Patients and samples
This study was approved by the Ethics Committee of Nanfang Hospital (Guangzhou, China). Fresh tissue samples were collected from Department of Pathology, Nanfang Hospital. All patients given informed consents. None of these patients received chemotherapy or radiotherapy before operation.
Murine CRC cell lines (MC38, CT26), murine melanoma cell lines (B16F10) and murine breast cancer cell line (4T1) were kindly gifted by W. Yang of Southern Medical University. All cell lines were cultured in DMEM medium with 10% fetal bovine serum (Gibco), containing at 37 °C, in the atmosphere of 5% CO2. In addition, mycoplasma contamination was detected via PCR technique.
Crypt isolation and organoid culture
Mouse intestinal crypts were dissociated and cultured according to the protocols of Hans Clever . We made a few modifications in isolation. Isolated small intestines from C57BL/6 mice were opened longitudinally, and rinsed with cold phosphate-buffered saline (PBS) until without any visible debris. The tissue was cut into 5 mm pieces and was collected in a 50 ml conical centrifuge tube containing 30 mL shaking buffer (1 unit/ml of penicillin, 1 μg/ml of streptomycin, and 2.5 ng/ml of amphotericin B in PBS). The tube was swung on a tube rotator (QB-208, Kylin-Bell) at 80 rpm for 10 min (4°C). Discarded supernatant, segments were rinsed with 5 ml cold shaking buffer. Repeated the previous steps. Intestinal segments were changed to 30 ml EDTA chelation buffer (15 mM EDTA in shaking buffer), shaken on a tube rotator for 30 min, and then shocked vigorously for 30 sec on VORTEX-5 (Kylin-Bell). The supernatant with villous debris was discarded; the sediment was resuspended with shaking buffer and 2 min oscillated shakily. Dissociated intestinal crypts were filtered through 70 mm strainers. Collected supernatant were rotated at 200 g for 10 min at 4°C to collect intestinal crypts. Sedimentation were resuspended in intestinal organoid growth medium, counted, and embedded in Matrigel (growth factor reduced, phenol red free; BD Biosciences) on ice, and planted 50 μl/well in 24-well plates (100 crypts / well) for 30 minutes at 37°C, and overlaid with 500 ul intestinal organoid growth medium. Intestinal organoid growth medium, also called ENR culture medium, it contains DMEM/F12(Invitrogen), 2 mM Glutamax, 10 mM HEPES, 100 U/ml penicillin, 100 μg/mL streptomycin (Invitrogen), 1 mM N-acetyl cysteine (Sigma), B27 supplement (Invitrogen), N2 supplement (Invitrogen), 50 ng/ml mouse EGF (Peprotech), 100 ng/ml mouse Noggin (Peprotech) and 100 ng/mL R-spondin-1 (R&D Systems) or 10% human R-spondin-1-conditioned medium from R-spondin-1-transfected HEK 293T cells).
Hematoxylin eosin & Immunofluorescence staining
The fixed colon tissue was rolled into a so-called “Swiss roll” and dehydrated. A thickness of 2.5 μm Paraffin-embedded sections were obtained and stained with H&E solution. For immunofluorescence staining, frozen colonic tissues and subcutaneous tumors were used to obtain 5 μm sections for subsequent staining. Frozen sections were incubated with anti-CD8 (1:200, #100716, Biolegend) and PDL-1(1:50, #13684S, Cell Signaling Technology). Followed by secondary antibodies and fluorescent reagents goat anti-rat IgG with fluorescein-Cy3 (Perkin Elmer). Nucleus were highlighted using DAPI. Positive expression was evaluated under inverted confocal microscope (LSM880, Olympus).
Total RNA extraction and real-time quantitative PCR
Total RNA of scraped colonic mucosa or cultured organoid/ cells were isolated from Trizol reagent (TaKaRa, Dalian China) following manufacturer's instruction. cDNA synthesis was performed according to the instruction of PrimeScript™ RT reagent Kit (TaKaRa, Dalian China). qRT-PCR was carried out using SYBR Premix Ex Taq™ II (TaKaRa, Dalian China) and 7500-fast instrument (Applied BioSystems). Data were normalized to the mean Ct values of housekeeping gene GAPDH and presented as 2-ΔΔCt.
