The role of extracellular vesicle cargo in chemotherapy resistance: liquid biopsy and in vitro studies in bone tumors

Osteosarcoma (OS) and Ewing sarcoma (EWS) are the most common primary bone tumours in children and adolescents, and they present major clinical challenges due to chemotherapy resistance. This significantly limits the effectiveness of standard treatments, which rely on intensive high-dose chemotherapy and surgical resection. Despite these aggressive approaches, survival rates have remained largely unchanged for decades, particularly in metastatic cases. The high toxicity and limited efficacy of current therapies highlight the urgent need to better understand resistance mechanisms and develop effective targeted treatment strategies. Recent studies have revealed that extracellular vesicles (EVs) may mediate chemoresistance by facilitating intercellular communication and transferring resistance-associated cargo, such as specific miRNAs and proteins. This suggests that EVs could serve as biomarkers for monitoring tumour behaviour and resistance patterns through minimally invasive approaches such as liquid biopsies. High-throughput techniques in transcriptomics, proteomics, and drug screening have transformed the analysis of drug resistance mechanisms in bone tumors. When integrated with 3D models that better replicate the tumor microenvironment than traditionally 2D cultures, these methods will provide deeper insights into EV-mediated interactions and tumour cell dynamics. To use this approach in bone tumours, this thesis explores 3D multiple myeloma organoids as a model for studying chemoresistance in a physiologically relevant setting. This PhD thesis investigates miRNA and protein signatures within EVs linked to chemoresistance in OS, focusing on mechanisms underlying resistance to chemotherapeutics like doxorubicin, cisplatin, and methotrexate. Using high-throughput technologies, this research characterizes EV-mediated communication and examines how resistant cell-derived EV cargo influences recipient cells, promoting drug resistance. Furthermore, proteomic analysis of EV cargo from CD99-silenced EWS cells has provided insights into tumour aggressiveness, identifying EVs as molecular players in sarcoma progression. By uncovering these molecular profiles and pathways, this work aims to identify potential biomarkers and therapeutic targets, advancing personalized treatment for pediatric bone tumors.