Cell lines and cell culturing

Human MDA-MB-231 and LoVo cells were purchased from the American Type Culture Collection (ATCC), MDA-MB-231 cells were maintained in L-15 medium (cat.no. KGL1801-500; KeyGEN BioTECH). LoVo cells were cultured in DMEM basal medium. FBS (cat.no.12484028; Gibco, Thermo Fisher Scientific) was added to the DMEM and L-15 basal culture medium. Cells were cultured at 37 °C in a 5% (v/v) CO2 incubator.

Specimen collection

The surgical tumor tissue specimens employed in this investigation were ethically sourced with adherence to the principles of informed consent. Written informed consent was obtained with written informed consent from a 46-year-old male Chinese patient with ascending colon cancer, who received right hemicolectomy at Tongji Hospital (Wuhan, China). The primary tumor measured 5.5 × 4 × 3 cm. Additionally, a 0.9 × 0.8 × 0.6-cm tumor nodule was located within the mesentery of the ascending colon and 3 cm away from the primary lesion. The patient was staged as T3N1M0 according to the TNM classification system, with the primary tumor histologically characterized as moderately differentiated adenocarcinoma during post-surgical pathological analysis.

Preparation of the single cell suspension

The procurement of fresh tumor specimens was initiated by a sequential washing procedure. Fresh tumor specimens were washed with 70% ethanol, followed by sterile PBS with an antibiotic (cat.no. 15240062; Gibco; Thermo Fisher Scientific) component. Subsequently, the tissues were sectioned into diminutive fragments using sterile scissors. These fragments were then enzymatically digested by incubation in serum-free DMEM/F12 (cat. no. 11320033; Gibco; Thermo Fisher Scientific) containing 1.5 mg/ml collagenase IV (cat. no. 17104019; Gibco; Thermo Fisher Scientific) and 20 μg/ml hyaluronidase (cat. no. H1115000; Sigma-Aldrich; Merck KGaA), and supplemented with 1X antibiotic/antimycotic (cat. no. 15240–062; Gibco; Thermo Fisher Scientific) at 37 °C for 1–2 h. The cells were incubated in red blood cell lysis buffer (cat. no. 00–4333-57; eBioscience; Thermo Fisher Scientific) on ice for 10 min to eliminate red blood cells. The isolated primary CRC cells were washed twice in PBS and resuspended for subsequent organoid culture establishment.

Organoid culture

For organoid establishment, freshly isolated primary CRC cells were resuspended in Matrigel (growth factor reduced, phenol-free; cat. no. 354263; Corning) and seeded into 24-well culture plates at the indicated density (1,000 cells/30 µl Matrigel/well). After Matrigel aggregation, cells were covered with human colorectal organoid culture medium and incubated at 37˚C in an incubator with 5% CO2. The composition of the human CRC organoid culture medium was as follows: DMEM/F12 (cat. no. 11320033; Gibco; Thermo Fisher Scientific) supplemented with 1X B27 supplement (cat. no. 17504044; Gibco; Thermo Fisher Scientific), 50 ng/ml recombinant human epidermal growth factor (EGF) (cat. no. 100–47; PeproTech), 10 nM gastrin (cat. no. G9145; Sigma-Aldrich; Merck KGaA), 500 nM A83-01 (cat. no. HY-10432; MedChemExpress), 1X N2 (cat. no. A1370701; Gibco; Thermo Fisher Scientific, Inc.), 50 ng/ml recombinant human fibroblast growth factor FGF (cat. no. 45033; PeproTech.), 4 mM niacinamide (cat. no. HYB0150; MedChemExpress), 10 μM Y27632 (cat. no. HY-10071; MedChemExpress), 500 ng/ml R-spondin-1 (cat. no. 12038; PeproTech) and 100 ng/ml Noggin (cat. no. 120-10C; PeproTech). The medium was changed every 3 days. For serial passages, after 10 days of culture, whole organoids were digested using TrypLE™ Express (cat. no. 12605010; Gibco; Thermo Fisher Scientific) at 37˚C for 10 min. Individual organoid-derived cells were resuspended in PBS and used for a new round of organoid culture in Matrigel.

Animal experiments

The execution of animal studies adhered to protocols and guidelines ratified by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology (Wuhan, China) (TJH-201901005). Briefly, 4-week-old female BALB/c nude mice were obtained from Gem Pharmatech (Jiangsu, China). The mice were randomly divided into different groups (n = 5). Organoid cells were washed twice with PBS and resuspended prior to injection. For subcutaneous xenografts, 5 × 105 cells were injected per mouse. When the tumor grew to a size of 0.3 × 0.3 cm, the mice in the experimental group were injected intraperitoneally with oxaliplatin (10 mg/kg) (cat. no.09512; Sigma aldrich), and mice in the control group were injected intraperitoneally with an equal volume of saline once a week. A939572 (cat. no. HY-50709; MedChemExpress) was re-suspended in sterilized H2O vehicle at 30 mg/kg in a 50 μl dose. Mice were orally fed by using a syringe to administer the 50 μl dose once daily/mouse. The mice were euthanized by cervical dislocation 4 or 5 weeks after tumor implantation before the tumors were removed. All animal experiments were conducted in accordance with institutional guidelines and ethics approval.

