Construction Of Organoid Culture Model Of Human Esophageal Squamous Cell Carcinoma In Vitro

Objective:Esophageal cancer is one of the most common thoracic malignancies.The incidence and mortality of esophageal cancer are increasing in recent years,which seriously affects the lives and health of Chinese residents.The main pathological types of esophageal carcinoma include esophageal squamous cell carcinoma and esophageal adenocarcinoma,while the main pathological type of esophageal carcinoma in our country is esophageal squamous cell carcinoma.At present,the clinical diagnosis and therapeutic effect of esophageal cancer is still not great,the prognosis of patients is poor,and the five-year survival rate is low.Therefore,it is particularly important to explore the pathogenesis of esophageal cancer and develop targeted drugs.Good experimental models often play important roles in the scientific research of malignant tumors.For a long time,there has been a lack of ideal and effective experimental models in the study of esophageal carcinoma,which has become one of the most important factors limiting the study of esophageal carcinoma.With the rise and development of organoid technology,organoid has become an important in vitro model for clinical and basic cancer research.Domestic research teams have successfully established culture systems for organoid models of gastric cancer,intestinal cancer,lung cancer and breast cancer.However,the relevant culture system of the organoid model of esophageal squamous cell carcinoma has not been reported in detail in China.The purpose of this study is to construct and optimize a mature organoid culture system for human esophageal squamous cell carcinoma,so as to provide a new technological platform for related research on esophageal carcinoma that is closer to the human internal environment.Materials and methods:The tissue samples used to construct the organoid model of human esophageal squamous cell carcinoma were collected from patients admitted to the Department of Thoracic Surgery of our hospital from 2020 to 2022.All enrolled patients in this study have signed informed consent,and this study has been approved by the ethics committee of the hospital.H&E staining and immunohistochemistry were used to evaluate whether the organoid model could maintain the characteristics of the original tumor tissues.The optimal culture scheme of WRN conditional medium was prepared based on L-WRN tool cell lines.The next generation sequencing technology was used to detect the similarity of genomic expression between tumor organoid models and original tumor tissues.The mice used in the experiment of subcutaneous tumor formation in nude mice were from the highly immunodeficient model NOG mice purchased from Beijing Vital River company to explore whether the organoid model still possessed the ability of tumor formation of the original tumor cells.Results:In this study,a set of organoid culture system for human esophageal squamous cell carcinoma was successfully established.We found that the organoid models of tumor cultured in vitro had a high degree of homology with the original tumor tissues according to histomorphology and tumor markers.By adjusting the proportion of cytokines in complete medium,we found that the growth efficiency of organoid decreased when removing any factor in Wnt-3A,R-spondin-1,Noggin,EGF,Ca Cl₂and N-Acetyl-L-cysteine,respectively,compared with complete medium condition.At the same time,the mean diameter of tumor organoids decreased significantly when any factor of Wnt-3A,R-spondin-1 and Noggin was removed.The removal of Gastrin,FGF10,Nicotinamide,SB202190 and A83-01 had no significant effect on the growth of tumor organoids.Meanwhile,WRN conditional medium containing Wnt-3A,R-spondin-1 and Noggin,prepared by L-WRN tool cell lines,was proved to be able to replace commercial cytokines,and its culture effect was even better than that prepared by commercial cytokines.Next generation sequencing technology was used to analyze the genome of esophageal squamous cell carcinoma organoids established in vitro and the original tumor tissues.The experiment of subcutaneous tumor formation in nude mice also proved that the tumor organoids cultured in vitro still had tumor formation ability.Conclusions:This study successfully constructed and optimized a complete organoid culture system for esophageal squamous cell carcinoma,which confirmed its potential to replace the traditional experimental model and become a new scientific research platform for research of esophageal carcinoma.Tumorigenesis in nude mice not only verified that organoid model still had good potential for tumorigenesis in vivo,but also laid a foundation for the application and popularization of organoid model constructed by this culture system.