Generation and Single-Cell Transcriptomic Analysis of Hepatocellular Carcinoma Organoids following Drug Treatment.

Hepatocellular carcinoma is a major cause of cancer-related mortality worldwide and is characterized by marked intratumoral heterogeneity, which contributes to variable treatment responses, tumor recurrence, and disease progression. A deeper understanding of how tumor cells change at the transcriptional level before and after drug treatment is therefore essential for improving therapeutic strategies. However, practical experimental workflows that connect organoid-based drug treatment with downstream single-cell transcriptomic profiling remain limited. In this study, a standardized workflow for generating hepatocellular carcinoma organoids, applying defined drug treatment, and performing single-cell RNA sequencing on samples collected before and after treatment is developed. The protocol includes organoid revival and expansion, pre-treatment quality assessment, drug exposure, organoid preparation for single-cell dissociation, library construction, and basic comparative analysis of single-cell transcriptomic data. Critical technical precautions are provided to improve reproducibility and sample quality. This workflow enables side-by-side characterization of cellular composition and transcriptional changes associated with drug treatment in hepatocellular carcinoma organoids. The protocol is robust, scalable, and adaptable across different organoid systems, providing a practical platform for investigating treatment-associated gene expression changes at single-cell resolution.