Grouped-seq for integrated phenotypic and transcriptomic screening of patient-derived tumor organoids

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.

Acoustic Printing of Patient‐Derived Organoids That Preserve Tumor Microenvironment for Personalized Drug Screening

“…The high‐throughput, precisely controllable droplets enable the organoids to be more suitable for personalized drug screening and parallel evaluation of multiple drug treatments. [ 57 ] We first established a mechanical sample processing and enzymatic digestion method to generate single cell suspension from patients' tumor tissues. The cell suspension derived from bladder cancer patient tissue was loaded into the sample chamber for subsequent droplet excitation (Figure 1a).…”

Function-associated scRNA-seq on single lung cancer organoids unravels the immune landscape of tumor parenchyma

“…To perform scRNA-seq on single cells digested form organoids, we upgraded our previously developed Group-seq methodology ( 17 ) to the single-cell level with a higher throughput. Since we need to record the barcode in each microwell to trace single cells, we employed a ligation-based barcode synthesis method to lower the costs of the long capture oligos that attach to magnetic beads for mRNA capture (Fig.…”

“… (A) Structure of the 3’ mRNA capture oligo for FascRNA-seq( 17 ). (B) Violin plots of the number of genes (average 5380), mapped reads (average 30522), and ratio of mitochondria-related genes (Mt.…”

Microwell array chip-based single-cell analysis

“…In addition, microwell chip-based sequencing and imaging linking analysis have enabled photophysiological analysis of microalgae 100 and integration of the phenotype and transcriptome of patient-derived tumor organoids. 101…”