Establish Esophageal Stratified Squamous Epithelial Organoids And Study The Role Of Stemness Markers Sox2 And P63 In Proliferation-differentiation Homeostasis,Damage Repair And Carcinogenesis

Esophageal squamous cell carcinoma(ESCC)is a common malignant tumor of the digestive tract that has a high rate of mortality and poor prognosis.The occurrence and development of ESCC involves progressive genomic alterations driven by inflammatory and other factors within the tumor microenvironment.The phenotype of ESCC develops from hyperplasia to papilloma,and then to squamous cell carcinoma.This progression involves the gradual imbalance between cellular proliferation and differentiation.However,the underlying mechanisms responsible for the transition from esophageal epithelial hyperplasia to ESCC remain to be fully understood.Most patients with ESCC present with advanced disease and so it is difficult to understand the driving genetic changes that lead to imbalances in proliferation and differentiation at the early stage of the disease.These processes are also difficult to experimentally model as tumor mutation burdens are high making it difficult to distinguish the driving genes involved in esophageal epithelial proliferation and differentiation.The ideal model system should have a defined genomic background and with good stability and reproducibility,simple operation.In vitro organoids are robust models that can be used to study tumor development and progression.Organoids consist of stem cells or organ-specific progenitor cells that self-organize into a three-dimensional structure that recapitulates the structure and function of specific organs.In this study,we established a rat esophageal organoid model that was used to explore the key genes involved in esophageal epithelial proliferation-differentiation.Our data provide a theoretical basis to better understand the pathological processes involved in ESCC.A detailed study was conducted on the growth patterns of rat esophageal organoids.We found that the organoids had an asymmetric growth pattern as the structure of the organoids was asymmetrical cell within 2-5 days mass structure before a stratified squamous structure appears on the sixth day.The growth pattern of the organoids was divided into two stages,the initial stage of development and the steady-state stage of proliferation-differentiation.Next,we used immunofluorescence to detect the localization and expression of Sox2 and p63 in the two growth stages.We found that subsets of p63-Sox2+cells appeared on day 4 and that this group of cells persisted to day 10 when organoids were mature.Next,we used single-cell sequencing technology(SCST)to determine that this group of cells was not present in the organoids.We then established an over-expression Sox2 model and a two-stage knockdown Sox2 model to confirm that Sox2 plays an important role in the initial development of the esophagus and the steady-state process of proliferation-differentiation.To clarify the function of p63-Sox2+cell subsets in the organoids,we established an esophageal organoid inflammatory injury model.Our data showed that this group of cells proliferated excessively in the organoids damaged by inflammation and this proliferation phenomenon was reversible.shRNA was used to knockdown Sox2 and p63 in the steady-state stage of the organoids,further confirming that p63-Sox2+cell subsets are involved in the inflammatory injury responses.Loss of the p63-Sox2+cell subsets led to the proliferation of the organoids.Under these conditions,the differentiation homeostasis is unbalanced and the response to inflammatory injury is lost.Conversely,overexpression of Sox2 enhanced the response of the p63-Sox2+cell subpopulation to inflammatory injury.Finally,the localization and expression of Sox2 and p63 in ESCC through the established organoids of human esophageal squamous cell carcinoma cell lines and rat esophageal squamous cell carcinoma organoid models were investigated.Our data showed that Sox2 and p63 are often highly expressed in SCC and no subpopulations of p63-Sox2+cells are present.In summary,this study has successfully established a rat esophageal organoid model that was used to investigate growth patterns of the esophagus and identified two stages of development,proliferation and differentiation.p63-Sox2+cells identified by immunofluorescence and SCST.Cell subpopulations were identified in an inflammatory injury model indicating that p63-Sox2+cells play an important role in the regeneration and repair of esophageal injury.The hyperproliferation of this population of cells drives the process of repairing damage and is reversible.Our results provide a theoretical basis for esophageal-related studies including esophageal stem cell markers and the etiology of ESCC.