2.1 Cell culture
Yanbian University Cancer Research Center supplied the human TNBC cell lines (MDA-MB-231 and Hs578T), human umbilical vein endothelial cells (HUVECs), and mouse macrophages (RAW264.7). All cells were cultured in RPMI-DMEM containing 10% FBS and 1% streptomycin-penicillin (100U/mL). Cells were incubated at 37°C in an atmosphere of 5% CO2.
2.2 Flow cytometry assay
Cells (5×105) suspension was washed twice with cell staining buffer. Fix the cells with fixation buffer, and break the cell membrane with permeabilization buffer. Cells were then stained with CD68-FITC (mouse), CD206-PE (mouse), and CD86-APC (mouse) antibodies (Biolegend, CA, USA). Cells were suspended with 500µL cell staining buffer and examined with a BD Accuri C6 flow cytometer (BD Biosciences, CA, USA). All tests are controlled by homologous isotype control antibodies.
2.3 Western blot assay
Cells were collected and appropriate amount of RIPA lysate (RIPA lysate: PMSF = 100:1) was added according to the number of cells. The protein concentration was determined and quantified using BSA kit (Roche, Basel, Switzerland). Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (Millipore, Bedford, MA). The membranes were incubated with the primary antibody at 4℃ overnight and then the secondary antibody were incubated at RT for 1 h. Enhanced chemiluminescence detects antibody signals by BioRad system (Hercules, CA, USA) and collects images.
2.4 Immunofluorescence (IF) staining
Slides with cells on the 6-well plates were fixed with methanol for 15min and permeated with 0.5% Triton X-100 (CWBIO, Beijing, China) for 10 min. Then, blocked with 3% BSA (Solarbio, Beijing, China) for 2 h. Cells were incubated with primary antibody in 3% BSA at 4°C overnight and incubated with Alexa Fluor 488 Goat Anti-Rabbit IgG or Alexa Fluor® 568 goat anti-mouse IgG (1:400; Invitrogen, CA, USA) for 1 h. Cells were counterstained with DAPI and imaged by Leica SP5II confocal microscope.
ELISA was performed according to the instructions. The concentrations of IL-10 (mouse) and IL-12 (mouse) in cell culture supernatants were determined using the kit (Mlbio, Shanghai China).
2.6 MTT assay
Cells (5×103) suspension was prepared and incubated on 96-well plates for 24 h. Conditional medium or drugs were added and incubated for 0, 24, 48 and 72 h, respectively. Add 100 µL MTT reagent (1mg/mL) to each well and incubate for 4 h under the same conditions. Then, add 100 µL DMSO to each well and the absorbance value (OD) at 490 nm was measured. Five wells per group at least were analyzed and repeated three times.
2.7 Apoptosis assay
Cells (1×106) suspension was washed twice with binding buffer. Cells were stained using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, CA, USA) according to the instruction. Cells were analyzed with a BD Accuri C6 flow cytometer.
2.8 Cell morphology
RAW264.7 cells with a 30% fusion rate were incubated on 6-well plates for 12 h and added with IL-13 and/or αPD-L1 for 48 h. Morphological changes of cells were observed under a microscope and images were collected.
2.9 Conditioned Medium (CM) Preparation
RAW264.7 cells were treated with TAM/M2 inducing factor Interleukin-13 (30 ng/mL) (IL-13, Biolegend, CA, USA) and αPD-L1 (15 µg/mL) (10F.9G2 monoclonal antibody, BioXcell, SKU: BE0101) for 48 h. Serum-free medium was cultured for 24 h, and three CM (RAW264.7, IL-13/RAW264.7 and IL-13 + αPD-L1/RAW264.7) were prepared. The CM can be directly used for assays or stored at -80°C. The CM was filtered through a screen and added with 2% FBS.
2.10 Migration assay
Cells (5×104) in 100 µL containing 10% serum RPMI-DMEM were seeded onto the upper chambers. The bottom chambers were filled with culture medium containing 10% FBS RPMI-DMEM, and the cells were incubated at 37°C for 6 h. Then, the upper chambers were replaced with different groups of CM. Cells passing through the subsurface of the filtration membrane were fixed with 4% paraformaldehyde for 20 min and then stained with crystal violet for 20 min. Three fields (200×) were randomly selected under the microscope to collect images. Image J software (NIH, Bethesda, MD, USA) counted the number of cells in each field.
2.11 Wound healing assay
Cells were incubated on 6-well plates for 24 h. Scratch the wound vertically with a 200 µL pipette tip and wash three times with PBS to remove dead cells in the well. The CM (containing IL-13 and/or αPD-L1) was incubated at 37°C in an atmosphere of 5% CO2. Take pictures at 0, 12, and 24 h.
2.12 Endothelial tube formation assay
The matrigel (BD Biosciences, San Diego, CA, USA) and RPMI-DMEM were diluted 1:1 in 96-well plates and solidify at 37°C for 4 h. HUVECs (2×104) were incubated in 2:1 diluted CM and culture medium at 37°C for 4 h. Capillary structure was collected under the microscope.
2.13 Establish cell co-culture system
RAW264.7 cells (5×104) in 600 µL containing 10% serum RPMI-DMEM were seeded onto the bottom chambers. MDA-MB-231 or Hs578T cells (1×105) in 100 µL containing 10% serum RPMI-DMEM were seeded onto the upper chambers, and the cells were incubated at 37°C for 6 h. The supernatant was harvested for ELISA assay, and RAW264.7 cells were harvested for western blot and flow cytometry assays.
2.14 Animal studies
4T1 cells (5×105) were injected into subcutaneous of 5-week-old BALB/c female mice to establish tumor model. Tumor size was measured every 3 days and tumor volume was calculated using the formula length×width2×0.5. The animals were sacrificed after 25 days of αPD-L1 treatment. For detecting lung metastases, 4T1 cells (1×105) were injected into the tail vein of BALB/c female mice. The lungs were collected and the surface nodules were counted. The tumor and lung tissues were fixed with formalin and sectionalized. The expression of the markers was confirmed by IHC staining. All experiments were performed in accordance with the procedures of the Animal Ethics Committee of the Yanbian University.
2.15 Immunohistochemistry (IHC) staining
Tumor and lung tissue sections were dewaxed and dehydrated. Then, the tissue sections were repaired with citrate buffer (pH 7.0) at 80°C for 20 min. The tissue sections were incubated with 3% H2O2 at RT for 30 min. The tissue sections were incubated with primary antibody at 4 ºC overnight. After incubation with secondary antibody at RT for 1 h. Slide immunostaining was advanced using DAB and counterstained with hematoxylin.
2.16 Statistical analysis
GraphPad Prism 8.0 software (GraphPad, La Jolla, CA, USA) was used for statistical analysis. T-test was used to compare the mean values of two samples. One-way ANOVA was used to compare the mean values of multiple groups. All experimental data were repeated for three times and their mean values were taken as mean ± standard deviation. Depending on the experiment, the significance was *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. P > 0.05, was non-significant (ns).