Three-dimensional Bio-printing Of Primary Human Hepatocellular Carcinoma For Personalized Medicine

Background and Aims Hepatocellular carcinoma(HCC)is the most common malignant tumor of the liver,the third leading cause of cancer-related death in China,and the fourth leading cause of cancer-related death in the world.Empirical targeted drugs are the main treatment measures for patients with advanced hepatocellular carcinoma.However,due to the extraordinary individual heterogeneity among patients with liver cancer,the efficacy of targeted therapy for different patients varies greatly,and a new model of individualized treatment for patients is imperative.Currently,the establishment of patient-derived xenograft(PDX)and in vitro tumor organoids is the fundamental way to explore individualized tumor models for specific treatment.However,the construction of patient-derived PDX and organoids is highly dependent on the proliferative capacity of the parental tumor.It is difficult for patients with low Ki-67 levels to achieve,and the overall success rate is rather low.In addition,the time and economic cost of constructing PDX and organoids are relatively high and cannot be practically applied to the individualized diagnosis and treatment of clinical patients.Here,for the first time,we adopted the most advanced interdisciplinary technology of three-dimensional(3D)bioprinting,and successfully established a patient-derived individualized liver cancer model system to realize drug screening for personalized treatment.Methods HCC specimens were obtained from six HCC patients after surgical resection.The liver cancer cell suspension is acquired through tissue digestion,and mixed with sodium alginate and gelatin to prepare the bio-ink with a designed cell concentration.Afterwards,a 3D cell bioprinter was used to construct three-dimensional printed HCC(3DP-HCC)models for all 6 patients.We performed histopathological analysis of liver cancer tissue samples and corresponding 3DP-HCC models,whole exon sequencing(WES)and RNA sequencing(RNA-sequencing,RNA-seq)to verify whether the key features of the original tumors,the consistency of genetic mutation and gene expression profile were well-maintained.In addition,we conducted xenograft experiment to evaluate whether the tumorigenic potential and histological characteristics of the parental liver cancer tissue were retained in 3DP-HCC models.Finally,based on the 3DP-HCC models,we conducted individualized screening of a variety of the most commonly used empirical targeted drugs for all 6 patients.Results After the HCC specimens of 6 patients were successfully digested into single cell suspension,the suspension was prepared for bio-ink.Then 3DP-HCC models were successfully established for all six patients.The in vitro 3DP-HCC model grows well during long-term culture and can retain the key characteristics of parental HCC tissues,including stable expression of the specific tumor marker,as well as the genetic variations and genetic expression profiles of the parental HCC tissues.Xenograft study confirmed that both tumorigenic potentials and histological features were retained in 3DP-HCC models after long-term culture.Finally,drug screening experiments confirmed that the 3DP-HCC model can quantitatively and intuitively display the anti-tumor effect of drugs.Conclusion The 3DP-HCC model is a reliable in vitro model system for liver cancer,which can be used for the screening of specific drugs and helps to formulate individualized treatment regimens for patients with liver cancer.