125 | Impact of 2D and 3D in vitro culture models on extracellular vesicles in human ovarian cancer

Giulia Di Fazio¹, Giuseppina Poppa¹, Giuseppe Cammarata², Sandra D’Ascenzo¹, Vincenza Dolo¹, Simona Taverna², Ilaria Giusti¹ | ¹Department of Life, Health and Environmental Sciences, University of L’Aquila, Italy; ²Institute of Translational Pharmacology IFT, National Research Council of Italy CNR, Palermo, Italy.

Extracellular vesicles (EVs) and their molecular cargo have garnered significant attention as potential biomarkers. Nevertheless, the identification of reliable candidate molecules necessitates comprehensive preliminary in vitro investigations. Historically, in vitro studies on EVs have primarily used two-dimensional (2D) culture models due to their ease of use and high reproducibility. However, these 2D systems have significant drawbacks, as they do not accurately replicate the complex structural organization and physiological features of tumors as seen in vivo. As a result, there has been a growing interest in three-dimensional (3D) culture models, including spheroids. Unlike traditional 2D approaches, 3D systems more closely resemble tumor architecture and allow for better interaction between cells and the surrounding extracellular matrix. Despite this advancement, the isolation of EVs from these 3D models still lacks standardized procedures, making consistent and comparable analyses challenging.[1,2] The present study aimed to analyze and compare EVs released by the ES-2 ovarian carcinoma cell line cultured in 2D and 3D models. Their molecular composition was assessed, and functional assays were performed using fibroblasts as target cells. The results revealed both molecular and functional differences between the two models. Proliferation assays were performed on fibroblasts treated with EVs derived from ES-2 cells cultured in 2D or 3D, showing increased proliferative activity following treatment with EVs from the 3D model. Western blot analysis of EV molecular content also revealed higher levels of TGF-β in EVs isolated from the 3D model. Fibroblast activation was further confirmed by Western blot analysis of α-SMA expression, a typical marker of cancer-associated fibroblasts, which was higher in fibroblasts treated with EVs from the 3D model. In parallel, analysis of circRNAs in EVs was initiated to evaluate their potential as tumor biomarkers. Differential expression of specific circRNAs was observed between EVs from the two culture models. Overall, these results indicate that EVs derived from 2D and 3D human ovarian cancer models exhibit distinct characteristics depending on the experimental system used. This highlights the importance of carefully selecting the culture model, as it can influence both functional study outcomes and the identification of novel biomarkers.
This work was supported by the Funding Program PRIN 2022 under grant agreement N°20224XB79P, funded by European Union, Next-generation EU, PNRR, M.4-C2-1.1.