This study was reviewed and approved by Medical Ethics Committee of the Suqian First People's Hospital. The approval of our ethics committees is available in Supplemental Data 1.
Patients and human tissue samples
Human CRC tissue microarrays (TMAs) were constructed from 100 cases of surgically resected colorectal tumor tissues (2016–2018), along with matched cases of adjacent normal tissues from the Pathology Department, Suqian First Hospital, Jiangsu Province, China. All tissue specimens were reviewed using hematoxylin and eosin staining; representative areas free from necrosis and hemorrhage were selected in the paraffin blocks. We took 1-mm diameter cylinders from intratumoral or peritumoral tissues (1–2 cm from the tumor edge) and transferred to the TMA by the Outdo Biotech Company, Shanghai, China. The relevant clinical data was collected using retrospective medical chart reviews. Survival data were collected every three months, with the final update on 10/31/2019. All protocols were reviewed and approved by the academic ethics committee.
The demographic data and post-surgical follow-up of the 100 CRC cases are shown in Table 1. The majority of patients were diagnosed (post-surgically) with stages II and III according to the American Joint Committee on Cancer (AJCC) staging 8th edition (72/100), 19 cases were diagnosed as AJCC stage I, and nine cases were AJCC stage IV as have been found with liver metastasis prior to the surgical resection.
Immunohistochemistry and scoring
Archived paraffin-embedded tumor tissues and adjacent normal tissues were constructed for tissue microarrays and IHC. IHC was performed using the polymer HRP detection system (Zhongshan Goldenbridge Biotechnology, Beijing, China) according to the manufacturer’s instructions. The primary antibody was anti-WNT7b (Catalog#: AF3460, R&D system) at 1:1000 dilution. The second antibody, was anti-goat (Catalog#: 6403-05, BioVision) at 1:2000 dilution. All TMA slides were scanned using the Leica Aperio AT2 digital slide scanner.
The scoring of WNT7b immunostaining was based on both the intensity and percentage of positively membranal staining cells. The final expression score of a single sample was equal to the intensity grade (0, 1, 2, and 3) multiplied by the percentage level (0–100%). Each CRC sample was compared with its paired adjacent normal tissue. TMA slides was evaluated by two independent pathologists who were blinded to patient information. If there were discrepancies, results were jointly assessed by both investigators and the final score was formed by consensus.
HCT116 and CCD-18co cells were purchased from Tongpai Biotechnology Company, Shanghai, China. Cells were maintained in 5% CO2 at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies/Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies/Gibco, NY, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin (Life Technologies/Gibco, MD, USA). Cells (1 X 106) were seeded into dishes (10-cm diameter) for 72 h at each passage.
For transient WNT7b gene knockdown, cell-lines were transfected with 10–20 nM of duplexed siRNAs using Lipofectamine 2000 (Invitrogen/Life Technologies, NY, USA). The RNA interference sequence is listed in the Supplementary Data 2.
Cells were harvested at 90% confluence. For regular immunoblots, cell lysates were obtained using RIPA Lysis Buffer (Millipore, MA, USA). Separate nuclear and cytoplasmic protein extraction was performed NE-PER Nuclear and Cytoplasmic Extraction Reagents (Millipore, MA, USA). Cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, MA, USA). The membranes were probed with primary antibodies overnight at 4 °C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immune complexes were detected using enhanced chemiluminescence (Cell Signaling Technology, MA, USA). GAPDH was used to correct for differences in loading of the proteins from the control and experimental groups. Detailed antibody information is displayed in Supplementary Data 3.
Cells grown on cover slips in 24-well plates were fixed in 4% paraformaldehyde for 20 min, then the cells were permeabilized with 0.5% Triton X-100 (Solarbio, Beijing, China) for 20 min at room temperature. The HCT116 cells and CCD-18co cells were incubated at 4 °C overnight with the primary antibody (Catalog #: AF3460, 1:1000 dilution, R&D system) after blocking with 3% nonfat dry milk in PBS for 1 h at room temperature. Then the cells were incubated for 1 h at 37 °C with Fluorescein (FITC)-conjugated AffiniPure Fab Fragment (1:200 dilution, Jackson ImmunoResearch Laboratories, PA, USA) as the secondary antibody. The nuclei were stained by DAPI (Sangon Biotech, Shanghai, China) for 5 min after washing with PBS. Images were captured using a confocal microscope (Olympus, Tokyo, Japan).
Transwell assays were performed using growth factor-reduced, Matrigel-coated filters (8 mm pore size, BD, Franklin Lakes, NJ, US) in 24-well plates. Cells were trypsinized and seeded onto the upper chambers of the Transwells (3 x 104 cells/well) in supplement-free DMEM medium. The lower chambers of the Transwells were filled with DMEM medium containing 100 ng/mL of EGF. The chambers were incubated at 37 °C with 5% CO2 for 24 h. At the end of incubation, cells on the upper surface of the filter were removed using a cotton swab. Cells migrating through the filter to the lower surface were fixed with 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 5 min. Migrated cells were viewed and photographed using a phase-contrast microscope (Olympus).
The χ2 test was used to analyze the significance of migrated cells in the Transwell assay using SPSS. Fisher's exact test were used to analyze the significance of lymphatic and remote metastasis between each group based on membrane upregulated expression of WNT7b by SPSS. For survival analysis, we compared Kaplan–Meier survival curves between different thresholds of membranal upregulated expression of WNT7b using the log-rank test on GraphPad Prism 7.