Prostate Organoid Cultures as Tools to Translate Genotypes and Mutational Profiles to Pharmacological Responses

Summary

Presented here is a protocol to study pharmacological responses in prostate epithelial organoids. Organoids closely resemble in vivo biology and recapitulate patient genetics, making them attractive model systems. Prostate organoids can be established from wildtype prostates, genetically engineered mouse models, benign human tissue, and advanced prostate cancer.

Abstract

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, three-dimensional (3D) cultures grown in a defined medium that resembles the prostatic epithelium. Prostate organoids can be established from wild-type and genetically engineered mouse models, benign human tissue, and advanced prostate cancer. Importantly, patient derived organoids closely resemble tumors in genetics and in vivo tumor biology. Moreover, organoids can be genetically manipulated using CRISPR/Cas9 and shRNA systems. These controlled genetics make the organoid culture attractive as a platform for rapidly testing the effects of genotypes and mutational profiles on pharmacological responses. However, experimental protocols must be specifically adapted to the 3D nature of organoid cultures to obtain reproducible results. Described here are detailed protocols for performing seeding assays to determine organoid formation capacity. Subsequently, this report shows how to perform drug treatments and analyze pharmacological response via viability measurements, protein isolation, and RNA isolation. Finally, the protocol describes how to prepare organoids for xenografting and subsequent in vivo growth assays using subcutaneous grafting. These protocols yield highly reproducible data and are widely applicable to 3D culture systems.

Introduction

Drug resistance is one of the major clinical problems in cancer treatment. Metastatic prostate cancer (PCa) treatment is primarily directed at the androgen-signaling axis. Next-generation anti-androgen therapies (e.g., enzalutamide and abiraterone) have showed great clinical success, but virtually all PCa eventually progresses towards an androgen-independent state, or castration resistant prostate cancer (CRPC).

Recent genomic and transcriptomic profiling of CRPC revealed there are three general mechanisms of resistance in prostate cancer: 1) activating mutations resulting in the restoration of androgen receptor (AR) signaling

Protocol

All work described in this protocol has been performed with previously established murine organoids and patient-derived organoids. All animal work was performed in compliance with the guidelines of Research Animal Resource Center of Memorial Sloan Kettering Cancer Center (IACUC: 06-07-012). All patient-derived tissues were collected in compliance with rules and regulations of Memorial Sloan Kettering Cancer Center (IRB: 12001).

1. Medium and buffer preparation

1. Thaw basement membrane matrix (e.g., Matrigel) at 4 °C overnight before starting the experiment. Keep it on ice during use.
2. Place culture plates at 37 °....

Results

Seeding efficiency
Organoid formation capacity is determined by phenotype and genotype. Wild-type (WT) prostate basal cells showed superior organoid formation capacity (30%-40%) compared to luminal cells (3%) (Figure 1A). After organoid establishment, the formation capacity increased drastically. Typically, 25%-30% of cells derived from a WT organoid can form a new organoid (Figure 1B.......

Discussion

Understanding the molecular mechanisms underlying anti-androgen resistance and discovering potential therapeutic vulnerabilities requires testing of pharmacological responses in model systems mimicking prostate cancer. Described here is a detailed protocol for the reliable analysis of pharmacological responses in patient-derived and genetically engineered prostate organoids and preparation of these organoid samples for downstream applications.

There are two critical steps in this protocol. The.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A83-01	Tocris	2939	Organoid medium component: Final concentration 200 nM
ADMEM/F12	Gibco/Life technologies	12634028	Organoid medium component
B27	Gibco/Life technologies	17504-044	Organoid medium component
Cell culture plates	Fisher	657185
Cell Titer Glo	Promega	G7571
DHT	Sigma-Aldrich	D-073	Organoid medium component: Final Concentration 1 nM
DMSO	Fisher	BP231-100
EGF	Peprotech	315-09	Organoid medium component: Final concentration 50 ng/ml for mouse, 5 ng/nl for Human
FGF10	Peprotech	100-26	Human specific organoid medium component: Final concentration 10 ng/ml
FGF2	Peprotech	100-18B	Human specific organoid medium component: Final concentration 5 ng/ml
Glutamax	Gibco/Life technologies	35050079	Organoid medium component
HEPES	MADE IN-HOUSE	N/A	Organoid medium component: Final concentration 10 mM
Matrigel (Growthfactor reduced & Phenol Red free)	Corning	CB-40230C	Organoid medium component
N-Acetylcysteine	Sigma-Aldrich	A9165	Organoid medium component: Final concentration 1.25 mM
Nicotinamide	Sigma-Aldrich	N0636	Human specific organoid medium component: Final concentration 10 mM
NOGGIN	Peprotech or stable transfected 293t cells with Noggin construct (Karthaus et al. 2014)	120-10C	Organoid medium component: Final Concentration 10% conditioned medium or 100 ng/ml
Penicillin/Streptavidin	Gemini Bio-Products	400-109	Organoid medium component
Phospatase inhibitors	Merck Millipore	524629
Prostaglandin E2	Tocris	3632464
Protease Inhibitors	Merck Millipore	539131
R-SPONDIN	Peprotech or stable transfected 293t cells with R-Spondin1 construct (Karthaus et al. 2014)	120-38	Organoid medium component: Final Concentration 10% conditioned medium or 500 ng/ml
RIPA buffer	Merck	20-188
RNA-easy minikit	Qiagen	74104
SB202190	Sigma-Aldrich	152121-30-7	Human specific organoid medium component: Final concentration 10 μM
TryplE	ThermoFisher	12605036
Y-27632	Selleckchem	S1049	Organoid medium component: Final Concentration 10 μM