M1 Marophages Induce Dormancy Of Hepatocellular Carcinoma Cells

BackgroundsHepatocellular carcinoma(HCC)is one of the most common malignant tumors in the world,and the mortality rate ranks fourth among cancer-related death cancers.It lacks early biomarkers and has a high recurrence rate and metastasis rate after surgery.HCC poses a serious threat to human health.For liver cancer patients,surgery is the most effective treatment,but the high recurrence and metastasis rate after surgery seriously affects the treatment effect.How to prevent tumor from the recurrence and metastasis is of great significance.TLumor metastasis is the main cause of cancer-related death.Although the primary tumor mass is reduced after surgery or chemotherapy,metastatic lesions emerges over months,years or decades.In the process of tumor metastasis,disseminated tumor cells(DTCs)play a critical role.As the biological characteristic of residual disseminated tumor seems quite different from that of the primary tumor,the disseminated tumor cells are in a state of slow proliferation for a long time when transferred to a new tissue.They have strong ability of immune escape and drug resistance,and remain clinically asymptomatic,entering a dormant state.The concept of tumor dormancy was first proposed by Rupert Willis and redefined by Geoffrey Hadfield in 1952 as temporary mitotic arrest of tumor cells.Tumor dormancy can be divided into three categories:cell dormancy,angiogenesis dormancy and immune-mediated dormancy.Based on the current treatments targeting the proliferation of tumor cells,a proposal of "awakening" dormant cells in order to kill them is raised.But this strategy may worsen the patient’s condition.Some scholars believe that the dormant cells are tumor stem cells.If the tumor stem cells can remain dormant or can be eradicated in the dormant state,it may be a new strategy to prevent liver cancer metastasis.Tumor immunity is a hot topic in tumor therapy nowadays.Tumor cells with gene mutation are supervised by immune surveillance mechanisms mediated by Tcells,NK cells and macrophages.In 2002,Schreiber et al.proposed the "tumor immune editing theory",which divided the process of tumor immune surveillance into three main stages:Elimination,equilibrium and escape.Equilibrium is directly related to tumor recurrence,during which the immune system may induce tumor cells to be dormant.Immune-induced tumor dormancy refers to the phenomenon of cell cycle arrest,down-regulation of proliferation-related gene expression and slow metabolism due to action of the immune system.Previous studies have found that tumor microenvironment can change tumor behavior.In the tumor microenvironment,there are a large number of tumor-associated macrophages(TAM)with complex functions.TAMs are divided into M1-like macrophages and M2-like macrophages according to their phenotypes and functions.Ml-like macrophages have strong antibacterial and antitumor activities.M2-like macrophages inhibit the anti-tumor cell immune response to promote tumor growth and metastasis.Previous literatures have shown that M1-like cells inhibit the growth of tumors and block the cell cycle of tumor cells,so we speculated that M1 macrophages may cause dormancy of tumor cells.In view of the above problems,we conducted the following studies according to the definition of tumor dormancy.The first part is in vitro experiments whereby M1 macrophages induced HCC cells dormancy.The second part is in vitro animal experiments whereby bone marrow derived M1 macrophages induced HCC cells dormancy.Methods and resultsⅠ:M1 macrophages induced dormancy of HCC cells in vitro.(Ⅰ)The supernatant of M1 macrophages culture inhibited the proliferation of HCC cells.1.THP-1 cells were treated with PMA to be adhered in the plate,then the cells were induced to M1 macrophages through using LPS and IFN-γ.After 24 hours,M1 macrophages were placed on the upper layer of transwell co-culture system.HuH-7 cells were cultured in the lower layer.After 24 hours of co-culture,HuH-7 cells were cultured in medium containing EdU for 2h.Then,the EdU positive cells was detected by flow cytometry detection.The results showed that the positive rate of EdU in the HCC cells stimulated by supernatant of M1 macrophages decreased compared to control HCC cells.2.SMMC-7721,BEL-7402 and HuH-7 cells were respectively cultured in the 96-well plate.After 48 hours of stimulation with M1 supernatant,the number of cells was counted twice with an interval of 24 hours.The results showed that M1 supernatant inhibited the proliferation of HCC cells.