Patients and specimens
76 patients were pathologically diagnosed with CRC and received surgical therapy at Fudan University Shanghai Cancer Center. Patients received neoadjuvant radiochemotherapy were excluded from our study. Specimens from the 76 patients were made into tissue microarrays (TMA). Our study was carried out in accordance with the requirements of the Biomedical Ethics Committee of Fudan University Shanghai Cancer Center.
Cell lines and culture
CRC cell lines (DLD1, HT29, LOVO, HCT116, RKO, SW480, SW620), normal colonic epithelial cell line (NCM460) and HEK-293T cell line were purchased from Type Culture Collection Cell Bank, Chinese Academy of Sciences. All cell lines were cultured in Dulbecco’s Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS), penicillin (107 U/L) and streptomycin (10 mg/L) and incubated at 37 °C in a humidified atmosphere containing 5% CO2 in Fudan University Shanghai Cancer Center.
Lentivirus production and transfection
Core plasmid was co-transfected with psPAX2 and pMD2.G into HEK-293T cells using Hieff TransTM Liposomal Transfection Reagent (Yeasen, Shanghai, China). 48 hours later, virus supernatant was collected. 3×105 cells were cultured in a 6-well plate. After incubation in virus supernatant for 48 hours, stable cell lines were selected with puromycin or flow cytometry and transfected efficiency was evaluated by immunoblot.
Flow cytometry
Apoptosis assay was conducted using Annexin V-PE/7-AAD apoptosis detection kit (Yeasen, Shanghai, China). Briefly, 3×105 cells were cultured in a 6-well plate with or without being treated with oxaliplatin for 48 hours. Adherent cells and cells in supernatant were collected and washed by phosphate buffer saline (PBS) twice. Cells suspended with 1× binding buffer with the addition of 5 μL Annexin V and 10 μL 7-AAD. After incubation protecting from light for 15 minutes, apoptosis rate was detected by Flow cytometer.
Western blotting
Total protein was extracted with NuPAGE® LDS sample buffer (Thermo Fisher Scientific, Waltham, USA). A certain amount of protein was separated by SDS-PAGE gel and transferred to PVDF membranes. After blockage with non-fat milk powder and incubation with primary antibodies and horseradish peroxidase-conjugated secondary antibodies, images were captured with ImageQuant™ biomolecular imager. All the antibodies used in our study were listed in additional file named Table S1.
Drug cytotoxicity assay
Drug cytotoxicity was evaluated by Cell Counting Kit-8 (CCK-8) assay. Cell suspension was seeded in 96-well plate and cells were treated with a gradient concentration of oxaliplatin for 48 hours after adhesion. Medium containing CCK-8 reagent (Yeasen, Shanghai, China) was added to each well of the plate. After two-hours’ incubation, absorbance at 450nm was detected by microplate reader.
Clone formation assay
1000 cells were cultured in a 6-well plate and treated with oxaliplatin for 48 hours after adhesion. The medium was replaced by the fresh every three days. 2 weeks later, cells were fixed with 4% paraformaldehyde for 30 minutes and stained with crystal violet for 30 minutes. Images were photographed and visible colonies were counted.
Co-immunoprecipitation (Co-IP) assays and mass spectrometry
Cells in culture dish were lysed with IP buffer containing protease inhibitors and phosphatase inhibitor cocktail (Topscience, Shanghai, China). IP buffer was a mixture of 1 mM EDTA, 20 mM HEPES, 150 mM NaCl, 0.05% sodium deoxycholate and 0.05% NP-40. Lysate was spun at the speed of 12,000 rpm at 4 ℃ for 12 minutes. 60 μL was kept for input and the rest of the extract was immunoprecipitated with anti-HA beads or anti-FLAG resins at 4 °C for more than 3 hours. After washing 5 times with IP buffer, beads or resins of protein bound were lysed with 1.25× SDS loading buffer and boiled at 100 °C for 10 minutes. Bands appeared after electrophoresis and silver staining in SDS-PAGE gel received mass spectrometry analysis (Wayen Biotechnologies, Shanghai, China) for protein identification. Interacted protein was further confirmed by immunoblot.
Mammosphere formation assay
Mammosphere formation assay was conducted as previously described [16]. Cells were digested by trypsin and washed by PBS twice. 300 cells suspended in 200 μL mammosphere medium were seeded in 96-well plate with ultra-low attachment surface. The formulation of mammosphere medium was shown in additional file named Table S2. 10 days later, images were photographed and mammosphere were counted.
Immunohistochemistry (IHC)
IHC was performed as previously described [17]. The results of IHC staining were determined by immunoreactive score (IRS), ranging from 0 to 12. IRS equals the number of positive cells multiplied by the staining intensity. The number of cells stained were divided into 5 groups: 0, 1-10%, 11-50%, 51-80% and 81-100%, corresponding to 0 - 4 score respectively. And staining intensity has 4 levels, negative, weak, moderate and strong, corresponding to 0 - 3 score respectively.
Immunocytochemistry (ICC)
Sterilized round coverslips were laid in a 24-well plate and cells were seeded on them. After 24 hours incubation, cells were fixed with 4% paraformaldehyde for 30 minutes and permeabilized on ice with 0.25% Triton X-100. Then cells were blocked with BSA and incubated with primary antibodies for 2 hours and fluorescent secondary antibodies protecting from light for 1 hour at room temperature. After mounting with DAPI Fluoromount mounting medium for 15 minutes, images were captured using Leica confocal system.
Xenograft experiments
5×106 cells were subcutaneously injected into four-week-old male BALB/c nude mice. 5 days later, mice were randomly divided into groups and treated with oxaliplatin (5mg/kg) or 5% glucose solution by intraperitoneal injection, twice per week for three weeks, respectively. All mice were sacrificed and tumors were collected and weighed. Tumor volume equaled length × width^2 × 0.5 and was measured twice a week. Xenografts were saved in 4% paraformaldehyde for following experiments. Tumor weight and volume were presented as means ± standard deviation (SD). Apoptosis rates in the xenograft were determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technology. Xenograft experiments were approved by the Committee on Animals Handling of Fudan University Shanghai Cancer Center.
Statistical analysis
Data are shown in mean ± SD. All analyses were performed by IBM SPSS 22.0 software. Quantitative variables were analyzed using Student’s t-test. The Log-rank test in the Kaplan-Meier method and the Cox regression model were used to assess patients’ survival outcome and prognostic factors. All the experiments were performed in triplicate. A two-tailed value of P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001).