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

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 cancers^1,2. 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 options^3,4,5.

One approach that may be useful in identifying new therapeutics is using patie....

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 desc....

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.......

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-.......

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