2.1 Isolation and culture of human hepatobiliary tumor organoids

All study procedures strictly adhered to the Declaration of Helsinki. Liver tumor tissues were obtained from patients undergoing surgical resection for PLC at the First Affiliated Hospital of Nanjing Medical University, detailed patient information is shown in Table S1. This study was approved by the Ethics Committee of Nanjing Medical University, and the patients provided written informed consents. The isolation and culture of human tumor organoids were performed as described previously [14]. Briefly, tissues were minced under sterile conditions, digested with liver digestion medium (Thermo Fisher Scientific, Waltham, MA, USA) at 37 ℃ for 30–60 min, and centrifuged at 200 × g for 5 min. After washing twice with cold Advanced DMEM/F12 (Gibco, Grand Island, NY, USA), the pellet was mixed with Type 2 Basement Membrane Extract (BME2; R&D, Minneapolis, MN, USA), and seeded into ultra-low attachment 24-well plates (Corning Inc., Corning, NY, USA) (2,000–5,000 cells/well). Following solidification of BME2, 600 µL of culture medium was added to each well. The tumor organoids culture medium was based on advanced DMEM/F12 medium supplemented with 30% (vol/vol) Wnt-3 A conditioned medium (CM), 10% (vol/vol) Rspo-1 CM, 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), 1% Glutamax (Invitrogen), 10 mM HEPES (Sigma-Aldrich, St. Louis, MO, USA), 1:50 B27 (Thermo Fisher Scientific), 1:100 N2 supplement (Thermo Fisher Scientific), 2.0 µM A8301 (R&D), 1.25 mM N-acetyl-l-cysteine (Sigma-Aldrich), 10 µM forskolin (R&D), 10 mM nicotinamide (Merck KGaA, Darmstadt, Germany), 10 µM rock inhibitor Y27632 (R&D), 10 nM recombinant human (Leu15)-gastrin I (Thermo Fisher Scientific), 50 ng/mL recombinant human EGF (R&D), 100 ng/mL recombinant human FGF10 (R&D), 25 ng/mL recombinant human HGF (R&D), and 50 ng/mL recombinant human noggin (R&D). After initial culturing for 10–14 days, organoids were dissociated with dispase for 15 min, followed by TrypLE Express (Thermo Fisher Scientific) digestion for 3–5 min. Organoids were then passaged at a ratio of 1:4–1:6 every 7–9 days.

To prepare frozen stocks, organoid cultures were dissociated and mixed with recovery cell culture freezing medium (GIBCO) and frozen following standard procedures. When required, the cultures were thawed using standard thawing procedures and cultured as described above. The culture medium was supplemented with Y-27,632 (10µM). Organoid pictures were taken with either a Leica M80 stereoscope and Leica MC170 HD camera or with an inverted microscope Leica DMIL and Leica DFC 450 C camera.

2.2 Organoid quantification and morphometric analysis

For drug sensitivity assays, organoid size and number were quantitatively assessed. Following treatment, organoids from three replicate wells per condition were collected. To obtain single-organoid suspensions for counting, organoids were dissociated using Dispase for 15 min at 37 °C, washed twice with PBS, and resuspended in 100 µL of PBS. Organoid counting was performed using an automated system: 20 µL of the suspension was loaded into a 3D counting chamber (Corning, Cat. No. 480201), and counts were obtained using a Corning® Cell Counter (Model 6749). Organoid size (diameter or cross-sectional area) was measured from bright-field images using ImageJ. Morphological disruption was defined by criteria such as loss of structural integrity, membrane blebbing, or cellular disintegration, as observed in high-resolution images.

2.3 Cancer cell culture

Human intrahepatic bile duct epithelial cell lines, along with human ICC cell lines (HUCCT1 and RBE) and HCC cell lines (HCCLM3 and Hep3B), were obtained from the Chinese Academy of Sciences type culture bank. These cells were cultured in DMEM (Gibco, USA) enriched with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were maintained in a temperature-controlled incubator set at a constant 37 °C with a 5% CO2 atmosphere.

