Establishment and Maintenance of Patient-derived Prostate Cancer Organoids: A Detailed Experimental Protocol.

Prostate cancer (PCa) is a highly heterogeneous malignancy and a leading cause of cancer-related mortality in men, with significant clinical challenges in addressing treatment resistance and disease progression. Existing preclinical models, such as two-dimensional (2D) cell lines and patient-derived xenografts (PDXs), have limitations in faithfully recapitulating the complexity of PCa, including tumor heterogeneity, androgen receptor (AR)-dependent signaling, and microenvironmental interactions. To address this gap, this study presents a robust and reproducible protocol for establishing Patient-derived organoids (PDOs) that preserve the genetic, phenotypic, and histological features of the parental tumors. This method enables the culture of organoids from both localized and advanced PCa, providing a biologically relevant model for studying disease mechanisms, drug response, and biomarker discovery. The results demonstrate the protocol's ability to generate organoids from a variety of clinical specimens, with applications in high-throughput drug screening and precision medicine. By offering a standardized workflow that addresses key technical challenges, this study highlights the importance of PDOs as versatile tools for advancing prostate cancer research and developing more effective therapeutic strategies.