Ethics statement

The acquisition and use of human specimens were in concordance with the International Ethical Guidelines for Research Involving Human Subjects, as stated in the Helsinki Declaration. Our studies were approved by the Medical Ethics Committee of Guangzhou Women and Children’s Medical Center (No.: 202019001). The EC tissue samples were collected after obtaining signed informed consent from the participants. The use of EC tissue microarray (TMA) (Cat. HUteA060CS01) was approved by the Ethics Committee of Shanghai Outdo Biotech Company (No.: SHYJS-CP-1504005). The specific information of the patients involved in this study was presented in Supplementary Table 1. The experiments involving animals were conducted according to the ethical policies and procedures approved by the Guangzhou Women and Children’s Medical Center Animal Welfare and Ethics Committee (KTDW-2024-00480).

Cell culture

Two human EC cell lines (AN3CA and Ishikawa) were obtained from Procell Life Science & Technology (Wuhan, China). The cell passage number was no more than 20, and the cells were tested and confirmed to be free of Mycoplasma. AN3CA cells were cultured in Minimum Essential Medium (Gibco, Grand Island, NY, USA) plus 10% fetal bovine serum (FBS) (Gibco). Ishikawa cells were cultured in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% FBS. All cells were cultured at 37 °C with 5% CO2.

Reagents

All antibodies used in this study were purchased from Proteintech Group, Inc (Rosemont, USA). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat. #60004-1-Ig) and beta-actin (ACTB) (Cat. #66009-1-Ig) were used at a 1:20,000 dilution for western blotting. Antibodies against c-Myc (Cat. #67447-1-Ig), G1/S-specific cyclin-D1 (Cyclin D1) (Cat. #26939-1-AP), ribosomal protein S6 (S6) (Cat. #66886-1-Ig), phospho-S6 ribosomal protein (Ser235/236) (Cat. #80130-2-RR), sequestosome 1 (P62) (Cat. #18420-1-AP), GTPase KRas (KRAS) (Cat. #12063-1-AP) and RAC-alpha serine/threonine-protein kinase (AKT) (Cat. #10176-2-AP) were used at a 1:5000 dilution for western blotting. Antibodies against cyclin-dependent kinase inhibitor 1B (P27) (Cat. #25614-1-AP), 3-Phosphoinositide Dependent Protein Kinase 1 (PDPK1) (Cat. #17086-1-AP), microtubule-associated protein 1 light chain 3 beta (LC3) (Cat. #14600-1-AP), HRAS (Cat. #18295-1-AP), and phospho-AKT (Ser473) (Cat. #66444-1-Ig) were used at a 1:1000 dilution for western blotting. The anti-c-Myc antibody (Cat. #67447-1-Ig), the anti-CHKA antibody (Cat. #13520-1-AP), the anti-phospho-mTOR (Ser2448) antibody (Cat. #67778-1-Ig), and the anti-Ki67 antibody (Cat. #27309-1-AP) were used at a 1:500 dilution for immunohistochemistry (IHC). The anti-BrdU antibody (Cat. #66241-1-Ig), the anti-phospho-mTOR (Ser2448) antibody (Cat. #67778-1-Ig), and the anti-c-Myc antibody (Cat. #10828-1-AP) were used at a 1:200 dilution for immunofluorescence (IF). Additionally, the anti-CD44 antigen antibody (Cat. #FITC-65117) was used at a 1:200 dilution for flow cytometry. Chemical compounds including PC (Cat. #HY-B2233B), BrdU (Cat. #HY-15910), N-Acetylcysteine (NAC) (Cat. #HY-B0215), cisplatin (DDP) (Cat. #HY-17394), paclitaxel (PTX) (Cat. #HY-B0015), and MHY1485 (Cat. #HY-B0795) were purchased from MedChemExpression (Shanghai, China). Recombinant human c-Myc protein (Cat. #Ab169901) was purchased from Abcam (Cambridge, UK).

