Three-dimensional Bioprinting In Constructing Tumor Microenvironment Model For Hepatocellular Carcinoma

Background and Objectives:Hepatocellular carcinoma(HCC)is one of the most common gastrointestinal tumors,which is the fourth leading cause of cancer-related death worldwide.The liver function of HCC patients is usually impaired,drug treatment can cause secondary damage to the patients'liver.Therefore,it is imperative to establish a new model in individualized treatment for HCC patients to reduce unnecessary chemotherapy exposure.Currently,the construction of patient-derived xenograft(PDX)and organoid models is the fundamental way to explore in vitro individualized models.However,the establishment of PDX and organoids is highly dependent on the proliferative capacity of parental tumor.In recent years,3D bioprinting models emerged and were confirmed that they could successfully establish in vitro tumor models with higher success rate.Here,we firstly utilized 3D bioprinting technology to construct a multi-cell type tumor model in vitro for tumor drug screening.Methods:Tissue specimens from four HCC patients were collected,and HCC cell suspensions were obtained by tissue digestion.Human primary HCC cells,human primary fibroblasts,human umbilical vein endothelial cells,and monocytes induced by fobotoxin were printed as concentric circle-like in vitro tumor models using SUNP BIOTECH bioprinter.Then,the models were functionally evaluated using morphological analysis,Calcein-AM/PI survival staining,and CCK-8 cell activity assays to clarify the survival and proliferation ability of hepatocellular carcinoma cells and mesenchymal cells.We performed immunofluorescent staining using AFP/CD68,CD68/CD163,AFP/CD31,AFP/? SMA to locate the different cell fractions and clarify the distribution of mesenchymal cells in relation to HCC cells.Results:After tissue digestion of liver cancer tissue specimens from four patients,HCC cell suspensions were obtained and in vitro HCC microenvironment models were successfully constructed.The tumor cells and mesenchymal cells were distributed in the inner and outer concentric circles as expected and showed scattered spherical clusters.The number and survival of the cells in each group were stable,and the infiltration of tumor cells from the inner circle to the outer circle of mesenchymal cells could be observed.Conclusion:This study successfully established an in vitro microenvironment model of HCC and demonstrated that tumor cells and mesenchymal cells could grow stably and maintain phenotype in the model.Histopathological analysis,whole-exome sequencing and RNA sequencing are required to verify whether the model can retain the characteristics of parental liver cancer tissues and to assess the consistency of gene mutations and gene expression in the further.Background:Autosomal dominant polycystic liver disease(ADPLD)is characterized by multiple cysts in the liver without(or only occasional)renal cysts.Though at least seven genes were identified to be associated with high risk for developing ADPLD,more than 50%of patients still lack clear genetic attributes.Methods:To identify additional ADPLD associated genes,we collected 18 independent ADPLD samples and performed whole exome sequencing on all samples.After filtering with Human Gene Mutation Database(HGMD)professional edition database,we identified a new mutation point in PKHD1.Results:Among the 18 ADPLD samples,we found two cases with PRKCSH mutation,two cases with PKD2 mutation,one cases with PKHD1/PKD1 co-mutation,one case with PKHD1 mutation,one case with PKD1 mutation,and one case with GANAB mutation.We identified a new PKHD1 missense mutation in an ADPLD family,in which both patients showed the innumerable small hepatic cysts as reported previously.In addition,we found the PRKCSH and SEC63 mutation frequencies are lower in the Chinese population than European and American population.Conclusion:In this study,we reported a family with ADPLD associated with a novel mutation in PKHD1(G1210R)gene.And we believed the genetic profile of Chinese population in ADPLD is different from European and American population,suggesting that the research on genetic mutation of ADPLD in Chinese population could fill the gap of previous research.