Name: Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing (Video) | JoVE Journal
Uploaded: 2023-09-15T15:00:00.000+00:00
Duration: 5 min 35 s
Description: Learn a low-cost method for drug viability assays using patient-derived ovarian cancer organoids. This accessible protocol enables pre-clinical testing before expensive high-throughput screening.

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5 minutes

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 established PDOs for viability assay
NOTE: Typically, these organoids are within the first five passages. As described previously, ovarian cancer PDOs are formed by resuspending primary cell suspension in basement membrane extract (BME) such as Cultrex or Matrigel17 (see Table of Materials).
<ol>
	<li>PDOs should have a perceived doubling time of 24-72 h for the best efficiency of the assay. Estimate the perceived doubling time by visually determining the number of days it takes for organoids to form after passaging/plating. 
	NOTE: In our experience, organoids that take longer than three days to form cannot be used for this growth inhibition assay.</li>
	<li>Identify drugs and select a range of concentrations to test (e.g., Carboplatin, 0-50 &#181;M, see Table of Materials).</li>
	<li>Determine the plate setup. This assay will be performed in triplicate, limiting the number of drugs and concentrations that can be tested on one plate. 
	NOTE: Selected PDO lines must be carefully observed before performing the assay. Established ovarian cancer PDOs form multicellular solid and hollow spheres with tumor-like budding shapes (Figure 1B). PDO morphology, genomic profile, etc., must be compared to that of the patient sample (when possible) before performing the assay29-33. Ovarian cancer PDOs can be generated from primary and metastatic tumor samples and ascites17.</li>
</ol>
2. Reagent preparation for the viability assay
<ol>
	<li>Base Media: To advanced DMEM/F12, add 1% (v/v) penicillin-streptomycin, 1x glutamax, and 1% (v/v)HEPES17 (see Table of Materials).</li>
	<li>Prepare Advance Organoid Media for ovarian cancer organoids following previously published report17.</li>
</ol>
3. Plating of organoids (~Day -2)
NOTE: This step must be performed 1-3 days before adding the drugs. Before beginning, warm all reagents (Base Media, Advance Organoid Media, and organoid dissociation reagent; see Table of Materials) to 37 &#176;C in a water bath. Thaw BME in an ice water bath.
<ol>
	<li>Use a brightfield microscope to confirm whether organoids of interest are 70%-90% confluent. 
	NOTE: It is recommended to save images of the organoids for future reference.</li>
	<li>Add 1-2 mL of warmed Base Media to the well containing organoids and pipette up and down to mechanically dissociate the BME-containing organoids. Transfer the entire solution to a 15 mL conical tube17. 
	NOTE: Typically, 1-2 mL of media is enough to properly dilute the BME. Organoids of the same patient and passage can be combined.</li>
	<li>Vortex for 5-10 s to further dissociate the cells, and centrifuge at 1107 x g for 5 min at room temperature (RT).</li>
	<li>Remove the supernatant with a single-channel pipette and discard. Resuspend organoids in 1 mL organoid dissociation reagent (see Table of Materials) and transfer to a 1.5 mL microcentrifuge tube. Incubate for 7&#160;min at 37 &#176;C.
	<ol>
		<li>If the pellet is still gelatinous because of the remaining BME, add an additional 1 mL of Base Media, vortex for 5-10 s, and repeat centrifugation.</li>
	</ol>
	</li>
	<li>Centrifuge at 1107 x g for 5 min at RT. Remove and discard the supernatant and resuspend the cell pellet in 1 mL of Base Media.</li>
	<li>Count the number of cells in each PDO culture using an automatic cell counter (see Table of Materials) or a hemocytometer.</li>
	<li>Resuspend cells at 20,000 cells per 10 &#181;L of 25% Base Media and 75% BME. Do this by resuspending the organoids in the media and mixing&#160;with BME.</li>
	<li>Plate one 3 &#181;L droplets of resuspended BME + PDO cells into one well of a black opaque 96-well plate (see Table of Materials). Place the droplets in the center of each well. Plate each drug concentration in triplicate. 
	NOTE: Since the cell viability assay is luminescence-based, it is best to use black opaque plates to avoid background. Transparent plates can be used to visualize organoids during the assay,&#160;but the bottom of the plates should be covered with opaque tape before measuring luminescence.</li>
	<li>Incubate plate in a cell culture incubator at 37 &#176;C for 15 min.</li>
	<li>Add 100 &#181;L of Advance Organoid Media to each well. Incubate plate for 24-72 h (determine according to perceived PDO doubling time). Add 100 &#181;L of Advance Organoid Media to an empty well for use as blank. 
	NOTE: The goal is to allow organoids to form.</li>
	<li>Optional: For growth rate analysis, plate additional triplicate PDOs in a separate plate. This will be used to count cells at Time = 0 and should be assayed on Day 0 using the viability assay described in Section 5.</li>
</ol>
4. Addition of drugs for the viability assay on Day 0
NOTE: Day 0 refers to the day the drugs are added to the fully formed organoids.
<ol>
	<li>Dilute selected drugs in Advance Organoid Media to desired concentrations. Dilutions can be made in 1.5 mL tubes or in a pre-set up 96-well plate, which would allow for the use of a multi-channel pipette to dispense the media. 
	NOTE: Drugs must be resuspended in the solvent suggested by the manufacturer, such as dimethyl sulfoxide. For this study, the following dilutions of carboplatin in Advance Organoid Media, were made: 1, 5, 10, 25, 50, and 75 &#181;M.</li>
	<li>Remove media from each well with a single or multi-channel pipette, being careful not to touch or disturb the PDO/BME droplet.</li>
	<li>Add 100 &#181;L of Advance Organoid Media and desired drug(s) to each well. Ensure to add fresh media to the three control wells. 
	NOTE: Drug concentrations will vary and depend on the scientific question and drug mechanism of action. When deciding what concentrations to use, we begin with previously established drug concentrations in comparable 2D cell lines (e.g., immortalized ovarian cancer cell lines). Concentrations may need to be increased if there is no observed effect on organoids.</li>
	<li>Refresh media and drugs as needed (repeat steps 4.1-4.3). Whether the media will need to be refreshed depends on the assay length, the drug&#39;s biological activity, and the drug&#39;s half-life. For assays over one week, the media will need to be refreshed at least once.</li>
</ol>
5. Ending the viability assay for readout (~Day 7)
NOTE: This step can be performed on Days 7-10. The assay length must be determined according to the half-life and pharmacodynamics of the drugs being tested.
<ol>
	<li>Allow the viability assay reagents to reach room temperature in dark. Store the reagents at 4 &#176;C overnight (in the dark) to reduce the thaw time.</li>
	<li>Remove the assay plate from cell culture incubator and allow it to acclimate to room temperature for 30 min. The plate does not have to acclimate in the dark.</li>
	<li>Add 100 &#181;L of viability assay reagent (see Table of Materials) to each well (for a total volume of 200 &#181;L), and place on a plate-shaker for 5 min (80 rpm). Remove the plate from the shaker and incubate for an additional 25 min at room temperature. Ensure the plate is always protected from light by covering it with foil or an opaque box.</li>
	<li>Turn on the bioluminescence plate reader and open the i-control software (see Table of Materials).</li>
	<li>Under Connect to: Instrument Name, select infinite 200Pro.</li>
	<li>Select Luminescence and Default Script.</li>
	<li>From the drop-down menu, choose plate type [BD96fb_Falcon-BD] Falcon 96 Flat Black.</li>
	<li>Determine which wells to read and highlight the corresponding wells under Part of Plate.</li>
	<li>Under Measurements on the left side of the screen, drag and drop Luminescence under the Part of Plate.</li>
	<li>Select the luminescence parameters: Attenuation: NONE; Integration time: 1000 ms; Settle time: 0 ms.</li>
	<li>Remove the cover from the plate and load it into the plate reader. Press Start to begin.</li>
	<li>On completion, export data and save.</li>
</ol>
6. Data analysis
<ol>
	<li>Calculate the percent cell viability.
	<ol>
		<li>Subtract the &#34;Blank&#34; from every well for the Blank Corrected readings. Next, calculate Control Average by averaging the three control wells.</li>
		<li>Use the following equation to calculate the percentage of viable cells in each well: (Experimental well / Control Average) x 100</li>
		<li>Graph the resulting data in the analysis software (see Table of Materials).</li>
	</ol>
	</li>
	<li>Examine Growth Rate (GR) metrics.
	<ol>
		<li>Manually calculate the GR metrics according to the published report34.</li>
		<li>Alternatively, use the online GR calculator (see Table of Materials) to generate the metrics35. Use the Day 0 measurements as &#34;cell_count_time0&#34;, which reflect the growth rate of the untreated PDOs.</li>
		<li>Export data and graph.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

