PATIENT-DERIVED ORGANOIDS AND EXTRACELLULAR VESICLES: UNLOCKING NEW FRONTIERS IN BREAST CANCER DRUG TESTING AND BIOMARKER DISCOVERY

Despite significant advancements in cancer research, Breast cancer (BC) is still the most common cancer diagnosed in women and the second leading cause of death. In fact, therapies used against BC are not satisfactorily tailored to individual patients and often result in failure. The main reason of that is tumor heterogeneity, resulting in different levels of aggressiveness and treatment sensitivity. To address this challenge, personalized medicine strategies are crucial; achieving this requires preclinical models that can replicate the complexity of BC in vitro. Furthermore, identifying new biomarkers through liquid biopsy could help in detecting and stratifying BC risk. In this context, the development of patient-derived organoids (PDOs) has revolutionized 3D culture models in oncology, offering a way to closely mimic the original tumor’s characteristics, including inter- and intra-tumoral heterogeneity. Moreover, their potential as a source of cancer biomarkers is also recently emerging. Besides this, liquid biopsy, a non-invasive method, shows promise in monitoring disease progression by analyzing Extracellular vesicles (EVs). EVs could help stratify patients by risk, allowing for targeted treatment. Despite their potential, traditional quantification methods remain challenging, necessitating further research to identify specific and reliable biomarkers for clinical use. In my PhD project, I explored innovative strategies in the study of BC, focusing on patient-derived organoids (PDOs) and extracellular vesicles (EVs). The primary goal was to develop PDO-based platforms that closely mimic tumor characteristics, providing a reliable in vitro model for several applications: (1) study of tumor evolution, (2) drugs screening, (3) nanodrugs development and (4) biomarkers discovery. Furthermore, I presented an emerging application of the PDO model in the biomarker discovery field, by setting up a protocol for isolating PDO-derived EVs (EVs-PDO) and defining their biochemical signature with the aim of identifying promising BC biomarkers. Six candidate biomarkers (HER2, EpCAM, EGFR, Survivin, and Galectin-3) were validated through biochemical signature analysis on EVs-PDO and confirmed on clinical plasma samples. Additionally, to demonstrate the potential of EVs as non-invasive biomarkers for the detection and risk stratification of BC, we used Raman spectroscopy (RS) to analyze the biochemical profile of EVs circulating in the blood. Although EVs interact with other extracellular particles, such as lipoproteins, in the bloodstream, RS has been shown to be a useful tool in defining the typical spectroscopic fingerprint of EVs and also to distinguish it from that of possible contaminants. Supporting the potential of RS as a diagnostic tool for BC, the results showed that this method can identify unique biochemical signatures in the BC group, specifically attributed to higher levels of nucleic acids and lipids. The findings underscore the potential of integrating PDOs and EVs in BC research, with significant implications for early detection, personalized treatment, and monitoring of therapeutic responses. The use of PDOs as platform for the identification of novel BC biomarkers through EV analysis contribute to the advancement of precision medicine. The study concludes that the PDOs and EVs together provide powerful tools for improving BC risk assessment and guiding tailored therapeutic approaches.