2.1 Cell Lines and Cell Cultures
Human HER2-positive BC cell lines SK-BR-3 and AU565 cells were maintained in DMEM medium supplemented with 10% FBS. The cells were cultured at 37℃ in a humidified incubator with 5% CO2.
2.2 Chemicals and Antibodies
Pyrotinib and SHR-1316 were obtained from Hengrui Medicine Co. Ltd. The following antibodies: PI3K,p-PI3K, AKT, p-AKT,GAPDH, FOXO1 were obtained from Cell Signaling Technology (Cell Signaling Technology, Inc, Shanghai, China).
2.3 Immunocytochemistry
Cells were seeded in cover glass at a density of 5000 cells/well and incubated for 16 hours. Cells were fixed with 4% formaldehyde solution for ICC assay. ICC was performed to detect PD-L1 expression of HER2-positive BC cells using DAB Kit. PD-L1 antibody was purchased by Cell Signaling Technology, Inc.
2.3 Cell Viability Assay
Cell viability and the cytotoxic effects of pyrotinib, SHR-1316, and pyrotinib combined with SHR-1316 against SK-BR-3 and AU565 cells were generally evaluated by MTT assay. Cells were plated in 96-well plastic culture plates in medium plates at a density of 3000–5000 cells/well and treated with PBS pyrotinib, SHR-1316, or both drugs in combination for 48 hours at 38℃. The absorbance of each well was measured at 570 nm by a microplate reader (Bio-Tek, Norcross, GA, U.S.A.). The cell viability was calculated according to the different absorbance values of each experimental group. The mean IC50 values were calculated by SPSS. Combination index (CI) values were calculated using CompuSyn (ComboSyn Inc.). GraphPad Prism 7.0 was used to plot dose-response inhibition curves.
2.4 Apoptosis Analysis
Cells (2.5×105 cells/well) were plated in 6-well plates for the apoptosis assay and treated with PBS, pyrotinib(0.5ug/ml), pyrotinib(5ug/ml), SHR-1316(0.1mg/ml), SHR-1316(1mg/ml), or pyrotinib(5ug/ml) combined with SHR-1316(1mg/ml) for 48h. Then, cells washed with PBS, adjusted to 1×106 cells/ml, treated with Annexin V-FITC/PI apoptosis detection kit based on the manufacturer’s protocol. Finally, the cells were detected by BD FACS caliber, using the BD CellQuest Pro software for analysis.
2.5 Wound Healing Assay
Wound-healing assay was used to analyze the cell migration ability. Cells were plated in 6-well plastic culture plates in medium plates at a density of 5000 cells/well and treated with PBS, pyrotinib(0.5ug/ml), pyrotinib(5ug/ml), SHR-1316(0.1mg/ml), SHR-1316(1mg/ml), or pyrotinib(5ug/ml) combined with SHR-1316(1mg/ml) in serum-free medium for 48 hours. The same observation site was photographed at 0h, 6h, 12h, 18h and 24h after treatment. The blank area of the same observation site at 0h and 24h was measured by ImageJ software (NIH, Bethesda, MA, U.S.A.), and the cell migration of different experimental groups was calculated. T test was used to analyze the difference of cell migration between the experimental groups.
2.6 Transwell Invasion Assay
The aperture of the bottom membrane of the Transwell chambers or wells (Corning Inc., Corning, NY, USA) was 8μm. The chambers were coated with Matrigel (Sigma-Aldrich, St. Louis, MO, USA), and were used for detecting the cell invasive ability. Cells were harvested and resuspended in serum-free DMEM, and 200μl of cell suspension (5 × 105 cells/ml) containing PBS,0.5μg/ml pyrotinib, 5μg/ml pyrotinib, 0.1 mg/ml SHR-1316, 1mg/ml SHR-1316, 5μg/ml pyrotinib and 1mg/ml SHR-1316. The cells were cultured in an incubator at 37˚C with 5% CO2 for 24 hours. The medium containing 20% fetal bovine serum was added to the outer chamber, and then 4% formaldehyde was added to each chamber for 1.5 hours at room temperature. Ammonium oxalate crystal violet staining was used. The Transwell chamber was placed under the microscope to take photos. The cells in the photos were counted by ImageJ software (NIH, Bethesda, MA, U.S.A.). SPSS was used to analyze the differences between the experimental groups.
2.7 Animals and tumor model
Pathogen free (SPF) BALB/c nude female mice, aged 5–6 weeks, were purchased from Changzhou Covens Experimental Animal Co. Ltd. (Changzhou, Jiangsu, China), and raised in animal facility (Temperature, 20-26°C; humidity, 40-60%; 12/12-h light/dark cycle; free access to food and water). Human peripheral mononuclear cells (PBMCs) were extracted from normal human blood, and expanded with T cells. SK-BR-3 cells mixed with PBMCs were injected subcutaneously into nude mice at a ratio of 4:1 to construct HER2-positive BC xenograft model. The experimental groups were divided into three groups: pyrotinib group (30mg/kg /d), SHR-1316 group (200 μg / d1.3.5), combined group (pyrotinib 30mg/kg /d+SHR-1316 200 μg / d1.3.5), The control group was not given any medication. The tumor volume and weight of mice were measured every 3 days. The volume calculation formula: Tumor volume = (width 2 × length) / 2. After 21 days of experiment, the mice were euthanized, tumor was stripped, and tumor size and doubling index were recorded and the statistical chart was drawn in GraphPad prism 7.0. The expression levels of CD3, CD4 and CD8 in tumor tissues of mice treated with different drugs were detected by immunocytochemistry (IHC). CD3、CD4、CD8 antibodies were obtained from Beijing T&L Biotechnology Co. Ltd. All of the animal experiments conformed to the requirements of the ethics committee.
2.8 Western Blotting
The cells were treated with PBS,0.5μg/ml pyrotinib, 5μg/ml pyrotinib, 0.1 mg/ml SHR-1316, 1mg/ml SHR-1316, 5μg/ml pyrotinib and 1mg/ml SHR-1316 for 48 h. The cells were harvested and prepared for cytosolic and nuclear protein extraction using a cytoplasmic and nuclear protein extraction kit (Cowin Bio, Beijing, China) according to the manufacturer’s instructions. Protein concentration was quantified using BCA protein assay kit (Solarbio, Beijing, China). Equal amounts of protein were separated by in 8-12% SDS-PAGE (Solarbio, Beijing, China), transferred to PVDF membrane (Millipore, Massachusetts, U.S.A), blocked with 5% skim milk, 0.1% Tween and phosphate buffered saline (PBST), then incubated overnight with 1:1,000 diluted antibodies against PD-L1, PI3K, AKT, FOXO1(Cell Signaling Technology). Next, the membranes were incubated with secondary antibodies at 37°C for 1 h. Finally, the membranes were washed with PBST and detected in Tanon 2500 chemiluminescence imaging system (Tanon, Shanghai, China). The protein band quantification was quantitated by ImageJ software (NIH, Bethesda, MA, U.S.A.).
2.9 Statistical analysis
Statistical analysis was performed using IBM SPSS 23.0 software (SPSS, Chicago, IL, USA). All the data were presented as mean value ± SD, and statistically significant differences between different experimental groups and control group were examined using one-way analysis of variance (ANOVA), P<0.05 was considered as statistically significant difference.