The New Mechanism Of Origin And Metastasis In Hepatoma Revealed By The Next Generation

Background and ObjectiveLiver cancer is the second most common cause of cancer-related death after lung cancer and is among the few solid tumours that are increasing in incidence and mortality rates world-wide.Hepatocellular carcinoma is the most common type of primary liver caner and is characterized by both phenotypic and molecular heterogeneity.The formation of intrahepatic metastasis and microsatellite lesions are the main forms of recurrence and metastasis of hepatoma.The distribution of intrahepatic sinusoids and small bile ducts provides favorable conditions for the intrahepatic dissemination of cancer cells.As the largest digestive and metabolic organ in the human body,the liver has a strong regeneration function.It had already been clearly confirmed that the liver stem cells,stellate cells,mature liver cells and exogenous mesenchymal stem cells could participate in liver damage repair and coordinate of liver tissue regeneration.In fact,each group of the renewable cells has the potential to occur mismatch in the process of injury repairation which causes the possibility of multiple initial origins of hepatoma.The complex origin further leads to internal heterogeneity of hepatoma which causes the complex signaling pathways crosstalk between the tumor cells and normal cells in the process of tumor development.There is a relation of mutual dependence and mutual promotion among different subgroups of the cells in hepatoma which is a big challenge for the clinical treatment of hepatoma.The combined hepatocellular carcinoma and intrahepatic-cholangiocacinoma(c HCC-ICC)is a unique type of hepatoma characterized by low incidence,highly malignancy and complex composition.Limited by the existing clinical and pathology diagnostic indicators of this type of hepatoma.The clinical diagnosis and treatment become more difficult.Based on the current research of liver cancer,our study hopes to make a breakthrough in the field of hepatoma research from the following aspects,aiming at providing new ideas for the clinical diagnosis and treatment of hepatoma through clinical-basic-clinical transformation: An important medium of signal transmission-exosomes,revealing new mechanisms of interaction between different tumor cells in the hepatoma;Using genome and exon sequencing analysis at the single cell level to explore the origin of hepatoma and determining the role of cancer stem cells in the development and recurrence of hepatoma;Hoping to find some new clinical markers to assist the evaluation of hepatoma through the genomics research;Hoping to find high-frequency mutant genes and specific driving mutation in c HCC-ICC through the genome and exon data analysis which can provide potential indicators and targets for clinical diagnosis and treatment.Methods 1)The exosomes of hepatoma were separated and purificated by ultracentrifugation and characterized by scanning electron microscopy,exosmes staining,quantitative analysis of proteins and mi RNAs.2)The exosomes of hepatoma were co-cultured with normal hepatocytes.The key enzyme activities of DNA damage and repair were detected by RT-PCR.The regulation role of hepatoma exosmes in DNA damage repair of normal recipient cells was clarified.3)The effects of exosomes secreted by hepatoma cells on the invasion and metastasis of tumor cells were investigated by wound healing assay,chemotaxis assay,invasion assay and clone formation assay.4)Establish a mouse model of allogeneic bone marrow transplantation after irradiation with lethal dose of radiation,then further induce primary hepatoma based on this model and explore the origin of hepatoma.5)The cancer stem cells and non-stem cancer cells in fresh hepatoma samples were separated by flow sorting technique.The biological characteristics of cancer stem cells were verified in vitro.The copy number alterations of cancer stem cells and non-stem cancer cells were analyzed by using single-cell whole genome sequencing to describe the tumor evolution model and to explore the role of cancer stem cells in the development of hepatoma.6)To explore the role of cancer stem cells in the process of tumor recurrence by comparing the tumor evolution models among the patients with different clinical features of hepatoma(newly diagnosed hepatoma and recurrent hepatoma).By comparing the copy number alterations of patients with different degrees of malignancy(with vascular invasion and no vascular invasion)to explore the effect of the degree of copy number alterations on the malignancy of hepatoma.7)Through the whole genome and exon sequencing of fresh frozen samples of c HCC-ICC,the copy number alterations and single nucleotide variations were analyzed,and the high frequency and specific mutation genes of c HCC-ICC were explored in order to assist the clinical diagnosis and treatment.8)Through the single-cell whole genome sequencing analysis and single cell mutation verification to reveal the tumor heterogeneity of c HCC-ICC and to explore the possible origin of c HCC-ICC.Results 1)Rab27a regulates the secretion of exosomes in hepatoma,and its expression intensity is positively correlated with the liver cirrhosis,serum level of AFP and vascular invasion of hepatoma patients.2)Hepatoma-related exosomes can affect DNA repair activity of the normal liver cells and induce malignant transformation of these cells.3)The exosomes secreted by hepatoma cells can promote chemotaxis,invasion and clonal formation of tumor cells in vitro.It is further confirmed that hepatoma-related exosomes can promote the proliferation and metastasis of hepatoma cells and are closely related with tumor recurrence in vivo.4)The mouse model of allogeneic bone marrow transplantation after irradiation with lethal dose of radiation revealed that bone marrow stem cells participated in the liver repairing process and then took part in the initiation and development of hepatoma.5)The analysis of single-cell copy number alterations showed that the development of cancer stem cells was closely related to the recurrence of hepatoma.6)There is heterogeneity within the hepatoma tissue,the complexity of copy number alterations from single cell level may help to reveal the degree of malignancy in hepatoma.7)The frequency of TP53 mutation in c HCC-ICC is significantly higher than that in HCC and ICC which could be used as a potential marker for the diagnosis of c HCC-ICC.8)There are specific high-frequency mutations in c HCC-ICC,including FSIP2,PKHD1L1,SVEP1 and LRP1 B which has never been reported in previous studies of hepatoma sequencing,and in-depth revelation of these high frequency mutations may help us to understand the occurrence of c HCC-ICC and can be used as a potential clinical diagnostic criteria or therapeutic target.9)Single-cell level analysis of copy number alterations and single nucleotide variations reveal that heterogeneity exisits within c HCC-CC and the tumor cells with hepatocyte phenotype and bile duct cell phenotype have the same origin.Conclusions 1)Rab27a could be used as a predictor of the liver cirrhosis and vascular invasion of hepatoma;Exosmes secreted by the hepatoma cells absorbed by recipient cells on one hand promoted its EMT transformation followed by enhancing their ability to migration and invasion,on the other hand affected the DNA repair process of which would induce malignant transformation of normal cells;The experimental results further showed that the exosomes secreted by hepatoma cells could also promote the intrahepatic and extrahepatic disseminated of hepatoma cells.2)Bone marrow mesenchymal stem cells participated in the liver repairing and regeneration and in some certain circumstances could act as the origin of hepatoma and participate in the development of hepatoma;Inhibition of bone marrow mesenchymal stem cells helped reduce the incidence of hepatoma(based on the mouse model established in our study).3)Cancer stem cells were involved in the development of hepatoma and played an important role in the recurrence of hepatoma;There was heterogeneity in hepatoma and different subtypes of cancer stem cells might have different regulatory functions;Copy number alterations could be used as a potential indicator of the degree of malignancy of hepatoma.4)Compared with HCC and ICC,c HCC-ICC had a higher frequency of TP53 mutation;c HCC-ICC had specific mutations different from the other two types of hepatoma,including: FSIP2,PKHD1L1,SVEP1 and LRP1B;The differentiated hepatocytes and cholangiocarcinoma cells in c HCC-CC had the same origin rather than the subsequent carcinogenic transformation.