Library preparation for transcriptome sequencing and data analysis

Library construction was performed using the Illumina Truseq™ RNA sample prep kit (Illumina, Inc.) according to the manufacturer’s protocol. Briefly, total RNA was extracted and mRNA was isolated from total RNA using magnetic beads. Under the action of reverse transcriptase, using mRNA as a template for reverse synthesis of one-stranded cDNA, followed by two-stranded synthesis, forming a stable double-stranded structure. The double-stranded cDNA structure had sticky ends, which were made up to flat ends by adding End Repair Mix. Finally, the cDNA library was sequenced on an Illumina platform.

Analysis of differential gene expression between the 45P and 45E organoid lines was performed using DESeq2 software using the quantified RNA sequencing (RNA-seq) data. Differentially expressed genes (DEGs) were identified using the following thresholds: |log2 fold-change (FC)|≥ 1 and adjusted P-value < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted on DEGs using the clusterProfiler R software package. GO terms and KEGG pathways with a corrected P-value < 0.05 were considered significantly enriched. Gene set enrichment analysis (GSEA) software was additionally utilized to perform GSEA of the full RNA-seq dataset.

Transwell, clonal culture, organoid culture and apoptosis assays

For migration assays, organoids were dissociated into single cells and 5 × 104 cells were added to the upper chamber of a Transwell insert (pore size, 8 μm; cat. no. 3422; Corning). The lower chamber contained 600 μl 30% FBS as a chemoattractant. After 24 h of incubation, paraformaldehyde was used to fix the migratory cells through the insert and these were stained with crystal violet (cat. no. C8470; Beijing Solarbio Science & Technology). Images of migrating cells were captured and cells were counted at a magnification of × 100 using an inverted microscope (Nikon Corporation).

For the colony formation assay, cells were spread in 6-well plates at a density of 200 cells/well and incubated for 1 week. After 1 week, 45P and 45E were treated with oxaliplatin (1 μM),5-fluorouracil (1 μM), A939572 (10 nM) or A922500 (10 μM) (cat. no. HY-10038; MedChemExpress) for a week. The medium was removed, the colonies were fixed in paraformaldehyde and stained with crystal violet, and images were captured.

For the organoid formation assay, cells were resuspended in Matrigel (growth factor reduced, phenol-free; cat. no. 354263; Corning) and seeded into 24-well plates at the indicated density (1,000 cells/30 µl Matrigel/well). After Matrigel aggregation, cells were covered with human colorectal organoid culture medium and incubated at 37˚C in an incubator with 5% CO2. After 1 week, 45P and 45E were treated with oxaliplatin (1 μM) or 5-fluorouracil (1 μM). The treatment duration was 5 days to induce organoid growth inhibition. Organoid’s apoptosis analysis, oxaliplatin (10 μM), A939572 (10 nM) or A922500 (10 μM) for 24 h to induce organoid apoptosis. Images of organoids were captured at a magnification of × 100 using an inverted microscope (Nikon Corporation).

For apoptosis analysis, the organoid was cultured for ~ 7 days and the cells were treated with oxaliplatin (10 μM), 5-fluorouracil (10 μM), A939572 (10 nM) and A922500 (10 μM) for 24 h. Next, cells were collected using TrypLE™ Express (cat. no. 12605010; Gibco; Thermo Fisher Scientific) with Phenol Red (cat. no. 12605; Gibco; Thermo Fisher Scientific) and stained using the annexin V-FITC/PI kit (cat. no. 559763; BD Biosciences) according to the manufacturer’s instructions, then analyzed using flow cytometry (FACSAria; BD Biosciences).

Western blotting

Total protein was extracted from organoids using RIPA lysis buffer with a protease inhibitor cocktail (cat. no. HY-K0010; MedChemExpress). Equal amounts of protein were separated by SDS-PAGE using 10% polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride membranes (cat. no. IPVH00010; Immobilon-P), which were blocked with 5% skim milk for 1 h at room temperature. Membranes were incubated overnight at 4 °C with primary antibodies against E-cadherin (ECAD), Vimentin (VIM), snail family transcriptional repressor 2 (Slug), twist family bHLH transcription factor (TWIST), zinc finger E-box binding homeobox 1 (ZEB1), β-Actin (ACTB), CD133, Sox2, Oct4, acetyl coenzyme A carboxylase 1(ACC1), SCD1, FASN at the dilutions recommended by the manufacturers. After washing with TBS with Tween 20 (TBST) buffer three times, the membranes were incubated with horseradish peroxidase-linked secondary antibodies (dilution, 1:5,000) for 2 h at room temperature. Finally, protein bands were visualized using enhanced chemiluminescence substrate after TBST washes 3 times.

