高级别浆液性卵巢癌患者来源类器官的生成和培养
Summary
患者来源类器官（PDO）是一种三维（3D）培养物，可以在 体外模拟肿瘤环境。在高级别浆液性卵巢癌中，PDO代表了研究新型生物标志物和治疗方法的模型。
Abstract
类器官是3D动态肿瘤模型，可以从患者来源的卵巢肿瘤组织，腹水或胸腔积液中成功生长，并有助于发现卵巢癌的新疗法和预测性生物标志物。 这些模型概括了克隆异质性、肿瘤微环境以及细胞间和细胞间基质相互作用。此外，它们已被证明在形态学、细胞学、免疫组织化学和遗传学上与原发肿瘤相匹配。因此，类器官有助于肿瘤细胞和肿瘤微环境的研究，并且优于细胞系。本协议描述了从患者肿瘤，腹水和胸腔积液样本中生成患者来源的卵巢癌类器官的不同方法，成功率高于97%。患者样品通过机械和酶消化分离成细胞悬浮液。然后使用基底膜提取物（BME）对细胞进行铺板，并用含有特异性用于培养高级别浆液性卵巢癌（HGSOC）的补充剂的优化生长培养基支持。在形成初始类器官后，PDO可以维持长期培养，包括传代以扩增以进行后续实验。
Introduction
2021 年，美国约有 21，410 名女性新诊断出患有上皮性卵巢癌，12，940 名女性死于这种疾病1。尽管在手术和化疗方面已经取得了足够的进展，但超过 70% 的晚期疾病患者会出现化疗耐药性，并在诊断后 5 年内死亡2，3。因此，迫切需要治疗这种致命疾病的新策略和具有代表性的、可靠的临床前研究模型。
由原发性卵巢肿瘤产生的癌细胞系和患者来源的异种移植物（PDX）是卵巢癌研究中使用的主要仪器。癌细胞系的一个主要优点是它们的快速扩增。然而，它们的持续培养导致表型和基因型改变，导致癌细胞系偏离原始原发性癌症肿瘤样本。由于癌细胞系和原发肿瘤之间存在差异，在细胞系中具有积极作用的药物测定在临床试验中无法具有相同的效果2。为了克服这些限制，使用了 PDX 模型。这些模型是通过将新鲜的卵巢癌组织植入免疫缺陷小鼠中创建的。由于它们是 体内 模型，它们更准确地类似于人类生物学特征，反过来，更能预测药物结果。然而，这些模型也有很大的局限性，包括生成它们所需的成本、时间和资源4.
PDO为临床前研究提供了一种替代模型，克服了癌细胞系和PDX模型的局限性。PDO概括了患者的肿瘤和肿瘤微环境，因此为临床前研究提供了理想的体外可处理模型2，3，5。这些3D模型具有模拟原发性肿瘤的自组织功能，这是它们的二维（2D）细胞系对应物所不具备的特征。此外，这些模型已被证明在遗传和功能上代表其亲本肿瘤，因此是研究新疗法和生物过程的可靠模型。简而言之，它们提供类似于细胞系的长期扩增和储存能力，但也包括小鼠模型固有的微环境和细胞间相互作用4，6。
本协议描述了从患者来源的肿瘤、腹水和胸腔积液样本中创建 PDO，成功率高于 97%。然后，PDO培养物可以扩增多代，并用于测试药物治疗敏感性和预测生物标志物。这种方法代表了一种可用于根据PDO的治疗反应进行个性化治疗的技术。
Protocol
为研究收集的所有人体组织标本都是根据机构审查委员会（IRB）批准的协议获得的。下面概述的方案是在无菌人体组织培养环境中进行的。从人类受试者那里获得了知情的书面同意。符合条件的患者必须有卵巢癌的诊断或推定诊断，愿意并能够签署知情同意书，并且至少年满18岁。肿瘤组织（恶性原发性肿瘤或转移部位）、腹水和胸腔积液是在手术时从同意的患者那里获得的。这些标本立即被运?…
Representative Results
为了产生PDO，将样品以机械和酶促方式消化成单细胞悬浮液。然后将细胞重悬于BME中，并补充专门设计的培养基（图3）。类器官通常在 10 天的时间范围内建立，之后它们在培养物中展示离散的类器官（图 4）。 图3</stron…
Discussion
卵巢癌由于其诊断时的晚期以及化疗耐药性的普遍发展而极其致命。通过利用癌细胞系和PDX模型，卵巢癌研究取得了许多进展;然而，显然需要一种更具代表性和负担得起的 体外 模型。PDO已被证明可以准确代表肿瘤异质性，肿瘤微环境以及其原发性肿瘤的基因组和转录组学特征，因此是各种研究方法的理想临床前模型，例如在药物治疗中实施类器官模型16。
<p class="jove_…Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Ron Bose，MD，PhD的指导，以及Barbara Blachut，MD的协助，以建立此协议。我们还要感谢华盛顿大学圣路易斯医学院妇产科和妇科肿瘤科、华盛顿大学院长学者计划和生殖科学家发展计划对该项目的支持。
Materials
| 1% HEPES | Life Technologies | 15630080 | |
| 1% Penicillin-Streptomycin | Fisher Scientific | 30002CI | |
| 1.5 mL Eppendorf Tubes | Genesee Scientific | 14125 | |
| 10 cm Tissue Culture Dish | TPP | 93100 | |
| 10 mL Serological Pipet | |||
| 100 µm Cell Filter | MidSci | 100ICS | |
| 15 mL centrifuge tubes | Corning | 430052 | |
| 2 mL Cryovial | Simport Scientific | T301-2 | |
| 2% Paraformaldehyde Fixative | Sigma-Aldrich | ||