Cell viability assay

EC cells were seeded into 96-well plates (5000 cells per well) and allowed to adhere overnight. To evaluate the inhibitory effect of PC accumulation on the proliferation ability of EC cells, the two EC cell lines were treated with gradient concentrations of PC (0, 10, 20, 40, 60, 80, and 100 mM) for 48 h or incubated in 0 mM, 20 mM and 40 mM PC for 5 days. To verify that PC accumulation inhibits EC cell viability by downregulating the mTOR-c-Myc signaling axis, AN3CA cells were incubated in PC (0, 20, 30, and 40 mM) with or without exogenous c-Myc protein (5 µg/ml) supplementation for 48 h, and Ishikawa cells were incubated in PC (0, 40, 50, and 60 mM) with or without exogenous MYC protein supplementation for 48 h. Moreover, the EC cell lines were incubated with PC (40 mM) in the presence or absence of MHY1485 (10 μM) for 48 h. To evaluate the effect of PC accumulation on the chemosensitivity of EC cells, cells were treated with vehicle, PC (20 mM), DDP (1 μg/mL), PTX (2 nM), PC (20 mM) combined with DDP (1 μg/mL), and PC (20 mM) combined with PTX (2 nM) for 48 h. To evaluate the effect of PC-mediated ROS production on EC cell viability, AN3CA cells were incubated with PC (40 mM) for 48 h, following a 6-h preincubation with NAC (1 mM). Physiological saline was used as a vehicle control. Cell viability and cell growth curves were determined using the Cell Counting Kit-8 (CCK-8) (Cat. #KGA317; KeyGen, Nanjing, China) assay, and the results are presented as background-subtracted relative light units, normalised to the vehicle control. Statistical analysis (mean ± SD) was performed using triplicate samples.

Colony formation assay

EC cells were seeded into 6-well plates at a density of 1 × 104 cells per well. To assess the inhibitory effect of PC on EC cell proliferation, the two EC cell lines were treated with 20 mM and 40 mM PC for 5 days, with physiological saline serving as the control. To examine the rescue effect of the mTOR agonist MHY1485, EC cells were treated with physiological saline, 40 mM PC, 10 μM MHY1485, or a combination of 40 mM PC and 10 μM MHY1485 for 5 days. After that, the cells were fixed with 100% methanol for 20 min and stained with 0.1% crystal violet for 20 min. Cell colonies were counted using ImageJ software (NIH, USA) and were statistically analyzed using GraphPad Prism version 6.0. Three independent experiments were performed.

Tumorsphere assay

Briefly, 1 × 104 EC cells were resuspended and plated on the ultra-low attachment six-well plates. The tumorspheres were cultured in Dulbecco's Modified Eagle Medium (DMEM)/F-12 (Gibco) with epidermal growth factor, insulin, hydrocortisone, and B27 supplement. EC cells were incubated in PC (0, 20, and 40 mM) for 48 h, and tumorspheres with a diameter > 100 μm were counted.

Western blotting

To evaluate the effect of PC accumulation in EC cells on c-Myc protein, cells were treated with 20 mM PC for 48 h, washed with phosphate-buffered saline (PBS), and subjected to centrifugation for cell pellet collection. To evaluate changes in the cell cycle induced by PC accumulation, AN3CA cells were treated with 20 mM PC and Ishikawa cells were treated with 40 mM PC, and the cell pellet was collected after 24 and 48 h of treatment. To evaluate the effect of PC accumulation on the HRAS–mTOR axis in EC cells, the two EC cell lines were treated with 40 mM PC for 48 h, washed with PBS, and subjected to centrifugation for cell pellet collection. To evaluate the effect of PC-mediated ROS production on c-Myc protein, AN3CA cells were incubated with PC (20 mM) for 48 h, following a 6-h preincubation with NAC (1 mM), washed with PBS, and subjected to centrifugation for cell pellet collection. All collected cell pellets were lysed with RIPA lysis buffer (Cat. #KGP703, KeyGen) according to the manufacturer’s instructions. Protein concentration was determined with a BCA kit (Cat. #KGP902, KeyGen). The next steps in the experimental process were carried out as previously described [22]. Briefly, samples were loaded on SDS-PAGE gel and then transferred onto a 0.2 µM polyvinylidene difluoride membrane (Cat. #ISEQ00010, Millipore, Bedford, MA, USA). The membrane was blocked and then incubated with a primary antibody at 4 °C overnight. Subsequently, the membranes underwent incubation with appropriate secondary antibodies for 1 h at room temperature. Protein bands were detected using the enhanced chemiluminescent reagents (Cat. #P10060, NCM Biotech, Nanjing, China). The normalized densitometry data were determined using ImageJ software. Each western blotting image is representative of three independently repeated results.

