Study On The Characteristics Of Immune Microenvironment And Anti-tumor Effect Of Tissue-resident Memory T Cells In Molecular Classification Of Endometrial Cance

Part one Immune microenvironment and inflammatory gene expression profiles of different molecular subtypes of endometrial carcinomaBackground Endometrial carcinoma is the second most common gynecologic malignancy.With the increase of life expectancy and the improvement of economic living standard,the incidence rate and mortality rate of the disease shows a rising trend.Based on the characteristics of tumor gene mutations and their relationship to prognosis,endometrial carcinoma has been classified into four types by molecular typing:POLE ultramutated,microsatellite instability-high(MSI-H),copy number high,and copy number low.Extensive researches have demonstrated the potential clinical use of molecular typing in prognostic evaluation and treatment selection of endometrial carcinoma.However,molecular typing also has certain limitations:The existing molecular typing system needs the participation of multiple test platforms,type MSI-H and copy number low have intermediate prognosis but the difference between these types is not significant.Thus,other markers need to be found to further layer the prognosis of these patients.Infiltrating immune cells,in addition to tumor cells,play an important role in the occurrence and development of tumor.Analysis of the characteristics of tumor-infiltrating lymphocytes and the RNA expression profile of T-cell inflammatory genes can further stratify the prognosis and treatment guidance from the perspective of tumor immune microenvironment.Objectives 1.To explore the feasibility and value of second-generation sequencing(NGS)for molecular typing of endometrial carcinoma.2.To detect the T cell inflammatory gene RNA expression profile and tumor immune cell infiltration characteristics in different molecular subtypes of endometrial carcinoma samples.3,To explore the factors or methods for further stratification of the immunotherapy and prognostic outcomes of endometrial carcinoma on the basis of molecular typing.Methods The patients who underwent surgical staging for endometrial carcinoma in Peking Union Hospital from March 2021 to July 2021 were prospectively included.The preoperative peripheral blood and surgical pathological specimens were collected for NGS sequencing,tumor immune-related RNA detection and pathological multiple immunofluorescence staining.The characteristics of immune microenvironment were analyzed and the differential gene expression related to prognosis and immunotherapy were detected among different molecular types of endometrial carcinoma.Results 1.The proportion of molecular subtypes detected by NGS sequencing is similar to that in previous studies.The results of TP53 gene mutation and MSI state have a high coincidence rate with the expressions of pathological immunohistochemical p53 and mismatch repair protein.NGS can simultaneously provide information on tumor mutation load and other gene mutations related to immunotherapy.2.The expression of immunerelated RNA and the infiltration state of immune cells in tumor immune microenvironment are different between and within the molecular typing groups.3.Three differentially expressed genes,CCR5,IL10 and TNFRSF14,which can stratify the prognosis within the group,were found from MSI type,TP53 wild-type and TP53 mutant,respectively.Conclusions NGS can accurately determine the molecular typing of endometrial carcinoma by one-stop.There were differences in the expression of tumor-related gene,immune microenvironment-related RNA and immune cell infiltration characteristics among the EC molecular typing groups.The expression of tumor immunity-related RNA such as CCR5,IL-10,and TNFRSF14 may become stratification factors that provide guidance for the endometrial carcinoma prognosis and immunotherapy on the basis of molecular typing.Part two Study of antitumor effect of CD8~+tissue resident memory T cells in endometrial carcinoma and their application as new immunotherapy targetsBackground Tumor-infiltrating immune cells play an important role in tumor development and immune escape in tumor microenvironment.Tissue resident memory T cells(TRM)are thought to be related to tumor prognosis.But its regulatory mechanism is still unclear.Previous studies have shown that there are a large number of CD103~+CD8~+TRM cells aggregate in tumor-infiltrating lymphocytes of endometrial carcinoma.The expression abundance of CD103~+ CD8~+ TRM cells is positively correlated with the prognosis and immunotherapy effect of various tumors,suggesting that CD103~+CD8~+TRM may be the important target cell for immunotherapy.A variety of immune checkpoints are highly expressed on tumor-infiltrating CD8~+T cells.Previous studies have found that PD-1/PD-L1 inhibitors have poor therapeutic effect on endometrial carcinoma.However,TIGIT,an immune checkpoint,and its ligands are also widely expressed in endometrial carcinoma.TIGIT may become a new target for immunotherapy of endometrial carcinoma.Objectives 1.To explore the expression abundance,tissue resident characteristics and functional depletion of tumor-infiltrating CD8~+T lymphocytes and the CD 103~+CD8~+TRM subpopulations in endometrial carcinoma by flow cytometry experiment.2.To determine the killing abilities of CD103~+CD8~+TRRM and CD103-CD8~+Tcells by in vitro cell experiments.3.To verify the reversal effect of PD-1 and TIGIT inhibitors on CD103~+CD8~+Tcells exhaustion.Methods Patients who underwent surgical staging for endometrial-like carcinoma of the uterus were included.Peripheral blood and tumor-infiltrating lymphocytes were separated and polychromatic flow cytometry experiment and mass spectral flow cytometry experiment were performed.The distribution,expression of immune checkpoints and the secretion characteristics of intracellular factors of CD8~+T cells and their subsets were analyzed.The peripheral circulating CD8~+T cells,tumor-infiltrating CD103-and CD103~+CD8~+Tcells were sorted by flow cytometry and then treated with PD-1 and TIGIT antibodies alone or in combination before coculture with tumor cells.The response of CD8~+T cells to immune checkpoint inhibitors was reflected through the analysis of the killing effect on tumor cells.Results 1.CD103~+CD8~+TRM is the main subgroup of CD8~+Tcells.Compared with the normal population,the proportion of this subgroup in endometrial carcinoma is higher,and varies with surgical staging and differentiation.2.This subgroup is characterized by tissue chemotaxis and resident,up-regulation of surface inhibitory immune checkpoint expression and down-regulation of cytokine secretion.3.TIGIT is widely expressed and highly co-expressed with PD-1 on CD8~+ Tcells of endometrial carcinoma.4.In vitro experiments confirmed that PD-1 and TIGIT monoclonal antibodies could enhance the killing effect of tumor-infiltrating CD8~+Tcells.The depletion state of CD103~+subgroup was more easily reversed by PD-1 and TIGIT monoclonal antibodies.Conclusions The majority of tumor-infiltrating T cells in endometrial carcinoma are CD103~+CD8~+TRM subsets with tissue chemotactic and resident characteristics and inhibited cell function.TIGIT is widely expressed therein and co-expressed at a high ratio with PD-1.The combined use of TIGIT and PD-1 antibodies can enhance the killing function of CD8~+Tcells.Tumor-infiltrating CD103~+CD8~+TRM is an important target cell population for this effect.However,other immunomodulatory molecules and cells are still needed for further enhancing the efficacy of this combined therapy.