The Establishment Of In Situ Syrian Hamster Pancreatic Cancer Model With Mutant KRas^G12D And SMAD4 Interference RNA Expressing Lentiviral Vectors

Background and AimPancreatic cancer is a highly malignant solid tumor of digestive tract,with a high morbidity and mortality worldwide.The five-year survival rate of pancreatic cancer is less than 10%.Pancreatic cancer is the tumor with the worst prognosis among digestive tract tumors,often diagnosed in the middle and late stage.The effect of conventional clinical surgery and chemotherapy is limited,resulting in poor prognosis.In order to improve the early diagnosis rate of pancreatic cancer and develop new treatment methods,it is necessary to conduct in-depth research on the pathogenesis and development of pancreatic cancer.Animal model is the most important method to study pancreatic cancer.Currently,few preclinical models can accurately reflect the pathogenesis,pathological manifestations and tumor metastasis of pancreatic cancer.It is of great significance for the study of human pancreatic cancer to establish a new animal model that highly fits the occurrence and development of human pancreatic cancer.Spontaneous pancreatic cancer can be induced by site-directed modification of mutated tumor suppressor gene Tp53 and oncogene KRas in KPC mice.However,the KPC mice express genetic changes in all cells from birth,which is different from the development of human pancreatic cancer.Chemical induction of mouse pancreatic cancer is low-cost and can induce PANIN lesion process,but the induced animal mortality is high,the tumorigenesis rate is low and the induction cycle is long.Chemical-induced hamster pancreatic cancer is less likely to have Tp53 mutations,which is inconsistent with the high frequency of Tp53 mutations in human pancreatic cancer.In addition,some patients with pancreatic cancer have genetic mutations caused by long-term inflammation of the pancreas.Under repeated stimulation of inflammation,cells in the pancreas have genetic mutations,thus developing pancreatic cancer.In order to better simulate in situ pancreatic gene mutations,animal models induced by lentiviral vectors were used.It may be a better method to establish pancreatic cancer model by injecting in situ the target gene changes into the pancreas of animals to make them express or reduce the target gene mutations in the pancreas.At the same time,injection of lentivirus can cause inflammation in the pancreas,which may promote pancreatic cancer.Mice are often used in animal studies.We and other researchers found that human granulocyte-macrophage colony-stimulating factor（GM-CSF）and interleukin-12（IL-12）had no function in mice,but it did in Syrian hamsters.Therefore,we established an animal model of pancreatic cancer by means of Tp53 gene knockout in Syrian hamster combined with lentivirus,which is similar to humans in terms of time and space and simulated inflammatory environment,and may be a good method.In this study,Syrian hamster pancreatic cancer model was stably and efficiently prepared by in situ injection of p53 gene knockout with KRas G12D lentivirus and SMAD4 interfering lentivirus,and then pathological identification of tumor tissues.It provides an ideal experimental tool for the study of biological behavior and regulation of human pancreatic cancer metastasis and the screening,efficacy and mechanism of anti-tumor metastasis drugs.MethodsHEK293T cells were co-transfected with mutant KRas ^G12Dplasmid and SMAD4interference plasmid with lentivirus packaging plasmids ps PAX2 and envelope plasmid PMD2.g,respectively,for lentivirus packaging.After the concentration of disease venom,the mixture of the two lentiviruses was situ injected into the pancreatic tail of the male Tp53 knockout Syrian hamster.Tumor occurrence and activity status of Syrian hamsters were observed regularly,animal weight was measured,and survival time was recorded.The tumor,liver,and spleen tissues of the hamsters were collected for fixation,and subsequently stained with HE to observe the tumor type,growth and metastasis ability.The expression of Ki67 in tumor tissues was detected by immunohistochemistry.Results1.Thirty days after lentivirus injection,Tp53 knockout Syrian hamsters felt protrusions（tumors）on their abdomen.With the extension of time,the size of the tumors grew larger with time,as did the number of animals with tumors.In the dual-virus group,two died before the end of the observation period,tissue lysis occurred at sampling.The remaining three Syrian hamsters had a tumor formation rate of 100%.2.Histopathological results showed that pancreatic tumors were successfully induced after 30 days by the combined injection of the mutant KRas G12D lentivirus and smad4-interfering lentivirus.During the 95-day observation period,tumor metastases occurred in the livers and spleens of the experimental hamsters.Immunohistochemical results showed that Ki67 was expressed in tumor tissues.ConclusionThe in situ model of hamster pancreatic cancer was successfully established based on Tp53 knockout Syrian hamster combined with co-injection of lentiviruses expressing KRas ^G12Dand SMAD4 interference RNA into pancreatic tail,and its clinicopathological manifestations were similar to those of human.This study provides a powerful tool for studying the pathogenesis of pancreatic cancer and developing novel therapeutics.