M1-like Macrophages Supernatant Induced High Expression Of Immune Checkpoint Molecules In Hepatocellular Carcinoma Cells

BackgroundHepatocellular carcinoma(HCC)is one of the most fatal tumor diseases because of its complexity,recurrence and metastasis.Hepatocellular carcinoma(HCC)is the sixth most common cancer in the world and the third most common reason of cancer related death.HCC in Asian and African countries is related to hepatitis B and C virus infection,chronic alcohol consumption,food intake contaminated with aflatoxin,other genetic diseases(such as hemochromatosis caused by iron overload in the body)and cirrhosis of the liver.The pathogenesis of hepatocellular carcinoma(HCC)is still unclear due to the complex factors,uncontrollable and multi-step progression of the pathogenesis,resulting in difficulties to the treatment of HCC.As we all know,cancer patients not only suffer from this disease,but also bear huge medical costs.Therefore,it is urgent to actively seek for more effective tumor treatment methods.With announcement of the 2018 Nobel Prize in physiology,tumor immunotherapy will enter a new era.Immune checkpoint-targeting therapy is also getting more and more attention.Immune checkpoint molecules programmed cell death-1(PD-1)and Cytotoxic T lymphocyte antigen 4(CTLA 4)have a negative regulatory role in the activation of T cells,reducing T cell activity and even leading to its apoptosis.The most important ligand of PD-1 is PD-L1.The PD-L1 expressed on tumor cells could bind to PD-1 expressed on T cells,resulting in inhibition of proliferation of T cells,secretion of cytotoxic mediators and killing tumor cells.Binding of CTLA 4 with the B7-1(CD80)/B7-2(CD86)ligand on the surface of antigen-presenting cells(APC)can inhibit the activation of T cells and reduce APC activated T cells,thereby reducing the T cell response.At present,antibodies against PD-1 and CTLA 4 immune checkpoints have been produced,which is a great boon for tumor patients.Although antibodies targeting the PD-L1/PD-1 pathway have good efficacy,there are still deficiencies.In addition to insensitivity or drug resistance to antibody therapy in some cases,antibody therapy can also bring more immune-related side effects,such as hypothyroidism,pneumonia colitis and hypophysitis.Moreover,the high cost of monoclonal antibody therapy brings an unbearable economic burden to patients.Due to the important role of PD-L1 in tumor immune escape and the existing problems in current clinical treatment,the research on the regulatory mechanism of PD-L1 expression in tumor cells is worthy of attention.Understanding the regulatory mechanism of PD-L1 expression in tumor cells may lead to the discovery of more and more effective new methods of tumor immunotherapy.Tumor microenvironment is a local environment of tumor tisses,including not only the tumor cells themselves,but also the surrounding fibroblasts,immune cells,inflammatory cells and the intercellular matrix,microvessels and biological molecules in the nearby area.Macrophages play an important role in tumor microenvironment.In the present study,we ask whether activated macrophages induce expression of checkpoint molecules(PD-L1,PD-L2,B7-H3,B7-H4,CD48,CD47,HVEM and Galectin9).Since these immune checkpoint molecules mediate tumor immune escape,we also ask whether activated macrophage induce immune escape of HCC cells.Research purposeMacrophages,as important immune cells in tumor microenvironment can help tumor cells to conduct immune escape and promote the survival and development of tumors.In this study,we investigated the expression of immune checkpoint molecules induced by M1-like macrophages supernatant.In the first part,the effect of M1-like macrophages supernatant on the expression of immune checkpoint molecules PD-L1,PD-L2,B7-H3,B7-H4,CD48,CD47,HVEM and Galectin-9 was detected.In the second part,we mainly explored that M1-like macrophages supernatant induced expression of immune checkpoint molecules in HCC cells with enhanced response by second-stimulation.In the third part we demonstrated that macrophages can promote the immune escape of hepatocellular carcinoma cells.Research methods and results1.M1-like macrophages supernatant induced high expression of immune checkpoint molecules in hepatocellular carcinoma cellsHuman monocyte THP1 was inoculated on the cell culture plate,and PMA was added to induce cell adhesion.Then LPS and IFN-? were added to stimulate the cells for 18h to obtain M1-like macrophages.The supernatant was discarded,washed with PBS,replaced with fresh medium,and obtained M1 supernatant after 24h.The M1 supernatant was used to stimulate Huh-7 cells,BEL-7402 cells and SMMC-7721 cells.This was the stimulation group.Normal cultured hepatocellular carcinoma cells were the control group.Then,the cells were collected.RNA was extracted and reversely transcribed into cDNA.RT-PCR was used to detect the expression of various immune checkpoint molecules in mRNA level.The expression of immune checkpoint molecules in protein level was detected by flow cytometry.(1)M1-like macrophages supernatant induced high expression of immune checkpoint molecules in hepatocellular carcinoma cells at mRNA levelThe expression of immune checkpoint molecules PD-L1,PD-L2,B7-H3,B7-H4,CD48,CD47,HVEM and Galectin-9 in HCC cells were detected by RT-PCR.In Huh-7 cells,compared with the control group,the expression of PD-L1,B7-H3,B7-H4,CD48,CD47 and Galectin-9 molecules in the stimulation group were significantly increased(P<0.05),while the expression of PD-L2 and HVEM molecules were also increased,but there was no significant difference(P>0.05).In BEL-7402 cells,the expression of PD-L1,PD-L2,B7-H3,B7-H4,CD48,CD47 HVEM and Galectin-9 molecules in the stimulation group were significantly higher than those in the control group(P<0.05).In SMMC-7721 cells,the expression of PD-L1,PD-L2,B7-H3,B7-H4,CD47,HVEM and Galectin-9 molecules in the stimulation group were significantly higher than those in the control group(P<0.05),but the expression of CD48 was not significantly different from this in the control group(P>0.05).