TGF-β1 Promotes The Occurrence And Development Of Hepatocellular Carcinoma By Inducing The Expression Of PD-L1 On Hepatic Stellate Cells

Primary liver cancer (PLC) is one of the most common and highly malignant cancer in the world. HCC, which account for more than 90% of PLC, is the main type of PLC and the fifth most common malignant tumor. Despite the development of diagnosis and treatment of HCC, it is still a highly lethal disease. Recent literature has highlighted that tumor cells and surrounding stromal cells are a fundamental role of the tumor microenvironment in the pathogenesis of HCC. Liver cancer microenvironment is a dynamic system, includes cancer cells, hepatic stellate cells, fibroblasts, immune cells, endothelial cells, vascular tissue as well as inflammatory cytokines and extracellular matrix (ECM) proteins. Hepatic stellate cells (HSCs), which are known as Ito cells, belong to the most important cell type and are responsible for collagen synthesis in the liver. Following liver injury, quiescent HSCs are activated and transdifferentiated into myofibroblast-like cells which are matrix-secreting myofibroblasts. Activated HSCs (aHSCs) secrete substantial amounts of ECM proteins, cytokines and chemokines into the stroma, leading to the development of HCC. It has been reported that aHSCs have a strong immunomodulatory activity, which are including up-regulation of programmed death ligand-1 (PD-L1) and expression the molecules involved in antigen presentation. Activated HSCs can promote HCC progression by enhancing of the immunosuppressive cell population of regulatory T cells (Tregs) and inducing T cells apoptosis. Further studies have frequently demonstrated that high expression of PD-L1 on HSCs promoted progression and invasion of HCC cells. TGF-β1, a soluble cytokine produced and secreted mainly by inflammatory cells and malignant hepatocytes, is overexpressed in tumor tissue of HCC patients. TGF-β1 plays a important role in the pathogenesis of HCC and inhibits the immune function, regulates cell growth and differentiation, promotes the development of HCC.In this study, we investigated whether TGF-β1 induced PD-L1 expression on LX-2 cells via activating ERK1/2 signaling. At the same time, whether TGF-β1-HSCs induced T cells apoptosis and decreased the cytotoxicity of T cells towards HepG2 cells. Next, subcutaneous cotransplantation of TGF-β1-HSCs and H22 cells were established. We investigated whether TGF-β1-HSCs promoted the development of HCC. HCC cells activated HSCs, at the same time, activated HSCs induced HCC cells, which promotes the the development of HCC. Then, we observed the expression of PD-L1 by establishing co-culture of HepG2 with HSCs and the level of TGF-β1 in HepG2 and revealed the correlation between TGF-β1.Objective:To investigate the effect of TGF-β1 on the induction of PD-L1 expression in LX-2 (human HSCs) and the effect of TGF-β1-LX-2 in apoptosis and proliferation of T cell in vitro. Investigating the expression of PD-L1 and the level of TGF-β1 in co-culture of HepG2 with LX-2, to reveal the correlation between the level of TGF-β1 and PD-L1 expression. H22 cells combination TGF-β1-HSCs cells were injected subcutaneously in mice, to analyze the effect of TGF-β1-HSCs on promoting the development of HCC in vivo.Methods:In vitro,5,10 and 20 ng/ml TGF-β1 was added to LX-2 cells and incubated for 24 h,48 h,72 h, and 72 h. Then cells were detected by flow cytometry and western blot assay. The expression of PD-L1, ERK1/2, p-ERKl/2 were detected. After treatment with TGF-β1, LX-2 were harvested and co-cultured with T lymphocytes 48 h, observing the apoptosis of T cell by flow cytometry and the proliferation of T cells by CCK-8. The T cell was collected from co-cultured and cultured with HepG2 cells for 24 h. MTT assayed the cytotoxicity of T cell on HepG2. Establish co-culture of HepG2 with LX-2 by transwell. After co-cultured 48 h, the expression of PD-L1 on LX-2 was tested by flow cytometry. The level of TGF-01 in the supernatant of HepG2 was tested by ELISA at 48,72 h.In vivo, H22, TGF-β1-HSC+H22, HSC+H22 were injected subcutaneously in the left armpit of C57BL/6J mice. On day 21 after implantation, mice were humanely sacrificed. Then, the tumors were removed and weighted. The apoptosis and the subgroup of T cells in spleens were analyzed by flow cytometry. HE staining was observed and the number of T cell and the expression of a-SMA in tumor tissue were detected by IHC. T cells in tumor tissue were stained APC-CD3 and TUNEL.Results:1 TGF-β1 increased PD-L1 expression on LX-2 which related with ERK1/2 signaling1.1 TGF-β1 increased PD-L1 expression on LX-2 