Human ovarian cancer cell-culture models (bi- and tri-dimensional) affect the biological properties of extracellular vesicles

Introduction: Ovarian cancer (OC) is a fatal malignancy in women. Identifying biomarkers for early detection is urgent; Extracellular vesicles (EVs) are a promising biomarker. Although most preliminary in vitro studies conducted on EVs to date have used the two-dimensional (2D) culture models, for the past few years research has been shifting toward three-dimensional (3D) models, which are more architecturally similar to in vivo tumours. Methods: EVs were isolated by differential centrifugation and ultracentrifugation from the ES-2 (CLS-Cytion) cell line grown (McCoy’s 5A medium with 5% FBS HyClone) as a monolayer or spheroids obtained by using Ultra Low Attachment BIOFLOAT 384-well plates (faCellitate). EV release from both models was verified by SEM, and theywere characterized according to MISEV 2023 guidelines (TEM, NTA,Western blot). The biological effects were tested on human umbilical vein endothelial cells (HUVEC as a target: proliferation andmotilitywere evaluated, respectively, by CCK8 and the wound healing assay. circ-RNAs analysis was performed with single assays with divergent primers by real-time quantitative PCR (RT-qPCR), using the SYBR Green RT-PCR Kit. Results: The protocol set generates three-dimensional, rounded spheroids with clinically relevant dimensions (> 400 nm). SEM images show the EV release from the cellmembrane in 2Dand 3D models. EV characterization demonstrated their identity, which is the same for both models: they appear intact and rounded (TEM), with amean size of 153.6–163.7nm(NTA), and are positive for CD63, CD9, and TSG-101 and negative for CNX (WB). 2D and 3D EVs did not stimulate HUVECs proliferation but were able to differentially induce their motility in a dose-dependent manner. EVs also have a different content of selected circRNAs: EVs released by OC cells growing in 3D model, compared to 2D, are enriched in hsa_circ_ITCHand hsa_circ_CDR1as; Xonversely, hsa_circ_HipK3 and hsa_circ_MuC-16 are less represented. Summary/Conclusion: When approaching the EVs study in search of novel OC biomarkers, the choice of in vitro model should be carefully evaluated since it can affect different functional and, most importantly, molecular tests. Funding: Project funded from Program PRIN 2022 under grant agreement N◦20224XB79P, funded by the European Union, Nextgeneration EU, PNRR, M.4-C2-1.1.