Evaluation of extracellular vesicles released by 3D spheroids and 2D conventional cell culture of the SKOV3 ovarian cancer cell line

Abstract

Cancer cells communicate with the surrounding tumour microenvironment by secreting extracellular vesicles (EVs), referred to as exosomes, which play a critical role in cancer progression by transferring bioactive cargo including proteins, lipids, mRNAs, miRNAs, and DNA fragments to healthy cells, enabling distant organ colonization and metastasis. SKOV3, a widely used human ovarian adenocarcinoma cell line, is traditionally cultured under anchorage-dependent (2D monolayer) conditions; however, suspension culturemore faithfully mimics the three-dimensional, non-adherent microenvironment encountered in vivo, giving rise to altered cell signalling, spheroid formation, and distinct exosome release profiles. This research compares exosomes secreted by SKOV3 cells under 2D and 3Dconditions, isolated through ultrafiltration and Size Exclusion Chromatography (SEC), and characterised using Scanning Transmission Electron Microscopy (STEM), Dynamic Light Scattering (DLS), Raman spectroscopy, and Flow Cytometry. Multivariate analysis across FastGrow and McCoy’s media identified the principal components governing exosome physical properties, while optimised SEC runs demonstrated how Sepharose 2B and 6B columns yield purer exosomal fractions. Additional investigations included refractive index analysis, storage effects, Surface-Enhanced Raman Scattering (SERS) of Rhodamine 6G and Crystal Violet, BCA protein estimation, and spheroid growth analysis. This work presents an accessible, reproducible isolation protocol to lower the technical barrier across laboratory settings, laying the groundwork for non-invasive exosome-based cancer screening.