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Updated: Jul 14, 2025
Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis
Published on: December 23, 2022
Automation, live-cell imaging, and endpoint cell viability for prostate cancer drug screens
Rolando D Z Lyles1,2, Maria J Martinez2,3, Benjamin Sherman2,3
1Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America.
Abstract:
Androgen deprivation therapy (ADT) is the standard of care for high risk and advanced prostate cancer; however, disease progression from androgen-dependent prostate cancer (ADPC) to lethal and incurable castration-resistant prostate cancer (CRPC) and (in a substantial minority of cases) neuroendocrine prostate cancer (NEPC) is common. Identifying effective targeted therapies is challenging because of acquired resistance to established treatments and the vast heterogeneity of advanced prostate cancer (PC). To streamline the identification of potentially active prostate cancer therapeutics, we have developed an adaptable semi-automated protocol which optimizes cell growth and leverages automation to enhance robustness, reproducibility, and throughput while integrating live-cell imaging and endpoint viability assays to assess drug efficacy in vitro. In this study, culture conditions for 72-hr drug screens in 96-well plates were established for a large, representative panel of human prostate cell lines including: BPH-1 and RWPE-1 (non-tumorigenic), LNCaP and VCaP (ADPC), C4-2B and 22Rv1 (CRPC), DU 145 and PC3 (androgen receptor-null CRPC), and NCI-H660 (NEPC). The cell growth and 72-hr confluence for each cell line was optimized for real-time imaging and endpoint viability assays prior to screening for novel or repurposed drugs as proof of protocol validity. We demonstrated effectiveness and reliability of this pipeline through validation of the established finding that the first-in-class BET and CBP/p300 dual inhibitor EP-31670 is an effective compound in reducing ADPC and CRPC cell growth. In addition, we found that insulin-like growth factor-1 receptor (IGF-1R) inhibitor linsitinib is a potential pharmacological agent against highly lethal and drug-resistant NEPC NCI-H660 cells. This protocol can be employed across other cancer types and represents an adaptable strategy to optimize assay-specific cell growth conditions and simultaneously assess drug efficacy across multiple cell lines.
Insights
A new semi-automated protocol streamlines drug discovery for advanced prostate cancer (PC) by optimizing cell growth and live-cell imaging. This method identified EP-31670 for androgen-dependent and castration-resistant PC, and linsitinib for neuroendocrine PC.
Area of Science:
- Oncology
- Drug Discovery
- Cell Biology
Background:
- Androgen deprivation therapy (ADT) is standard for advanced prostate cancer (PC), but progression to castration-resistant (CRPC) and neuroendocrine (NEPC) forms is common.
- Acquired resistance and tumor heterogeneity complicate the identification of effective targeted therapies for advanced PC.
- Developing robust and reproducible methods is crucial for discovering novel therapeutics against lethal PC subtypes.
Purpose of the Study:
- To develop an adaptable, semi-automated protocol for optimizing cell growth and drug screening in diverse prostate cancer cell lines.
- To enhance the robustness, reproducibility, and throughput of in vitro drug efficacy assays using live-cell imaging.
- To validate the protocol's effectiveness in identifying potential therapeutic agents against different PC subtypes.
Main Methods:
- Established optimized 72-hour culture conditions for a panel of 9 human prostate cell lines (non-tumorigenic, ADPC, CRPC, NEPC) in 96-well plates.
- Integrated live-cell imaging and endpoint viability assays for real-time monitoring and drug efficacy assessment.
- Validated the protocol by confirming known drug effects and identifying novel therapeutic candidates.
Main Results:
- Optimized cell growth and confluence for 72-hour drug screens across multiple prostate cancer cell lines.
- Demonstrated the protocol's reliability by validating EP-31670 (BET and CBP/p300 inhibitor) efficacy against androgen-dependent (ADPC) and castration-resistant (CRPC) prostate cancer cells.
- Identified linsitinib (IGF-1R inhibitor) as a potential agent against highly lethal and drug-resistant neuroendocrine prostate cancer (NEPC) cells.
Conclusions:
- The developed semi-automated protocol offers an adaptable and efficient strategy for optimizing cell growth and assessing drug efficacy across various cancer cell lines.
- This pipeline facilitates the streamlined identification of novel or repurposed drugs for challenging and heterogeneous cancers like advanced prostate cancer.
- The findings highlight potential therapeutic avenues, including linsitinib for NEPC, addressing unmet needs in lethal prostate cancer treatment.
