Exploring The Role And Mechanism Of MUC16 In The Development Of Ovarian Cancer Based On Organoid Model

Background and objectives: Epithelial ovarian cancer(EOC)is one of the common malignant tumors of the female reproductive system,with incidence increasing year by year.The 5-year survival rate of patients with advanced ovarian cancer is only 29%,which is a serious threat to women’s life and health.Currently,tumor reduction surgery combined with chemotherapy is the standard treatment for ovarian cancer,but the recurrence rate of ovarian cancer is still high and the prognosis remains poor.The application of targeted drugs such as PARP inhibitors and bevacizumab is also quite limited due to target limitations and increased side effects of combination drugs.Immunotherapy may provide new ideas and approaches for EOC treatment.However,due to the complexity of the immune microenvironment of solid tumors,the efficacy of immunotherapies such as PD-1/PD-L1 in EOC is also not ideal.The patient-derived EOC organoid provides a platform for personalized immunotherapy research for exploring the immune microenvironment of EOC,which might improve the effectiveness of EOC immunotherapy and improve the prognosis of EOC patients.This study aims to investigate the function of mucin 16(MUC16)acting on neutrophils via Siglec-9 to regulate the immune microenvironment of EOC and its potential mechanisms,and to provide new ideas of immunotherapy for EOC.Contents and methods:(1)Construction and identification of patient-derived EOC organoids and validation of the MUC16 expression of the EOC organoids;(2)Detection the presence of neutrophils in EOC with immunohistochemical(IHC)staining;Analyse of the expression of Siglec-9(the MUC16 receptor)on neutrophils in EOC based on TCGA database and RNA sequencing data;Validation of Siglec-9expression on the surface of neutrophils by flow cytometry and immunofluorescence(IF).(3)Determination of immunophenotype of neutrophils alteration with flow cytometry and q PCR;Validation of Siglec-9 on the neutrophils binding to MUC16 stimulated by EOC organoids with IF.(4)Determination of the immunosuppressive phenotype of neutrophils after MUC16 stimulation with flow cytometry and q PCR;Determination of the altered secretory cytokines with MUC16-stimulated neutrophils by multiplex liquid-phase protein quantification;RNA sequencing and bioinformatics analysis were applied to explore the potential mechanism of the altered immune phenotype of MUC16-treated neutrophils;Cell killing assay was applied to determine whether MUC16-treated neutrophils affect the ability of NK cells to kill ovarian cancer cells.Results:(1)We successfully constructed EOC organoids that can be frozen,passaged,and have certain structures that are consistent with the source tumor tissues.The EOC organoids expressed a high level of MUC16.(2)Neutrophils were present in ovarian cancer tissues;Bioinformatics analysis suggested that Siglec-9,the receptor of MUC16,was highly correlated with the neutrophil infiltration level and highly correlated with neutrophil surface markers in pan-cancer including ovarian cancer;Experimental validation indicated the presence of Siglec-9 on the surface of neutrophils.(3)MUC16 bound to EOC organoid-stimulated neutrophils and resulted in altered neutrophil immunophenotype.(4)MUC16 protein-stimulated neutrophils showed altered immunophenotype and secretory cytokine similar to those of EOC organoid-stimulated neutrophils;RNA sequencing analyses results suggested that MUC16-stimulated neutrophils activated inflammatory-related pathways and highly expressed PD-L1 and immunosuppressive molecules;MUC16-stimulated neutrophils were able to attenuate the ability of NK cells to kill ovarian cancer cells.Conclusions: MUC16 can cause neutrophil activation of inflammatory response and high expression of immunosuppressive factors by acting on Siglec-9 on the surface of neutrophils,resulting in suppression of the ovarian cancer immune microenvironment,which in turn leads to inhibition of NK cell killing of ovarian cancer cells.Thus,MUC16/Siglec-9 is a potential target for immunotherapy and provides a new direction for further study of immunotherapy in ovarian cancer.