Patients and specimens
A total of 63 CRC samples and 20 normal colorectal tissues were collected from patients who were diagnosed as primary CRC by histopathology and underwent curative resection at the Zhongnan Hospital of Wuhan University (Wuhan, China) between January 2013 and April 2015. No patients had received adjuvant preoperative chemotherapy or radiotherapy. Moreover, all enrolled patients had available clinicopathological characteristics information and follow-up data. Formalin-fixed, paraffin-embedded tumor specimens were prepared after surgical resection. Informed consents were obtained from these patients. The study protocol was approved by the research ethics committee of Zhongnan Hospital of Wuhan University.
The Paraffin-embedded specimens were sectioned at 3μm thickness. After dewaxing, antigen retrieval was made in the microwave of citrate buffer. The slices were then blocked with 3% BSA and stained overnight at 4°C with the primary antibodies against human Wnt5a (1:100, Abcam), CD68 (1:100, Abcam), HLA-DR (1:100, Abcam), CD163 (1: 100, Abcam), IL-10 (1:100, Abcam). Subsequently, specimen sections were incubated with fluorochrome-conjugated secondary antibodies. Nucleus staining was performed using Diaminophenylindole (DAPI).
Before immunofluorescence staining, all samples were stained with hematoxylin and eosin (H&E) to ensure that all specimens contained representative CRC tissue. For each section, at least 6 fields of view (at 200× magnification) were used to calculate positive cells stained with indicated antibody. A quantitative analysis was performed to determine the numbers of infiltrating CD68+ and Wnt5a+CD68+ cells. All of the above-mentioned cells were counted manually. Only cells with macrophage-like morphology were included. Cell counting was carried out in a blinded manner by two research fellows familiar with histomorphology and immunofluorescence staining. If there was a disagreement, the analyses would be discussed with the authors.
Cell culture and reagents
Human CRC cell lines (SW480, HT29, HCT116, DLD-1) were obtained from the cell bank of Chinese Academy of Sciences in Shanghai. Human monocyte cell line THP-1 was purchased from ATCC. Cells were cultured in RPMI 1640 medium (Hyclone) with 10% heat-inactivated fetal bovine serum (Wisent) and incubated in a humidified atmosphere with 5% CO2 at a constant temperature of 37°C. Human recombinant Wnt5a and IL-10 protein were purchased from R&D Systems and were used at the final concentration of 500ng/ml and 50 ng/ml respectively. Box5 (Wnt5a inhibitor, Merck, USA), U0126 (p-ERK inhibitor, Merck, USA), S3I-201 (p-STAT3 inhibitor, Merck, USA), CK59 (p-CaMKII inhibitor, Merck, USA) were used at 100μM, 20ng/ml, 100μM, 50μM, respectively. Human neutralizing IL-10 antibody and isotype IgG were purchased from Biolegend, USA.
Macrophage generation and differentiation
THP-1 cells were first treated with 100 ng/ml PMA (Sigma-Aldrich, USA) for 24h to generate THP-1 macrophages (M0 macrophages), followed by induction with IL-4 (50 ng/ml, R&D Systems)/IL-13 (20 ng/ml, R&D Systems) or LPS (100 ng/ml, Absin)/ IFN-γ (20 ng/ml, Absin) for 48h to differentiate into M2 or M1 macrophages. To mimic TAMs formation, M0 macrophages were co-cultured with colorectal cancer cells (HCT116 or DLD-1) in a 6-well transwell co-cultivation system (0.4μm pore size, Corning, USA). After 48h, the co-cultured macrophages were collected to obtain TAMs.
The level of Wnt5a and IL-10 was estimated in different culture media by an ELISA kit (R&D Systems) according to the manufacturer’s instructions. The presented value represented the average of 3 independent experiments.
Macrophages were harvested from 6-well plates, washed twice with PBS, made into single-cell suspensions, and then incubated with antibodies (FITC Mouse anti-Human CD68, PE Mouse Anti-human CD163, APC Mouse anti-Human HLA-DR, all from BD Biosciences, USA) for 60 min at 4°C. The stained cells were then washed twice and resuspended in the 200μl flow buffer for analysis on a FACS Calibur flow cytometer (BD Biosciences, USA) using FlowJo software (FlowJo, USA).
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNAs were extracted using the Trizol Reagent (Invitrogen, USA) according to the manufacturer's instructions. Then 2ug RNA was reverse transcribed to synthesize cDNA using the Primescript™ RT reagent Kit (Vazyme, Nanjing, China), followed by RT-qPCR using the SYBR-Green PCR Master Mix (Vazyme, Nanjing, China), which was performed on the BIO-RAD CFX96 Real-time PCR machine. The relative expression level of mRNA was computed by using 2-ΔΔCt method. The sequences of all primers are listed in Table S2.
