Analisi delle Vescicole Extracellulari come potenziali biomarkers nelle patologie tumorali

Extracellular Vesicles (EVs) are non-replicating particles released by cells, crucial for cellular communication, and implicated in cancer progression. Their content mirrors their parent cells, making them potential candidates for cancer biomarker discovery. While most studies relied on two-dimensional (2D) cell cultures, these do not accurately represent in vivo tumor microenvironments. In contrast, three-dimensional (3D) cultures provide a more realistic model, though standardized EV isolation protocols remain undeveloped. This study aimed to develop a 3D culture model using the human ovarian cancer cell line CABA I to isolate EVs. Using the hanging drop method, 3D spheroids were optimized for size and morphology, evaluated for EV release, and showed multilayered structures and vasculogenic mimicry phenomenon. The study was then extended to 2 breast cancer cell lines with different aggressiveness grades (MDA-MB-231 and MCF-7), adapting methods to optimize 3D culture conditions and EV isolation. Since the main purpose of the present study was to identify potential biomarkers associated with EVs, concurrently, studies were initiated on 3 blood samples from healthy donors, aiming to identify the best biological fluid between serum and plasma for EV isolation: both biofluids contained EVs but each one carries pros and cons. In the end, plasma was chosen for further study. Preliminary in vitro evaluations compared 2D and 3D breast cancer cell lines-derived EVs using Fourier Transform Infrared Spectroscopy (FT-IR) and ELISA for glycan profiling. Notably, FT-IR analysis revealed differences in the molecular composition of EVs from MDA-MB-231 2D and 3D models, reflecting, hypothetically, different biological activities, whereas MCF-7 showed no significant differences. Glycan profiling indicated variations in 2D and 3D breast cancer-derived EVs, with differences in galactose, fucose, and N-acetylglucosamine levels. Parallel analyses on the ES-2 ovarian cancer cell line supported these findings, highlighting the dynamic EV glycan composition. FT-IR and glycan profiling were also applied to plasma-EVs from breast cancer patients and healthy donors. FT-IR requires further refinement for plasma-derived EV analysis, while the glycan profile evaluation revealed few differences between patients and healthy donors in the level expression of N-acetyl galactosamine and fucose, respectively, which were higher and more stable in healthy donors compared to patients. However, further investigations with larger cohorts are needed to achieve statistical significance. In summary, the results of this thesis showed that the molecular composition of EVs varies in the different cell culture models, emphasizing the importance of selecting an appropriate in vitro model to accurately reflect in vivo conditions. FT-IR spectroscopy and glycan profiling are promising techniques for identifying EV-related biomarkers, but further research is required to refine FT-IR protocols, particularly for plasma-derived EVs. Nevertheless, glycan analysis offers promising initial results for potential clinical applications.