippet: CTC number, CTCs and cfDNA concentrations following SEMS placement. (A) Fluorescence-stained CTCs captured by the CTC chip after SEMS placement. CTCs were stained with DAPI and CK19 (scale bar, 50 µm). (B) Differences in the numbers of CTCs before and after SEMS placement. The number of CTCs increased at 24 h after SEMS placement and decreased 4 days after SEMS placement in three cases (patient 1, 4 and 5). The number of CTCs was slightly increased at 4 days after SEMS placement compared with those before SEMS placement in three cases (patient 2, 7 and 13). The number of CTCs before SEMS placement was not tested in two cases (patient 10 and 12). In the remaining five cases (patient 3, 6, 8, 9 and 11), CTC numbers remained unchanged or decreased. (C) cfDNA concentrations in plasma were determined in patients 1–6 before and after SEMS placement. Pre, before SEMS placement; POD1 and POD4, 24 h and 4 days after SEMS placement, respectively; n.s., no significance; CTC, circulating tumor cells; cfDNA, cell-free DNA; SEMS, self-expandable metallic stents.

Article Snippet: After fixation, CTCs were incubated with sheep anti-human CK19 antibodies (#sc-33119; Santa Cruz Biotechnology), which were diluted 1:200 and incubated for 1 h. Then, donkey anti-sheep antibodies conjugated with Alexa Fluor 488 (#1807723; Thermo Fisher Scientific), which were diluted 1:1,000, were incubated for 30 min as secondary antibodies to visualize CTCs.

Techniques: Fluorescence, Staining, Clinical Proteomics

Journal: Oncology Letters

Article Title: Detection of circulating colorectal cancer cells by a custom microfluid system before and after endoscopic metallic stent placement

doi: 10.3892/ol.2019.11047

Figure Lengend Snippet: Images of circulating cancer stem-like cells stained with CD133. (A) CD133-positive pancreatic cancer Capan-1 cells were captured and stained with DAPI (blue), CK19 (green) and CD133 (red). A number of cells were CD133 positive (scale bar, 50 µm). (B) Representative images of the captured circulating tumor cells stained with DAPI, CK19 and CD133 in a sample obtained from patient 7 (scale bar, 50 µm). (C) The number of CD133-positive cells in the blood did not change significantly before and after SEMS placement. In seven cases (patients 7–13), CD133-positive cells were determined around stenting. The number of CD133-positive cells did not change significantly in response to stenting. SEMS, self-expandable stents; Pre, before SEMS placement; POD1 and POD4, 24 h and 4 days after SEMS placement, respectively; n.s., no significance.

Article Snippet: After fixation, CTCs were incubated with sheep anti-human CK19 antibodies (#sc-33119; Santa Cruz Biotechnology), which were diluted 1:200 and incubated for 1 h. Then, donkey anti-sheep antibodies conjugated with Alexa Fluor 488 (#1807723; Thermo Fisher Scientific), which were diluted 1:1,000, were incubated for 30 min as secondary antibodies to visualize CTCs.

Techniques: Staining