Establishment and Maintenance of Patient-derived Prostate Cancer Organoids: A Detailed Experimental Protocol

Summary

Prostate cancer heterogeneity and treatment resistance remain challenges. We describe a protocol for generating patient-derived organoids (PDOs) that preserve key parental tumor characteristics. This standardized model supports drug screening and precision medicine advancements.

Abstract

Prostate cancer (PCa) is a highly heterogeneous malignancy and a leading cause of cancer-related mortality in men, with significant clinical challenges in addressing treatment resistance and disease progression. Existing preclinical models, such as two-dimensional (2D) cell lines and patient-derived xenografts (PDXs), have limitations in faithfully recapitulating the complexity of PCa, including tumor heterogeneity, androgen receptor (AR)-dependent signaling, and microenvironmental interactions. To address this gap, this study presents a robust and reproducible protocol for establishing Patient-derived organoids (PDOs) that preserve the genetic, phenotypic, and histological features of the parental tumors. This method enables the culture of organoids from both localized and advanced PCa, providing a biologically relevant model for studying disease mechanisms, drug response, and biomarker discovery. The results demonstrate the protocol's ability to generate organoids from a variety of clinical specimens, with applications in high-throughput drug screening and precision medicine. By offering a standardized workflow that addresses key technical challenges, this study highlights the importance of PDOs as versatile tools for advancing prostate cancer research and developing more effective therapeutic strategies.

Introduction

Prostate cancer (PCa) remains one of the most prevalent malignancies in men worldwide, second only to lung cancer in cancer-related mortality. According to recent epidemiological studies, the global incidence of PCa has been steadily rising, and it is projected that the number of new cases of prostate cancer annually will rise from 1.4 million in 2020 to 2.9 million by 2040¹. Despite advancements in early detection and the development of novel therapies, including androgen deprivation therapy (ADT) and targeted agents, prostate cancer remains a significant clinical challenge due to its heterogeneous nature and the inevitable development of trea....

Protocol

This study was reviewed and approved by the Medical Ethics Committee of Wuxi No. 2 People's Hospital (Approval Number: Y-38 (2023)). All participants provided written informed consent forms. Patient data underwent de-identification to ensure privacy protection in compliance with ethical standards.

1. Sample collection

1. Tissue acquisition
   1. Preparation before sampling
      1. Inclusion Criteria: Ensure all participants have a confirmed diagnosis of prostate cancer, as determined by pathological or imaging examination. Derive tumor samples from surgical resection, biopsy, or other clinical speci....

Results

Morphological and dynamic growth features of patient-derived prostate cancer organoids
Patient-derived prostate cancer organoids were successfully established from fresh tumor tissues collected during clinical procedures. Initial organoid formation was typically observed within 5-7 days of culture (Figure 2). Under brightfield microscopy, organoids exhibited distinct morphological features, forming compact, well-defined spheroids or glandular-like structures. Organoids d.......

Discussion

The establishment of PDOs for PCa represents a critical advancement in preclinical modeling, offering significant advantages over conventional 2D cell cultures and PDXs¹⁶. The method presented in this study enables the generation of organoids that faithfully recapitulate the histological, genetic, and phenotypic characteristics of the original tumor. By preserving tumor heterogeneity, including AR signaling and cellular architecture, the organoids provide a more biologically relevant platform for .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A 83-01	TOCRIS	2939
adMEM/F12	Gibco	12634010
biosafety cabinet	Jinan Xinbeixi Biotechnology Co., Ltd
Cell Recovery Solution	Corning	354253
centrifuge	Shenzhen Ruiwode Life Technology Co., Ltd	M1416R
centrifuge tubes	Nanjing iResearch Biotechnology Co., Ltd
Cultrex PathClear BM	R&D	3533-010-02
Cultrex Reduced Growth Factor BME, Type 2	R&D	3533-005-02
culture containers (24-well low-adhesion plates)	thermo	Nunc-174930-ZX
DHT	ISOREAG	521-18-6
electronic balance	Nanjing iResearch Biotechnology Co., Ltd
Fetal Bovine Serum	Gibco	10099141
GlutaminePlus-200mM	R&D	B90210
HEPES	R&D	1254/10
humidified cell culture incubator (37°C, 5% CO2)	Shanghai Hetian Scientific Instrument Co., Ltd	HY-160SY
ice box	Nanjing iResearch Biotechnology Co., Ltd
medium-sized culture dishes	absin	abs7005
N21-MAX Media Supplement (50X)	R&D	AR008
N-Acetylcysteine	Sigma	A9165-5G
Nicotinamide	R&D	3533-010-02
Normocin	Invivogen	ant-nr-2
Pipet-aid pipetting device	Nanjing Hotspot Scientific Instrument Co., Ltd
pipettes	Nanjing Hotspot Scientific Instrument Co., Ltd
Primocin	Invivogen	ant-pm-2
rhEGF	R&D	236-EG-01M
rhFGF	R&D	233-FB-025
rhFGF-10	R&D	345-FG-025
rhNoggin	R&D	6057-NG-100
rhR-Spondin 1	R&D	4656-RS-100
SB 202190	TOCRIS	1264
Single-cell suspension preparation apparatus	RWD	DSC-400
Tissue scissors/scalpel	Nanjing iResearch Biotechnology Co., Ltd
TryplE Express	Gibco	126A04013
Tumor Dissociation Kit, Human	Miltenyi Biotec	130-095-929
tweezers	Nanjing iResearch Biotechnology Co., Ltd
water bath	Changzhou Zhongjie Experimental Instrument Manufacturing Co., Ltd	HH-600
Y-27632 dihydrochloride	R&D	1254/10