| BackgroundsAs we all know, hepatocellular carcinoma (HCC) has became one of the most harmful disease for people’s healthy in china. The concept of tumor microenvironment is gradually perceived with the deep research to tumor. Tumor microenvironment refers to the local environment of tumor tissue, including many immune cells, stromal cells and many inflammatory factors, as well as oxygen content and pH. Because it contains polyphyletic constituent, tumor microenvironment also has complex functions. Both pro-inflammatory cells (Th1 lymphocytes and Th17 lymphocytes) and anti-inflammatory cells (Th2 lymphocytes and Tregs) are in tumor microenvironment. There are both also pro-inflammatory cytokines (TNF-a, IL-6 and IL-12) and anti-inflammatory cytokines (TGF-(3, IL-4 and IL-10) in tumor environment. In fact, no matter those pro-inflammatory cells and cytokines or anti-inflammatory cells and cytokines all play a critical role in the development and progression of tumors. Tumor associated macropharess (TAMs) is a group of cells which are abundant and have complex functions in tumor microenvironment. Tumor associated macrophages were divided into Ml-like macrophages and M2-like macrophages on the basis of its phenotypes and different functions by researchers. The classification of tumor associated macrophages can help researchers to analyze the different functions of TAMs more subtly.With the development of immunotherapy in the fields of tumor research, drugs targeting the famous molecule PD-L1 were approved to treat melanoma and non-small cell lung cancer by FDA in the year of 2014 following the approval of the monoclonal antibody of CTLA-4 in the year of 2009. As a significant immunosuppressive molecule, PD-L1 is expressed on APC cells and many tumor cells. PD-1 is mainly expressed on T lymphocytes. When the PD-L1 molecule engages its receptor PD-1, the transduced signaling suppresses the proliferation and differentiation of T lymphocytes. As for the regulation of the PD-L1 expression, some cytokines such as IFN-y and IL-27, which are included in the tumor microenvironment, can upregulate the expression of PD-L1 in the tumor cells, as well as hypoxia. However, there may be more mechanisms of the inducible expression of PD-L1 on tumor cells.TAMs includes M1-like macrophages and M2-like macrophages. They have different functions in the development and progression of tumor. We ask whether the TAMs promote the expression of PD-L1 in the tumor cells in the present study. If they do, what are the mechanisms for the promotion of PD-L1 expression. In addition, we wonder whether those tumor cells with high PD-L1 expression induced by tumor associated macrophages suppress the functions of CD8+T lymphocytes.Objective1.To investigate the relationship between expression of PD-L1 on HCC cells and infiltration of CD68+HLA-DR+M1-like macrophages.2. To explore the mechanism by which the Ml-like macrophages promote the expression of PD-L1 on HCC cells.Methods and Results1. There are abundant Ml-like macrophages in the HCC tissues..We used double immunohistochemical staining to detect the amount of M1-like or M2-like macrophages in HCC tissue. The CD68 and HLA-DR were used as markers to identify M1-like macrophages. Our results revealed that there are 49.76% of CD68+HLA-DR+macrophages in tumor and 65.48% of CD68+HLA-DR+ macrophages in mesenchyme.2. The expression of PD-L1 on HCC tissues is heterogeneous and is associated with the infiltration of Ml-like macrophages on the HCC tissues.We used immunohistochemical staining to detect the expression of PD-L1 in 30 HCC specimens. The result showed that the positive rate of PD-L1 expression in hepatocellular carcinoma was 20%. Moreover, we found that the PD-L1 expression was heterogeneous in the same HCC tissue. We analyzed the relationship between PD-L1 expression on HCC cells and the infiltration of M1-like macrophages using serial sections from the same HCC tissue. The result demonstrated that the PD-L1 expression of hepatocellular carcinoma tissue was associated with the number of M1-like macrophages infiltrating in the hepatocellular carcinoma tissue.3. Murine RAW264.7 derived M1-like macrophages promote the expression of PD-L1 on murine HCC cells.To investigate whether the different phenotypes of macrophages have significant impact on the expression of PD-L1 in vitro, we used the supernatants of M1-like macrophages and M2-like macrophages of RAW264.7 stimulated respectively by LPS and IL-4 to culture murine hepatocellular carcinoma cell Hepal-6. The expression of PD-L1 was detected at mRNA level by RT-PCR and at protein level by flow cytometry. Interestingly, results demonstrated that the supernatants of M1-like macrophages but not the supernatants of M2-like macrophages dramatically induced the high-expression of PD-L1 on HCC cells at mRNA level and protein level.4. Murine bone marrow derived M1-like macrophages promote the expression of PD-L1 on murine HCC