2.4 Whole-exome sequencing (WES)

Genomic DNA was extracted from hepatobiliary tumor organoids, corresponding tumor tissues, and 3 healthy livers using the Hipure Universal DNA kit (Magen, Guangzhou, China). The heathy liver samples (0.25 cm3) were obtained from explant livers during liver transplantation performed at the First Affiliated Hospital of Nanjing Medical University. The DNA libraries were prepared by shearing the samples, followed by end repair, phosphorylation, and ligation to barcoded sequencing adaptors. Ligated DNA fragments with lengths between 200 and 350 bp were selected and enriched using the SureSelect Human All Exon V6 Kit (Agilent Technologies, Palo Alto, CA). Sequencing was conducted using the Illumina Novaseq 6000 (San Diego, CA). The reads were aligned to the GRCh38 reference using the Burrows-Wheeler Aligner, and variant calling was performed using the Genome Analysis Toolkit (GATK) Unified Genotyper with local realignment and base quality score recalibration. SNPs and InDels were filtered using GATK’s Variant Filtration with appropriate standards, and variants exhibiting segregation distortion or sequencing errors were discarded. Cancer-related variants were determined by performing the following filtering steps as described previously [15]: (i) filtering out variants with reads supporting variations of ≥ 2 in our sequenced 3 healthy liver samples. (ii) filtering out variants present in dbSNP, (iii) filtering out variants with a frequency of > 0.01 in the ExAC database (v03), (iv) filtering out synonymous and intronic variants, and (v) filtering out variants with SIFT scores > 0.05.

2.5 RNA sequencing (RNA-seq)

Total RNA was extracted from different type of samples, including PLC organoids, tumor tissues, and their corresponding adjacent non-tumor liver tissues. 500ng total RNA was used for RNA-seq library construction with Hieff NGS® Ultima Dual mode mRNA Library Prep Kit (Yeasen #12309ES) followed the manufacture. All libraries were quantified with Qubit and analysis using Agilent 2100 bioanalyzer. All quality-controlled libraries were sequenced with Illumina Novaseq X Plus. Quality control of raw sequencing data was performed using Fastp (v0.18.0), and clean reads were aligned to human reference genome Grch38 by Hisat2 (v2.1.0) using default setting. Transcript quantification was performed using RSEM (v1.2.19)software. Differential expression genes analysis was performed by DESeq2(v1.24.0) software between two different groups, and principal component analysis (PCA) was also performed using the same R package. The correlation between samples were calculated using cor () function in R.

2.6 ScRNA-seq analysis

Single-cell suspensions were prepared from dissociated tumor organoids, and cell concentration was quantified and adjusted to 1 × 10⁶ cells mL⁻¹ using a Countess II Automated Cell Counter. Sequencing libraries were constructed employing the 10x Genomics Chromium platform (Chromium Single Cell 3′ Reagent Kit v3). Briefly, cells were combined with barcoded gel beads and master mix to form Gel Bead-in-Emulsions (GEMs) for single-cell isolation. Within GEMs, gel beads dissolved to release primers containing unique molecular identifiers (UMIs) and cell barcodes for reverse transcription. The resulting barcoded cDNA was amplified via PCR, and the library was prepared through fragmentation (sheared to 200–300 bp using a Bioruptor), end-repair, adapter ligation, and final PCR enrichment. Sequencing was performed on an Illumina platform. Raw data were processed using Cell Ranger to generate a gene expression matrix. Stringent quality control was applied using Seurat: cells were retained only if they exhibited UMI counts between 3,000 and 40,000 and a mitochondrial gene percentage below 10%. Following this QC, high-quality transcriptomes were obtained from all organoid samples, with final cell yields per sample ranging from 5,535 to 21,829 cells (specifically: HCC118_O, 21,829 cells; HCC12_O, 7,679; HCC13_O, 12,192; HCC133_O, 7,047; HCC77_O, 7,582; ICC42_O, 10,890; ICC61_O, 7,843; ICC79_O, 11,069; CHC11_O, 10,884; CHC142_O, 7,730; CHC63_O, 5,535). All subsequent analyses were based on this rigorously filtered dataset.

2.7 In vitro testing of targeted drugs using patient-derived liver tumor organoids

Tumor organoids were harvested, dissociated, mixed with BME2, and seeded (1.2 × 103/well) into 96-well ultra-low attachment plates. After culturing for 3 days, each well was added with drugs and cultured for further 5 days. Meanwhile, 10% Triton X-100 was used as positive control, and DMSO as negative control. Then, 100 µL CellTiter-Glo 3D solution (Promega Corporation, Madison, WI, USA) was added to each well, and incubated for 30 min at room temperature. Luminescent signal was measured with a plate reader (PerkinElmer, Inc.,Waltham, MA, USA), and cell viability was calculated by dividing the amount of ATP in the test wells by that in the vehicle-control wells, after subtracting the background levels. All drug screens were performed in triplicates.