Total cellular proteins were extracted using RIPA lysis reagent (Aspen) containing a protease inhibitor cocktail (Aspen) with or without a phosphatase inhibitor (Bio-swamp). The protein concentration was measured by BCA protein assay reagent kit (Aspen) and mixed with 4 × SDS loading buffer (Bio-swamp) for denaturation in a 100°C boiling water bath for 8 min. The proteins were then separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Merck). The PVDF membranes were blocked with 5% skim milk for 90 min, washed three times, and then incubated with primary antibody at 4°C overnight. Thereafter, the PVDF membranes were washed three times for 30 min and incubated with HRP-conjugated secondary antibodies for 90 min at room temperature. Finally, the membranes were again washed five times for 30min, exposed to ECL developer (Aspen) and analyzed by Bio-Rad Image Lab software. The following primary antibodies used in this study were listed: Wnt5a (1:1000, Abcam), ERK1/2 (1:1000, Protein Tech), p-ERK1/2 ( 1:1000, CST), STAT3 (1:1000, Protein Tech), p-STAT3 (1:1000, CST), NF-κB p65 (1:1000, Protein Tech), p-NF-κB p65 (1:1000, CST), CaMKII (1:1000, Protein Tech), p-CaMKII ( 1:1000, CST), GAPDH (1:5000, Protein Tech), α-Tubulin (1:5000, Protein Tech).
Lentiviral-mediated shRNA interference was performed as described previously(26). Wnt5a expression was knocked down in SW480 or TAMs by transduction of a lentiviral vector expressing a short hairpin RNA–Wnt5a shRNA (sh-Wnt5a) or negative control shRNA (sh-NC) (Genechem, China). Transfections were performed using X-treme GENE HP reagent (Roche, USA) according to the manufacturer's instructions.
CCK-8 and clone formation assay
Cell viability was estimated using a CCK-8 reagent kit (Vazyme, Nanjing, China). 5000 CRC cells were planted in 96-well plates and cultured for indicated time, followed by addition of CCK-8 solution. Then, the spectral absorbance in each well was quantified at 450 nm using a microplate reader (Molecular Devices, USA). For colony formation detection, co-cultured HCT116 or DLD-1 cells were washed, digested, counted and seeded with 500 cells per well in a 6-well plate. After 2 weeks, colonies were fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet (Sigma). Each clone that had more than 50 cells was counted. All experiments were performed in triplicate.
Transwell migration and invasion assay
The experiments were performed using a 24-well transwell system (8μm pore size; Corning, USA). For the migration assay, co-cultured 5 × 104 HCT116 or DLD-1 cells were suspended in 600 μl RPMI 1640 containing 1% FBS and seeded into the upper chamber, while 700 μl RPMI 1640 containing 10% FBS was added to the lower chamber. After incubation for 48h, the chamber was fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet. Cells in the upper chamber were removed with a cotton swab. Cells in 4 randomly microscopic fields (at ×100 magnification) were counted and photographed. For the invasion assay, the upper chamber was coated with Matrigel (BD Biosciences, USA) before inoculation. The remaining steps were the same as the migration assay. Each experiment was performed in triplicates.
In vivo experiments
Twenty-four BALB/c female nude mice (6-8 weeks old and 20-25 g) were purchased from the Hubei Research Center of Laboratory Animals (Wuhan, China). All animal experiments were conducted strictly in compliance with the guidelines of the Institutional Animal Care and Use Committee of the Zhongnan Hospital of Wuhan University. Nude mice were randomly divided into 4 groups (n=6 per group). HCT116 cells alone (5 × 105), TAMs alone (1 × 105), HCT116 cells (5 × 105) + sh-NC TAMs (1 × 105), HCT116 cells (5 × 105) + sh-Wnt5a TAMs (1 × 105) were separately mixed, resuspended with 200ul PBS, and then subcutaneously injected into the right flank of mice. After 10 days, the size of the tumors was measured with digital vernier calipers once a week. The volume of the tumors was calculated according to the formula: volume = (length × width2)/2. After 45 days of cells inoculation, 1ml of blood per mouse was harvested by cardiac puncture into EDTA-containing tubes, and then nude mice were sacrificed. Tumor tissues were collected and measured for weight and volume, followed for further analysis by RT-qPCR assay or IHC staining. In addition, liver and lung tissues of mice were collected to further evaluate the metastatic burden by H&E staining.
The sample sections were subjected to dewaxing, antigen retrieval, incubation with 3% hydrogen peroxide solution and 3% BSA blocking. Slides were subsequently incubated with primary antibodies against human Wnt5a (1:100, Abcam), CD163 (1:100, Abcam), IL-10 (1:100, Abcam), Ki67(1:500, Abcam) at 4°C overnight, followed by incubation with HRP-labeled secondary antibodies. Immunoreactions were visualized using enzymatic Avidin-Biotin Complex (ABC)-diaminobenzidine (DAB).
Circulating tumor cells (CTCs) were captured, isolated, and identified by a novel CTCBIOPSY method as reported by our previous study(8, 9, 27). Briefly, 1ml of blood of mice was first diluted with 4 ml of 0.9% physiological saline and then totally transferred to CTCBIOPSY tubes with an 8-μm diameter aperture membrane. The diluted blood samples were filtered by positive pressure through the membrane, where CTCs were captured. CTCs on the membrane were stained with the primary antibodies (CK, CD45, Hoechst, all from Abcam) and then counted under a microscope (BX51-Olympus, Japan).
All results were presented as means ± SEM from at least three independent experiments. Statistical analyses were performed using SPSS statistical software (version 19.0, IBM, USA). Means of continuous outcome variables were appropriately tested with one-way analyses of variance or two-tailed Student's t-test. The clinical feature correlation analysis was performed using chi-squared test. Kaplan–Meier curves were applied to survival analysis, and log-rank test was used to assess differences in different subgroups of patients. Univariate and multivariate Cox-regression analyses were performed to determine independent prognostic factors. Differences were considered statistically significant when p values <0.05.