(Ⅱ)M1 supernatant inhibited the expression of MKI67 in HCC cells.M1 macrophages were cultured for 48 hours to obtain the M1 supernatant.SMMC-7721,BEL-7402 and HuH-7 cells were treated with M1S for 24h.Then,their RNA was extracted and subjected to RT-PCR assay for the expression of MKI67.Using unstimulated HCC cells as the control group,the results showed that M1 supernatant inhibited the expression of MKI67 in HCC cells.(Ⅲ)M1 supernatant inhibited glucose consumption in HCC cells.HuH-7 cells were treated with M1 supernatant for 48h.After the M1 supernatant were washed,fresh media was added for 48 hours.The supernatant was collected and concentration of glucose was detected using glucose assay kit.The difference value between the concentration of glucose in the culture supernatant and that in fresh medium serves as an index of glucose consumption.Using unstimulated HCC cells as the control group,the total number of cells in each group was counted,and the glucose concentration consumed by each cell was calculated.The results showed that the glucose consumption in the M1 supernatant stimulated group was significantly lower than that of the control group.This suggests that M1 supernatant inhibited glucose consumption in HCC cells.(Ⅳ)M1 supernatant blocked HCC cell cycle.1.M1 supernatant leaded to change in the cell cycle of HCC cells.BEL-7402 cells were treated with M1 supernatant for 48h.The HCC cells treated with M1 supernatant and untreated HCC cells were stained with PI.The cell cycle change was detected by flow cytomertry.The results showed that M1 macrophages blocked HCC cells at G2/M phase.2.M1 supernatant leaded to the change of cyclin expression in HCC cells.SMMC-7721,BEL-7402 and HuH-7 cells were treated with M1 supernatant for 24h.The expression of CCNB1 was detected by RT-PCR.Using unstimulated HCC cells as the control group,the results showed that M1 supernatant inhibited the expression of CCNB1 in HCC cells.(Ⅴ)M1 supernatant induced changes in the activity of signaling pathways related to cell proliferation in HCC cells.SMMC-7721,BEL-7402 and HuH-7 cells were treated with M1 supernatant for 48h.Western blot detects the expressions of ERK1/2,p-ERK1/2,p38,and p-p38 in the treatment group and the untreated group.The results showed that M1 macrophages up-regulated the expression of p-ERK1/2,but had no effect on the expression of p-p38.SMMC-7721 and BEL-7402 cells were treated with M1 supernatant for 48h.Western blot detects the expressions of p-AKT.The results showed that M1 macrophages down-regulated the expression of p-AKT.(Ⅵ)M1 supernatant inhibited the growth of HCC cells for a long-term.1.M1 macrophages inhibited the growth of HCC cells for a long-term.HuH-7 cells were treated with M1 supernatant for 48h.Then,HuH-7 cells were cultured in the 96-well plate again,and the number of cells was counted every day for a total of 4 days.The results showed that HCC cells stimulated by M1 supernatant continued to proliferate slowly.2.M1 supernatant inhibited the clonal formation of HCC cells.SMMC-7721 and BEL-7402 cells were treated with M1 supernatant for 48h.Then SMMC-7721 and BEL-7402 cells were respectively cultured in 6-well plates.After 1 week,SMMC-7721 and BEL-7402 cells were washed three times with PBS,fixed with methanol for 30min,stained with crystal violet for 30min,and photographed.The results showed that M1 supernatant inhibited the clonal formation of HCC cel s.(Ⅶ))M1 macrophages did not cause cell senescence and apoptosis of HCC cells.1.M1 macrophages did not cause cell senescence of HCC cells.SMMC-7721,BEL-7402 and HuH-7 cells were treated with M1 supernatant for 48h.The senescence of untreated HCC cells and HCC cells stimulated by M1 supernatant were detected with β-galactosidase kit respectively.The results showed that Ml macrophages did not cause cell senescence of HCC cells.2.M1 macrophages did not cause cell apoptosis of HCC cells.Western blot detected the expression of apoptotic protein Bax in untreated HCC cells and M1 supernatant stimulated HCC cells,and the results showed that Bax did not change,suggesting that M1 macrophages did not cause cell apoptosis of HCC cells.