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Updated: Oct 10, 2025
Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis
Published on: December 23, 2022
Grouped-seq for integrated phenotypic and transcriptomic screening of patient-derived tumor organoids
Yushuai Wu1, Kaiyi Li1, Yaqian Li2
1Department of Biomedical Engineering, School of Medicine, Tsinghua University, Beijing 100084, China.
Abstract:
Patient-derived tumor organoids (PDOs) have emerged as a reliable in vitro model for drug discovery. However, RNA sequencing-based analysis of PDOs treated with drugs has not been realized in a high-throughput format due to the limited quantity of organoids. Here, we translated a newly developed pooled RNA-seq methodology onto a superhydrophobic microwell array chip to realize an assay of genome-wide RNA output unified with phenotypic data (Grouped-seq). Over 10-fold reduction of sample and reagent consumption together with a new ligation-based barcode synthesis method lowers the cost to ∼$2 per RNA-seq sample. Patient-derived colorectal cancer (CRC) organoids with a number of 10 organoids per microwell were treated with four anti-CRC drugs across eight doses and analyzed by the Grouped-seq. Using a phenotype-assisted pathway enrichment analysis (PAPEA) method, the mechanism of actions of the drugs were correctly derived, illustrating the great potential of Grouped-seq for pharmacological screening with tumor organoids.
Insights
A new pooled RNA-seq method, Grouped-seq, enables high-throughput drug screening using patient-derived tumor organoids (PDOs). This cost-effective approach links genomic data with drug response, advancing personalized cancer therapy research.
Area of Science:
- Biotechnology
- Genomics
- Cancer Research
Background:
- Patient-derived tumor organoids (PDOs) are valuable for drug discovery.
- High-throughput RNA sequencing of PDOs is limited by sample quantity.
Purpose of the Study:
- To develop a high-throughput RNA sequencing method for PDOs.
- To enable cost-effective analysis of drug effects on PDOs.
- To link genomic and phenotypic data for drug screening.
Main Methods:
- Pooled RNA-seq methodology adapted to a superhydrophobic microwell array chip.
- Development of a ligation-based barcode synthesis for cost reduction.
- Application of Grouped-seq to patient-derived colorectal cancer organoids treated with anti-CRC drugs.
Main Results:
- Achieved over 10-fold reduction in sample and reagent consumption.
- Reduced cost to approximately $2 per RNA-seq sample.
- Successfully derived drug mechanisms of action using phenotype-assisted pathway enrichment analysis (PAPEA).
Conclusions:
- Grouped-seq offers a cost-effective, high-throughput solution for PDO drug screening.
- The method integrates genomic and phenotypic data for comprehensive analysis.
- Grouped-seq shows significant potential for pharmacological screening in tumor organoids.
