Sch B (cat. no. B21327-1g) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. Sch B was prepared as a 125 mM stock solution in 100% DMSO [the final concentration of DMSO in culture medium did not exceed 0.1% (v/v)] and stored in the dark at −20°C in our laboratory. Ferrostatin-1 (Fer-1; cat. no. HY-100579), deferoxamine mesylate (DFO; cat. no. HY-B1625), Z-VAD-FMK (cat. no. HY-16658B), N-acetylcysteine (NAC; cat. no. HY-B0215) and necrostatin-1 (NEC-1; cat. no. HY-15760) were purchased from MedChemExpress. Anti-acyl-CoA synthetase long-chain family member 4 (ACSL4; cat. no. 22401-1-AP), anti-GSH synthetase (GSS; cat. no. 67598-1-Ig) anti-solute carrier family 3 member 2 (SLC3A2; cat. no. 15193-1-AP), anti-transferrin (TF; cat. no. 17435-1-AP), and anti-α-tubulin (cat. no. 66031-1-Ig) were purchased from Proteintech Group, Inc. Anti-glutathione (GSH) peroxidase 4 (GPX4; cat. no. ab125066), anti-γ-glutamyl-cysteine ligase catalytic subunit (GCLC; cat. no. ab207777), anti-solute carrier family 7 member 11 (SLC7A11; cat. no. ab307601) and anti-Ki67 (cat. no. ab16667) were purchased from Abcam.

Human pancreatic ductal adenocarcinoma cell lines MIA PaCa-2 (cat. no. CRL-1420), PANC-1 (cat. no. CRL-1469) and PL45 (cat. no. CRL-2558) were purchased from American Type Culture Collection. These cell lines were cultured in DMEM (cat. no. C11995500BT; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 1% penicillin-streptomycin (PS; cat. no. 15140122; Gibco; Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum (FBS; cat. no. FSP500; Shanghai ExCell Biology, Inc.). Human pancreatic duct epithelial cells (hTERT-HPNE) were purchased from Guangzhou Jennio Biotechnology Co., Ltd. and cultured in RPMI-1640 medium (cat. no. C11875500BT; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 1% PS and 10% FBS. All cells were maintained at 37°C with 5% CO₂ in a sterile incubator. All cell lines tested negative for mycoplasma using the Mycoplasma test kit (cat. no. G1901; Wuhan Servicebio Technology Co., Ltd.).

MIA PaCa-2, PANC-1, PL45 and hTERT-HPNE cells were seeded in 96-well plates at a density of 3×10³ cells/well and incubated overnight. The cells were then treated with Sch B at concentrations of 0, 7.8, 15.6, 31.3, 62.5 or 125 µM for 24, 48 or 72 h at 37°C. The control groups received an equivalent concentration of DMSO (≤0.1%). For inhibitor studies, the cells were pre-treated with Fer-1 (10 µM), DFO (20 µM), Z-VAD-FMK (50 µM), NEC-1 (50 µM) or NAC (2 mM) for 4 h at 37°C, followed by treatment with Sch B (20 µM) for 24 h at 37°C. Subsequently, 10 µl Cell Counting Kit-8 (CCK-8) reagent (cat. no. CK04; Dojindo Laboratories, Inc.) dissolved in 100 µl fresh medium was added to each well and incubated for 2 h. Absorbance was measured at 450 nm using a microplate reader (Tecan Group, Ltd.).

MIA PaCa-2 and PANC-1 cells were seeded in 6-well plates at a density of 1×10³ cells/well and incubated overnight. The cells were then treated with Sch B (5, 10 or 20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. Subsequently, the cells were cultured for 10 days, with the medium changed every 3 days. Colonies were then fixed in 4% paraformaldehyde for 10 min at room temperature and stained with 0.1% crystal violet for 15 min at room temperature, after which, images were captured under an inverted light microscope (Zeiss AG). A colony was defined as a cluster of ≥50 cells and the number of colonies was semi-quantified using ImageJ version 1.4.3 software (National Institute of Health).