Flow cytometry

Flow cytometry was used to detect the cell cycle, apoptosis rate, ROS level and CD44 protein expression. For cell cycle analysis, EC cells were incubated with 20 mM PC for 24 h, and then cells were stained with a cell cycle staining kit (Cat. #CCS012, MultiScience, Shanghai, China) according to the manufacturer's instructions. For CD44 detection, AN3CA and Ishikawa cells were treated with 20 mM PC for 48 h. For apoptosis analysis, AN3CA cells were treated with 20 mM PC for 24 h or 48 h and Ishikawa cells were treated with 40 mM PC for 24 h or 48 h, and then cells were stained with Annexin V-FITC reagent (Cat. #KGF001, KeyGen) according to the manufacturer's instructions. For ROS detection, both two EC cell lines were treated with 20 mM PC for 0, 24, and 48 h. ROS detection kit (Cat. #S0033S, Beyotime, Shanghai, China) was used to evaluate ROS levels according to the manufacturer's protocol. Cell cycle distribution was measured using ModFit LT 3.1 software (Verity Software House, USA), and the remaining flow cytometry results were analyzed using FlowJo software (Tree Star Inc., USA).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay

To evaluate the effect of PC accumulation in EC cells on MYC gene expression, cells were treated with 20 mM PC for 48 h, washed with phosphate-buffered saline (PBS), and subjected to centrifugation for cell pellet collection. To evaluate the changes in cancer cell stemness-related genes expression induced by PC accumulation, EC cells were treated with 40 mM PC for 24 h, washed with phosphate-buffered saline (PBS), and subjected to centrifugation for cell pellet collection. Total RNA was isolated from cells using the AG RNAex Pro RNA reagent (Cat. #AG21101, Accurate Biotechnology, ChangSha, China). The cDNA synthesis was performed following the manufacturer's protocol using the Evo M-MLV RT Kit with gDNA Clean for qPCR (Cat. #AG11705, Accurate Biotechnology). The resulting cDNA was then amplified using SYBR Green Premix Pro Taq HS qPCR (Cat. #AG11701, Accurate Biotechnology) on the Biosystems 7500 Real-time PCR system. Real-time PCR was performed with the following primers: MYC-fw 5′-CGTCCTCGGATTCTCTGCTC-3′, MYC-rev 5′-CTCATCTTCTTGTTCCTCCTCAGA-3′; GAPDH-fw 5′-TCTGACTTCAACAGCGACAC-3′, GAPDH-rev 5′-CGTTGTCATACCAGGAAATGAG-3′. ALDHA1A-fw 5′-GCACGCCAGACTTACCTGTC-3′, ALDH1A1-rev 5′-CCTCCTCAGTTGCAGGATTAAAG −3′; NANOG-fw 5′-CAATGGTGTGACGCAGAAGG-3′, NANOG-rev 5′-GAAGGTTCCCAGTCGGGTTC-3′; SOX2-fw 5′-ATGGGTTCGGTGGTCAAGT-3′, SOX2-rev 5′-ATGTGTGAGAGGGGCAG-3′; EPCAM-fw 5′-CTGGCCGTAAACTGCTTTGT-3′, EPCAM-rev 5′-AGCCCATCATTGTTCTGGAG-3′. The gene expression levels were normalized to GAPDH gene expression levels.

