Name: ovarian cancer patient-derived organoid models for pre-clinical drug testing
Uploaded: 2023-09-15T15:00:00
Description: Watch this Scientific Journal Video about Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing at JoVE.com

Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R01CA243511. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank Deborah Frank for her editorial comments.

The collection of human specimens for this research was approved by the Washington University School of Medicine Institutional Review Board. All eligible patients over the age of 18 years had a diagnosis or presumed diagnosis of high-grade serous ovarian cancer and were willing and able to provide informed consent. The tumor tissue from either primary or metastatic sites, in addition to ascites and pleural fluid, were obtained from consented patients at the time of care.
1. Selection of ...

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This article describes a method that can be used to assess the therapeutic effects of conventional or novel drugs on ovarian cancer PDOs. Researchers must consider several issues before conducting the viability assay in the PDO model.
First, when selecting a PDO to use in the viability assay, one must determine the ideal organoid type (tumor vs. ascites) and passage number for their needs. In our experience, ascites-derived PDOs grow more rapidly and are easier to generate than tumor-...

Although rare, ovarian cancer is one of the most lethal gynecological cancers1,2. A challenge in developing new treatments is that ovarian cancer is heterogeneous, and the tumor microenvironment differs greatly among patients. Additionally, many ovarian cancers develop resistance to platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors, highlighting the need for greater therapeutic options3,4,5.
One approach that may be useful in identifying new therapeutics is using patient-derived organoids (PDOs). Organoids are three-dimensional clusters of multiple cell types that self-organize and form in vitro&#160;&#34;mini-organs&#34;6,7,8,9,10. Organoids can recapitulate important tissue morphology and gene expression profiles11,12. Some of the first organoids were derived from intestinal, gastric, and colon cancer cells from both mice and humans8,9,13. Long-lived organoid cultures have been established from a wide range of benign and malignant tissues, including the bladder, colon, stomach, pancreas, brain, retina, and liver14,15,16. We previously demonstrated methods to establish PDOs from ovarian cancer tumors and ascites samples17. PDOs can be used to study molecular characteristics, cellular mechanisms, and novel drug treatments18,19,20. PDOs have several advantages over traditional two-dimensional primary cell cultures for drug screening. Although primary two-dimensional cultures are a low-cost method for drug screens, primary cell cultures are single-cell types and lack the three-dimensional architecture of tumors21,22,23. Nevertheless, PDOs are a precious resource, and cost-effective protocols are needed to optimize their use in therapeutic drug screening.
This article describes an in vitro method to use ovarian cancer PDOs to test the effects of known or candidate drugs. Whereas current medium- and high-throughput drug screens using PDOs require expensive automated dispensing instruments24,25,26, this cost-effective method uses readily available basic lab supplies and an ATP-based cell viability assay in a standard 96-well plate format (Figure 1A). This method will facilitate preliminary tests of novel ovarian cancer drugs prior to scaling up to larger screens27,28. Although ovarian cancer PDOs are used here, this method can be applied to other cancer organoid models.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Plastic Tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Plastic Tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96 well Flat Black Plates</td>
			<td>MidSci</td>
			<td>781968</td>
			<td></td>
		</tr>
		<tr>
			<td>Advance Organoid Media&#160;</td>
			<td></td>
			<td></td>
			<td>see Graham et al 2022 (Jove)</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Thermo Fisher</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Brightfield Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carboplatin&#160;</td>
			<td>Teva&#160;Pharmaceuticals&#160;USA</td>
			<td>NDC&#160;00703-4246-01</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Viability&#160;</td>
			<td>Promega</td>
			<td>G9681</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex</td>
			<td>R &#38; D Systems</td>
			<td>3533-010-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>GR Calculator&#160;</td>
			<td>http://www.grcalculator.org</td>
			<td></td>
			<td>Online calculator</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Rocker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile P10, P200, and P1000 Barrier Sterile Pipette Tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile P10, P200, and P1000 Pipettes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tecan Infinte 200Pro Plate Reader; i-Control Software</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Thermo Fisher</td>
			<td>12605010</td>
			<td>Organoid dissociation reagent</td>
		</tr>
	</tbody>
</table>

ovarian cancer patient-derived organoid models for pre-clinical drug testing

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 is crucial to advancing healthcare and improving patient outcomes. Organoids are in-vitro three-dimensional multicellular miniature organs. Patient-derived organoid (PDO) models of ovarian cancer may be optimal for drug screening because they more accurately recapitulate tissues of interest than two-dimensional cell culture models and are inexpensive compared to patient-derived xenografts. In addition, ovarian cancer PDOs mimic the variable tumor microenvironment and genetic background typically observed in ovarian cancer. Here, a method is described that can be used to test conventional and novel drugs on PDOs derived from ovarian cancer tissue and ascites. A luminescence-based adenosine triphosphate (ATP) assay is used to measure viability, growth rate, and drug sensitivity. Drug screens in PDOs can be completed in 7-10 days, depending on the rate of organoid formation and drug treatments.