MIA PaCa-2 and PANC-1 cells were seeded in 8-well chamber slides at a density of 9×10³ cells/well and were incubated overnight. The cells were then treated with Sch B (5, 10 or 20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. For inhibitor experiments, the cells were pre-treated with Fer-1 (10 µM) for 4 h followed by treatment with Sch B (20 µM) for 24 h at 37°C. Subsequently, 500 µl EdU working solution (cat. no. C10310; Guangzhou RiboBio Co., Ltd.) was added to each well and incubated for 2 h at 37°C. The cells were then permeabilized with 0.5% Triton X-100 for 15 min at room temperature and were stained with Apollo 567 for 30 min at room temperature. After washing, the slides were mounted with DAPI-containing Fluoroshield (cat. no. ab104139; Abcam). Images of EdU-positive cells were captured using a confocal microscope (Zeiss AG).

Cell death was measured using the LDH cytotoxicity assay kit (cat. no. C0017; Beyotime Biotechnology). MIA PaCa-2 and PANC-1 cells were seeded in 35-mm dishes at a density of 9×10⁴ cells/dish and incubated overnight. The cells were then treated with Sch B (5, 10 or 20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. For inhibitor experiments, the cells were pre-treated with Fer-1 (10 µM) for 4 h followed by treatment with Sch B (20 µM) for 24 h at 37°C. After treatment, the culture medium was collected and centrifuged at 800 × g for 5 min at 4°C to remove cell debris. Subsequently, 120 µl cell culture supernatants were transferred to a new 96-well plate and mixed with 60 µl LDH working solution. After incubation for 30 min at 37°C in the dark, the absorbance was measured at 490 nm using a microplate reader. The percentage of cell death was calculated as follows: (treated cell LDH-control cell LDH)/(maximum LDH release-control cell LDH) ×100.

The Trypan blue assay (cat. no. C0040; Beijing Solarbio Science & Technology Co., Ltd.) was performed to confirm the results of the LDH assay. MIA PaCa-2 and PANC-1 cells were seeded in 35-mm dishes at a density of 9×10⁴ cells/dish and incubated overnight. The cells were then treated with Sch B (5, 10 or 20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. Subsequently, the cells were stained with 0.04% Trypan blue solution for 3 min at room temperature. The unstained (viable) and blue-stained (dead) cells were counted using an automatic cell counter (Guangzhou BodBoge Technology Co., Ltd.) and the percentage of dead cells was calculated.

Intracellular Fe²⁺ levels were measured using the FerroOrange fluorescent probe (cat. no. F374; Dojindo Laboratories, Inc.). MIA PaCa-2 and PANC-1 cells were seeded in 8-well chamber slides at a density of 9×10³ cells/well and incubated overnight. The cells were then treated with Sch B (5, 10 or 20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. For inhibitor experiments, the cells were pre-treated with Fer-1 (10 µM) for 4 h followed by treatment with Sch B (20 µM) for 24 h at 37°C. Subsequently, the cells were stained with 1 µM FerroOrange in serum-free medium for 30 min at 37°C in the dark. Fluorescence intensity was acquired using a confocal microscope.

MIA PaCa-2 and PANC-1 cells were seeded in 150-mm dishes at a density of 6×10⁵ cells/dish and incubated overnight. The cells were then treated with Sch B (20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. Subsequently, the cells were harvested and fixed in a 2.5% glutaraldehyde solution (pH 7.4) for 6 h at 4°C. After washing with 0.1 M phosphate buffer (pH 7.2), the samples were postfixed with 1% OsO₄ for 4 h at 4°C. Following dehydration, the samples were embedded in Epon-Araldite resin and polymerized at 60°C for 48 h. Semithin sections (1 µm) were stained with toluidine blue for positioning, after which, ultrathin sections (70 nm) were collected on copper grids, stained with 2% uranyl acetate for 8 min at room temperature, followed by 2.7% lead citrate for 8 min at room temperature, and examined under a transmission electron microscope (Thermo Fisher Scientific, Inc.).

The levels of GSH and MDA were measured using ELISA kits (cat. nos. S0052 and S0131S; Beyotime Biotechnology, Inc.) according to the manufacturer's protocol. MIA PaCa-2 and PANC-1 cells were seeded in 150-mm dishes at a density of 6×10⁵ cells/dish and incubated overnight. The cells were then treated with Sch B (5, 10 or 20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. For inhibitor experiments, the cells were pre-treated with Fer-1 (10 µM) for 4 h followed by treatment with Sch B (20 µM) for 24 h at 37°C. GSH and MDA levels in cell lysates were measured using a microplate reader.

