An Exploratory Study Of Residual Disease-associated Circulating Small Extracellular Vesicles MicroRNAs In Ovarian Cancer

Objectives: No residual disease after debulking surgery is the most critical independent prognostic factor for advanced ovarian cancer(AOC).What determines the resectability of tumors is internal characteristics of tumor biology.Active debulking surgery can make up for tumor biological differences between OC patients to a great extent.As a communication medium between cells,small extracellular vesicles(s EVs)transmit carcinogenic activity by transferring tumor-related cargoes.This study investigated the molecular characteristics of circulating s EVs that can predict residual disease risk in AOC,and their tissue source.Methods: The differential expression profile of pre-treatment plasma s EVs micro RNAs(mi RNAs)between AOC patients with no residual disease(R0)and any residual disease(non-R0)after debulking surgery were analyzed using high-throughput sequencing.The candidate mi RNAs expression of plasma and tissue samples was measured by Taq Man q RTPCR.Tissue s EVs were extracted from primary tumor tissue,metastatic tumor tissue,adjacent tissue,or non-tumor tissue via differential ultracentrifugation and size exclusion chromatography(SEC).Cell-typespecific markers(EpCAM,FAP,CD45,CD31)in tumor tissue homogenate and tumor s EVs pool were evaluated by western blot analysis.Plasma and tumor tissue s EVs from different cell sources were captured by the improved magnetic bead sorting system(MACS)using the above cellspecific surface proteins as labeled antibodies.s EVs uptake and co-culture assays evaluated the biological efficacy of s EVs 4-mi RNA in OC cells.Results: 151 differentially expressed circulating sEVs miRNAs were found between R0 and non-R0 patients;Among them,122 were upregulated and 29 were down-regulated.Taq Man q RT-PCR analysis further confirmed 4-mi RNA signature(mi R-320a-3p,mi R-378a-3p,mi R-1307-3p,let-7d-3p)with consistent expression trends in plasma s EVs and tumor tissues.The results showed that circulating s EVs 4-mi RNA was mainly derived from tumor tissue;The proportion of Ep CAM+ s EVs in s EVs pool of tumor tissue was the highest.The MACS data suggested that epithelial OC cells-derived s EVs captured on EpCAM+ magnetic beads were the major vehicles of circulating s EVs 4-mi RNA.In addition,the expression of s EVs 4-mi RNA was not affected by circulating confounders.Target gene prediction and pathway enrichment analysis based on residual diseaserelated GEO dataset identified the correlation between 4-mi RNA and residual disease,and extensive metastasis of OC.In vitro experiments suggested that mi R-320a-3p,mi R-378a-3p,and mi R-1307-3p enhanced migration and invasion of OC cells,while let-7d-3p inhibited it.The function experiments of s EVs showed that mi R-320a-3p,mi R-1307-3p,and let-7d-3p could also regulate migration and invasion of OC cells after transmission by s EVs.Conclusion: High expression of miR-320a-3p,miR-1307-3p,and mi R-378a-3p,and low expression of let-7d-3p in circulating s EVs suggested the high risk of residual disease after primary debulking surgery in AOC.Differential expression of these four circulating extracellular vesicles 4-mi RNA between R0 and non-R0 patients was dominated by OC cells.4-mi RNA could affect the biological function of OC cells through s EVs transport.Tumor-derived s EVs 4-mi RNA has the opportunity to become predictive biomarkers for residual disease risk in AOC patients and therapeutic targets related to tumor progression.