2.8 Nile red staining

HCC cell lines (HCCLM3 and Hep3B) were seeded into six-well plates. After 24 h, the cells were stained with Nile Red, a fluorescent dye from Solarbio Life Sciences, China, following the manufacturer’s instructions to ensure consistency. Frozen sections of HCC organoids from two patients were also stained with Nile Red according to the guidelines. Micrographs were then captured using an inverted fluorescence microscope from Nikon, Japan.

2.9 Filipin staining

Following inoculation of HCC cell lines (HCCLM3, Hep3B) into six-well plates, cholesterol levels were assessed using Filipin fluorescent staining, as per the manufacturer’s instructions from Shanghai Jianmei Gene Medical Technology, china. HCC organoids were prepared as frozen sections, stained immediately upon coverslip application, and observed under a fluorescence microscope (Nikon, Japan). Stained cholesterol deposits exhibit blue fluorescence.

2.10 Mitochondrial membrane potential

JC-1 is an ideal and widely employed fluorescent probe for assessing mitochondrial membrane potential. Follow the instructions for the JC-1 test kit (Beyotime, China), 1 mL of cell suspension was taken, 2µL JC-1 working liquid (5µM) was added, mixed evenly, and incubated under 37 °C and 5% CO₂ for 20 min away from light. The stained cells were collected, washed once with 1 mL PBS, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were re-suspended with 1 mL PBS and visualized under a fluorescence microscope.

2.11 Histology and immunohistochemistry (IHC) analysis

Tissues and organoids were fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, and sectioned. The sections were subjected to H&E staining, and blindly evaluated by two experienced pathologists who specialized in hepatopathology. IHC analysis was conducted as described previously [14] using primary antibodies targeting AFP (1:250, Abcam, USA), HepPar1 (1:250, Abcam, USA), EPCAM (1:250, Abcam), KRT19 (1:1000, Abcam). The sections were incubated with primary antibodies overnight at 4 ℃. The sections were rinsed with washing buffer and incubated with goat anti-rabbit IgG secondary antibody (1:1,000, Abcam, USA) for 1 h. Reactivity was detected using a Dako EnVision Detection System (Dako, Hamburg, Germany) following manufacturer’s instructions.

2.12 Immunofluorescence

Immunofluorescent cells and organoids were fixed for 20 min with 4% paraformaldehyde at room temperature, followed by 5 min of permeabilization with 0.05% Triton X-100 in PBS. After overnight blocking in PBS containing 2% BSA, the samples were incubated with primary antibodies at 4 ℃ before being conjugated with Alexa Fluorite or HRP. Paraffin-embedded sections were heated in 10 mM sodium citrate buffer (pH 6.0) to restore antigenicity. These sections were blocked with PBS containing 10% fetal calf serum (FCS) and 1% BSA for 1 h at room temperature, then incubated with primary antibodies overnight at 4℃. Subsequently, the fluorophore-conjugated secondary antibodies combined with 3 µM DAPI were applied at room temperature for 1 h. primary antibodies: E-cadherin (1:250, Servicebio, China), N-cadherin (1:250, Servicebio, China).

2.13 Transmission electron microscopy

The PLC organoids were seeded onto 6 cm plates and treated with erastin (5 µM) or RSL3 (0.2 µM) for 12 h. Following treatment, the organoids were prefixed with 3% glutaraldehyde and then post-fixed in 1% osmium tetroxide. Subsequently, the organoids were dehydrated through a series of acetone washes, infiltrated with Epoxy 812 (Structure Probe Inc, PA, USA), and embedded accordingly. Semithin sections were stained with methylene blue, while ultrathin sections were cut using a diamond knife and stained with uranyl acetate and lead citrate. Finally, the sections were examined using transmission electron microscopy (TEM; Hitachi, HT7700, Japan).

2.14 Statistical analysis

Statistical analyses were conducted using GraphPad Prism 9.0. P<0.05 indicated statistical significance. Continuous variables between two groups were compared using an independent t-test, while ANOVA was employed to compare continuous variables across multiple groups(ns p ≥ 0.05, *p < 0.05, **p < 0.01,***p < 0.001, ****p < 0.0001.).