Lipid peroxidation was assessed using the C11-BODIPY (581/591) assay (cat. no. L267; Dojindo Laboratories, Inc.). MIA PaCa-2 and PANC-1 cells were seeded in 8-well chamber slides at a density of 9×10³ cells/well and incubated overnight. The cells were then treated with Sch B (5, 10 or 20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. For inhibitor experiments, the cells were pre-treated with Fer-1 (10 µM) for 4 h followed by treatment with Sch B (20 µM) for 24 h at 37°C. Subsequently, the cells were incubated with 2 µM C11-BODIPY (581/591) solution for 30 min at 37°C. Fluorescence intensity was measured using a confocal microscope.

PANC-1 cells were treated with Sch B (20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C and then lysed using M-PER mammalian protein extraction reagent (cat. no. 78505; Thermo Fisher Scientific, Inc.) with protease inhibitor cocktail (cat. no. HY-K0010; MedChemExpress) on ice for 10 min. The lysates were centrifuged at 12,000 × g for 20 min at 4°C, after which, the supernatants were collected and mixed with 10X TNC buffer (500 mM Tris-HCl, pH 8.0; 500 mM CaCl₂; 100 mM NaCl). Protein concentration was then quantified using a BCA assay. The lysates (4 µg/µl) in 1X TNC buffer were digested with pronase (0.25 µg/µl; cat. no. 63163824; Roche) at a pronase/protein ratio of 1:200 for 30 min at room temperature and the reaction was terminated by adding 5X SDS-PAGE loading buffer. Subsequently, the proteins were separated by SDS-PAGE and visualized by Coomassie staining; specific protein bands were excised for label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.

Nanoflow LC-MS/MS analysis of peptides was performed using a quadrupole Orbitrap Exploris^™ 480 Mass Spectrometer (Thermo Fisher Scientific, Inc.) equipped with an EASY nLC 1200 ultra-high pressure system (Thermo Fisher Scientific, Inc.) via a nano-electrospray ion source. Briefly, peptides (500 ng) were loaded on a 25-cm column (150 µm inner diameter, packed with ReproSil-Pur C18-AQ 1.9-µm silica beads; Guangzhou Promegene Biotechnology Co., Ltd.). The peptides were eluted over a 60-min gradient at a flow rate of 600 nl/min using buffer B (80% acetonitrile, 0.1% formic acid) as follows: 8–12% B for 5 min, 12–30% B for 30 min, 30–40% B for 9 min, 40–95% B for 1 min, followed by incubation with 95% B for 15 min.

The mass spectrometer was operated in positive ion mode with the FAIMS Pro^™ Interface (Thermo Fisher Scientific, Inc.) cycling between compensation voltages of-45 V and −65 V every 1 sec. Full-scan MS spectra (350–1,200 m/z, 120,000 resolution) were acquired in the Orbitrap analyzer with an automatic gain control (AGC) target of 300%, and a maximum injection time of 50 msec. The most intense ions from the full scan were isolated with an isolation width of 1.6 m/z and fragmented using higher-energy collisional dissociation with a normalized collision energy of 33%. MS/MS spectra were collected in the Orbitrap analyzer (15,000 resolution) with an AGC target of 75% and a maximum injection time of 22 msec.

The resulting MS/MS data were processed using Proteome Discoverer version 2.4 (Thermo Fisher Scientific, Inc.). Tandem mass spectra were searched against the UniProt human protein database (https://www.uniprot.org/taxonomy/9606). Trypsin/P was specified as a cleavage enzyme allowing up to two missing cleavages. The precursor mass tolerance was ±15 ppm, and fragment mass tolerance was ±0.02 Da. Carbamidomethyl on cysteine was defined as a fixed modification, and acetylation at the protein N-terminus and oxidation on methionine were specified as variable modifications. The peptide ion score threshold was set to >20 and high confidence in peptides was required. Search outcomes underwent filtering via a linear discriminant algorithm to achieve an estimated false discovery rate of 1%. Candidate proteins were selected based on the following criteria: i) Intensity ratio (compound/control) ≥2.0, ii) a molecular weight of ~70 kDa (matching the excised band) and iii) protein score ≥10.

The protein structure of ACSL4 was initially predicted using AlphaFold3 (https://github.com/deepmind/alphafold) and was then processed with AutoDockTools 1.5.6 (34) to preserve its original charges and generate a pdbqt file for docking. The 2D structure of Sch B was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/compound/108130). Structural optimization was performed using the MOPAC program (https://openmopac.net/) and PM3 atomic charges were calculated. The structure of Sch B was also prepared using AutoDock Tools 1.5.6 to create a pdbqt file. Docking simulations were carried out using AutoDock 4.2.6 (34). A grid box of 100×100×100 points was defined with the center at x=71.637, y=70.446, z=104.536. The grid spacing was 0.375 Å and the exhaustiveness was set to 50. Other docking parameters were set to default and are therefore not listed.