RNA-sequencing (RNA-seq)

EC cells were either incubated with physiological saline (control) or 40 mM PC for 24 h and then subjected to total RNA extraction for RNA-seq analysis. A minimum of 5 μg total RNA was used for RNA-seq. Novogene (Beijing, China) conducted RNA sequencing. Three biological replicates were used for each group. Gene Set Enrichment Analysis (GSEA) was performed for gene functional annotation [23]. We used the DESeq2 [24] for differential gene analysis and a difference of |log2 fold change|≥ 1 and adjusted P value ≤ 0.05 was defined as differential expression. Compared with the control group, the differentially expressed genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (https://www.kegg.jp/), and protein–protein interaction (PPI) analysis. The RNA sequencing results were shown in Supplementary Table 2.

IHC staining

After deparaffinization with xylene and rehydration with decreasing concentrations of ethanol, tissues or organoids sections were boiled in 10 mM, pH 6.0 sodium citrate buffer for antigen retrieval, followed by incubated with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Subsequently, sections were incubated with primary antibodies for 2 h at 37 °C, followed by incubation with secondary antibodies for 30 min at room temperature. After treatment with diaminebenzidine (Cat. #ZLI-9017, ZSGB-BIO, Beijing, China), sections were counterstained with hematoxylin for 1 min, differentiated with 1% hydrochloric acid ethanol for 5 s, dehydrated with increasing concentrations of ethanol, made transparent with xylene, and sealed with neutral gum. Published standards were referred to evaluate c-Myc and CHKA IHC scores [25] and the IHC score less than 5 is considered “low” expression.

Hematoxylin–eosin (H&E) staining

After deparaffinization with xylene and rehydration with decreasing concentrations of ethanol, organoid sections were stained with hematoxylin for 5 min. After washing away residual hematoxylin with flowing water, the sections were differentiated with 1% hydrochloric acid ethanol for 5 s and then stained with eosin for 5 min. Finally, the sections were dehydrated with increasing concentrations of ethanol, made transparent with xylene, and sealed with neutral gum.

Animal experiments

Female BALB/c nude mice (3–4 weeks of age) were purchased from the Guangdong Medical Laboratory Animal Center. Mice were housed in autoclaved, ventilated cages and provided with autoclaved water. They were subcutaneously injected with 5 × 106 AN3CA cells to establish the EC cell line-derived xenograft model. When tumors reached an average volume of 100 mm3, the mice were randomly divided into two groups (n = 6/group): (1) the experimental group, which received intratumoral injections of 80 mM PC every 2 days; and (2) the control group, which received intratumoral injections of equal volume of physiological saline. PC accumulation treatment was terminated after six intratumoral injections. After anesthetizing the mice, cervical dislocation was performed and subcutaneous tumors were removed. Tumor growth was monitored using callipers, and tumor volume was calculated according to the following formula: length × width2/2. Images of the subcutaneous tumors from both groups were taken, and tumors were weighted. The maximal tumor size/burden permitted by our institutional review board is 10% of body weight and mean tumor diameter = or > 15 mm in adult mice (~ 25 g). The maximal tumor size/burden permitted by our institutional review board was not exceeded.

