Name: creating matched in vivo/in vitro patient-derived model pairs of pdx and pdx-derived organoids for cancer pharmacology research
Uploaded: 2021-05-05T13:00:00
Description: Watch this Scientific Journal Video about Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research at JoVE.com

作者要感谢乔迪·巴博博士、费德里卡·帕里西博士和拉金德拉·库马里博士对手稿的批判性阅读和编辑。作者还要感谢皇家生物科学肿瘤学体外和体内团队的巨大技术努力。

所有涉及动物护理和使用的协议和修正或程序在开展研究之前都经过皇家生物科学机构动物护理和使用委员会 （IACUC） 的审查和批准。根据国家研究理事会《实验室动物护理和使用指南》（2011年）中报告的《实验室动物护理和使用指南》，对动物的护理和使用是根据AALAC（实验室动物护理评估和认证协会）国际准则进行的。所有动物实验程序均在SPF（特定无病原体）设施的无菌条件下进行，并严...

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本报告中PDX-/PDXO-CR2110的初步数据支持PDX与其衍生物PDXO在基因组学、组织病理学和药理学方面的生物学等价性，因为这两种模型都代表了从患者原始CSC衍生的疾病形式。这两种模型都是患者衍生的疾病模型，可能预测患者10、11、12、21的临床反应。匹配的体外模型和体内模型可以相辅相成，在体内进行...

癌症是各种遗传和免疫学疾病的集合。有效治疗的成功开发高度依赖于有效预测临床结果的实验模型。长期以来，大型患者衍生异种移植（PDX）库一直被视为体外系统的转化系统，以测试化疗和/或靶向疗法，因为他们能够重新概括患者肿瘤特征、异质性和患者药物反应1，从而使II期样小鼠临床试验能够提高临床成功率。PDX通常被认为是癌症干细胞疾病，具有遗传稳定性，与细胞系衍生的异种移植2形成鲜明对比。在过去的几十年里，世界各地都建立了大量的PDX系列，成为当今癌症药物开发的主力军。这些动物模型虽然应用广泛，转化价值巨大，但内在成本高、耗时、吞吐量低，因此不足以进行大规模筛选。PDX也不适合免疫肿瘤学（IO）测试，由于免疫损害的性质4。因此，充分利用现有的大型 PDX 库是不切实际的。
最近由汉斯·克莱弗斯实验室5号开创的发现，在大多数上皮起源5的人类器官中，建立了由成人干细胞产生的器官体外培养。这些协议已经进一步完善，使有机体从假定的CSC在人类癌的各种迹象6，7的增长。这些患者衍生的器官（PDOs）是基因稳定的8，9，并已被证明是高度预测临床治疗结果10，11，12。此外，PDOs 的体外性质使高通量筛查 （HTS）13能够实现，从而有可能提供比体内模型更优越的优势，并利用大型器官库作为患者群体的代名词。PDOs 有望成为重要的发现和转化平台，克服上述 PDX 的许多局限性。
PDO 和 PDX 都是患者衍生和 CSC 驱动的模型，能够在个性化治疗或临床试验格式的背景下评估治疗。现有的大型PDX库，如专有收藏的>3000 PDXs 14，15，16，17，因此适合肿瘤器官库（PDX衍生器官，或PDXO），从而形成了一个匹配的配对PDX和PDXO模型库。本报告描述了创建和描述结肠直肠癌PDXO-CR2110与其父母PDX-CR2110模型16相关的程序。

<table>
	<tbody>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Life Technologies</td>
			<td>12634028</td>
			<td>Base medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Hyclone</td>
			<td>SH30243.01</td>
			<td>Washing medium</td>
		</tr>
		<tr>
			<td>Collagenese type II</td>
			<td>Invitrogen</td>
			<td>17101015</td>
			<td>Digest tumor</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Organoid culture matrix (Basement Membrane Extract, growth factor reduced)</td>
		</tr>
		<tr>
			<td>N-Ac</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10C</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma</td>
			<td>S7076</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Gastrin</td>
			<td>Sigma</td>
			<td>G9145</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Rspondin</td>
			<td>Peprotech</td>
			<td>120-38-1000</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Life Technologies</td>
			<td>15630056</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Abmole</td>
			<td>M1817</td>
			<td>Organoid culture medium</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Life Technologies</td>
			<td>17105041</td>
			<td>Screening assay</td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D</td>
			<td>Promega</td>
			<td>G9683</td>
			<td>Screening assay (luminescent ATP indicator)</td>
		</tr>
		<tr>
			<td>Multidrop dispenser</td>
			<td>Thermo Fisher</td>
			<td>Multidrop combi</td>
			<td>Plating organoids/CellTiter-Glo 3D addition</td>
		</tr>
		<tr>
			<td>Digital dispener</td>
			<td>Tecan</td>
			<td>D300e</td>
			<td>Compound addition</td>
		</tr>
		<tr>
			<td>Envision Plate reader</td>
			<td>Perkin Elmer</td>
			<td>2104</td>
			<td>Luminescence reading</td>
		</tr>
		<tr>
			<td>Balb/c nude mice</td>
			<td>Beijing HFK Bio-Technology Co</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RNAeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>tRNA purification kit</td>
		</tr>
		<tr>
			<td>DNAeasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>Histogel</td>
			<td>Thermo Fisher</td>
			<td>HG-4000-012</td>
			<td>Organoid embedding</td>
		</tr>
	</tbody>
</table>

creating matched in vivo/in vitro patient-derived model pairs of pdx and pdx-derived organoids for cancer pharmacology research