PANC-1 cells were seeded in 150-mm dishes at a density of 6×10⁵ cells/dish. The cells were then treated with Sch B (20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. Total RNA was extracted using RNeasy plus Mini Kit (cat. no. 74134; Qiagen, Inc.) according to the manufacturer's protocol. RNA quality and RNA integrity number (>8.0) were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) with the Agilent RNA 6000 Nano Kit (cat. no. 5067-1511; Agilent Technologies, Inc.). RNA-seq libraries were constructed using the NEBNext^® Ultra^™ RNA Library Prep Kit (cat. no. E7770; New England BioLabs, Inc.). The libraries were then sequenced on an Illumina NovaSeq 6000 System platform (Illumina, Inc.) with 150 bp paired-end reads. Prior to sequencing, the RNA concentration of the library was determined using a Qubit^®2.0 fluorometer (Thermo Fisher Scientific, Inc.) with a Qubit RNA Assay Kit (cat. no. Q32852; Thermo Fisher Scientific, Inc.) and was then diluted to 4 nM. Insert size was assessed using the Agilent Bioanalyzer 2100 system, and qualified insert size was accurately quantified using StepOnePlus^™ Real-Time PCR System (Thermo Fisher Scientific, Inc.; library valid concentration >10 nM). mRNA libraries were sequenced on the Illumina sequencing platform by Guangzhou Promegene Biotechnology Co., Ltd.

For data analysis, differential expression analysis was performed using DESeq2 package v1.20.0 (https://bioconductor.org/packages/DESeq2) and the heatmap was generated using the cluster package v2.0.3 (https://cran.r-project.org/web/packages/cluster/) in R v3.6.2 (https://www.r-project.org/). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed using the R software clusterProfiler package v3.12.0 (https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html). The raw sequencing data have been deposited into a publicly available database.

MIA PaCa-2 and PANC-1 cells were seeded in 150-mm dishes at a density of 6×10⁵ cells/dish and incubated overnight. The cells were then treated with Sch B (5, 10 or 20 µM) or vehicle control (<0.1% DMSO) for 24 h at 37°C. For inhibitor experiments, the cells were pre-treated with Fer-1 (10 µM) for 4 h followed by treatment with Sch B (20 µM) for 24 h at 37°C. Total cellular protein was extracted using RIPA lysis buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) on ice for 30 min and quantified with a BCA assay kit (cat. no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.). The lysates (30 µg) were then separated by SDS-PAGE on 4–15% gels, followed by transfer of proteins to PVDF membranes. After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies overnight at 4°C: Anti-ACSL4 (1:5,000), anti-GSS (1:5,000), anti-SLC3A2 (1:5,000), anti-TF (1:1,000), anti-α-tubulin (1:20,000), anti-GPX4 (1:1,000), anti-SLC7A11 (1:1,000) and anti-GCLC (1:1,000). The membranes were then washed three times with Tris-buffered saline containing 0.1% Tween-20 and were subsequently incubated with HRP-conjugated secondary antibodies (1:10,000; cat. nos. 31430 and 31460; Thermo Fisher Scientific, Inc.) for 2 h at room temperature. Protein bands were visualized using an ECL reagent (cat. no. P1010-500; Applygen Technologies, Inc.). Protein band intensities were semi-quantified using ImageJ version 1.4.3 software and normalized to the corresponding α-tubulin loading control.

MIA PaCa-2 and PANC-1 cells were seeded in 6-well plates at a density 2×10⁶ cells/well and incubated overnight. When the cells reached 50–60% confluence, small interfering (si) RNAs (150 pmol) were transiently introduced into the cells for 6 h at 37°C using Lipofectamine^® RNAiMAX reagent (cat. no. 13778150; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 48 h post-transfection, the cells were harvested for WB to verify ACSL4 knockdown efficiency. Cell viability assay, EdU assay, LDH assay and lipid peroxidation assay were carried out as functional experiments. The target and negative control siRNAs were procured from Shanghai GenePharma Co., Ltd. The sequences were as follows: ACSL4 siRNA, 5′-GGGUUGAUAUCUGCAAUAATT-3′ (sense), 5′-UUAUUGCAGAUAUCAACCCTT-3′ (antisense). Negative control siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense), 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense).

