Viability Analysis and High-Content Live-Cell Imaging for Drug Testing in Prostate Cancer Xenograft-Derived Organoids

Area of Science:

• Oncology
• Biotechnology
• Pharmacology

Background:

• Tumor organoids are advanced models for in vitro oncology drug testing aiming for personalized cancer treatments.
• Current drug testing methods for organoids face challenges due to variable experimental conditions and limited read-outs, often overlooking inter-organoid heterogeneity.
• Existing bulk viability assays lose crucial information on specific biological responses and cell death modalities.

Purpose of the Study:

• To develop a systematic and standardized approach for prostate cancer (PCa) patient-derived xenograft (PDX) organoid drug testing.
• To establish an imaging-based procedure for high-content fluorescence microscopy to detect various cell death modalities in living PCa organoids.
• To identify essential conditions and quality checks for consistent and reliable organoid drug testing results.

Main Methods:

• Developed a systematic processing method for PCa PDX organoids for viability-based drug testing.
• Implemented an imaging-based drug testing procedure using high-content fluorescence microscopy on living PCa organoids.
• Utilized a dye combination (Hoechst 33342, propidium iodide, Caspase 3/7 Green) for segmentation and quantification of organoids and cell nuclei.
• Identified cytostatic and cytotoxic treatment effects by analyzing individual organoids and cell nuclei.

Main Results:

• Established essential conditions and quality checks for consistent organoid culturing and drug testing.
• Generated a high-content fluorescence microscopy-based drug testing procedure for PCa organoids.
• Successfully detected and quantified various modalities of cell death, distinguishing cytostatic from cytotoxic effects.
• Provided insights into the mechanistic actions of tested drugs on organoids.

Conclusions:

• The developed systematic approach enhances the validity of organoid-based drug testing for prostate cancer.
• The imaging-based procedure offers detailed mechanistic insights into drug responses, including specific cell death pathways.
• These methods are adaptable for other cancer types, potentially accelerating the clinical implementation of organoid drug testing.