Relationship between IL-10 and PD-L1 in liver hepatocellular carcinoma tissue and cell lines CURRENT STATUS: POSTED

Background Programmed death-ligand1 (PD-L1) plays a critical role in host immunity in the setting of cancer progression. Interleukin 10 (IL-10) is a multi-cellular, multi-functional cytokine that regulates cell growth and differentiation and participates in inflammatory and immune responses. The purpose of this study was to clarify the relationship between PD-L1 and IL-10 and their clinical importance in liver hepatocellular carcinoma(LIHC). Methods LIHC patients (n=100) who underwent surgery with preoperative therapy were included in the study. By immuno-histochemical staining, PD-L1, IL-10 and CD8 positive cells were examined in resected specimens. The gene expression levels of PD-L1, IL-10 and Met were detected by qRT-PCR and Western blots, and differentially compared in cancer, adjacent and normal tissues. In cell experiments, the Bel7405 and MHCC97-H cell-lines were incubated with IL-10 or anti-IL-10 antibody, and then PD-L1 and Met expression levels were compared by ELISA and Western blots. The effect of crizotinib and/or IL-10 on the proliferation, invasion and migration of LIHC cell-lines was estimated by CCK8 and transwell assay. Results In tumor tissues, the mRNA and protein levels of PD-L1, IL-10 and Met were higher than those in adjacent tissues. The high expression levels of PD-L1 and IL-10 indicated a poor prognosis. IL-10 reduced the expression of PD-L1 in LIHC cell-lines via Met signaling pathway. Over-expression of PD-L1 in increased the levels of IL-10, and Met in in LIHC tissue and cell lines. The combination of crizotinib and IL-10 were more effective in inhibiting the proliferation, migration and invasion of LIHC cell lines. Conclusions The combination of IL-10 and PD-L1 monoclonal antibody may have therapeutic promise in treating LIHC.


Introduction
Primary liver cancer (referred to as liver cancer) is the second leading cause of death worldwide, with histological types of hepatocellular carcinoma, intrahecholangio carcinoma, hepatocytes-cholangio carcinoma, and fibrolameloid liver cancer. Liver hepatocellular carcinoma (LIHC) accounts for about 90% of primary liver cancer. The prevalence of liver cancer varies among regions, with Central and and plays an important inhibitory role in inflammatory and immunological responses. By inhibiting the activation of natural killer (NK) and T cells, IL-10 enables tumor cells to escape immune surveillance (24,25). However, IL-10 also functions in immune activation, which promotes tumor-specific immune surveillance and reduces the occurrence of pathogenic inflammatory reactions by activating T and NK cells. Therefore, IL-10 plays a key role in this two-way immune regulatory system (26)(27)(28). Some articles indicate that the expression level of PD-L1 is correlated with the expression level of IL-10 in the tumor micro-environment (29)(30)(31)(32)(33). However, the relationship between both factors remains unclear.
Mesenchymal-epithelial transition factor (Met) is a tyrosine kinase (TK) receptor for hepatocyte growth factor (HGF) (34,35) (Fig. 1A). Activation of the Met gene leads to multiple downstream pathways that promote a tumorigenic phenotype.
In our study, we found that in LIHC tissue, PD-L1 expression levels were positively correlated with IL-10 expression levels. Over-expression of PD-L1 upregulates IL-10 expression in LIHC tissue and cells lines by positive feedback regulation. IL-10 down-regulates the expression levels of PD-L1 in LIHC cells-lines by negative feedback regulation. We have also discovered that PD-L1 and IL-10 are linked by the Met signaling pathway ( Fig. 1B and C).
Thus, our study provides new insights into the functional role of IL-10. The efficacy of combined IL-10 and PD-L1 inhibitor therapy was shown to be better than use of the PD-L1 inhibitor alone.

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 sixwell plate at a density of 3×10 4 -10 5 per well.When the cells reach 70% to 80% confluency, add

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 2-5×10 3 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 pathwayrelated 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 X 2 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.

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Patient characteristics and immunohistochemistry.
The demographic and clinicopathologic characteristics of 100 LIHC patients are summarized in Tables 1 and 2. By the immunohistochemistry (IHC) evaluation, PD-L1 was mainly expressed in the membrane and cytoplasm ( Fig. 2A, and B) and IL-10 in the cytoplasm of cancer cells (Fig. 2C, and D).
Among the 100 LIHC cases, the high expression level of PD-L1 was observed in 72 tumor tissues (72.0%) and in 28 adjacent tissues (28.0%). In tumor tissues, the expression of PD-L1 was significantly higher than that of adjacent tissues (P = 0.000; Table 3). Meanwhile, the high expression levels of IL-10 were observed in 67 tumor tissues (67.0%) and in 35 adjacent tissues (35.0%). In tumor tissues, the expression of IL-10 was significantly higher than that of adjacent tissues (P = 0. 000; Table 4). According to TCGA database,there was a positive correlation between PD-L1 and IL-10 expression (Fig. 3A),Furthermore,there was also positive correlation between PD-L1 and CD8 expression (Fig. 3B). In our study,by Pearson correlation analysis, there was a positive correlation between the expression levels of PD-L1 and IL-10 in tumor and adjacent tissues ( Fig. 3C and D ), and a positive correlation was also found between the expression levels of PD-L1and CD8 in tumor and adjacent tissues (Fig. 3E, and 3F). Moreover, when analyzing both mRNA by PCR and protein expression by Western blots, we found expression levels of PD-L1 and IL-10 in tumor tissues were higher than those found in adjacent and normal tissues, and the expression levels of Met in the tumor tissues were higher than those found in adjacent tissues (Fig. 3G-J).