Male Balb/c-nude mice (age, 4–5 weeks; weight, 18.5–22.8 g) were purchased from GemPharmatech Co., Ltd. All mice were housed under specific pathogen-free conditions at the Laboratory Animal Center of Qingyuan People's Hospital, The Sixth Affiliated Hospital of Guangzhou Medical University (Qingyuan, China). The housing conditions were as follows: Temperature, 20–26°C; humidity, 40–70%; 12-h light/dark cycle; standard rodent chow diet and autoclaved water provided ad libitum. Before the experiment, the mice were acclimated for 1 week. All animal procedures were ethically reviewed and approved by the Ethics Committee on Laboratory Animal Care of The Affiliated Qingyuan Hospital (Qingyuan People's Hospital) of Guangzhou Medical University (approval no. LAEC-2023-027). The study was performed in strict compliance with the UK Animals (Scientific Procedures) Act 1986 (https://www.legislation.gov.uk/ukpga/1986/14/contents) and its associated guidelines. PANC-1 cells (5×10⁶ cells in 100 µl saline) were injected subcutaneously into the right flank of the mice. Mice were monitored by visual observation and palpation until tumors were detected. On day 10 post-injection, the mice were randomly divided into three treatment groups (n=5 mice/group): Model group (20% SBE-β-CD), a low-dose Sch B group (50 mg/kg) and a high-dose Sch B group (100 mg/kg). Mice received daily oral gavage of Sch B or vehicle for 6 weeks. The body weight and tumor volume were recorded weekly. Tumor volume was calculated using the formula: V=1/2 × (longest axis) × (shortest axis)².

At the end of the study, mice were euthanized by CO₂ exposure at an air displacement rate of 30%/min following anesthesia with isoflurane (2.5% for induction and 1.5% for maintenance in oxygen) to ensure humane conditions. Death was confirmed by cessation of heartbeat and respiration, absence of reflexes and limb paralysis. Humane endpoint criteria followed the Laboratory Animal Guidelines for euthanasia in China (GB/T 39760-2021; http://std.samr.gov.cn/gb/search/gbDetailed?id=BD89DE8E080A3D08E05397BE0A0A4FAD), including a maximum tumor diameter exceeding 15 mm or tumor volume exceeding 1,500 mm³, weight loss exceeding 20% of initial body weight, and an inability to access food or water, as well as difficulties in movement or respiration. No animals required euthanasia or died before the end of the study.

LSL-Kras^G12D/+ (3 male mice; age, 8 weeks; weight, 24.3–26.7 g), Pdx1-Cre (3 male mice; age, 10 weeks; weight, 24.6–25.8 g) and Tp53^flox/flox (2 male mice and 2 female mice; age 6 weeks; weight, 21.6–24.5 g) strains on a C57BL/6J background were obtained from Shanghai Model Organisms Center, Inc. The housing conditions were as follows: Temperature, 20–26°C; humidity, 40–70%; 12-h light/dark cycle; standard rodent chow diet and autoclaved water provided ad libitum. Before the experiment, the mice were acclimated for 1 week. To generate KPC mice, LSL-Kras^G12D/+ mice were first crossed with Trp53^flox/flox mice to generate LSL-Kras^G12D/+; Trp53^flox/+ offspring. These offspring were then crossed with Pdx1-Cre mice to generate KPC mice, which were genotyped by PCR. At weaning (21–28 days old), KPC mice were anesthetized with isoflurane (2.5% for induction and 1.5% for maintenance in oxygen). A 1–2 mm segment of the tail tip was excised using sterile scissors, after which, the incision was pressed against a sterile gauze pad to achieve hemostasis. For post-operative analgesia, buprenorphine hydrochloride (0.05 mg/kg, subcutaneous) was administered immediately after the procedure, and the mice were placed on a heating pad for recovery. Genomic DNA was extracted from mouse tail samples using a rapid DNA isolation kit (cat. no. NE0400; Leagene; Beijing Regen Biotechnology Co., Ltd.) according to the manufacturer's protocol. PCR was performed in a 20-µl reaction mixture containing 10 µl 2X Taq Master Mix (cat. no. P112-02; Vazyme, Inc.), 0.5 µl each primer (10 µM), 7 µl ddH₂O and 2 µl DNA template. The thermal cycling conditions comprised an initial denaturation step at 94°C for 3 min; 35 cycles of denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec and extension at 72°C for 30 sec; followed by a final extension at 72°C for 5 min. The PCR products were electrophoresed on a 1.5% agarose gel (cat. no. BY-R0100; Biowest, Inc.) with Goldview nucleic acid stain (cat. no. AG11915; Hunan Accurate Bio-Medical Technology Co., Ltd.) and visualized under UV light. The primer sequences are listed in Table SI. All animal procedures were ethically reviewed and approved by the Ethics Committee on Laboratory Animal Care of The Affiliated Qingyuan Hospital (Qingyuan People's Hospital) of Guangzhou Medical University (approval no. LAEC-2021-024). The study was performed in strict accordance with the UK Animals (Scientific Procedures) Act 1986 and its associated guidelines. KPC mice (age, 10 weeks; weight, 20.0–27.8 g; 6 male mice and 4 female mice) were randomly divided into two treatment groups (n=5 mice/group): A model group (20% SBE-β-CD) and a Sch B group (100 mg/kg). Mice received daily oral gavage of Sch B or vehicle for 8 weeks and body weight was monitored weekly. Tumor volume was calculated using the formula: V=1/2 × (longest axis) × (shortest axis)².

