Bioinformatics Analysis
A bioinformatics analysis was conducted by the TCGA database (https://www.cancer.gov/) and the TCGA-based visualization website GEPIA (http://gepia.cancer-pku.cn/).The expression correlation of PD-L1 and IL-10 genes of LIHC was analyzed. The analyses also included the relationship between IL-10 and overall survival (OS) or disease-free survival (DFS) .
Clinical samples
One hundred patients that comprised 55 males and 45 females, with liver hepatocellular carcinoma (LIHC) in the Third Affiliated Hospital of Soochow University from September 2013 to September 2018 were included in the study, which was approved by the Hospital Ethical Review Committee. No patients were treated with any relevant chemotherapy, radiotherapy, targeted therapy, or immunotherapy before surgery. Among 100 LIHC surgical resections, 50 cases were highly differentiated and 50 cases moderately or poorly differentiated. From each patient, three specimens were collected from liver hepatocellular carcinoma tissues, adjacent tissues to the tumor, and normal tissues. The specimens were confirmed by pathological diagnosis as LIHC. A portion of the tissues was embedded in paraffin and another part was freshly resected surgical tissues, and stored in liquid nitrogen for further experiments.
Patient follow-up
Patients were instructed to return for follow-up, including a clinical examination, thoracic computed tomography, and abdominal ultrasonography, at 3-6 month intervals for one year, and then every 6-12 months thereafter.
Immunochemistry
The paraffin-embedded tumor samples were sectioned to 5 μm slices. The tissue section slides were deparaffinized and rehydrated. For PD-L1, IL-10 and CD8 were stained by corresponding antibodies. Detailed experimental processes can be seen in the supplementary materials. The cell intensity and the rate of positive cells were recorded (Table S1). For data analysis, the final scores of less than eight were defined as low expression and scores of eight or more, were defined as high expression.
Quantitative RT-PCR
The tissue pieces were first cut into small sections, and then a homogenizer was used to extract the total RNA using the Trizol method. We compared the differences in IL-10, PD-L1, and Met mRNA expression among cancerous tissues, adjacent tissues, and normal tissues by quantitative RT-PCR using the described primers (please see Table S2). Detailed experimental processes can be seen in the supplementary materials.
Cell culture
Human liver hepatocellular carcinoma Bel7405 and MHCC-97-H cell-lines were obtained from the Cell Research Center, Third Affiliated Hospital of Soochow University for in vitro studies. Detailed culture conditions can be found in the supplementary materials.
SiRNA construction of liver hepatoellular carcinoma cell-lines
We used products of obtained from the Gene Pharma Company to build siMET and siPD-L1. Detailed experimental process can be seen in the supplementary materials. SiRNA sequences are listed in Table S3.
Over-expression of PD-L1
The hepatocellular carcinoma cell lines over-expressing PD-L1(Lentivirus-mediated) is from Cell Research Center ,the Third Affiliated Hospital of Soochow University.In the article, it is abbreviated as LV-PD-L1.
ELISA
One day before drug treatment, cells in logarithmic growth phase were removed and seeded on a six-well plate at a density of 3×104-105 per well.When the cells reach 70% to 80% confluency, add different concentrations of IL10(Creative BioMart,New York,USA) or anti-IL10 antibody (Creative Biolabs,New York,USA)to the cells.When the cells were cultured for 24 to 48 hours, the cells were counted by a hemocytometer to ensure that the number of cells per well was approximately the same.PD-L1 quantikine elisa kit(R&D systems,Minneapolis,MN,USA) was used to test the PD-L1 expression level in cell culture supernate or cell lynate of after 24 hours and 48h.
At the same time, After successful transfection of si PD-L1 Bel7405,si PD-L1 MHCC-97H, and LV PD-L1 Bel-7405, LV PD-L1 MHCC-97H cell culture supernatant was taken to test the IL-10 expression level after 24,48 hours.Human IL-10 high sensitivity Elisa kit(Multi Sciences,Hangzhou,Zhejiang,China) was used to test the IL-10 expression level.
CCK8
We used the CCK8 assay (Dojindo, Tokyo, Japan) to detect the proliferation of cells after treatment with Crizotinib (Cell Signaling Technology, Danvers, MA, USA) and Crizotinib combined with IL-10. Lastly, we added Crizotinib or Crizotinib combined with IL-10 to the constructed siMet Bel7405 and siMet MHCC-97-H. The proliferation of cells was detected by CCK8. Detailed experimental processes can be seen in the supplementary materials.
Transwell Test
Analysis of tumor cell migration and invasion was performed by a transwell chamber assay (Corning, New York, USA). About 2-5×103 cells,Bel7405 or siMet-Bel7405 cells and MHCC-97H or siMet-MHCC-97H cells, were seeded onto the upper chamber in serum-free medium, and then 500 µl of medium containing 1% FBS was added to the lower chamber. Crizotinib or crizotinib combined with IL-10 were added to the lower chamber. After 24 or 48 h incubation, the cells were fixed by incubating in 4% formaldehyde at room temperature for 5 min. After staining with crystal violet, the number of cells that migrated or invaded were observed under the light microscope.
Western blots
PD-L1 expression levels, Met signaling pathway-related molecules (Met and phospho-Met) and their downstream MAPK signaling pathway-related molecules (akt, phospho-akt, Mek, phospho-Mek, Erk, phospho-Erk) were analyzed by Western blotting. Meanwhile, we performed Western immunoblotting on LIHC cells that over-expressed or knocked-down PD-L1 gene expression to detect differences in IL-10 expression levels.
After that, Crizotinib or Crizotinib combined with IL-10 were added to the cells. Met signaling pathway-related molecules (Met and phospho-Met) were analyzed. In our tissue experiments, the tissue pieces were first cut into small sections, and then a homogenizer was used to extract tissue protein using the protein extraction agent. We compared the differences in IL-10, PD-L1, and Met expression among cancerous tissues, adjacent tissues, and normal tissues. Detailed experimental processes can be seen in the supplementary materials.
Statistical Methodology
Quantitative data were presented as mean ± SD. Comparison of the differences between the two groups of data used the X2 test. Kaplan-Meier curves were generated for OS and DFS. SPSS version 25.0 statistical software was used to assess Cox regression analyses. Alpha values of P <0.05 were considered statistically significant. All data was analyzed by SPSS version 25.0 or Graphpad 5.0 software. Western blotting gray values were analyzed by Image J software.