Comparison of high-throughput single-cell RNA-seq methods for ex vivo drug screening

Area of Science:

• Oncology
• Genomics
• Pharmacology

Background:

• Functional precision medicine (FPM) seeks to personalize cancer treatment by matching drugs to individual patient tumor cell characteristics.
• High-throughput ex vivo drug profiling is advancing FPM, requiring sophisticated experimental systems.
• Single-cell gene expression analysis offers a powerful lens for understanding cellular heterogeneity in drug response.

Purpose of the Study:

• To present a proof-of-concept for an integrated experimental system combining ex vivo drug treatment with single-cell RNA sequencing (scRNA-seq).
• To evaluate different scRNA-seq methods for their ability to profile drug responses at the single-cell level.
• To identify transcriptional responses to specific drugs in a relevant cancer model.

Main Methods:

• Developed an integrated system for simultaneous ex vivo drug treatment and barcoding of multiple drug conditions within a single scRNA-seq experiment.
• Applied the system to the glucocorticoid-resistant acute lymphoblastic leukemia (ALL) Reh cell line.
• Evaluated three distinct scRNA-seq approaches for cell recovery, drug condition tagging, library complexity, sensitivity, and differential gene expression analysis.

Main Results:

• All three evaluated scRNA-seq methods demonstrated high cell recovery and accurate tagging of drug conditions.
• Significant variations in library complexity, gene detection sensitivity, and differential gene expression analysis were observed across the methods.
• A consistent and substantial transcriptional response to fludarabine, a key drug for high-risk ALL, was identified across all tested methods.

Conclusions:

• The integrated approach holds promise for studying drug responses at the single-cell level in the context of FPM.
• Methodological choices in scRNA-seq significantly impact the analysis of drug response data.
• The generated dataset of 27,327 cells is publicly available for further methodological comparisons and research.