At the end of the study, mice were euthanized by CO₂ exposure at an air displacement rate of 30%/min following anesthesia with isoflurane (2.5% for induction and 1.5% for maintenance in oxygen) to ensure humane conditions. Death was confirmed by cessation of heartbeat and respiration, absence of reflexes and limb paralysis. Humane endpoint criteria included the emergence of hemorrhagic abdominal ascites, severe cachexia, weight loss exceeding 20% of initial body weight, and inability to access food or water, as well as difficulties in movement or respiration. Mice were observed daily for signs of inactivity and the presence of abdominal ascites. Before the end of study, two KPC mice in the model group developed hemorrhagic abdominal ascites and were euthanized based on the veterinarian's recommendation.

At the end of the study, ~200 µl blood samples were immediately collected from Balb/c-nude mice via cardiac puncture following CO₂-induced euthanasia. After clotting at room temperature for 30 min, serum was obtained by centrifuging the blood at 1,000 × g for 10 min at 4°C and was stored at −80 until further use. Serum samples were used for biochemical analysis. Six biochemical indices, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), LDH, creatinine (CREA) and uric acid (UA) (cat. nos. 105-020579, 105-020580, 105-020585, 105-00004A, 105-020587 and 105-00023A; Shenzhen Mindray Bio-Medical Electronics Co., Ltd.), were determined according to the manufacturer's protocol using an automatic biochemical analyzer (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.) to evaluate the effects of Sch B on liver, heart and kidney function.

Tumor tissues and major organs were fixed in 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and sectioned into 3-µm slices. Following deparaffinization and rehydration, the sections were stained with hematoxylin for 10 min and eosin for 10 sec at room temperature, then dehydrated and mounted for examination. For immunohistochemistry, the paraffin-embedded sections were deparaffinized and rehydrated, followed by immersion in citrate buffer (pH 6.0) at 100°C for 2.5 min for antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 min at room temperature. The sections were then permeabilized with 0.5% Triton X-100 solution for 10 min and blocked with 10% normal goat serum (cat. no. SL038; Beijing Solarbio Science & Technology Co., Ltd.) for 30 min at room temperature. Subsequently, the sections were incubated with primary antibodies against ACSL4 (1:500) and Ki67 (1:200; cat. no. ab16667; Abcam) overnight at 4°C. After rinsing, the sections were incubated with an undiluted HRP-conjugated secondary antibody (cat. no. PR30009, Proteintech Group, Inc.) for 2 h at room temperature. The reaction was visualized using DAB (cat. no. PK001; Abcarta, Inc.) for 0.5–1 min followed by counterstaining with hematoxylin for 5 min at room temperature. Finally, the sections were sealed using an automatic sealing machine (Tissue-Tek^® Film; Sakura Finetek Japan Co., Ltd.), and images were captured using a light microscope (Olympus Corporation) and semi-quantitatively analyzed with ImageJ version 1.4.3 software.

All data are presented as mean ± standard error of the mean. All in vitro experiments were performed three times and representative experiments are shown. Comparisons between two groups were performed by unpaired Student's t-test. Multiple comparisons were analyzed using one-way analysis of variance followed by Dunnett's post hoc test. Data were analyzed using GraphPad Prism 8 (Dotmatics). P<0.05 was considered to indicate a statistically significant difference.

