前列腺有机体培养作为工具，将基因型和突变谱转化为药理反应
Summary
这里介绍的是一个协议，研究在前列腺上皮器官的药理反应。有机体与体内生物学非常相似，并重述了患者基因，使它们成为有吸引力的模型系统。前列腺器官可以从野生型前列腺、基因工程小鼠模型、良性人体组织和晚期前列腺癌中建立。
Abstract
这里介绍的是研究前列腺上皮器官的药效动力学、干细胞潜力和癌症分化的一个方案。前列腺有机体是雄激素反应，三维（3D）培养生长在定义的介质，类似于前列腺上皮。前列腺器官可以从野生型和基因工程小鼠模型、良性人体组织和晚期前列腺癌中建立。重要的是，患者衍生的有机物在遗传学和体内肿瘤生物学中与肿瘤非常相似。此外，有机物可以使用CRISPR/Cas9和shRNA系统进行基因操作。这些受控遗传学使有机体培养具有吸引力，成为快速测试基因型和突变型对药理反应的影响的平台。然而，实验协议必须特别适应有机体培养的3D特性，以获得可重复的结果。此处介绍了用于执行种子测定以确定有机体形成能力的详细协议。随后，本报告展示了如何通过可行性测量、蛋白质分离和RNA分离来进行药物治疗和分析药理反应。最后，该协议描述了如何使用皮下移植制备异种移植和后续体内生长测定的有机物。这些协议产生高度可重复的数据，并广泛适用于3D培养系统。
Introduction
耐药性是癌症治疗的主要临床问题之一。转移性前列腺癌（PCa）治疗主要针对雄激素信号轴。下一代抗雄激素疗法（例如，苯甲酰胺和阿比拉酮）在临床上取得了巨大的成功，但几乎所有PCa最终都朝着雄激素独立状态，即抗雄激素前列腺癌（CRPC）发展。
最近CRPC的基因组和转录组谱显示前列腺癌有三种一般抗药性机制：1）激活突变，导致雄激素受体（AR）信号1的恢复;2） 旁路信号激活，如下一代抗雄激素治疗抗性临床前模型，其中糖皮质激素受体（GR）的激活可以补偿AR信号2的损失;3） 最近确定的谱系可塑性过程，其中肿瘤细胞通过从依赖药物靶向的细胞类型转换到不依赖于此的另一种细胞类型（在PCa中，表现为AR阴性）获得抗药性和/或神经内分泌疾病[NEPC]）3，4.然而，引起耐药性的分子机制并不为人所知。此外，获得抗雄激素耐药性可能导致治疗脆弱性，可以加以利用。因此，在模拟患者表型和基因型的模型系统中评估药物反应至关重要。
前列腺有机体是在具有定义介质的3D蛋白质基质中生长的器官培养物。重要的是，前列腺器官可以从鼠的良性和癌性组织或人类起源建立，它们保留在体内5，6中发现的体型和基因状特征。重要的是，抗雄激素敏感PCa和CRPC细胞都表现在当中有机物简编中。此外，前列腺器官很容易使用CRISPR/Cas9和shRNA5进行基因操作。因此，前列腺器官是测试药物反应和阐明抗药性机制的合适模型系统。在这里，详细的协议描述，以执行药物测试和分析使用前列腺有机体的药理反应。
Protocol
Representative Results
Discussion
了解抗雄激素耐药性的分子机制并发现潜在的治疗弱点，需要测试模仿前列腺癌的模型系统中的药理反应。本文介绍的是一个详细的方案，用于可靠地分析患者衍生和基因工程前列腺器官中的药理反应，并制备这些有机体样品用于下游应用。
此协议中有两个关键步骤。第一是确定有机物的播种效率和生长速度。有机体生长速度差异很大。这依赖于物种，因为鼠源的有机物的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
K.P. 由 NIH 1F32CA236126-01 提供支持。C.L.S. 由 HHMI 支持;CA193837;CA092629;CA224079;CA155169;CA008748;和斯塔尔癌症联盟W.R.K. 由荷兰癌症基金会/KWF Buit 2015-7545 和前列腺癌基金会 PCF 17YOUN10 提供支持。
Materials
| A83-01 | Tocris | 2939 | Organoid medium component: Final concentration 200 nM |
| ADMEM/F12 | Gibco/Life technologies | 12634028 | Organoid medium component |
| B27 | Gibco/Life technologies | 17504-044 | Organoid medium component |
| Cell culture plates | Fisher | 657185 | |
| Cell Titer Glo | Promega | G7571 | |
| DHT | Sigma-Aldrich | D-073 | Organoid medium component: Final Concentration 1 nM |
| DMSO | Fisher | BP231-100 | |
| EGF | Peprotech | 315-09 | Organoid medium component: Final concentration 50 ng/ml for mouse, 5 ng/nl for Human |
| FGF10 | Peprotech | 100-26 | Human specific organoid medium component: Final concentration 10 ng/ml |
| FGF2 | Peprotech | 100-18B | Human specific organoid medium component: Final concentration 5 ng/ml |
| Glutamax | Gibco/Life technologies | 35050079 | Organoid medium component |
| HEPES | MADE IN-HOUSE | N/A | Organoid medium component: Final concentration 10 mM |
| Matrigel (Growthfactor reduced & Phenol Red free) | Corning | CB-40230C | Organoid medium component |
| N-Acetylcysteine | Sigma-Aldrich | A9165 | Organoid medium component: Final concentration 1.25 mM |
| Nicotinamide | Sigma-Aldrich | N0636 | Human specific organoid medium component: Final concentration 10 mM |
| NOGGIN | Peprotech or stable transfected 293t cells with Noggin construct (Karthaus et al. 2014) | 120-10C | Organoid medium component: Final Concentration 10% conditioned medium or 100 ng/ml |
| Penicillin/Streptavidin | Gemini Bio-Products | 400-109 | Organoid medium component |
| Phospatase inhibitors | Merck Millipore | 524629 | |
| Prostaglandin E2 | Tocris | 3632464 | |
| Protease Inhibitors | Merck Millipore | 539131 | |
| R-SPONDIN | Peprotech or stable transfected 293t cells with R-Spondin1 construct (Karthaus et al. 2014) | 120-38 | Organoid medium component: Final Concentration 10% conditioned medium or 500 ng/ml |
| RIPA buffer | Merck | 20-188 | |
| RNA-easy minikit | Qiagen | 74104 | |
| SB202190 | Sigma-Aldrich | 152121-30-7 | Human specific organoid medium component: Final concentration 10 μM |
| TryplE | ThermoFisher | 12605036 | |
| Y-27632 | Selleckchem | S1049 | Organoid medium component: Final Concentration 10 μM |
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