Cultured cells or organoids were lysed with SDS lysis buffer (KeyGEN, Jiangsu, China). Equal amounts of protein extracts were separated by electrophoresis in 10%SDS-PAGE gel and then transferred to PVDF membrane (Merck Millipore, MA, US). After 5% fully Skimmed milk blocking, the PVDF membrane were incubated with the primary antibody anti-GAPDH (1:1000, #60004-1-Ig, Proteintech), anti-Phospho-STAT3(1:300, #9145S, Cell Signaling Technology), anti-STAT3 (1:100, #124H6, Cell Signaling Technology), anti-Phospho-STAT1 (1:100, #9167S, Cell Signaling Technology), anti-STAT1 (1:500, #9172S, Cell Signaling Technology) and anti-IRF1 (1:500, #8478S, Cell Signaling Technology). Signals were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies and Super Signal West Femto Chemiluminescent Substrate (34096, Thermo Fisher Scientific). Images were captured and analyzed using the Image Lab Software (Tanon 5200).
Female and male C57BL/6, BAL b/c (Guangdong Medical Laboratory Animal Center) and IL-6-/- mice (Jackson) were used at 8–12 weeks of age. Mice were housed in pathogen free environments and were allowed for free access to water and food. Animal related research protocols were conformed to the U.S. Public Health Service Policy on Use of Laboratory Animals. All treatments described were approved by the Ethics Committee on Use and Care of Animals of Southern Medical University.
Chemically-induced colorectal cancer model was achieved by a single intraperitoneal injection of 10 mg/ kg azoxymethane (AOM, Sigma-Aldrich,) followed by addition of dextran sulfate sodium salt (DSS，MP Biomedical, M.W. 36,000-50,000 kDa) to the drinking water at a concentration of 2.5% for 5 days (first DSS cycle). This was followed by 10 days of regular sterile water for recovery, and this DSS cycle was repeated twice. The mice were sacrificed by cervical dislocation on day 100 and the colons were removed.
For colitis induction, mice were supplied with 2% of DSS dissolved in distilled drinking water for 5 days, followed by 5 days of regular drinking water, and sacrificed for colon tissue on day 21. Histopathological analyses and PR-PCR were performed according to standard methods.
Subcutaneous tumor models
MC38 cells (1×106) suspended in 200 µl PBS were injected respectively subcutaneously into the right hind limb of 8-12 week male wild type (WT) C57BL/6 mice and IL-6-/- mice (n = 6 each group).
MC38 cells (1×106), CT26 cells (1×106), B16F10 (5×105) cell and 4T1 (1×106) were resuspended in 200 µl PBS and implanted subcutaneously into the flank of 8-12 week male C57BL/6 mice for MC38 and B16F10 and BALB/c for CT26 and 4T1 (n = 8 each group).
Tumor size was measured every day with a Vernier caliper, and tumor volume was counted as 0.5×L×W2 (cm3; L stands for length and W for the width of the tumor).
Mice were killed at indicated days after injection. Tumors were detached, measured and recorded. Each tumor divided into two part. One part was made into frozen section for further immunofluorescence assessment and other was fixed with formalin and embedded in paraffin for HE.
Treatment of mice with anti-IL-6 and anti-PD-1
Treatment with IL-6 antibodies (#BE0046, BioXcell) or/and PD-1 antibodies (#BE0146, BioXcell) was started when the tumor volume in mice reached 100 mm3, while the control group injected isotype (BE0089, BioXcell ). The treatments were administered by intraperitoneal injection, 100 μg per mouse every three day until sacrifice.
Mice were anesthetized by inhaling 1.5 % isoflurane (RWD Life Science Co, Ltd, Shenzhen, China). Optical colonoscopy was performed using a Karl Storz (Tuttlingen, Germany) Image 1 HD Camera System. After AOM/DSS induction or DSS-induced model, colitis or tumor were monitor and scored by endoscopy as described previously.
Data were all summarized with mean ± standard error of mean (SEM) unless stated otherwise. The unpaired two-tail student’s t-test was used for experiments where two means were compared, unless indicated. For three or more groups with two parameters, one-way or two-way analysis of variance (ANOVA) was used. Datasets used for IL-6 expression paired comparison and survival analysis in R (version 4.0.3) were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GSE20916, GSE17538, and GSE41258). Survival analysis was performed using “survminer” package. In brief, “surv_cutpoint” function to find optimal cutpoint and log-rank P value based on IL-6 expression, and “survfit” function was used to fit overall survival time and status. Statistical analyses were performed using GraphPad Prism software 5.0 (CA, US) and SPSS software (Version 22.0, IL, US).