The chemical structure of Sch B is illustrated in Fig. 1A. PC cell lines (MIA PaCa-2, PANC-1 and PL45) and a normal pancreatic epithelial cell line (hTERT-HPNE) were treated with varying concentrations of Sch B for the indicated times, and cell proliferation was assessed by CCK-8, colony formation, and EdU assays. The CCK-8 results showed that Sch B significantly inhibited the viability of all PC cell lines in a concentration- and time-dependent manner; the half maximal inhibitory concentration values at 24 h were 11.8 µM for MIA PaCa-2 cells, 11.5 µM for PANC-1 cells and 15.27 µM for PL45 cells (Fig. 1B). Notably, hTERT-HPNE cells were substantially less sensitive than the cancer cells. Because MIA PaCa-2 and PANC-1 exhibited greater sensitivity to Sch B, they were selected for all subsequent mechanistic and functional experiments. Colony formation assays (Fig. 1C) and EdU staining (Fig. 1D) confirmed these findings. To determine whether Sch B induces cell death, LDH release and Trypan blue staining assays were performed. The LDH assay indicated that Sch B significantly increased the cell death rate in a concentration-dependent manner (Fig. 1E). Similar results were obtained from the Trypan blue staining assay (Fig. 1F). Together, these findings indicated that Sch B may effectively inhibit PC cell proliferation and induce cell death.

To investigate the mechanisms underlying Sch B-induced cell death in PC cells, RNA sequencing was performed on PANC-1 cells treated with 20 µM Sch B. A total of 4,101 significant differentially expressed genes (DEGs) were identified between the Sch B group and the control group; among them, 2,525 DEGs were upregulated and 1,576 DEGs were downregulated (Fig. 2A and B). KEGG pathway enrichment analysis revealed that the upregulated DEGs were most significantly enriched in the ‘ferroptosis’ pathway (Fig. 2C). Downregulated DEGs were predominantly enriched in ‘Oxidative phosphorylation’, ‘Chemical carcinogenesis-reactive oxygen species’, ‘Alanine, aspartate and glutamate metabolism’, and ‘Glutathione metabolism’ pathways; these pathways are closely associated with ferroptosis (Fig. 2D). To further investigate the mode of cell death induced by Sch B, specific inhibitors of cell death were utilized in MIA PaCa-2 and PANC-1 cells. As shown in Fig. 2E and F, the ferroptosis inhibitors Fer-1 and DFO, the apoptosis inhibitor Z-VAD-FMK and the ROS scavenger NAC effectively reversed the inhibitory effects of Sch B on cell viability. By contrast, the necroptosis inhibitor NEC-1 did not affect Sch B-induced cell viability. These findings indicated that ferroptosis may be partially involved in Sch B-induced PC cell death.

Intracellular iron overload and lipid peroxidation of membranes are hallmark features of ferroptosis. The results of FerroOrange staining and the C11-BODIPY assay showed that Sch B dose-dependently increased the levels of Fe²⁺ (Fig. 3A) and lipid peroxidation (Fig. 3B) in both MIA PaCa-2 and PANC-1 cells. Sch B also significantly elevated MDA, a marker of lipid peroxidation, while concurrently reducing GSH levels (Fig. 3D). Morphologically, ferroptotic cells exhibit distinct ultrastructural changes in the mitochondria; therefore, subcellular changes in Sch B-treated cells were observed. Transmission electron microscopy revealed that mitochondria in the Sch B group appeared rounder and smaller, with denser membranes and fewer cristae, which is a typical feature of ferroptosis (Fig. 3C). Subsequently, the expression levels of proteins involved in iron metabolism, GSH metabolism and lipid peroxidation were assessed. The results showed that Sch B upregulated the expression levels of ACSL4 and SLC3A2, and downregulated GPX4, GCLC and GSS, whereas TF and SLC7A11 exhibited no notable changes in PC cells (Fig. 3E). Collectively, these findings indicated that Sch B may induce ferroptosis in PC cells, at least in part by increasing iron availability and promoting lipid peroxidation, while suppressing glutathione synthesis and antioxidant defenses.