患者衍生的肿瘤异种移植（PDXs）被认为是最具预测性的前科模型，主要被认为是由癌症干细胞（CSC）驱动的常规癌症药物评估。一个大型的PDX库反映了患者群体的多样性，从而能够进行基于人群的临床前试验（"第二阶段类似小鼠的临床试验"）：但是，PDX 具有吞吐量低、成本高、持续时间长的实际局限性。肿瘤器官，也是患者衍生的CSC驱动模型，可以被认为是PDX的体外等价物，克服了某些PDX限制，用于处理大型器官库或化合物库。这项研究描述了一种创建PDX衍生器官（PDXO）的方法，从而产生了体外和体内药理学研究的配对模型。皮下移植的PDX-CR2110肿瘤是从肿瘤携带小鼠身上采集的，当肿瘤达到200-800毫米3时，按照批准的尸检程序，然后切除相邻的非肿瘤组织，分离成小肿瘤片段。小肿瘤碎片被洗净，并通过一个100μm细胞过滤器去除碎片。细胞簇被收集并悬挂在地下室膜提取物 （BME） 溶液中，并镀在 6 井板中，作为与周围液体介质一起生长的固体液滴，用于 CO2 孵化器的生长。在光显微镜下每周监测两次器官生长，并记录摄影，然后每周进行2次或3次液体介质变化。通过机械剪切破坏BME嵌入器官，辅之以添加滴虫素和添加10μM Y-27632，进一步通过（7天后）1：2的比例。器官在离心从BME释放后，在低温管中冷冻保存，用于长期储存，并取样（如DNA、RNA和FFPE块）以作进一步鉴定。

PDXO 的形态学，典型的光显微镜下的器官，与父母的 PDX 每 H&E 染色一致 在光显微镜下，PDXO-CR2110演示了典型的囊性形态（图1A），如以前描述的患者衍生器官（PDO），支持PDXO和PDO在相同培养条件下相似的证据。
H&E染色组织的病理学检查显示，PDXO-CR2110（图1B）的组织结构和细胞类型反映了原始的PDX-CR2110（<strong class="x...

Watch this Scientific Journal Video about Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research at JoVE.com

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

描述使用患者衍生的异种格拉夫特 （PDX） 进行体外筛查的方法，从而形成匹配的体内/体外模型对。PDX 肿瘤被机械或酶地收获/加工成小块，然后是 Clevers 的生长肿瘤器官的方法，这些器官是通过、冷冻保存和针对原始 PDX 进行特征的。

为癌症药理学研究创建匹配的体内/体外患者衍生模型对 PDX 和 PDX 衍生器官

Patient-derived tumor xenografts (PDXs) are considered the most predictive preclinical models, largely believed to be driven by cancer stem cells (CSC) ...

此协议可用于建立配对患者衍生的异种移植物（PDX）和相应的患者衍生器官线的匹配库。这种方法的优点是，它可以用来创建使用PDX进行体外筛查的器官，从而产生匹配的体外/体外模型对。首先收获肿瘤并处理文本手稿中描述的组织。组织消化后，用五毫升无菌塑料移液器上下吹笛。然后将 20 毫升 AD+添加到同质化中，然后用 100 微米细胞过滤器进行过滤。用 AD+清洗两次，然后将其转移到塑料管中，然后以 450 倍 G 的离心处理，为期五分钟。将 BME 中的细胞重新保存并保存在冰上。为了准备器官培养，将细胞悬浮物的200微升转移到六井板的每口井中，并在37摄氏度下孵育板30分钟。在每口井中加入两毫升的有机介质，并图像代表在显微镜下滴落。将器官培养物保持在37摄氏度和5%的二氧化碳中，每三到四天发生一次中等变化，每七天以一比二的比例通过。分离器官后，通过 70 微米滤镜运行到 50 毫升塑料管中。在显微镜下数一数器官，并在冰上将其重新注入BME，最终浓度为5%，在每384井板中加入50微升的器官悬浮剂，并配有液体分配器，每口油井的播种密度为200 CR2110 PDXO。使用数字分配器在串行稀释中将 SN38 添加到每个油井中。使用数字分配器软件工具创建板图。治疗应包括负控制车辆与100%的生存能力和积极控制五微摩尔葡萄球菌素与0%的可行性。完成后，将经过药物处理的384井板放回37摄氏度的孵化器中。在光显微镜下，PDXO CR2110 显示了典型的囊性形态，表明 PDX 衍生器官与患者衍生器官在相同培养条件下的相似性。H&E染色组织的病理学检查表明，PDXO CR2110的组织结构和细胞类型与原始的PDX CR2110相似。PDX 和 PDXO 的基因组特征比较表明转录体表达的相关性为 94.92%，DNA 突变的一致性为 97.67%，这表明这两个模型之间的整体基因组相似性。在384井板的PDXO CR2110上进行了药物敏感性检测。PDXO CR2110对伊里诺特坎敏感，对西斯铂具有抗药性，与PDX治疗结果一致。匹配的体外模型和体内模型可以互相称赞体外筛查和体内验证，提高药物发现的成功率，并可能降低临床开发中的自然减员率。

Research

JoVE Journal

Cancer Research

A Melanoma Patient-Derived Xenograft Model

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

科研

癌症研究

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

使用患者源性肿瘤类器官的高通量 体外 检测

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

Establishment and Culture of Patient-Derived Breast Organoids

患者来源乳腺类器官的建立和培养

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

通过免疫荧光测定 可视化 患者来源的卵巢癌类器官中的 DNA 损伤修复蛋白

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

建立胰腺癌斑马鱼患者来源的异种移植物进行化学敏感性测试

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

用于临床前药物测试的卵巢癌患者来源的类器官模型

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...