Construction Of Liver Cancer Model And Cell Lines In Syrian Hamster

BackgroundLiver cancer is a common malignancy worldwide.Most patients with liver cancer are already in the middle and advanced stage when they are diagnosed.The recurrence and metastasis rate of liver cancer after surgical resection is high,which seriously affects the prognosis of patients.Therefore,early screening,early diagnosis and treatment are essential to improve the survival rate of liver cancer.Now,the new treatment methods of liver cancer depends on the big progressions of preclinical research.The molecular mechanisms of liver cancer is the theoretical basement of treatment,which provide new idea for treatment.Models and cancer cell lines are critical for the investigation of molecular mechanisms.Therefore,more and more models and cell lines have been established to explore the pathogenesis and treatment of human liver cancer.In recent years,the advantages of the Syrian hamster as an animal model have come to the fore.Syrian hamsters play an important role in evaluating the efficacy of COVID-19 vaccines.Syrian hamsters have many unique advantages over other experimental animals.They share characteristics with humans in such aspects as tissue morphology,biology and immunology.Some human adenoviruses can replicate in Syrian hamsters,and many human cytokines have biological function in Syrian hamsters but not in mice.They are susceptive to tumor-inducing viruses and can be infected with a variety of human viruses.In addition,they are perennial estrus animals with accurate estrus cycle.All in all,they are promising experimental animals.At present,there is limited published literature related to the establishment of liver cancer models using Syrian hamsters.Therefore,the establishment of a suitable Syrian hamster model and cell lines of liver cancer is valuable for the study of cancer biology and new strategies for the treatment of the disease.ObjectiveThe aim of this study is to use Syrian hamsters with homozygous mutation of Tp53 gene(Tp53^-/-)to construct liver cancer model and tumor cell lines.In this study,lentivirus carrying Syrian hamster KRAS gene mutant KRAS^G12Dwas injected Syrian hamster liver in situ,so as to establish Syrian hamster liver cancer model.Primary tumor cells were isolated from tumor tissues.Stable hamster liver cancer cell lines were obtained by different purification methods.The pathological characteristics of the model and molecular biological characteristics of the cell lines were identified.Methods（1）The sequences of the target plasmid FUB-KRAS^G12D-P2A-EGFP and the package plasmid ps PAX2and p MD2.G were verified by sequencing.（2）Lentiviruses expressing KRAS^G12Dmutant and EGFP were produced using second-generation packaging system.After concentrated using ultra-high-speed centrifuge,virus titers were detected by flow cytometry.（3）Golden hamsters(Tp53^-/-)were injected with PBS,empty vector lentivirus and Lentivirus that carry the KRAS^G12Drespectively.The status of hamsters were closely observed and basic palpation of the liver region was performed after surgery.（4）The tumor tissues were collected for pathological analysis.Subsequently,the expression of liver cancer related genes in tumor tissues were assayed by reverse transcription PCR.（5）The collagenase digestion method was used to isolate the tumor cells from the tumor tissues.Subsequently,the differential adhesion method and monoclonal method were used to purify the primary cells.（6）Biological characteristics of three stable cell lines were analyzed by morphological analysis,tumor growth curve analysis,monoclonal formation experiment,chromosome karyotype analysis and other experiments.（7）5×10⁶cells were injected into the right flank of wild-type hamsters.Tumor growth of three groups were monitored and tumor metastasis of three groups were analyzed.Results（1）The sequencing results showed that the sequences of target plasmid and package plasmid were correct.（2）After packaging and concentration,the titer of control lentivirus was 1.5×10⁷IU/m L and the titer of lentivirus expressing the KRAS^G12Dwas 2×10⁷IU/m L.（3）The Group injected with lentivirus expressing the KRAS^G12Dall showed liver nodules with palpable hardness different from the texture of other liver regions and poor mobility.However,hamsters in the the other two groups had no liver abnormalities.（4）Tumor tissues were preliminarily identified as sarcomatoid hepatocellular carcinoma（SHC）by macroscopic morphological observation combined with the results of reverse transcription PCR and HE.（5）Three stable cell lines named SHC805,SHC905 and SHC105 were obtained after purification.They all had abnormal karyotypes,which was consistent with the characteristics of tumor cells.There are similarities and differences in morphology,tumor cell growth curve and plate clone formation among them.For example,their cell doubling time were 31 h,21 h and 22 h in sequence,and the plate clone formation rates were 13%,20%and 33%.（6）The tumorigenesis rate of the three cell lines in hamsters was 100%,but the tumor growth rate of the three cell lines were different.The tumor of SHC805 group grew slower than that of the other two groups.And the tumor mass reach to 3500mm³need longer.In addition,the metastatic potential of SHC905 and SHC105 was higher than that of SHC805 through preliminary observation.Conclusion（1）in situ injection of lentivirus expressing the KRAS^G12Dcan induce the occurrence of liver tumors in Tp53^-/-Syrian hamsters,which were preliminarily identified as SHC through molecular biology and pathology methods.（2）Three stable tumor cell lines SHC805,SHC905 and SHC905 with different morphology were obtained.All of them have the characteristics of malignant cells and 100%tumorigenic ability in wild type hamsters.And SHC905 and SHC105 have higher metastasis potential than SHC805.