(Ⅷ)The inhibitory effect of M1 supernatant on HCC cell proliferation was reversible.1.We stimulated HCC cells with M1 supernatant for 48 hours,counting every day.The results showed that the HCC cells did not return to proliferating state until the sixth day.These cells are referred to as restorative cells.The control group was regular HCC cells.The HCC cell stimulated with M1 supernatant for 48h is designated as M1 supernatant group,which are dormant.The number of all the cultured cells was counted twice with internal of 24 hours.The results showed that the cell number in M1 supernatant group was in a state of slow proliferation.The cell proliferation in the restorative cells group was restored.The results showed that the inhibition of M1 supernatant on the proliferation of HCC cells was reversible and M1 macrophages induced the dormancy of HCC cells.2.The EdU positive rates of SMMC-7721 cells in three groups were detected by flow cytometry.The results showed that the positive rates of EdU in HCC cells stimulated by M1 supernatant were decreased compared to the control group,there was no significant difference in the EdU positive rate of restorative cells group.3.SMMC-7721,BEL-7402 and HuH-7 cells in three different group were stained with PI,and cell cycle changes were detected by flow cytometry.The results showed that M1 macrophages blocked the cell cycle of HCC lines in G2/M phase,there was no significant difference in the cell cycle of restorative cells group.(Ⅹ)M1 supernatant inhibited proliferation by testing EdU in cancer stem cells.HuH-7 cells were treated with M1 supernatant for 48h,the stem cell mark CD13 was used to detect the positive rate of HuH-7 stem cell by flow cytometry analysis.The results showed that the EdU positive rate of HuH-7 stem cell by M1 macrophages decreased.Ⅱ.Ml macrophages derived from murine bone marrow induced dormancy of HCC cells in vivo.(Ⅰ)M1 supernatant derived from bone marrow of mice leaded to cell cycle arrest of H22 cells.Mouse bone marrow cells were induced into macrophages with M-CSF,then,polarized into M1 macrophages through using LPS.The Ml macrophage supernatant was acquired from the 24h culture of the M1 macrophages.After stimulated with the Ml macrophage supernatant for 48 hours,H22 cells were stained with PI.The cell cycle change were detected by flow cytometry.The results showed that M1 macrophages from mouse bone marrow blocked the cell cycle of H22 cells at G2/M phase.(Ⅱ)M1 macrophages from mouse bone marrow induced HCC cell dormancy and inhibited tumor growth.1.H22 cells or a mixture of M1 macrophages from mouse bone marrow and H22 cells were injected into BaLB/C mice subcutaneously.The size of tumors was measured every three days for a total of six times.Statistical analysis showed that M1 macrophages from mouse bone marrow inhibited tumor growth.2.The regular H22 cells or the H22 cells stimulated with M1 supernatant from mouse bone marrow for 48 hours were subcutaneously injected to mice.The size of tumors was measured every three days for a total of six times.Statistical analysis was carried out for the data from sixth measurement of tumor size.The tumor mass was isolated after the mice was sacrificed,photographed and weighed.The results showed that H22 cells treated with M1 supernatant formed slow tumor growth.ConclusionsⅠ:M1 macrophages induced tumour dormancy in hepatocellular carcinoma cells.M1 macrophages can inhibit the proliferation and metabolism of HCC cells.This proliferation inhibition is due to cell cycle arrest,not cell senescence or apoptosis.Moreover,this proliferation inhibition is reversible and sustainable,suggesting that M1 macrophages induced tumour dormancy in hepatocellular carcinoma cells.Ⅱ:M1 macrophages inhibited HCC growth by inducing HCC cell dormancy.The tumor inoculated with M1 macrophages and HCC cells in mice grow slower than that with HCC cells alone.In addition,the tumor inoculated with the HCC cells treated with M1 supernatant grow slower than that with the HCC cells without treatment.These results suggests that M1 macrophages can inhibit tumor growth by inducing dormancy of hepatocellular carcinoma cells.