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 ...

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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 ...

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids (Video) | JoVE

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...