Reverse transcription‑quantitative PCR (RT-qPCR)

Total RNA was extracted using RNA isolater reagent (cat. no. R401-01; Vazyme Biotech) and reverse transcribed using the HiScript II Q RTSuper Mix for qPCR kit (cat. no. R222; Vazyme Biotech.) according to the manufacturer's instructions. A ChamQ SYBR qPCR Master Mix kit (cat. no. Q311; Vazyme Biotech) was used to perform qPCR using an ABI 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific). qPCR results were analyzed using the comparative Ct method (2−ΔΔCq) with ACTB expression as an endogenous control. The primer sequences used in the present study are provided in Supplementary Table 3.

Determination of ATP, free fatty acid, triglyceride and cholesterol content

For the quantification of ATP, different volumes of lysate were added depending on the number of organoids, and after lysis, the supernatant was centrifuged at 4˚C at 12,000 × g for 5 min and used for subsequent assays. The ATP assay was performed using a kit (cat. no. S0026; Beyotime Institute of Biotechnology) according to the manufacturer’s protocol.

For the free fatty acid measurement, organoids were lysed by ultrasonication directly in PBS then centrifuged at 8,000 rpm for 10 min at 4 °C. The supernatant was taken for the subsequent assay. Free fatty acid levels were examined using a kit (cat. no. BC0595; Beijing Solarbio Science & Technology) according to the manufacturer's protocol.

Triglyceride and cholesterol levels were determined by lysing organoids directly by ultrasonication in PBS. The lysed fluid was measured directly without centrifugation. The determination was performed using the kits (cat. nos. A111-1–1 and A110-1–1; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol.

Oil red O and BODIPY staining

To assess lipid accumulation, organoids were fixed in Oil Red O fixative, rinsed in 60% isopropanol and stained with Oil Red O solution according to the kit instructions (cat. no. G1262; Beijing Solarbio Science & Technology). After washing, nuclei were counterstained with Mayer's hematoxylin.

BODIPY 493/503 was diluted in PBS and configured as a 5 µM working solution (cat. no. MX5403; MKBio). According to the manufacturer’s protocols, organoids were incubated with BODIPY 493/503 for 30 min at room temperature, washed, counterstained with DAPI for 5 min and imaged by fluorescence microscopy.

Short tandem repeat (STR) analysis for authentication

To validate the established organoids, STRs of 45P and 45E organoids and original tumor tissues were analyzed. STR profiles were compared with STR data of cell lines from databases such as American Type Culture Collection, RIKEN BioResource Center, DSMZ and Japanese Collection of Research Bioresources Cell Bank.

Cell proliferation and viability assays

Cell proliferation assays were performed by adding cells (1,000 cells/well) to matrix gel, spreading them evenly in 96-well plates and incubating them in organoid medium for different durations. Cell numbers were examined using a Cell Counting Kit 8 (CCK8) assay (cat. no. HY-K0301; MedChemExpress), which was performed according to the manufacturer's protocol. The cell viability assay was performed by adding the desired drugs (10 μM oxaliplatin, 10 nM A939572 or 10 μM A922500) for 24 h according to the experimental requirements, and then the cell viability was measured using the CCK8 assay, which was performed according to the manufacturer’s protocol.

Immunofluorescence staining of paraffin sections

Paraffin sections were dewaxed to water and repaired at high temperature. The sections were closed with a liquid containing donkey serum (10%) for 30 min. The serum on the sections was blotted up with absorbent paper, and a primary antibody 50 µl drop of pan-cytokeratin (Pan-CK) (cat. no. C2931; Sigma aldrich) (1:200) was placed on the sections. The sections were transferred to a wet box and placed in a refrigerator at 4 °C overnight. The next day, sections were brought to room temperature and incubated with fluorophore-conjugated secondary antibody (594 donkey anti-mouse IgG; 1:400) (cat. no. abs20017. Absin) for 1 h protected from light. After washing, nuclei were counterstained with DAPI (1:100) (cat. no. abs47047616. Absin) for 10 min. Finally, the sections were sealed and stored in a wet box at 4 °C.

5-Ethynyl-2’-deoxyuridine (EdU) proliferation assay

Cells (1,000 cells/well) were added to the matrix gel, spread evenly in a 96-well plate and incubated in organoid medium. Cells were incubated with EdU (100 µl per well; diluted 1:1,000 in complete medium) for 2 h. The EdU proliferation assay (cat. no. C10310-1; Guangzhou RiboBi) was performed according to the manufacturer's protocol of the kit.

Statistical analysis

Statistical analysis was performed using the SPSS software package (Windows version 19.0; IBM Corp.) and Prism 8 (GraphPad; Dotmatics). All measurement data are presented as the mean ± standard deviation of at least three independent experiments and were statistically analyzed using Student's t-test (two-tailed) or ANOVA. Enumeration data were analyzed using Fisher's exact test. P < 0.05 was considered to indicate a statistically significant difference.