Animals
Female BalB/c mice (6 weeks old) were obtained from the Weitonglihua Company (Beijing, China). All animals were housed in a specific pathogen free facility. The experimental protocol was approved by the Animal Care and Research Committee of Huazhong University of Science and Technology.
Colitis-associated colorectal cancer model
CAC in mice was induced as previously described. To induce CAC, each mouse was injected intraperitoneally (i.p.) with 10mg/Kg azoxymethane (AOM, Sigma-Aldrich Chemical, Germany). Seven days later, they began to receive three cycles of 2.5% dextran sodium sulfate (DSS, MP Biomedicals, USA) in drinking water for one week and two weeks of regular drinking water throughout a 10-week observation period. A group of age- and gender-matched healthy control mice received vehicle (sterile water) injection and plain drinking water. Samples (colon, spleen, PB, BM) were harvested at weeks 5,7, 8 and 10 (W5, W7, W8 and W10) of the protocol.
MyD88 inhibitor treatment
All mice were randomly divided into the normal control (NC), CAC model (CAC), and MyD88 inhibitor-treated groups (I, n=4-5 per group). The mice in the MyD88 inhibitor group were treated with 50mg/kg TJ-M2010-5 (i.p.) daily beginning two days before the first DSS administration throughout the 10-week-observation period. The mice in the control group were injected with the same volume of sterile water.
Evaluation of colorectal tumors
The entire colons of each mouse was opened longitudinally, and the numbers of tumors in each colon was counted. The paraffin-embedded colon tissue sections (4 µm) were stained with hematoxylin-eosin (HE) staining.
Preparation of single cell suspensions
Lamina propria mononuclear cells (LPMCs)
LPMCs were isolated from colonic tissues using the Lamina Propria (LP) Dissociation Kit, according to the manufacturer's instructions (Miltenyi Biotec, Germany, in Supplementary Methods).
BM cells
Femurs of mice were aseptically removed and debrided of surrounding muscle tissue. BM cells were flushed from the femur using phosphate buffer saline (PBS).
Splenocytes
The spleen of the mice were excised and sliced into small pieces. The excised pieces were pressed through a strainer, and the harvested cells were washed using PBS.
PBMCs
PBMCs were isolated from freshly obtained whole blood from mice using density gradient separation (TBD sciences, China, in Supplementary Methods).
Antibodies (Abs)
The Abs used in this study are shown in Supplementary Methods.
Flow cytometry analysis
Quantitative flow cytometric analyses were performed using standard procedures. For analysis of intracellular cytokine production, cells were permeabilized for 20 min at 4 ℃. Data acquisition was performed using FACS Celesta flow cytometer and analyzed by the FlowJo software (Version 10.0).
IHC and IF
The paraffin-embedded colon tissue sections (4 µm) were incubated with Abs. The bound Abs were detected sequentially with biotin-conjugated secondary antibody and streptavidin-HRP and visualized using a DAB Kit (Beyotime Biotec, China) for IHC. For IF, Alexa Flour 488 anti-rabbit or Cy3 anti-rat Abs (Servicebio, China) were used. The slides were mounted with DAPI (Servicebio, China) and examined under fluorescence microscope.
Magnetic beads sorting cells
CD3- splenocytes, CD11b- BM cells, CD11b+Gr-1+ MDSC and CD4+ splenic T cells were purified by magnetic bead negative/positive selection according to the manufacturers’ instructions (Miltenyi Biotec., Germany).
Western blot
Colon tissue samples were homogenized and separated on SDS-PAGE for Western blot analysis. Cell lysates from sorted CD11b+Gr-1+ splenocytes were prepared and diluted using a sample preparation kit (Protein Simple) for the automated capillary western blot system, WES System (Protein Simple, in Supplementary Methods).
RAW264.7 cell culture and stimulation
RAW 264.7 cell line was obtained from the China Center for Type Culture Collection (CCTCC #GDC143, Wuhan, Hubei). Frozen aliquots were used in the experiments within six months of culture period, after the first thawing of the cells. The cell line has been tested and authenticated. Cells were cultured in a RPMI medium 1640 (Gibco, USA). After stimulation with 100 ng/mL of LPS (Sigma-Aldrich Co., Germany) for four hours with 40 μM TJ-M2010-5 one-hour pretreatment; subsequently, cells and the supernatant in individual wells were harvested for mRNA extraction and ELISA, respectively.
Quantitative real-time polymerase chain reaction (RT-qPCR)
The total RNA was extracted from mouse colon tissues or RAW264.7 cells as described using the Trizol reagent (Invitrogen, USA). cDNA was synthesized using the ThermoFisher Kit (USA). RT-qPCR was performed using the Hieff qPCR SYBR Green Master Mix (Yeasen Biotech., China) and a StepOne System (Life Technologies, USA). Relative fold changes were determined using the ΔΔCT calculation method. Values were normalized to internal control β-actin. The sequences of the primers were given in Supplementary Methods.
ELISA
The concentrations of GM-CSF, IFN-γ, IL-1β, IL-6, and TGF-β1 in the supernatant were analyzed using ELISA, according to the manufacturers’ instructions (eBioscience, USA).
In vitro MDSC differentiation from immature myeloid cells
CD11b- BM cells flushed from the femur using PBS grown in RPMI medium supplemented with 10% heat-inactivated FBS (Yeasen Biotech., China), 1 mM sodium-pyruvate (Meilunbio, China), and 50 μM β-mercaptoethanol (Solarbio, China). Cultures were supplemented with 10 ng/mL of GM-CSF (Peprotech, USA) and 1 μg/mL LPS, and were incubated at 37 ℃ for 8 days. At the end of the culture period, the population of CD11b+Gr-1+ MDSC that differentiated from immature myeloid cells was detected by flow cytometry.
In vitro suppressive activity of MDSC on CD4+ T cell proliferation
CD11b+Gr-1+ MDSCs were obtained from splenocytes of mice with CAC with or without the MyD88-inhibitor treatment. For evaluation of MDSC suppressive activity, 2×105 CD11b+Gr-1+ MDSC and 2×105 CD4+ splenic T cells from naive BalB/c mice were cultured in flat-bottom 96-well plates in complete RPMI medium. The proliferation assay of CD4+ T cells were performed using a carboxy-fluorescein diacetate succinimidyl ester (CFSE, Invitrogen, USA) assay, according to the manufacturer’s protocol. T cells were stimulated by the addition of anti-CD3/anti-CD28-coated microbeads (Invitrogen, USA) at a bead-to-cell ratio of 1:75 for 72 hours.
Statistics
Data are expressed as the mean±standard deviation (SD). Comparisons between groups were analyzed using Student’s t-test or the log rank test. Individual differences versus various controls were assessed using one-way ANOVA. All statistical analyses were performed using the SPSS software package (version 17.0; IBM Corp, Armonk, NY, USA). Statistical significance was set to P < 0.05.