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

Summary

This protocol outlines a streamlined method for generating hepatocellular carcinoma organoids, applying drug treatment, and performing single-cell RNA sequencing before and after treatment to characterize treatment-associated transcriptional changes.

Abstract

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.

Introduction

Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults and a major cause of cancer-related deaths worldwide^1,2,3. Its poor clinical outcome is due to significant intratumoral heterogeneity, leading to treatment resistance and recurrence⁴. A recent study indicates that developmental diversity and cell-state plasticity driven by key transcriptional programs such as the FOXM1/CEBPB axis contribute to therapeutic resistance in HCC⁵. These findings highlight the need to understand how tumor cell populations ....

Protocol

Studies using human tissues were reviewed and approved by the Committees for Ethical Review of Research of Guangzhou Medical University (approval no. GYZL-2024-KY31). All procedures involving human specimens were conducted in accordance with the institutional guidelines for human research. Written informed consent was obtained from all patients for the use of their clinical specimens for medical research. The reagents and equipment used are listed in the Table of Materials.

This workflow includes the recovery and expansion of cryopreserved hepatocellular carcinoma organoids, with minor adaptations for downstream therapeutic....

Results

Figure 1 outlines the generation and characterization of hepatocellular carcinoma (HCC) organoids. In Figure 1A, patient-derived tumor tissues were used to establish organoid cultures for histological analysis and treatment studies. Figure 1B shows bright-field images of organoids at days 1 and 6 post-recovery, demonstrating survival and expansion under standard three-dimensional culture conditions. At day 1, organoids appear as sma.......

Discussion

To ensure reproducibility, efficiency, and high-quality single-cell transcriptomic analysis, several technical steps in this workflow require careful control. Consistency during revival and expansion of patient-derived HCC organoids is essential, because variations in seeding density, basement membrane matrix composition, and culture timing can influence organoid morphology, growth, and downstream treatment responses^13,14. In addition, high cell viability during .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
[Leu15]-gastrin I human	Merck	G9145
1.5 mL Microtubes	Merck	AXYMCT150LC
1X RBC Lysis Buffer	Thermo Fisher Scientific	00-4333-57
2.0ml Internal Thread Cryogenic Vials	Nest	607001
A8301 (TGFb inhibitor)	Tocris Bioscience	2939
AFP Polyclonal antibody	Proteintech	14550-1-AP
Anti-Albumin antibody [ALB/2144]	abcam	ab236492
Anti-HNF-4-alpha antibody [EPR16885] - ChIP Grade	abcam	ab181604
Anti-Prealbumin antibody [EPR3219]	abcam	ab92469
Anti-SOX9 antibody [EPR14335-78]	abcam	ab185966
B27 Supplement (503), minus vitamin A	Thermo Fisher Scientific	12587010
Cell Recovery Solution, 100 mL	Corning	354253	Reagent used to dissolve basement membrane matrix and recover organoids before replating or downstream processing
CellTiter-Glo® 3D Cell Viability Assay	Promega	G9681
CHIR99021	Merck	SML1046
Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1	10x Genomics	CG000227
Corning Cell Strainer, 40 μm, Blue, S, IND, 1/50	Corning	431750
Costar 24-well Clear Flat Bottom Ultra-Low Attachment Multiple Well Plates, Individually Wrapped, Sterile	Corning	3473
Cultrex Reduced Growth Factor BME, Type 2 PathClear (BME)	Merck	3533-005-02	Extracellular matrix used to support three-dimensional organoid culture and dome formation
DMSO	Merck	C6164	Solvent used to prepare drug stock solutions and vehicle control
Dulbecco's Modified Eagle Medium/Ham's F-12	Thermo Fisher Scientific	12634028	Advanced DMEM/F-12
E7080 (Lenvatinib)	Selleck	S1164	Tyrosine kinase inhibitor used for organoid treatment and therapeutic perturbation
Fetal Bovine Serum Value FBS	Gibco	A5256701
Forskolin	Tocris Bioscience	1099
GlutaMAX supplement	Thermo Fisher Scientific	35050061
Hematoxylin and Eosin Staining Kit	Beyotime	C0105S
HEPES, 1 M	Thermo Fisher Scientific	15630080
Leica DM6 B Fluorescence Motorized Microscope	Leica	N/A
N2 supplement (1003)	Thermo Fisher Scientific	17502048
N-acetylcysteine	Merck	A0737-5MG
Nicotinamide	Merck	N0636
Nunc 15 mL Conical Sterile Polypropylene Centrifuge Tubes	Thermo Fisher Scientific	339651
Penicillin/streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122
Phosphate-Buffered Saline	GENOM BIO	GNM20012	Buffer used for washing organoids and preparing samples for downstream processing
Recombinant human EGF	Peprotech	AF-100-15
Recombinant human FGF10	Peprotech	100-26
Recombinant human HGF	Peprotech	100-39
Recombinant human Noggin	Peprotech	120-10C
Rho kinase inhibitor Y-27632 dihydrochloride	Merck	Y0503	ROCK inhibitor used to enhance organoid survival after thawing or passaging
R-spodin1-conditioned medium	(Broutier et al.)	N/A	Secretion of cell lines
Trypan Blue Stain, 0.4% membrane filtered Prepared in 0.85% saline	Gibco	15250061
TrypLE Express Enzyme (1x), no phenol red	Thermo Fisher Scientific	12604013	Trypsin substitute
Wnt-3a-conditioned medium	(Broutier et al.)	N/A	Secretion of cell lines