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

Abstract

Presented here is a protocol to study pharmacodynamics, stem cell potential, and cancer differentiation in prostate epithelial organoids. Prostate organoids are androgen responsive, threedimensional (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.

Development of Prostate Cancer Organoid Culture Models in Basic Medicine and Translational Research

“…However, loss of PTEN enhanced the resistance to the tested anti-androgens, as demonstrated previously [130]. Furthermore, they showed that dual loss of TP53 and PTEN resulted in complete resistance to the second-generation anti-androgens [131].…”

“…Recently, transcriptomic and genomic profiling of androgen-independent PC showed three general mechanisms of resistance in PC [131]. The first of which is activating mutations that lead to the restoration of androgen receptor signaling [132].…”

“…Furthermore, the acquired resistance to ADT may drive to therapeutic vulnerabilities that can be tackled. Therefore, it is necessary for developing a model system to evaluate drug responses that simulate patient phenotypes and genotypes [131].…”

“…Since organoids can be genetically handled using CRISPR/Cas9 and shRNA systems, it can be a promising platform for elucidating the mechanisms of resistance to anti-cancer drugs [110]. Pappas et al, studied the effects of several anti-androgen molecules on PC murine organoids with different genotypes including Wild type (WT) genotype, PTEN loss, TP53 loss, or dual PTEN and TP53 loss [131]. They found that loss of TP53 did not cause resistance to the anti-androgenic molecules.…”

“…In the same study carried out by Pappas et al, a growth of MSKPCA2 organoids was substantially inhibited by anti-androgenic molecules. MSKPCA2 organoids expressed high levels of androgen receptor and luminal cell marker (CK8), while MSKPCA3 organoids showed resistance and expressed basal cell marker (CK5) [131].…”