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

Bisiayo E. Fashemi; Lillian van Biljon; Jeimmy Rodriguez; Olivia Graham; Mary Mullen; Dineo Khabele

doi:10.3791/65068

Cancer Research

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

Published: September 15, 2023 doi: 10.3791/65068

Bisiayo E. Fashemi¹, Lillian van Biljon¹, Jeimmy Rodriguez¹, Olivia Graham¹, Mary Mullen¹, Dineo Khabele¹

¹Washington University of Medicine in St. Louis

Summary

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

Abstract

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.

Introduction

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 "mini-organs"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.

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Protocol

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).

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.
Identify drugs and select a range of concentrations to test (e.g., Carboplatin, 0-50 µM, see Table of Materials).
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.

2. Reagent preparation for the viability assay

Base Media: To advanced DMEM/F12, add 1% (v/v) penicillin-streptomycin, 1x glutamax, and 1% (v/v)HEPES¹⁷ (see Table of Materials).
Prepare Advance Organoid Media for ovarian cancer organoids following previously published report¹⁷.

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 °C in a water bath. Thaw BME in an ice water bath.

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.
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.
Vortex for 5-10 s to further dissociate the cells, and centrifuge at 1107 x g for 5 min at room temperature (RT).
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 min at 37 °C.
1. 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.
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.
Count the number of cells in each PDO culture using an automatic cell counter (see Table of Materials) or a hemocytometer.
Resuspend cells at 20,000 cells per 10 µL of 25% Base Media and 75% BME. Do this by resuspending the organoids in the media and mixing with BME.
Plate one 3 µL 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, but the bottom of the plates should be covered with opaque tape before measuring luminescence.
Incubate plate in a cell culture incubator at 37 °C for 15 min.
Add 100 µL of Advance Organoid Media to each well. Incubate plate for 24-72 h (determine according to perceived PDO doubling time). Add 100 µL of Advance Organoid Media to an empty well for use as blank.
NOTE: The goal is to allow organoids to form.
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.

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.

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 µM.
Remove media from each well with a single or multi-channel pipette, being careful not to touch or disturb the PDO/BME droplet.
Add 100 µL 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.
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's biological activity, and the drug's half-life. For assays over one week, the media will need to be refreshed at least once.

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.

Allow the viability assay reagents to reach room temperature in dark. Store the reagents at 4 °C overnight (in the dark) to reduce the thaw time.
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.
Add 100 µL of viability assay reagent (see Table of Materials) to each well (for a total volume of 200 µL), 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.
Turn on the bioluminescence plate reader and open the i-control software (see Table of Materials).
Under Connect to: Instrument Name, select infinite 200Pro.
Select Luminescence and Default Script.
From the drop-down menu, choose plate type [BD96fb_Falcon-BD] Falcon 96 Flat Black.
Determine which wells to read and highlight the corresponding wells under Part of Plate.
Under Measurements on the left side of the screen, drag and drop Luminescence under the Part of Plate.
Select the luminescence parameters: Attenuation: NONE; Integration time: 1000 ms; Settle time: 0 ms.
Remove the cover from the plate and load it into the plate reader. Press Start to begin.
On completion, export data and save.

6. Data analysis

Calculate the percent cell viability.
1. Subtract the "Blank" from every well for the Blank Corrected readings. Next, calculate Control Average by averaging the three control wells.
2. Use the following equation to calculate the percentage of viable cells in each well: (Experimental well / Control Average) x 100
3. Graph the resulting data in the analysis software (see Table of Materials).
Examine Growth Rate (GR) metrics.
1. Manually calculate the GR metrics according to the published report³⁴.
2. Alternatively, use the online GR calculator (see Table of Materials) to generate the metrics³⁵. Use the Day 0 measurements as "cell_count_time0", which reflect the growth rate of the untreated PDOs.
3. Export data and graph.

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Representative Results

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 Media: 1, 5, 10, 25, 50, and 75 µM. At the conclusion of the experiment on Day 7, the viability assay reagent was added to the plate, and the results were analyzed. Figure 2A depicts the percentage of live cells after carboplatin treatment.