These results illustrate the response of two PDOs to the chemotherapy drug carboplatin, which is used to treat ovarian cancer. Organoids were derived from tumor biopsy (PDO #1) and from ascites (PDO #2). These organoids were selected based on their perceived doubling time (1-2 days) and morphological appearance (formation of many large organoids). Both PDO #1 and PDO #2 were plated on Day -2, at passage two, and carboplatin was added on Day 0. We tested the following carboplatin concentrations diluted in Advance Organoid...

Watch this Scientific Journal Video about Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing at JoVE.com

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

We present a protocol that can be used to conduct therapeutic drug testing with patient-derived ovarian cancer organoids.

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 ...

该协议描述了一种使用患者来源的卵巢癌类器官进行药物活力测定的低成本方法。该技术的主要优点是使用低成本和现成的材料和实验室设备。这种方法可以在进行昂贵的高通量药物筛选之前使用。尽管该方法涉及在患者来源的卵巢癌类器官中进行药物活力测定，但它可以应用于任何类器官系统，包括那些源自小鼠的类器官系统。首先，在明场显微镜下观察含有类器官的基底膜提取物或 BME，以确保 70% 至 90% 的汇合度。使用移液管，将一到两毫升预热的基础培养基加入含有类器官的孔中，并通过上下移液来机械解离类器官。将整个类器官悬浮液转移到 15 毫升锥形管中，并在管中涡旋 5 至 10 秒以进一步分解细胞。然后，在室温下以1,107G离心细胞五分钟。使用单通道移液管，吸出并弃去上清液。将沉淀重悬于预热至37°C的一毫升类器官解离试剂中，并将悬浮液转移到1.5毫升微量离心管中。将样品在 37 摄氏度下孵育 7 分钟。然后在室温下将结果在1,107G的细胞悬浮液中离心五分钟。弃去上清液后，将细胞沉淀重悬于一毫升预热的基础培养基中。在基础培养基和BME的混合物中复溶细胞，保持每10微升20， 000个细胞的终浓度。在黑色不透明的96孔板的每个孔中，铺板一个三微升重悬细胞液滴。将板在 37 摄氏度的细胞培养箱中孵育 15 分钟。最后，在将板在 37 摄氏度下孵育 24 至 72 小时之前，向每个孔中加入 100 微升预热的高级类器官培养基。在接种后两天对类器官进行药物治疗。药物治疗后 7 天对类器官进行活力测定。向每个孔中加入 100 微升活力测定试剂，使总体积达到 200 微升，并以 80 RPM 摇动板五分钟。然后让板在室温下再孵育 25 分钟。同时，打开生物发光酶标仪并打开眼控软件。导航连接仪器“，选择Infinite 200Pro”选择发光“和默认脚本”接下来，从下拉菜单中选择BD 96 FB Falcon BD Falcon 96 Flat Black“作为板类型来设置发光参数。将衰减指定为无。积分时间为 1000 毫秒，建立时间为 0 毫秒。最后，揭开板，将其装入读板器，然后按开始“开始”。为方案中使用的两种不同的患者来源类器官（PDO）获取明场图像。PDO 1 来源于肿瘤活检，PDO 2 来源于腹水。活力测定的结果显示，在用不同浓度的卡铂处理后，PDO 1 和 PDO 2 中的活细胞百分比。将计算出的生长速率或GR值与相应的卡铂浓度作图，GR指标显示PDO 1的GR50值远高于PDO 2的GR50值。这表明PDO一对铂化疗的抵抗力更强 在铺板时，将细胞悬液的三微升液滴直接放置在孔的中心至关重要。如果将液滴放置在靠近孔的边缘，可能会导致液滴塌陷，从而影响测定结果。

Research

JoVE Journal

Cancer Research

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

患者衍生的胰腺癌异质异种移植模型,使用斑马鱼幼虫作为比较药物评估的宿主

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

科研

癌症研究

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

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. ...

Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

患者衍生胰腺腺腺腺腺癌有机体模型的分离和特征

Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

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 ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

患者源性肿瘤外植体作为预测患者耐药性的"实时"临床前平台

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

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 ...

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 ...