5.1 Cell culture
Mouse colon adenocarcinoma (CT-26) cells were cultured in RPMI-1640 containing 10% fetal bovine serum in a 37°C, 5% CO2 cell culture incubator. When cell fusion reached 80%~90%, cells were digested using 0.25% trypsin digestion solution for passaging or freezing.
5.2 Plasmid construction and verification
The shControl plasmid, shSTAT3 plasmid, shPD-L1 plasmid, and shSTAT3/shPD-L1 recombinant plasmid containing PLKO backbone were designed and synthesized by settlebio, and the plasmids contain ampicillin (Amp) resistance gene for exclusion of heterobacterial interference. The four plasmids were transfected into CT26 cells individually by lipo3000. After successful transfection, the cells were incubated in the incubator for 4 ~ 6 h, then replaced with culture medium and continued to incubate for 48 h. Cells were collected for qPCR and Western Blot assay.
5.3 Electroporation of attenuated Salmonella
After preparing electrocompetent attenuated Salmonella phoP/phoQ mutant strains, the electrocompetent bacteria was performed by adjusting the parameters of the electrotransformer. The electrocompetent products were sequenced by Sangon to detect the presence of the target gene fragment. The plates were coated with the successfully electrocompetent products by electroporation and incubated overnight at 37°C. Pick a monoclonal colony into LB medium (Amp) and continue to incubate for 14 ~ 16 h. Extract the plasmid and preserve the bacteria.
5.4 Plasmid Extraction
Plasmid extraction was performed according to the Omega Plasmid Extraction Kit instructions. The plasmid concentration was determined by UV spectrophotometer and the plasmids were stored at -20°C in the refrigerator for backup.
5.5 Cell viability assay
CT26 cells were inoculated into 96-well plate seeds (7×103 cells). The cells were transfected after 24h incubation in the incubator. After transfection for 24 h, 48 h, and 72 h, 10 µL of CCK-8 solution was added to each well and incubated for 1 h. The absorbance at A450 nm was measured by enzyme marker.
5.6 qPCR assay
Cellular RNA extraction: Discard the cell culture solution from the six-well plate, add Trizol lysate, and blow repeatedly. Transfer Trizol to a new centrifuge tube and add chloroform, isopropanol, and 75% ethanol as needed. Dried and precipitated with sterile DEPC water to detect RNA concentration; Reverse transcription-PCR: Sample RNA, reverse transcription Mix (TransGen Biotech, AE341), and RNase-free water was mixed in proportion. cDNA was synthesized by reverse transcription and its concentration was detected; qPCR: The cDNA, target gene primers, qPCR Mix (TransGen Biotech, AE341) and RNase-free water were mixed in proportion and amplified against the target gene.
5.7 Western blotting analysis
Protein extraction: Collect the treated cells, add RIPA cell lysate containing 1% PMSF protease inhibitor, centrifuge the supernatant after sonication and collect the supernatant, and prepare 30µg/20µl of the upper sample protein by measuring the protein concentration according to the instructions of the BCA kit; Protein electrophoresis and detection: SDS-PAGE gel was prepared, and electrophoresis was performed after loading the sample. Methanol-activated PVDF membranes were wet-transformed under ice bath conditions and then closed with 5% skimmed milk, incubated overnight at 4°C with antibody at room temperature for 1 h. Membranes were subsequently washed with TBS-T (0.05% Tween-20) buffer and immunoreactive proteins were visualized on a chemi-luminescence developer (Chemiscope 5300; Clinx Science Instruments, Co., Ltd., Shanghai, China). The grayscale values of protein bands were analyzed using Image J software (Image J ,46r, USA).
5.8 Colony forming cell (CFC) assay
After transfecting CT26 cells with plasmids for 24h, the cells were inoculated in six-well plates(5×103) and cultured for 7 ~ 9 days. After different treatments, cells were counted and seeded in six-well plates and cultured for 14 days with different treatments. Discard the culture solution and wash and dry the well plates with PBS. Then we used 10% formaldehyde to fix colonies for 10 min and stained with 0.5% crystal violet for 30 min. Finally, cells were photographed and counted.
5.9 Cell cycle and cell apoptosis analysis
After transfecting CT26 cells with plasmids for 24 h, 1 ~ 5×105 cells were collected per group. Add reagents sequentially according to the Annexin V-FITC/PI Apoptosis Assay Kit (BD Pharmingen, MI, Shanghai, China) manufacturer’s protocols. Cells were incubated in the dark before analysis by flow cytometry.
5.10 Cell invasion assay
After transfecting CT26 cells with plasmids for 24 h, cells were seeded in the upper chamber of a 24 well transwell at a density of 1 ~ 2×105 cells per well. Cells were cultured with serum-free medium in the upper chamber, and the lower chamber contained 10% FBS. After 48 h, the culture solution was aspirated. Invaded cells on the lower surface were fixed with 600 µL of 4% paraformaldehyde for 5 min and stained with 0.1% crystal violet (Sigma-Aldrich) for 10 min. The invaded cells were then imaged and counted under a microscope. For the migration assays, the procedures were performed as for the invasion assays but the filters were not precoated with Matrigel. The cells on the upper surface of the filters were gently wiped with a cotton swab and take pictures under the microscope. The crystalline violet dye could also be eluted with 3% acetic acid, and the eluate was measured on an enzyme marker for OD (570 nm) to indirectly reflect the difference in the number of migrating cells between groups.
5.11 Cell migration
Before cells were transfected, a straight line of cells was scraped off with a 1 mL gun in the wells of a six-well plate. unadhered cells were washed and removed with PBS and photographed under the microscope. After transfected cells for 24 h, microscopic photographs were taken to observe the healing of the scratches.