To elucidate the involvement of ferroptosis in the antitumor effects of Sch B, the ferroptosis inhibitor Fer-1 was used in vitro. Fer-1 pre-treatment effectively prevented Sch B-induced cell death (Fig. 4A) and significantly diminished the inhibitory effect of Sch B on cell proliferation (Fig. 4B). Moreover, Fer-1 pre-treatment significantly decreased Sch B-induced Fe²⁺ levels (Fig. 4C), lipid peroxidation (Fig. 4D), and MDA levels (Fig. 4E), whereas it significantly increased GSH levels (Fig. 4E). WB revealed that Fer-1 pre-treatment also reversed the Sch B-induced upregulation of ACSL4 and downregulation of GPX4 (Fig. 4F). Collectively, these findings suggested that ferroptosis may be the predominant mechanism underlying Sch B-induced PC cell death.

To explore the molecular mechanism of Sch B-induced ferroptosis, DARTS and mass spectrometry analyses conducted in PANC-1 cells were. The results showed that a protein band with a molecular weight of ~70 kDa was distinctly protected by Sch B (Fig. 5A). Mass spectrometry identified 136 proteins as potential targets with significant differences (intensity ratio ≥2.0). KEGG pathway enrichment analysis revealed that the potential target proteins were enriched in the ‘ferroptosis’ pathway. Based on the combination of intensity ratio ≥2.0, molecular weight matching the protected band (~70 kDa), and protein score ≥10.0, ACSL4 was identified as a promising candidate (Fig. 5B). Molecular docking was performed between Sch B and the predicted protein structure of ACSL4; the binding energy was-6.043 kcal/mol, indicating a potential interaction (Fig. 5C). Based on these results, it was hypothesized that Sch B may promote ferroptosis by interacting with ACSL4. To test this hypothesis, ACSL4 was silenced using siRNA in PANC-1 cells before Sch B treatment (Fig. 5D). ACSL4 knockdown effectively reduced the sensitivity of PC cells to Sch B, as evidenced by restored cell viability (Fig. 5E) and proliferative capacity (Fig. 5F). Additionally, ACSL4 knockdown protected against Sch B-induced cell death (Fig. 5G) and reduced lipid peroxidation (Fig. 5H). These findings confirmed that Sch B induces ferroptosis by activating the ACSL4-regulated ferroptosis pathway in PC cells.

To assess the therapeutic efficacy of Sch B in vivo, two murine models were established: A subcutaneous xenograft tumor model and a genetically engineered orthotopic spontaneous pancreatic tumor model (KPC). In the xenograft model, Sch B treatment led to a significant dose-dependent inhibition of tumor growth; this was evidenced by reduced tumor weight and volume compared with in the model group (Fig. 6A and B). Immunohistochemical analysis showed a decrease in Ki67-positive cells and an increase in ACSL4 expression after Sch B treatment (Fig. 6C). Furthermore, the role of Sch B in cancer development and metastasis was investigated using the KPC model. Starting at 10 weeks of age, KPC mice received daily oral administration of Sch B (100 mg/kg) or vehicle. Gross pathological examination revealed a substantial pancreatic tumor burden occupying most of the pancreatic space in the model group. By contrast, mice treated with Sch B displayed significantly smaller tumors with preserved pancreatic architecture (Figs. 6D and S1). Histological analysis indicated that mice in the model group developed poorly differentiated tumors characterized by a prominent desmoplastic stroma and complete loss of normal acinar structure. Conversely, Sch B-treated mice exhibited moderately differentiated tumors with decreased stromal density and residual normal pancreatic tissue (Fig. 6E). Immunohistochemical analysis confirmed that Sch B treatment significantly reduced Ki67-positive cells and increased ACSL4 expression compared with in the model group (Fig. 6F). Notably, all model mice (5/5) developed abdominal metastases (mesenteric: 5/5; liver: 3/5; diaphragmatic: 3/5; bile duct: 2/5; peritoneal: 2/5); by contrast, only one Sch B-treated mice showed metastatic lesions (mesenteric: 1/5) (Fig. 6G). Additionally, toxicological assessments in xenograft mice indicated that Sch B treatment significantly increased relative liver weight, but had no effect on body weight or the relative weights of other organs (Fig. 7A). Histological examination revealed no observable systemic organ toxicity compared with in the model group (Fig. 7B). Serum biochemical analysis revealed no significant differences in ALT, AST, CK, LDH, CREA and UA levels between the Sch B-treated group and the model group (Fig. 7C). Overall, Sch B effectively suppressed tumor growth in vivo by promoting ferroptosis.