A Preliminary Study On The Polarization Mechanism Of Tumor-Associated Macrophages In Primary Liver Cancer

Primary liver cancer(PLC)is one of the common tumors in China.With the development of treatments such as surgery and minimally invasive treatment,patients has achieved good outcomes,but their 5-year survival rate is still less than 20%.The immunotherapeutic drugs such as PD-1 antagonists may benefit patients.However,their treatment effect is still limited.Today,the immunotherapy has become one of the most important areas of research on PLC.Tumor associated macrophages(TAMs)are important immune cells,and they can be polarized to M1 or M2 cells in the tumor microenvironment.The functions of the two typs of cells are different.The former can kill tumors.And the latter is characterized by promoting tumor growth,promoting tumor metastasis,and promoting tumor angiogenesis.TAMs is also of great significance in primary liver cancer.Relevant clinical studies have found that Patient prognosis is related to the polarization type of TAMs infiltrated in tumor.However,the mechanism of TAMs polarization is not clear.To explore the mechanism may find new ways for immunotherapy of primary liver cancer,which may be of great clinical significance.In our study,three patients with early-stage liver cancer were enrolled.All of cases were male,aged 53,51,and 46 years old,respectively.All patients were chronic hepatitis B and cirrhosis patients,their tumor sizes were 2.5 cm,2.5 cm,and 1.0 cm,respectively.Two cases were treated by liver transplantation and one were treated by laparoscopic resection.Postoperative pathology indicated moderate differentiation of hepatocellular carcinoma.After obtaining the specimen,the cell suspension were prepared by honing and digesting.After multiantibody labeling,the cells were sorted by flow cytometry,and then ATAC-seq and RNA-seq were performed.Bioinformatics analysis was performed after obtaining the original sequencing data.In experiments,we successfully established the approach of multiantibody labeling of TAMs.That is: M0: CD68+CD80-CD86-CD163-CD206-,M1: CD68+CD80+ CD86+CD163-CD206-,M2: CD68+CD80-CD86-CD163+CD206+.After that,cells were sorted out M0,M1,and M2 types of TAMs.At the same time,a special type of cells was found,and all antibody markers are positive in their surfaces,recorded as M12 type cells,which were sorted.All of sorted cells were analyzed by flow cytometry and sequenced,and the cells sorted was right by analysis.After obtaining the raw data for sequencing,bioinformatics analysis was carried out.Firstly,the data quality was good by bioinformatics analysis.Secondly,by the analysis of the transcriptome and chromatin openness: it is confirmed that M12 cells are also a type of polarized TAMs,unlike M1 and M2,but its function is not clear,and it may be closer to M1 cells.We focused on the transcriptome and chromatin openness of M0,M1,M2.The transcriptional map and chromatin accessibility map of TAMs were firstly characterized by bioinformatics tools,revealing TAMs polarization in the molecular level.Among these samples,TAMs' heterogeneity was obvious.Secondly,the chromatin openness had changed significantly among of them and were great different to different type of TAMs.Highly open genes were fewer in M0 cells than others,while in M1 and M2 cells,there were a large number of highly open genes.Subsequently,the correlation analysis between gene expression levels and chromatin openness showed that the correlation was low in M0 cells,but in M1,M2 and M12 cells,the correlation was significantly increased.It indicates that chromatin openness plays an important role in the TAMs polarization process.In addtion,the number of accessibility regions of chromatin M is significantly different among the four types cells,which may become a new direction of TAMs polarization research.In conclusion,in the tumor microenvironment,the chromatin openness and gene expression of TAMs have changed significantly,which may discover new ways for the immunotherapy of PLC.