cells.To deeply investigate whether those different phenotypes of macrophages have significant impact on the expression of PD-L1 in vitro, we used the supernatants of M1-like macrophages and M2-like macrophages derived from bone marrow macrophages to culture murine hepatocellular carcinoma cell Hepal-6. The expression of PD-L1 was detected at mRNA level by RT-PCR and at protein level by flow cytometry. Similarly, results demonstrated that the supernatants of M1-like macrophages but not the supernatants of M2-like macrophages dramatically induced the high-expression of PD-L1 on HCC cells at mRNA level and protein level.5. Human THP-1 derived M1-like macrophages promote the expression of PD-L1 on human HCC cells.We used the supernatants of M1-like macrophages and M2-like macrophages derived from THP-1 cells to culture human hepatocellular carcinoma cell HuH-7. The expression of PD-L1 was detected at mRNA level by RT-PCR and at protein level by flow cytometry. The result demonstrated that the supernatants of M1-like macrophages but not the supernatants of M2-like macrophages dramatically induced the high-expression of PD-L1 on HCC cells at mRNA level and protein level.6. RAW264.7 derived-M1 like macrophages induce the expression of PD-L1 on murine HCC cells via MEK/ ERK signaling pathway.M1-like macrophages were prepared by simulating murine RAW264.7 cells with LPS. Seven inhibitors were used, including the inhibitor of NF-KB, PI3K, ERK, P38, JAK, JNK and STAT3. Before we added the supernatants of M1-like macrophages to culture murine hepatocellular carcinoma cell Hepal-6, the inhibitors were added to the tumor cell culture 1 hour in advance. Then, the supernatants of M1-like macrophages including inhibitors were added to the cell culture. The expression of PD-L1 was detected by flow cytometry. The result demonstrated that the inhibitor of ERK can inhibit the expression of PD-L1 induced by Ml-like macrophages.7. RAW264.7 derived Ml like macrophages induce the expression of PD-L1 on murine HCC cells via the cytokine TNF-α.The supernatants of M1-like macrophages including neutralizing antibodies against IFN-γ, TNF-α or IL-6 were used to culture Hepa1-6 cells. The expression of PD-L1 was detected by flow cytometry. The result demonstrated that the expression of PD-L1 induced by M1-like macrophage supernatants can be down-regulated by the neutralizing antibody against TNF-α.8. THP-1 derived Ml like macrophages induce the expression of PD-Ll on HCC cells via JAK/STAT signaling pathway.M1-like macrophages were prepared by simulating THP-1 derived macrophages with LPS. Seven inhibitors were used, including the inhibitor of NF-KB, PI3K, ERK, P38, JAK, JNK and STAT3. Before we added the supernatants of M1-like macrophages to culture human hepatocellular carcinoma cell HuH-7, the inhibitors were added to the tumor cell culture 1 hour in advance. Then, the supernatants of M1-like macrophages including inhibitors were added to the cell culture. The expression of PD-L1 was detected by flow cytometry. The result demonstrated that the inhibitor of JAK can inhibit the expression of PD-Ll induced by M1-like macrophages.9. Murine HCC cells stimulated by M1-like macrophages promote the apoptosis of CD8+T lymphocytes.We used the supernatants of RAW264.7 derived M1-like macrophages as cell culture medium to culture murine hepatocellular carcinoma cell Hepal-6. Then, prepared activated T lymphocytes by stimulating murine spleen cells with magnetic beads conjugated with anti-CD3ε and anti-CD28 antibodies. The activated T cells were cocultured with Hepal-6 cells for 24h. Then, the apoptosis of CD8+T lymphocytes was analyzed by flow cytometry. The result revealed that murine hepatocelluar carcinoma cells stimulated by Ml-like macrophages promoted the apoptosis of CD8+T lymphocytes.Conclusions1. The expression of PD-L1 on HCC tissues is heterogeneous and is associated with the infiltration of M1-like macrophages.2. M1-like macrophages promote the expression of PD-L1 on HCC cells.3. M1-like macrophages promote the expression of PD-L1 on murine HCC cells and human HCC cells via different signaling pathway.4. HCC cells stimulated by M1-like macrophages promote the apoptosis of CD8+ T lymphocytes.Innovation and SignificanceWith the development of tumor immunotherapy, the therapy targeting the molecule of PD-L1 have made a great progress. But, the mechanism of the expression regulation of PD-L1 is not very clear. Our study focused on the relationship between the tumor associated macrophages and the expression of PD-L1 in HCC cells. We found that the M1-like macrophages can promote the expression of PD-L1 of hepatocellular carcinoma cells via certain important signaling pathways. Those findings provided a new idea to tumor immunotherapy. Because of the important role of tumor associated macrophages in upregulating the expression of PD-L1, some therapies should be cautious when targeting the tumor associated macrophages. |