cellsLX-2 cells treated by TGF-β1 (5,10 and 20 ng/ml) at 24 h,48 h and 72 h, then were assessed by flow cytometry. Compare with control group, PD-L1 expression was greatly increased in LX-2 treated by TGF-β1.1.2 TGF-β1 induces p-ERKl/2 expression in LX-2 cellsTGF-β1 can activate MAPK signaling pathway persistently including ERK1/2 pathway, the c-Jun amino terminal kinase (JNK) pathway and the p38 MAPK pathway. TGF-β1 can activate ERK1/2 and p38 MAPK in HSCs and the expression of PD-L1 was shown to be dependent on the MEK/ERK signaling pathway. Therefore, we assessed the effect of TGF-β1 on the expression and phosphorylation of ERK1/2 in LX-2 cells. These results demonstrate that the expression of p-ERKl/2 was obviously increased in LX-2 cells treated by TGF-β1 compare with control group.1.3 U0126 downregulated TGF-β1-induced PD-L1 expression on LX-2 cellsTo investigate whether TGF-β1 induced PD-L1 expression on LX-2 cells via activating ERK1/2 signaling. U0126 (5μM) was added to LX-2 cells before TGF-β1 Then, PD-L1 expression in ERK1/2-blockaded LX-2 cells was examined by flow cytometry and western blot. The expression of p-ERK1/2 was decreased significantly by the inhibitor U0126. The results indicated that TGF-β1 increased the expression of PD-L1 and p-ERK1/2. U0126 inhibited TGF-β1 induced PD-L1 and p-ERK1/2 expression.1.4 TGF-β1-LX-2 induced T-cell apoptosisPrevious studies reported that PD-L1 is a co-inhibitory molecule, and its receptor PD-1 is expressed mainly on the surface of T cells. Therefore, we further investigated the effect of TGF-β1-LX-2 cells on T-cell apoptosis. Compared with control group, TGF-β1-LX-2 cells obviously induced the apoptosis of T cells1.5 TGF-P-LX-2 inhbited proliferation of T cellsCompared with control group, TGF-β1-LX-2 cells markedly inhibited proliferation of T cells.1.6 TGF-β1-LX-2 suppressed the cytotoxicity of T cellsThe above data demonstrated that LX-2 cells treatment with TGF-β1 results in T-cell incompetence. The result indicated that after co-cultured with TGF-β1-LX-2 cells T cells cytotoxic activity were decreased compared with that of T cells co-cultured with control LX-2 cells.2 TGF-β1-HSCs cells promoted H22 tumor growth, promoted apoptosis of T cell and reduced the proportion of CD4+、CD8+T2.1 TGF-β1-HSCs cells promoted H22 growthResults indicated that compared with H22 cells, TGF-β1-HSCs significantly developed much larger tumors. The HE staining revealed that the stroma of tumors appeared irregular and invaded skin in TGF-β1-HSC plus H22 group. H22 group contain clearly demarcated bands of fibrosis within tumor tissue and these bands separated and surrounded small tumor nodules.2.2 TGF-β1-HSCs do not change spleen index, thymus index, liver index.The results showed that TGF-β1-HSCs cells do not change the spleen index, thymus index, liver index.2.3 TGF-β1-HSCs promoted a-SMA expressionThe results showed that TGF-β1-HSCs promoted the expression of a-SMA in tumor tissue compared with H22 group.2.4 TGF-β1-HSCs promoted apoptosis of T cell in spleen and tumor tissueThe apoptosis rate of T cells in the spleen and tumor was quantified using flow cytometry and TUNEL. The results showed that compared with control H22 group, TGF-β1-HSCs promoted the apoptosis of T cells in the spleen and tumor tissue.2.5 TGF-β1-HSCs reduced the expression of T-cell subsets in the spleen and tumorThese results indicate that the CD4+T cells and CD8+T cell subpopulations were downregulated in the H22 plus TGF-β1-HSCs group compared with control H22 group. IHC results indicate that CD3+T markedly decreased in the H22 plus TGF-β1-HSCs group compared with H22 group.3 Co-culture of HepG2 with LX-2 promoted PD-L1 expression3.1 The effect of HepG2 in expression of PD-L1 on LX-2 cellsLX-2 cells were co-cultured with HepG2 by transwell for 48 h. The highest level of HepG2-mediated PD-L1 expression on LX-2 was observed at a ratio of 10:1 (p<0.001).3.2 The level of TGF-β1 in supernatant of HepG2The level of TGF-β1 in HepG2 group was obviousely gradually increased.3.3 The correlation analysis between TGF-β1 and PD-L1 expression on LX-2 cellsResults showed that the level of TGF-β1 was positively correlated with the expression of PD-L1.Conclusion:1. TGF-β1 increased the expression of PD-L1 on LX-2 via ERK1/2 signaling.2. TGF-β1-LX-2 induced T cell apoptosis, inhbited T cell viability, inhibited T cells cytotoxicity on HepG2.3. TGF-β1-HSCs cells promoted H22 tumor growth by reducing the expression of T cell subsets and promoting apoptosis of T cell in spleen and tumor tissue.4. HepG2 increased the expression of PD-L1 on HSCs. TGF-β1 was positively correlated with the expression of PD-L1.