Objective: To investigate the heterogeneity of epithelial cells,T cells and Cancer-associated fibroblasts(CAF)between and within endometrial carcinoma.To explore the effect of CAF heterogeneity on the prognosis of endometrial carcinoma and explore the role and mechanism of vascular CAF(vCAF)in vasculogenic mimicry of endometrial cancer cells.Methods: Based on the Illumina Nova Seq6000 platform,single-cell transcriptome sequencing was performed on endometrial carcinoma and normal endometrial tissue respectively.Cell quality control,detection,reference genome comparison and diversity integration are carried out through Cell Ranger software.Use Scan software to infer cell cycle.Scrublet software performs multiple cell prediction and removes multiple cells.The dimensionality reduction analysis of data is carried out through principal component analysis(PCA),uniform manifold approximation and projection(UMAP)or T-distribution random neighborhood embedding(t-SNE).Single R package is used for cell type annotation.Infer CNV was used to identify malignant cells.Cellphone DB was used to analyze the intercellular communication.Enrichment analysis was carried out by GO,KEGG and GSVA.Hematoxylin-eosin staining(HE)and Sirius red staining were used to detect the desmoplastic reaction of EC.Immunohistochemistry(IHC)was used to detect the presence of different subtypes of CAF in endometrial carcinoma.The presence of vCAF in endometrial carcinoma tissue was detected by flow cytometry and multiple immunofluorescence.The effect of vCAF on the migration function of umbilical vein endothelial cells(HUVEC)was detected by scratch test.The effect of vCAF on the tubule formation ability of HUVEC was tested by tubule formation experiment.Cord formation assay(CFA)was used to detect the effect of vCAF on the formation ability of endometrial cancer cells.The cytokines secreted by vCAF were detected by real-time fluorescence quantitative polymerase chain reaction(q RT-PCR),immunoblotting(WB)and enzyme-linked immunosorbent assay(ELISA).Cell counting kit(CCK8)was used to detect the semi-inhibitory concentration of the drug niclosamide.Chromatin immunoprecipitation(CHIP)was used to detect the binding ability of transcription factor STAT3 and CDH5 gene promoter.The effect of vCAF on the progression of endometrial carcinoma was detected by subcutaneous tumor transplantation in nude mice.Results: 1.Endometrial carcinoma is composed of epithelial cells,T cells,fibroblasts,macrophages,natural killer(NK)cells,endothelial cells,B cells,monocytes,and dendritic(DC)cells.2.Epithelial cells are in the active stage of division,malignant epithelial cells have intratumoral heterogeneity,and different subsets of malignant epithelial cells play different biological functions in the tumor.3.The T cells in the immune microenvironment are highly heterogeneous,in which the proliferating T cells are characterized by depletion;The interaction between malignant epithelial cells and T cells may participate in the formation of the inhibitory immune microenvironment of endometrial carcinoma.4.CAF has obvious heterogeneity,which can be divided into matrix CAF(m CAF),inflammatory CAF(i CAF),vascular CAF(vCAF)and antigen presentation CAF(ap CAF),and play different biological functions respectively.5.vCAF is an important risk factor for poor prognosis of endometrial cancer patients.6.vCAF can significantly promote the migration and tubule formation of HUVEC,which fully explains its angiogenic regulation characteristics.7.vCAF can promote the formation of VM of endometrial cancer cells in vitro.8.vCAF secretes interleukin 10(IL-10)to promote epithelial-endothelial transformation(EET)of endometrial cancer cells through JAK1/STAT3 pathway.9.IL-10 promotes the expression of endothelial cell adhesion molecule(VE-Cadherin)through the JAK1/STAT3 signal pathway,thus promoting the formation of VM in endometrial cancer cells,while the JAK1/STAT3 pathway inhibitor niclosamide can significantly inhibit this process.10.vCAF accelerates its progression by promoting VM formation of endometrial cancer cells in vivo.Conclusions: 1.endometrial carcinoma has obvious heterogeneity between and within tumors,and different cell subsets play different roles in the occurrence and development of tumors.2.vCAF is an important factor for poor prognosis of endometrial carcinoma,and can promote angiogenesis and VM formation of endometrial carcinoma in vivo and in vitro.3.vCAF secretes IL-10 to promote the EET of endometrial cancer cells through JAK1-STAT3 pathway,thus promoting the formation of VM. |