Construction and culture of patient-derived organoid (PDO) models

The fresh EC tumor tissue samples were obtained from patients diagnosed with EC who had not received preoperative chemotherapeutics before surgery from June 2023 to February 2024 at Women's and Children's Medical Center, Guangzhou, China. The experiment was performed as previously described [26]. The fresh EC tumor tissues were obtained during surgery and washed twice with PBS containing 1% penicillin streptomycin. Then, the fresh EC tumor tissues were cut into small pieces of 1–2 mm3 volume. The tissue fragments were digested with type IV collagenase (Cat. #17104019, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 1 h. The above mixture was filtered through a 100-μm filter (Cat. #352360, Corning, New York, USA), and the filtrate was centrifuged at 300×g for 5 min. The cell precipitate was resuspended in the specific organoid culture medium (Cat. #OGCP-03–62, Guangzhou Orgen biotechnology Co., Ltd., Guangzhou, China) mixed with growth factor-reduced Matrigel (Cat. #CB-40230, Corning, USA). The cell concentration was 10,000 to 20,000 cells/50 μl. EC PDOs were transferred to 24-well plates when the Matrigel solidified. EC PDOs were incubated at 37 °C with 5% CO2 and the specific organoid culture medium were changed every 3 days. The cell viability of EC PDOs treated with gradient concentrations PC (0, 20, 40, 60, 80, and 100 mM) for 96 h was determined by the Cell Counting-Lite 3D Luminescent Cell Viability Assay (Cat. #DD1102-01, Vazyme, Nanjing, China).

IF staining

To determine the effect of PC accumulation on phospho-mTOR, AN3CA cells were treated with 20 mM PC for 48 h while Ishikawa cells were treated with 40 mM PC for 48 h. To evaluate the effect of PC-mediated ROS production on phospho-mTOR, AN3CA cells were incubated with PC (20 mM) for 48 h, following a 6-h preincubation with NAC (1 mM). Appropriately treated EC cells were fixed with 4% paraformaldehyde for 20 min, and then permeabilized with 0.1% Triton X-100 for 20 min after washing three times with PBS, and blocked with 5% bovine serum albumin for 30 min at room temperature after three times washing with PBS. Then, cells were incubated with primary antibodies overnight at 4 °C. After washing three times with PBS, the corresponding fluorescent secondary antibody was added and incubated at room temperature for 1 h at room temperature in the dark, followed by staining with 4ʹ,6-diamidino-2-phenylindole (DAPI) (Cat. #D-9106, Bioss, Beijing, China) for 5 min. Stained cells were imaged by fluorescence microscope. After deparaffinization and hydration, the EC organoid model sections were boiled in 10 mM, pH 6.0 sodium citrate buffer for antigen retrieval. After blocking with 10% goat serum for 1 h at room temperature, organoid sections were incubated with primary antibodies overnight at 4 °C. After washing three times with PBS, the corresponding fluorescent secondary antibodies were added and incubated for 1 h at room temperature. After washing three times with PBS, DAPI was added and incubated in the dark for 5 min. The EC organoid model sections were imaged by fluorescence microscopy.

Bioinformatics analysis and Statistical analysis

The gene expression levels of MYC, CHKA, and PDPK1 in The Cancer Genome Atlas database (TCGA) were obtained from the UCSC Xena project [27]. The Log-rank test was used to access the significance of survival data. The Kruskal–Wallis test was used to determine the significant differences in MYC gene expression among patients with different EC stages or grades. Pearson’s correlation and Spearman’s correlation were used to determine the correlation between the gene expression levels of MYC, CHKA, and PDPK1. The survival analysis based on MYC expression in patients with EC was implemented using the Kaplan-Meier plotter database [28]. Student's t-test was used for comparison between the two groups and the Kolmogorov–Smirnov test was applied to ascertain the normality of the data for the application of Student's t-test. The significance of three or more groups was compared by one-way ANOVA, followed by Dunnett's post hoc test. Comparisons among cell growth curves were conducted using two-way ANOVA, followed by Dunnett's post hoc test. Comparisons between tumor volume growth curves were conducted using two-way ANOVA, followed by Sidak's post hoc analysis. The Wilcoxon matched-pairs signed rank test was used to analyse c-Myc IHC score data. Except for IHC score result, which was presented as median with interquartile range, and MYC gene expression levels among patients with different EC stages or grades, which were presented as min to max, the other data were presented as mean ± standard deviation (SD). All data in this study were statistically analyzed using GraphPad Prism Version 6.0. P values < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001) were considered significant, otherwise non-significant (ns).