Extracellular vesicles derived-microRNAs predicting enzalutamide-resistance in 3D spheroid prostate Cancer model

Enzalutamide (ENZ) has emerged as a major treatment advance in castration-resistant prostate cancer (CRPC) patients; however the development of resistance remains a key challenge. The extracellular vesicles (VEs)-derived miRNAs play crucial roles tumor microenvironment cell communication, thereby influencing resistance mechanisms. Considering the urgent need for molecular biomarkers to monitor ENZ response and predict resistance, we intend to identify an EV-derived miRNA profile associated with ENZ resistance using an innovative 3D-spheroid in vitro model. Through the generation of this model, we provide a comprehensive platform for elucidating the molecular alterations involved in the process. An in vitro model of ENZ resistance was established through continuous exposure of LNCaP to increasing ENZ concentrations. A screening of 799 miRNAs from resistant and normal LNCaP cells were quantified. A bioinformatic analysis was performed using miRTarbase and Cytoscape and top 5 overexpressed miRNAs were selected, that will be analyzed in extracellular vesicles derived from ENZ resistance 3D spheroid models. We identified 12 up- and 13 downregulated miRNAs in LNCaP 30 μM ENZ cells compare to LNCaP·In silico analysis led to the construction of a 76 proteins cluster and functional enrichment revealed terms like PI3K/AKT, TFG-β and FOXO. hsa-miR-22-3p was significantly decreased at 5 and 20 μM ENZ concentration intracellularly, but significantly increased at 20 μM ENZ in EVs. hsa-miR-221-3p and miR-222-3p were upregulated in all concentrations both intracellularly and in EVs. The developed 3D-spheroid model effectively replicated the ENZ resistance to ENZ in an AR-independent manner, underscoring the importance of EVs-derived miRNAs in this adaptive process.