(2)M1-like macrophages supernatant induced high expression of immune checkpoint molecules in hepatocellular carcinoma cells at protein levelThe expression at protein level of PD-L1,CD47,HVEM and Galectin9 in Huh-7 and BEL-7402 cells was detected by flow cytometry.In Huh-7 and BEL-7402 cells,the expression at protein level of PD-L1,HVEM and Galectin9 in the stimulation group was significantly higher than those in the control group(P<0.05).In Huh-7 cells,the expression of CD47 in the stimulation group was significantly higher than that in the control group(P<0.05).2.Ml-like macrophages supernatant induced expression of immune checkpoint molecules in HCC cells with enhanced response by second-stimulationThe Huh-7,BEL-7402 and SMMC-7721 cells were stimulated by M1 supernatant for 48h,the supernatant was discarded,the cells were washed with PBS,and incubated with fresh medium for 6 days(resting group).The resting HCC cells were stimulated again by M1 supernatant for 24h(second stimulation group).Meanwhile,M1 supernatant was used to directly stimulate normal HCC cells for 24h,which were first stimulation cells(first stimulation group),and normal HCC cells were used as unstimulated cells(non-stimulation group).Collected cells,extracted RNA,and reversed RNA transcription into cDNA.(1)M1-like macrophages supernatant induced expression of immune checkpoint molecules in HCC cells with enhanced response by second-stimulation at mRNA levelAt mRNA level,the expression of PD-L1,PD-L2,B7-H3,B7-H4,CD48,CD47,HVEM and Galectin9 were detected by RT-PCR.In Huh-7 cells,the expression at mRNA level of PD-L1,B7-H3,B7-H4,CD48,CD47 and Galectin9 in the second stimulation group were significantly increased compared with those in the first stimulation group(P<0.05).The expression at mRNA level of PD-L2 and HVEM were not significantly different from that in the first stimulation group(P>0.05).In BEL-7402 cells,the expression of PD-L1,PD-L2,B7-H3,B7-H4,CD48,CD47,HVEM and Galectin9 in the second stimulation group were significantly higher than those in the first stimulation group(P<0.05).In SMMC-7721 cells,the expression of PD-L1,PD-L2,B7-H3,B7-H4,CD47,HVEM and Galectin9 in the second stimulation group were significantly higher than those in the first stimulation group(P<0.05),but CD48 was not significantly different from the first stimulation group(P>0.05).(2)M1-like macrophages supernatant induced expression of immune checkpoint molecules in HCC cells with enhanced response by second-stimulation at protein levelAt protein level,the expression of PD-L1 and CD47 in Huh-7 cells and the expression of PD-L1 in BEL-7402 cells were detected by flow cytometry.In Huh-7 cells,PD-L1 and CD47 in the second stimulation group were significantly higher than those in the first stimulation group(P<0.05).In BEL-7402 cells,PD-L1 in the second stimulation group was significantly higher than this in the first stimulation group(P<0.05).3.The immune escape of hepatocellular carcinoma cells promoted by macrophages was studied in mouse hepatocellular carcinoma model(1)The Hepal-6 cells were inoculated on the cell culture plate,then the macrophages supernatant activated by LPS and mouse-IFN-r was added to stimulate for 24h.The supernatant was discarded,and fresh supernatant was added to stimulate for 24h again(48h in total),the supernatant was discarded,and the cells were washed with high-pressure sterile PBS buffer for three times and resting for six days with fresh medium(resting group).The macrophages supernatant activated by LPS and mouse-IFN-r was used to stimulate resting cells again for 24 h(second stimulation group).On sixth day,the macrophages supernatant was used to directly stimulate normal Hepal-6 cells for 24h,which was the first stimulation group.The normal cultured Hepal-6 cells was used as the non-stimulation group.The single cell suspension was prepared and stained with fluorescent antibody.The expression at protein level of PD-L1 and CD47 were detected by flow cytometry.The expression of PD-L1 and CD47 in the second stimulation group was significantly increased compared with that in the first stimulation group(P<0.05).(2)The C57BL/6 mice were immunized with normal cultured Hepal-6 cells in the groin,and Hepal-6 cells in normal culture(control group)and hepl-6 cells stimulated by supernatant(stimulation group)were injected into the abdomen of immunized mice two weeks later.The mice in the control group and the stimulation group developed tumors on the third day,and the tumor volumes of mice in two groups were measured every three days afterwards.The tumor volumes of the mice in the stimulation group were significantly larger than those in the control group from the 6th day to the 12th day(P<0.05).Conclusions1.M1-like macrophages supernatant could induce high expression of various immune.checkpoint molecules in hepatocellular carcinoma cells2.M1-like macrophages supernatant induced expression of immune checkpoint molecules in HCC cells with enhanced response by second-stimulation3.Macrophages could promote immune escape of hepatocellular carcinoma cells...