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><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&amp;nbsp;</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&amp;nbsp;</td><td>Teva&amp;nbsp;Pharmaceuticals&amp;nbsp;USA</td><td>NDC&amp;nbsp;00703-4246-01</td><td></td></tr><tr><td>CellTiter-Glo 3D Viability&amp;nbsp;</td><td>Promega</td><td>G9681</td><td></td></tr><tr><td>Cultrex</td><td>R &amp;amp; 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&amp;nbsp;</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 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...

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 ...

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

Research

JoVE Journal

Cancer Research

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers’ method to grow tumor organoids that were passaged, cryopreserved and characterized against the original PDX.

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

Patient-derived organoids (PDO) are a three-dimensional (3D) culture that can mimic the tumor environment in vitro. In high-grade serous ovarian cancer, PDOs represent a model to study novel biomarkers and therapeutics.

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

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

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

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Establishment and Culture of Patient-Derived Breast Organoids

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

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

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is located.

A Hepatocellular Cancer Patient&#45;Derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

This protocol describes establishing three-dimensional (3D) tissue organoids from primary human ovarian surface epithelium (hOSE) cells. The protocol includes isolation of hOSE from freshly collected ovaries, cellular expansion of the hOSE, cryopreservation-thawing procedures, and organoid derivation. Immunofluorescence, quantitative analysis, and showcasing utility as a screening platform are included.

Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration

This protocol details the development of high-grade serous ovarian carcinoma (HGSOC) mouse models in vitro and in vivo. From the derivation of murine fallopian tube organoids, their genetic modification to recapitulate human HGSOC genetics, and their introduction in the ovarian bursa of syngeneic mice for in vivo development of tumors.

Development of Mouse-Derived Organoid Lines from Fallopian Tube Epithelial Cells for High Grade Serous Ovarian Carcinoma Modeling

Learn a low-cost method for drug viability assays using patient-derived ovarian cancer organoids. This accessible protocol enables pre-clinical testing before expensive high-throughput screening.

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

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