Relationship between the IL-10 expression level and survival periods
According to TCGA database,the expression level of the IL-10 gene did not affect overall survival (OS) or disease free survival (DFS) of patients with LIHC( Fig. 4A and 4B ).However,in our study ,the high IL-

Correlation between IL-10 and PD-L1 expression levels in LIHC cell-lines
ELISA studies showed that in both Bel7405 and MHCC 97-H cells, the over-expression of PD-L1 expressing cells increased the IL-10 expression levels. The IL-10 expression levels in siPD-L1 Bel7405 and MHCC 97-H cells was lower than that found in the LV-PD-L1 and control group (Fig 5A and 5C). increased the levels and appearance of IL-10, Met and phospho-Met cells, when compared with control cells (Fig. 5B, and D).
Detecting by ELISA, PD-L1 was expressed in both cell culture supernatant and cell lysates of Bel7405 and MHCC 97-H . The PD-L1 expression level was significantly higher in cell lysates than that found in the supernatant (Fig. 6A-D). At the protein level, IL-10 inhibited PD-L1 expression In both cell-lines, and anti-IL-10 antibody promoted the expression of PD-L1 in both cell-lines (Fig. 6A-F). IL-10 acted by inhibiting the Met signaling pathway and the downstream MAPK signaling pathway (Fig. 6E, and F).
Knockdown of Met gene expression in both cell-lines had no significant effect on PD-L1 expression levels after different treatments (Fig. 6G). The above phenomena were concentration and timedependent.

Effect of IL-10 and Crizotinib on proliferation, migration and invasion of LIHC cells.
When compared with crizotinib treatment alone, the combination of IL-10 and Crizotinib more potently inhibited proliferation, migration and invasion of LIHC cells ( Fig. 7A and B; Fig. 8A  At the transcriptional and translational levels, the higher expression of IL-10 and PD-L1 were observed in cancer tissues, when compared with those in normal tissues. In tumor and adjacent tissues, the PD-L1 expression level was positively correlated with IL-10 expression levels, and was also positively correlated with the CD8 + T cells. These results are consistent with previous studies (36,37).Moreover, in cancer tissues, the expression levels of Met at both the transcriptional and translational levels was significantly higher than those in adjacent tissues.
We also discovered that higher IL-10 expression levels were associated with higher age, advanced T stage, N pathological stage and higher PD-L1 expression On the other hand, higher PD-L1 expression levels tended to be related to patients of higher age group, poor tumor differentiation, advanced T stage, higher IL-10 expression levels and higher CD8 expression levels .In addition, PD-L1 combined with IL-10 might also be an independent prognostic factor in patients with LIHC. Patients in the low IL- ,the high IL-10 expression level group tended to have shorter survival periods than those with low IL-10 expression levels.These different reasons might be best described as follows. First, the TCGA database generally collects tumor information from European and American research studies.Differences in expression levels of related factors were due to ethnic differences. Second, the TCGA database generally uses high-throughput sequencing to detect the expression at the gene level, which might not be different at the protein level. Third, due to the difference in sample numbers, data reported in the current study was different from the resulst of TCGA.
In tissue experiments, we found that the expression levels of PD-L1 and IL-10 were higher in cancer tissues than in adjacent tissues. There was also a positive correlation between the two factors.
Similarly, in cellular experiments, we found that over-expression of PD-L1 can over-activate the Met signaling pathway, thereby up-regulating IL-10 expression. Thus, we infer that whether in cancer tissues or cells, over-expressed PD-L1 can activate the Met signaling pathway via positive feedback regulation, thereby up-regulating IL-10 expression. Instead, we also found that IL-10 down-regulates the expression of PD-L1, and anti-IL-10 up-regulates the expression of PD-L1 through the Met signal pathway. Thus we infer that IL-10 can down-regulate PD-L1 expression by inhibiting the Met signaling pathway and the down-stream MAPK signaling pathway via negative feedback regulation ( Fig. 1B and   C).
Some previously published studies suggest that IL-10 is an immunological negative regulator and inhibits the function of T, B and dendritic cells and up-regulates PD-L1 expression (29)(30)(31)45). Recent data have shown that IL-10 release might be mediated following PD-L1 blockade, which sustains immunosuppression in ovarian cancer (30). HGF/MET signaling is essential for cancer survival (46) and important in the interaction between IL-10 and PD-L1, which is involved in the immune response,

Data availability
All the data were available upon appropriate request .

Disclosure
Qian Qian and Jianping Chen contributed equally to this work.

Authors' Contributions
Qian Qian wrote the manuscript.Changping Wu revised the manuscript.Qian Qian,Jianping Chen and Weibing Wang did the data analysis.Qian Qian and Jianping Chen did the data collection.

Supplementary Files
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