Lee Sanghee, Burner Danielle N, Mendoza Theresa R, Muldong Michelle T, Arreola Catalina, Wu Christina N, Cacalano Nicholas A, Kulidjian Anna A, Kane Christopher J, Jamieson Christina A M
Department of Urology, University of California, San Diego; Moores Cancer Center, University of California, San Diego.
Moores Cancer Center, University of California, San Diego; Department of Medicine, University of California, San Diego.
J Vis Exp. 2020 Feb 3(156). doi: 10.3791/60367.
Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient's bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.
来自人类患者肿瘤标本的类器官三维（3D）培养以及前列腺癌的患者来源异种移植（PDX）模型，即患者来源类器官（PDO），是研究前列腺癌发生和转移机制的宝贵资源。它们的主要优势在于，与传统细胞系不同，它们保留了原始组织独特的基因组和功能异质性。此外，PDO的3D培养可用于预测药物治疗对个体患者的效果，是迈向个性化医疗的重要一步。尽管有这些优势，但很少有研究团队常规使用这种方法，部分原因是针对不同患者样本可能需要对PDO培养条件进行大量优化。我们之前证明，我们的前列腺癌骨转移PDX模型PCSD1能够重现供体患者骨转移对抗雄激素治疗的耐药性。我们使用PCSD1 3D类器官进一步表征抗雄激素耐药的机制。在概述目前已发表的关于PDX和PDO模型的研究后，我们描述了一种在优化培养条件下使用圆顶或漂浮基底膜（如基质胶）球体进行PDO 3D培养的分步方案。还介绍了体内缝线成像和用于组织学的细胞处理方法。该方案可针对其他应用（包括蛋白质免疫印迹、共培养等）进一步优化，并可用于探索3D培养的PDO在耐药性、肿瘤发生、转移和治疗方面的特性。
