Cell line and cell culture
The murine CRC cell line CT26 was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in a humidified incubator with 5% CO2 at 37 °C.
Animals
Female BALB/c mice (6–8 weeks, 18–22 g) were purchased from the Animal Center of Yangzhou University (Yangzhou, China) and the Animal Center of Jiangsu University (Zhenjiang, China). Mice were housed in cages and bred in pathogen-free rooms with a temperature of 23 °C ± 2 °C and relative humidity of 55%±10%. All animal experiments complied with the protocol of Jiangsu University Animal Ethics and Experimentation Committee.
CRC patient tumor tissue and frozen section
The CRC patient tissue was obtained from Department of General Surgery, Affiliated Hospital of Jiangsu University (Zhenjiang, China) and the CRC tissue and adjacent normal tissue frozen sections were obtained from the Department of Pathology, Jiangsu Cancer Hospital (Nanjing, China).
Construction of the CRC model and the CT26 cell transplantation model
To construct the CRC model, mice (6-weeks-old) received a single intraperitoneal injection of AOM (Ray Biotech, Guangzhou, China) at a dose of 10 mg/kg body weight. At 7 days after the AOM injection, mice were administered drinking water with 2% DSS (Novus, Centennial, USA) for 7 days, followed by DSS-free drinking water for 14 days. After this 21-day treatment was repeated for five cycles, the CRC mice model were sacrificed and tumor tissues were harvested for subsequent analysis.
For the CT26 transplantation model, 1 × 106 CT26 cells were implanted in mice by subcutaneous injection. Five days after CT26 transplantation, anti-CD90.2 (Biolegend, CA, USA), anti-IL-9 (Biolegend), or IL-9 (Peprotech, Rocky Hill, USA) were administrated to the CT26 cell-bearing mice to intervene the tumor growth through tail vein injection. The tumor length (L), diameter and width (W) were tested with a vernier calipers, and tumor volumes were calculated using the formula V = π × L × W2/6.
Isolation of CD8+ T cells and ILC2s
Murine CD8+ T cells were isolated from tumor tissue using the tumor infiltration CD8+ T-cell magnetic bead sorting kit (Miltenyi Biotec, Belgish-Gladbach, Germany). Cell purity was confirmed by flow cytometry (96%). Sorted CD8+ T cells were cultured in RPMI 1640 medium supplemented with 10% FBS (Gibco, NY, USA), 2 µg/ml anti-CD3 and anti-CD28 (Biolegend), 10 U/ml penicillin and 10 U/ml streptomycin (Gibco).
ILC2s were sorted from murine spleen by a combination of microbeads and flow cytometry. Briefly, the murine splenic cell suspension was incubated with biotin-binding antibodies (anti-CD21/CD35, anti-Ter-119, anti-CD45R, anti-CD3, anti-CD11b, and anti-CD49; Miltenyi Biotec) to enrich lineage negative cells. The enriched lineage negative cells were further stained with FITC-lineage, PE-CD90.2 and APC-KLRG1 (Biolegend) and then sorted by flow cytometry. The purity of the sorted ILC2s was approximately 90% and the sorted ILC2s were cultured in RPMI 1640 medium supplemented with 10% FBS, 10 ng/ml IL-7, 10 ng/ml IL-33 (Peprotech), 10 U/ml penicillin and 10 U/ml streptomycin.
Flow cytometry analysis
Tumor tissues were minced and incubated in digestion solution containing 0.5 mg/ml collagenase V, 0.2 mg/ml hyaluronidase and 0.015 mg/ml DNase I (Sigma, St. Louis, USA) in a 37 °C water bath for 1 h. The samples were then filtered through a 70 µm strainer before staining with fluorescence antibodies. Mouse Percy5-CD45, FITC-lineage, PE-CD90.2, and APC-KLRG1 (Biolegend) were used to analyze the percentage of ILC2s in the mouse tumor tissue and human Percy5-CD45, FITC-lineage, PE-CRTH2, and APC-CD127 (Biolegend) were used to analyze the percentage of ILC2s in the human tumor tissue. Mouse CD8 cells were stained with Percy5-CD45, FITC-CD3, and PE-CD8 (Biolegend). For analyzing the percentage of MDSCs, FITC-CD11b and APC-Gr-1 (Biolegend) were used. All surface stained samples were kept at 4 °C for 30 min.
For intracellular staining of IL-9, tumor tissue single-cell suspensions were first stimulated with 50 ng/mL phorbol myristate acetate (PMA), 1 µg/mL ionomycin, and 2 µg/mL monensin (eBioscience, San Diego, USA) for 6 h. Samples were fixed, permeabilized, and stained with IL-9 antibody on a shaker at 4 °C for 45 min.
The apoptosis kit FITC-Annexin V and APC-7-AAD (Multisciences, Hangzhou, China) was used following the manufacturer’s protocols to analyze the apoptosis of CT26 cells.
Immunofluorescence analysis
The frozen sections were incubated at room temperature for 1 hour and treated with 4% paraformaldehyde for 20 min. Sections were then washed three times with 1 × PBS and incubated with primary antibodies anti-lineage and anti-CRTH2 at a dilution of 1:200 in blocking buffer (PBS with 5% BSA, 1% saponin and 1% Triton 100) at 4 °C overnight. The sections were then washed three times in 1 × PBS and incubated with DAPI (1:1000; eBioscience) for 30 min, followed by three more washes in 1 × PBS. Neutral resin was used to seal the sections, which were left to air dry. Sections were then visualized directly with a confocal microscope.
Enzyme-linked immunosorbent assay (ELISA)
IFN-γ, IL-5, IL-9 and IL-13 in mouse serum and IFN-γ in culture supernatant were measured using an ELISA kit (MultiSciences) following the manufacturer’s protocols. All samples were measured in triplicate and the mean concentration was calculated from the standard curve.
Cell proliferation assays
IL-9-stimulated CT26 cells were cultured in 96-well plates (8 × 103 cells/well) in 5% CO2 at 37 °C for 24 h. The proliferation ability of CT26 cells was detected using a MTT proliferation assay kit (MultiSciences) as recommended by the manufacturer.
Migration assay
For the tumor migration assay, 2 × 105 IL-9-stimulated CD8+ T cells were co-cultured with 1 × 105 CT26 cells in wells of a 24-well plate for 24 h. Adherent CT26 cells were collected and 2 × 104 cells in 0.2 ml of serum-free medium were added to the upper chamber of a transwell system; the lower chamber was filled with 0.6 ml serum-containing culture medium. The plate was incubated in 5% CO2 at 37 °C for 20 h, and then the chamber was removed and cells were fixed with 4% paraformaldehyde for 15 min and stained with purple crystal for 10 min. Migration ability was quantified using direct microscopic visualization and cell counting.
Gene Expression Profiling Interactive Analysis (GEPIA)
GEPIA was used to analyze the expression of CXCL16 and CCL25 genes in the CRC tissue and adjacent normal tissue.
Statistical analysis
GraphPad Prism Version 7 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to analyze all the data. The data are expressed as the mean ± SD. Comparisons between groups were assessed by unpaired Student's t-test or analysis of variance (ANOVA). A P value less than 0.05 was considered to indicate a statistically significant difference.