Pappas Kyrie J, Choi Danielle, Sawyers Charles L, Karthaus Wouter R
Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center.
Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center; Howard Hughes Medical Institute.
J Vis Exp. 2019 Oct 24(152). doi: 10.3791/60346.
Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.
本文介绍了一种用于研究前列腺上皮类器官药效学、干细胞潜能和癌症分化的方案。前列腺类器官对雄激素有反应，是在类似于前列腺上皮的特定培养基中生长的三维（3D）培养物。前列腺类器官可从野生型和基因工程小鼠模型、良性人类组织以及晚期前列腺癌中建立。重要的是，患者来源的类器官在遗传学和体内肿瘤生物学方面与肿瘤非常相似。此外，类器官可以使用CRISPR/Cas9和shRNA系统进行基因操作。这些可控的遗传学特性使类器官培养成为一个有吸引力的平台，可用于快速测试基因型和突变谱对药理反应的影响。然而，实验方案必须专门适应类器官培养的三维性质，以获得可重复的结果。这里描述的是用于进行接种试验以确定类器官形成能力的详细方案。随后，本报告展示了如何进行药物处理，并通过活力测量、蛋白质分离和RNA分离来分析药理反应。最后，该方案描述了如何准备用于异种移植的类器官以及随后使用皮下移植进行体内生长试验。这些方案产生高度可重复的数据，并广泛适用于三维培养系统。