Next, the online GR Calculator was used to analyze the data. In the absence of cell count data, the luminescence values measured in the viability assay were used. After exporting the GR metrics, the GR values were graphed, which are the ratios between perceived growth rates in treated and untreated conditions normalized to a single cell division³⁴. These values were then plotted against the carboplatin concentrations (Figure 2B). Table 1 summarizes the GR metrics, including the respective control and treated cell doubling times and the GR Area Over the Curve (GR_AOC), which integrates the dose-response curve over a range of carboplatin concentrations tested³⁴. Sensitivity to a particular drug can be determined by interpreting the GR₅₀ value, corresponding to the concentration at which the drug has a half-maximal effect. For example, the GR₅₀ value for PDO #1 is much higher than that for PDO #2 (4.85 µMvs. 0.97 µM), indicating that PDO #1 is more resistant to platinum chemotherapy than PDO #2.

Figure 1: Patient-derived organoids before drug screening. (A) Experimental outline for PDO drug screen. Given the doubling time of the PDO line and the drug exposure time, the experimental plan may need to be adjusted. (B) Representative brightfield images (40x) of two ovarian cancer PDO lines (#1 and #2). Scale bar = 50 µm. Abbreviations: PDO = patient-derived organoids. Please click here to view a larger version of this figure.

Figure 2: Representative results of ovarian cancer PDO following carboplatin treatment. (A) Two PDO lines were treated with increasing concentrations of carboplatin for seven days. The X-axis shows carboplatin concentration; the Y-axis displays the % of live cells normalized to control organoids (no carboplatin). Assays were completed in triplicate with two biological replicates. The error bars denote the standard deviation. (B) GR-value graph representing the logarithmic carboplatin concentration (x-Axis) and the GR value (Y-Axis). These values were generated in the online GR calculator. The error bars indicate the standard deviation. Please click here to view a larger version of this figure.

	Treatment	Control Cell Doubling Time	Treated Cell Doubling Time	GR50	GR_AOC
PDO #1	Carboplatin	0.744	0.112	4.85	1.28
PDO #2	Carboplatin	0.972	0.0532	0.97	1.51

Table 1: Table depicting the control cell doubling time, treated cell doubling time, GR50, and GR_AOC values generated in the GR calculator. Abbreviations: PDO = patient-derived organoids, GR = growth rate, GR_AOC = growth rate area over the curve.

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Discussion

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-derived PDOs. Because this assay depends on the growth rate, using organoids that take a long time to form/grow may be difficult. We have only successfully used PDO lines with doubling times of less than 4 days.

Second, opaque black 96-well plates were used in this study. Since the ATP viability assay is luminescence-based, transparent wells will reduce the signal intensity and generate signal contamination. Opaque-walled, transparent bottom plates allow for the visualization of organoids during the assay and may help monitor treatment-induced changes in cell morphology. Although it is a strength related to cost, a limitation of this assay is using a 96-well plate, which limits the number of samples and drugs that can be evaluated simultaneously.

Third, the number of cells plated must be carefully considered and optimized for viability assays. This is especially true because of the variable growth rate of PDO lines; too few cells will not form organoids, and too many cells will lead to organoid overgrowth. To ensure the uniform distribution of cells, an automatic cell counter was used and maintained the same BME-to-media ratio (75:25). This high percentage of BME ensures the droplets remain solidified for the entirety of the assay. Here, 3 µL droplets of BME were placed in the center of the well. Although the size of the droplet can be increased, we caution against coating the whole well. Coating the whole well will cause the organoids to settle in the edges of the well, which will both impede their overall growth and affect the viability assay results. Off-center placement of the droplet is fine as long as it does not touch the edges of the well.

Fourth, self-pipetting introduces human error, but this can be overcome by attention to detail and the inclusion of additional control wells.

Finally, the length of the assay must be carefully selected. Prolonged exposure to drugs will affect the viability of PDOs, independent of the drug mechanism of action. For this reason, it is important to test a range of drug concentrations over a minimum of five days. It is important to decide whether the media will need to be changed for longer periods because reduced levels of growth factors will impede the effects of the drugs³⁶. To examine whether the media will need to be changed during the assay, one needs to compare the results from Day 0 and Day 7 controls. Untreated PDO controls should continue to grow throughout the assay continuously.

As PDOs continue to advance in complexity and better recapitulate their tissues of origin, their use should improve drug discovery. However, PDOs are likely to remain a precious resource and require cost-effective methods to preserve and optimize their use. Unlike medium- and high-throughput techniques, this protocol can be used to test known and novel compounds at lower cost with readily available materials and equipment. Finally, this method can be readily adapted to different cancer organoid models.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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.

Materials

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

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