| 37 °C water bath | NEST | 602052 | |
| 3dGRO R-Spondin-1 Conditioned Media Supplement | Millipore Sigma | SCM104 | |
| 6 well plates | TPP | 92006 | |
| 70% Ethanol | Sigma-Aldrich | R31541GA | |
| A83-01 | Sigma-Aldrich | SML0788 | |
| Advanced DMEM/F12 | ThermoFisher | 12634028 | |
| Agar | Lamda Biotech | C121 | |
| B-27 | Life Technologies | 17504044 | |
| Centrifuge | |||
| Cultrex Type 2 | R&D Systems | 3533-010-02 | basement membrane extract |
| DNase I | New England Bio Labs | M0303S | |
| DNase I Reaction Buffer | New England Bio Labs | M0303S | |
| EGF | PeproTech | AF-100-15 | |
| FBS | Sigma-Aldrich | F2442 | |
| FGF-10 | PeproTech | 100-26 | |
| FGF2 | PeproTech | 100-18B | |
| gentleMACS C Tubes | Miltenyi BioTech | 130-096-334 | |
| gentleMACS Octo Dissociator with Heaters | Miltenyi BioTech | 130-096-427 | We use the manufacturers protocol. |
| GlutaMAX | Life Technologies | 35050061 | dipeptide, L-alanyl-L-glutamine |
| Hematoxylin and Eosin Staining Kit | Fisher Scientific | NC1470670 | |
| Histoplast Paraffin Wax | Fisher Scientific | 22900700 | |
| Microcentrifuge | |||
| Mr. Frosty Freezing Container | Fisher Scientific | 07202363S | |
| N-acetylcysteine | Sigma-Aldrich | A9165 | |
| Nicotinamide | Sigma-Aldrich | N0636 | |
| p1000 Pipette with Tips | |||
| p200 Pipette with Tips | |||
| Pasteur Pipettes 9" | Fisher Scientific | 1367820D | |
| PBS | Fisher Scientific | MT21031CM | |
| Pipet Controller | |||
| Prostaglandin E2 | R&D Systems | 2296 | |
| Puromycin | ThermoFisher | A1113802 | |
| Recombinant Murine Noggin | PeproTech | 250-38 | |
| Recovery Cell Culture Freezing Medium | Invitrogen | 12648010 | |
| Red Blood Cell Lysis Buffer | BioLegend | 420301 | |
| ROCK Inhibitor (Y-27632) | R&D Systems | 1254/1 | |
| SB202190 | Sigma-Aldrich | S7076 | |
| T75 Flask | MidSci | TP90076 | |
| Tissue Culture Hood | |||
| Tissue Embedding Cassette | |||
| TrypLE Express | Invitrogen | 12604013 | animal origin-free, recombinant enzyme |
| Type II Collagenase | Life Technologies | 17101015 | |
| Vortex |
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Cite This Article
Graham, O., Rodriguez, J., van Biljon, L., Fashemi, B., Graham, E., Fuh, K., Khabele, D., Mullen, M. Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids. J. Vis. Exp. (191), e64878, doi:10.3791/64878 (2023).