Elaborating The Composition Of Myeloid Cell Subsets And Its Association With The Prognosis In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the solid tumors with high morbidity and mortality.For patients with HCC,resection is associated with survival only 5-7%at 5 years and as many as 60-70%have tumor recurrence at 5 years.The factors driving the evolution of HCC include internal and external factors.Intrinsic factors refer to mutations that occur in hepatocyte and external factors refer to the interaction of tumor cells with surrounding stromal cells,the immune system,and non-cellular components.For the emerged tumor cells,the immune system or extracellular matrix will be contributed to elimination of tumor cells,thereby inhibiting the development and metastasis of the tumor.Therefore,the tumor and its external environment,which constitutes the tumor microenvironment,plays an important role in tumor evolution,recurrence and metastasis.The immune microenvironment composed of tumor-infiltrating immune cells and secreted cytokines is an essential component that affects the tumor microenvironment.Owing to technical limitations,most of the previous studies on the complex immune microenvironment in HCC is based on the level of bulk cells to study a specific cell type in situ or after cell sorting.The development of single cell sequencing technology provides a powerful tool for the systematic study of tumor microenvironment.At present,the study of the immune microenvironment of HCC at the single cell level has a preliminary understanding of the composition and characteristics of T lymphocyte subtypes.However,the characteristics of myeloid cell subtypes in HCC and its effect on tumors are still poorly understood.In addition,the similarities and differences between the immune microenvironment in primary and recurrent HCC are unknown.Based on the current research status of HCC immune microenvironment,this study used single-cell RNA sequencing and analysis techniques to identify the immune cell composition and gene expression characteristics of the immune microenvironments from 6 primary and 12recurrent HCC.Especially for myeloid cells,we identified cell subsets that were related to the clinical phenotype of HCC and play a key role in the evolution of HCC.Combined with immunohistochemical technology and compared with the myeloid cells in peripheral blood mononuclear cell(PBMC)and other tumors from published data,the composition and function of the identified myeloid cells were verified.The results in this study are as follows:(1)In this study,a total of 12,870 CD45~+immune cells were obtained by sequencing after quality control,and a total of 14 cell subtypes and their characteristic genes were identified.Including T lymphocyte,natural killer cell(NK)and B lymphocyte,tumor-associated macrophages(TAM),monocytes and dendritic cells(DC).Further,the cell subtype identification of myeloid cells was performed and obtained 7 myeloid cell subtypes and their characteristic genes.Including TAM,two types of monocyte subtypes(FCN1＿mono and Mono4)and three DC subtypes(DC1,DC2 and DC3).(2)There are high infiltrations of CD4~+na?ve/CD8~+cytotoxic T cells and CD160+NK cells in the adjacent normal tissues,showing an activated immune microenvironment.In tumor tissues,TAM,regulatory T cells and CD8~+exhausted T cells related to immune regulation have a higher infiltration than those adjacent normal tissues.This indicates that the immune microenvironment in the tumor tissue shows an immunosuppressed and inactive state.Among myeloid cell subtypes,the Mono4 is enriched in tumor tissues.Comparing the cell composition of primary and recurrent HCC,we found that only DC is relatively high in recurrent HCC,especially the DC2 subtype,while other cell compositions have no significant difference in primary and recurrent HCC.It suggests that the immune cell composition is relatively consistent in relapsed and primary HCC.(3)The DC subtypes in HCC microenvironment was compared with in PBMC from healthy people.The results show that both DC1 and DC2 are present in the HCC and PBMC,while DC3(LAMP3+CCR7+)identified in this study is a new DC subtype in tumors and the existence of DC3 has been validated in melanoma and head and neck cancer from public data.DC cells in tumors highly express major histocompatibility complex class II(MHC-II)molecules and have migration-related characteristics.This indicates that the DC in the tumor has the function of migrating to the tumor-draining lymph node or tumor and presenting the tumor antigen through MHC-II which mediate the activation of CD4~+T cells.In addition,our study also found that there are different recruitment pathways for DC1 in tumor and adjacent tissue respectively.(4)Correlation analysis of myeloid subtypes and clinical phenotypes found that the high infiltration levels of TAM and FCN1＿mono are significantly associated with microvascular invasion and high alpha-fetoprotein(AFP)levels in HCC.The infiltration of TAM is significantly higher in HCC with higher antigen levels of hepatitis B virus(HBV)and larger tumor size.The high infiltration of DC2 is associated with a small tumor size in HCC.(5)Analysis of the correlation between the characteristics of each myeloid cell subtype and prognosis of HCC found that TAM tends to be M2-type polarization and indicates poor prognosis in primary HCC.The two subtypes of monocytes(FCN1＿mono and Mono4)exhibit two distinct immune functions:FCN1＿mono expresses the reported feature genes of myeloid-derived suppressor cells(MDSC),which is related to the secondary recurrence of relapsed HCC.Mono4 highly expresses the cytotoxic gene and is related to favorable prognosis in HCC.The three DC subtypes are also associated with favorable prognosis in both primary and recurrent HCC.In summary,this study analyzed and elaborated the association of myeloid cell subtypes and their functions with the clinical phenotype and prognosis of HCC by single cell RNA-seq.Our study reveals the unique immune microenvironment in HCC,identifies cell subtypes related to clinical phenotype and prognosis,and provides potential therapeutic targets and prognostic predictors for future clinical diagnosis and treatment of HCC.