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

Summary

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.

Abstract

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 discovery of novel therapeutics and predictive biomarkers for ovarian cancer. These models recapitulate clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Additionally, they have been shown to match the primary tumor morphologically, cytologically, immunohistochemically, and genetically. Thus, organoids facilitate research on tumor cells and the tumor microenvironment and are superior to cell lines. The present protocol describes distinct methods to generate patient-derived ovarian cancer organoids from patient tumors, ascites, and pleural fluid samples with a higher than 97% success rate. The patient samples are separated into cellular suspensions by both mechanical and enzymatic digestion. The cells are then plated utilizing a basement membrane extract (BME) and are supported with optimized growth media containing supplements specific to the culturing of high-grade serous ovarian cancer (HGSOC). After forming initial organoids, the PDOs can sustain long-term culture, including passaging for expansion for subsequent experiments.

Introduction

In 2021, approximately 21,410 women in the United States were newly diagnosed with epithelial ovarian cancer, and 12,940 women died of this disease¹. Although sufficient advancements have been made in surgery and chemotherapy, over 70% of patients with advanced disease develop chemotherapeutic resistance and die within 5 years of diagnosis^2,3. Thus, new strategies to treat this deadly disease and representative, reliable models for preclinical research are urgently needed.

Cancer cell lines and patient-derived xenografts (PDX) created from primary ovarian tum....

Protocol

All human tissue specimens collected for research were obtained according to the Institutional Review Board (IRB)-approved protocol. The protocols outlined below were performed in a sterile human tissue culture environment. Informed written consent was obtained from human subjects. Eligible patients had to have a diagnosis or presumed diagnosis of ovarian cancer, be willing and able to sign informed consent, and be at least 18 years of age. Tumor tissue (malignant primary tumor or metastatic sites), ascites, and pleural fluid were obtained from consenting patients at the time of their procedure. These specimens were immediately transported to the laboratory and proces....

Results

To generate PDOs, the samples were digested mechanically and enzymatically into single-cell suspensions. The cells were then resuspended in BME and supplemented with specifically engineered media (Figure 3). Organoids are typically established over a time frame of 10 days, after which they demonstrate discrete organoids in culture (Figure 4).

Discussion

Ovarian cancer is extremely deadly due to its advanced stage at diagnosis, as well as the common development of chemotherapy resistance. Many advances in ovarian cancer research have been made by utilizing cancer cell lines and PDX models; however, there is an evident need for a more representative and affordable in vitro model. PDOs have proven to accurately represent the tumor heterogeneity, the tumor microenvironment, and the genomic and transcriptomic features of their primary tumors and, thus, are ideal pre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1% HEPES	Life Technologies	15630080
1% Penicillin-Streptomycin	Fisher Scientific	30002CI
1.5 mL Eppendorf Tubes	Genesee Scientific	14125
10 cm Tissue Culture Dish	TPP	93100
10 mL Serological Pipet
100 µm Cell Filter	MidSci	100ICS
15 mL centrifuge tubes	Corning	430052
2 mL Cryovial	Simport Scientific	T301-2
2% Paraformaldehyde Fixative	Sigma-Aldrich
37 °C water bath	NEST	602052
3dGRO R-Spondin-1 Conditioned Media Supplement	Millipore Sigma	SCM104
6 well plates	TPP	92006
70% Ethanol	Sigma-Aldrich	R31541GA
A83-01	Sigma-Aldrich	SML0788
Advanced DMEM/F12	ThermoFisher	12634028
Agar	Lamda Biotech	C121
B-27	Life Technologies	17504044
Centrifuge
Cultrex Type 2	R&D Systems	3533-010-02	basement membrane extract
DNase I	New England Bio Labs	M0303S
DNase I Reaction Buffer	New England Bio Labs	M0303S
EGF	PeproTech	AF-100-15
FBS	Sigma-Aldrich	F2442
FGF-10	PeproTech	100-26
FGF2	PeproTech	100-18B
gentleMACS C Tubes	Miltenyi BioTech	130-096-334
gentleMACS Octo Dissociator with Heaters	Miltenyi BioTech	130-096-427	We use the manufacturers protocol.
GlutaMAX	Life Technologies	35050061	dipeptide, L-alanyl-L-glutamine
Hematoxylin and Eosin Staining Kit	Fisher Scientific	NC1470670
Histoplast Paraffin Wax	Fisher Scientific	22900700
Microcentrifuge
Mr. Frosty Freezing Container	Fisher Scientific	07202363S
N-acetylcysteine	Sigma-Aldrich	A9165
Nicotinamide	Sigma-Aldrich	N0636
p1000 Pipette with Tips
p200 Pipette with Tips
Pasteur Pipettes 9"	Fisher Scientific	1367820D
PBS	Fisher Scientific	MT21031CM
Pipet Controller
Prostaglandin E2	R&D Systems	2296
Puromycin	ThermoFisher	A1113802
Recombinant Murine Noggin	PeproTech	250-38
Recovery Cell Culture Freezing Medium	Invitrogen	12648010
Red Blood Cell Lysis Buffer	BioLegend	420301
ROCK Inhibitor (Y-27632)	R&D Systems	1254/1
SB202190	Sigma-Aldrich	S7076
T75 Flask	MidSci	TP90076
Tissue Culture Hood
Tissue Embedding Cassette
TrypLE Express	Invitrogen	12604013	animal origin-free, recombinant enzyme
Type II Collagenase	Life Technologies	17101015
Vortex