5.12 Cell cycle detection by flow cytometry
After transfecting CT26 cells with plasmids for 48h, 1–5×105 cells were collected per group. The following day, the sample precipitate was collected by centrifugation (1050 rpm, 5 min). Add 100 µl of RNaseA solution to the precipitated species and resuspend the precipitate. Add 400 µL of PI staining solution and mix well, incubate for 30 min protected from light. The cell cycle was detected by a flow cytometer (FC500, Beckman Coulter, FL, USA).
5.13 Colon cancer subcutaneous tumor model
We selected CT26 cells of the logarithmic growth phase to prepare cell suspension (3×105/100 µL). The left side of the back of the mice was dehaired and the cell suspension was injected into the subcutaneous part of the mice. When the tumor grew to the size of a soybean grain, treatment was performed by intraperitoneal injection of an attenuated Salmonella phoP/phoQ mutant strain at a dose of 100 µL and a concentration of 107 CFU/ml. After 12 days, the mice were euthanized. The main organs of mice were preserved by fixation with 4% paraformaldehyde. Some of the tumor tissues were fixed with 4% paraformaldehyde and some were frozen in liquid nitrogen.
Tumor volume was calculated with the following formula: V = (L) ×(W)2/2.
5.14 Colon cancer in situ model
Each mouse was injected intraperitoneally with AOM at a dose of 10 mg/kg and fed 3% DSS water for 7 d after one week, after which it was changed to normal water for 14 d, then 1% DSS water for 7 d and normal water for 14 d. The modeling was repeated with 1% DSS water for 7 d and normal water for 14 d. Treatment was performed on days 60 and 67 after intraperitoneal injection of AOM with an attenuated Salmonella phoP/phoQ mutant at a dose of 100 µL at a concentration of 5 × 105 cfu/ml. all mice were executed on day 74 and sampled as in the subcutaneous load tumor model. All animal experiments shall comply with the rules and regulations of the Ethics Committee of Jilin University (#2021 − 289).
5.15 Quantify cytokines in mouse serum by ELISA
We took blood from the mouse eye, left it for 30 min at 4°C, and centrifuged the supernatant. Add reagents sequentially according to the ELISA test kit (Proteintech) manufacturer’s protocols. After the reaction was terminated, the absorbance was detected at 450 nm using an enzyme marker.
5.16 immunohistochemistry (IHC) staining
The tumor tissues were fixed, embedded, and sectioned, and then the sections were stained, hydrated, and epitope/antigen repaired. After closing the sections with sheep serum, the sections were incubated overnight at 4°C with primary antibody, and the next day, the sections were incubated for 1 h at 37°C with secondary antibody. After PBS washing, HRP-labeled streptavidin incubation, DAB staining, hematoxylin re-staining, 1% hydrochloric acid alcohol weathering, and tap water anti-blue were performed sequentially. After dehydration and transparency, the films were sealed and microscopically observed and photographed after being ventilated and dried.
5.17 Hematoxylin-eosin(H&E) staining
The mouse organs were fixed, embedded, and sectioned. Tissue sections were placed in a thermostat at 60°C for 60 min and stained with xylene for 15 min before being placed in the thermostat for 60 min and stained with xylene for 15 min; Tissue sections were washed with 100% ethanol, left in room temperature for 10–15 min, repeatedly washed and left once; tissue sections were hydrated with 90% and 70% ethanol gradients, left at room temperature for 5 min, and soaked in distilled water for 3 min; Hematoxylin staining for 1 min, tap water washing for 5 min, distilled water washing for 30 s, 95% ethanol soaking for 5 s, eosin staining solution for 30 s; 90% and 70% ethanol washing the sections twice each, xylene transparency for 5 min, repeat the washing and transparency steps once; seal the sections to be air-dried and observe them under the microscope for photographs.
5.18 Terminal transferase-mediated DNA end labelling (TUNEL) assay
The tumor tissues were fixed, embedded, and sectioned. Xylene staining, ethanol gradient hydration, and antigen repair were performed sequentially on the tissue sections. TUNEL staining was performed after PBS washing. After the reaction was completed, DAB staining, hematoxylin re-staining, tap water anti-blue, ethanol gradient hydration, and toluene staining were performed sequentially. After dehydration and transparency, the films were sealed, left to air dry, and microscopically observed and photographed.
5.19 Detection of immune cells in mouse spleen by flow cytometry
The spleens of euthanized mice were placed in a Petri dish containing 2 ml of PBS. The spleen was squeezed with a strainer, discarding the supernatant liquid after centrifugation, adding erythrocyte lysate, and the spleen immune cells were collected by centrifugation again. The cells were incubated on ice for 30 min with antibodies and protected from light, the supernatant was discarded by centrifugation, and the cells were resuspended in PBS and assayed. Cells were incubated on ice with antibody for 30 min, then the supernatant liquid after centrifugation was discarded and the cells were resuspended with PBS and assayed. (Treg cell surface staining as above, followed by breaking the membrane on ice for 45 min with the membrane breaking reagent and staining the nucleus with the corresponding antibody)
5.20 Statistical analysis
All data was obtained from at least three independent experiments. Statistical analyses were performed with GraphPad Prism v9.00 software (GraphPad Software, Inc., USA). Comparison of the sets of data, one-way ANOVA was conducted. and all data are expressed as the mean ± standard deviation. P < 0.05 was considered statistically different, P < 0.01 was considered statistically significantly different, and P < 0.001 was considered extremely statistically significantly different.