SUPPORTING INFORMATION
Highly efficient isolation and release of circulating tumor cells based on size-dependent filtration and degradable ZnO nanorods substrate in a wedge-shaped microfluidic chip
Songzhan Li Yifan Gao Xiran Chen Luman Qin Boran Cheng Shubin Wang Shengxiang Wang Guangxin Zhao Kan Liu Nangang Zhang
S. Li Y. Gao X. Chen L. Qin S. Wang G. Zhao N. Zhang (*)
College of Electronic and Electrical Engineering, Wuhan Textile University, Wuhan, Hubei, 430200, People’s Republic of China
Email:
B. Cheng S. Wang
Department of Oncology, Peking University Shenzhen Hospital, Shenzhen, Guangdong, 518036, People’s Republic of China
K. Liu (*)
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan, 610054, People’s Republic of China
Email:
* Those two are co-corresponding authors.
1. The designed size of the microfluidic channel
Fig S1 The designed size of the microfluidic channel with (a) top view and (b) cross-section view along M-M’.
2. Clinical sample processing
Blood samples from 7 breast cancer patients and 5 health donors were used for CTC isolation and detection. Blood samples (2mL for each test) were collected in blood collection tubes (BD, USA) containing EDTA-K2 and finished the experiments within 6 hours. Just prior to each test, 4% paraformaldehyde (PFA) (MP Biomedicals, USA) solution was added into whole blood samples in a 1:10 v/v ratio, and incubated for 15 minutes at room temperature.
3. Staining and enumeration
Three-color immunocytochemistry method was applied to identify CTCs and white blood cells. The staining protocol was used in detail as followed: First, 0.3 mL of fixation/permeabilization standard solution (Cytofix/CytopermTM, BD Biosciences, USA) was introduced into the microchamber and incubated for 10 minutes. Second, 0.3 mL of blocking solution (PBS with 1% bovine serum albumin (BSA)) (Sigma-Aldrich, USA) was loaded and incubated for 15 minutes to prevent non-specific binding. Third, 0.3 mL of mixture solution of primary antibodies (PBS with 1% mouse monoclonal IgG to Pan-CK (Abcam, USA) and 0.5% rat monoclonal IgG to CD45 (Santa Cruz, USA) ) was introduced and incubated at 37℃ for 3 hours, and then wash buffer (BD Biosciences, USA) was introduced to remove unbonded antibody at flow rate of 200 μL min-1 for 15 minutes. Fourth, 0.3 mL of mixture solution of secondary antibodies (PBS with 0.2 % Alexa Fluor® 546 goat anti-mouse IgG(H+L) antibody and 0.5% Alexa Fluor® 488 donkey anti-rat IgG(H+L) antibody (Molecular Probe®, Life Technologies, USA)) was introduced and incubated at 37℃ for 30 minutes. Finally, DAPI solution (0.3mL，5μg/mL) was added and incubated for 15 minutes to identify nuclei.
After rinsing microchamber with PBS at 200 μL min-1 for 10minutes, microfluidic devices were fitted on a high-content screening system (CellInsightTM, CX5, ThermoFisher Scientific, USA) for imaging and enumeration.
4. Isolation of CTCs from clinic blood samples
As shown in Fig. S2 (a), CTCs exhibited high CK expression and negligible CD45 signals, but WBCs presented low CK and high CD45 expression levels. The combined information was utilized to delineate CTCs cells (CK+, CD45-, DAPI+) from WBCs (CK-, CD45+, DAPI+) and cellular debris. As shown in Fig. S2 (b), 4 to 14 CTCs were identified in 7 breast cancer patients (100%), Whereas, no cell was characterized as CTC in 5 healthy blood samples (0%), which suggested that our method was effective and reliable to capture and identify CTCs from peripheral blood sample.
Fig.S2 (a) Fluorescent images of cells isolated from breast cancer patient. CTCs (red arrow) was identified by the combined information (CK+, CD45-, DAPI+) and WBCs (green arrow) were identified by the predefined criteria (CK-, CD45+, DAPI+). Scale bars, 10 μm. (b) CTC enumeration results obtained from 2 mL whole blood samples of healthy donors (n=5) and breast cancer patients (n=17), respectively.