Cell lines and cell culture. The U2OS human osteosarcoma cells and 293FT human embryonic kidney cells were grown in the DMEM medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). All the cell lines were free of mycoplasma contamination and were authenticated by short tandem repeat (STR) fingerprinting at the Medicine Lab of Forensic Medicine Department of Sun Yat-Sen University (China). The reagents used in cell experiments in the article include ferroptosis inducer RSL3 (Cat: S8155, Selleck), Erastin (Cat: S7242, Selleck), ML210 (Cat: S0788, Selleck), FIN56 (Cat: S8254, Selleck), Sorafenib (Cat: 7397, Selleck), Sulfasalazine (SAS, Cat: S8254, Selleck); ferroptosis inhibitors ferrostatin-1 (Fer-1, Cat: S7243, Selleck); apoptosis inhibitors Z-VAD-FMK (Cat: S7023, Selleck); necroptosis inhibitors Necrostatin-1 (Nec-1, Cat: S8037, Selleck); pyroptosis inhibitors VX765 (Cat: S2228, Selleck); free fatty acids Palmitic acid (PA, Cat: GN10676, GLPBIO), Oleic acid (OA, Cat: GC30110, GLPBIO), α-Linolenic Acid (ALA, Cat: GC19540, GLPBIO), Arachidonic acid (AA, Cat: GC31725, GLPBIO).
Establishment of ferroptosis-resistant cell lines. 293FT-RSL3-Resistant cell, U2OS-RSL3-Resistant cell, 293FT-Erastin-Resistant cell, and U2OS-Erastin-Resistant cell were generated by growing parental cells in the presence of increasing concentrations (up to a maximum concentration of 5 μM of RSL3 or 10 μM of Erastin) for 3 months. Initially, cell numbers were markedly reduced, and for the following 2 months, the surviving cells were passaged approximately once 7 days. Surviving clones were collected and further expanded. After ferroptosis-resistant cells were established, they were continuously cultured in the presence of RSL3 or Erastin. The responses of these surviving cells to RSL3 or Erastin were tested by cell viability assay. Their ferroptosis resistance was confirmed.
Plasmids, retroviral infection, and transfection. The human LPCAT1 (Full, H135A) was cloned into the pSin-EF2 vector and pLVX-TRE3G-IRES vector. ShRNAs targeting LPCAT1 were cloned into the pSupper retroviral vector and pLVX-TRE3G-IRES vector. Transfection of siRNAs or plasmids was performed using the Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instruction. All primers and oligonucleotides are listed in Supplementary Table1. Stable cell lines expressing LPCAT1 or LPCAT1 shRNAs were generated via retroviral infection using 293FT cells and selected for 10 days with 0.5 µg/mL puromycin 48 h after infection.
CRISPR–Cas9-mediated gene knockout. We used CRISPR–Cas9 technology to knock out LPCAT1. The sgRNA was cloned into the empty backbone of lenti-CRISPR v2. The following sgRNA sequences were used: sgLPCAT1, 5’-GCACGTTGCTGGCATACAGCG-3’, 5’-GTGGTTCGCCGGCGGCTTCCA-3’ and 5’-TTACGATATCCAAATAAACTG-3’. Plasmids containing the sgRNA sequences were transfected into HEK293FT cells with the psPAX2 packaging plasmid and pMD2.G VSV-G envelope-expressing plasmid. The virus was collected for 72 h, and then 293FT-RSL3-Resistant cell, U2OS-RSL3-Resistant cell, 293FT-Erastin-Resistant cell, and U2OS-Erastin-Resistant cell were infected with virus and 0.8μg ml−1 polybrene and selected with puromycin (2μg ml−1; Invivogen) for 3 d
RNA extraction, reverse transcription, and real-time PCR. The total RNA was extracted from an indicated cell using the Trizol (Life Technologies) reagent according to the manufacturer’s instructions. Real-time reverse transcription-polymerase chain reaction (PCR) primers and probes were designed with the assistance of the Primer Express v 2.0 software (Applied BioSystems, Foster, CA, USA). Expression data were normalized to the geometric mean of the housekeeping gene GAPDH to control the variability in expression levels and calculated as 2−[(Ct of the gene) – (Ct of GAPDH)], where Ct represents the threshold cycle for each transcript. All primers are listed in Supplementary Table1.
Immunoblotting analysis (IB). IB was performed according to a standard protocol with the following antibody: anti-LPCAT1 (1:1000, Cat: HPA022268, Sigma-Aldrich), anti-FSP1 (1:1000, Cat: ab155326, Abcam), anti-GPX4 (1:3000, Cat: ab125066, Abcam), anti-DHODH (1:1000, Cat: ab174288, Abcam), anti-GCH1(1:1000, Cat: ab259882, Abcam), anti-LPCAT3 (1:1000, Cat: ab239585, Abcam), anti-GAPDH (1:10000, Cat: 60004-1-Ig, Proteintech). Uncropped images of immunoblotting were provided in Supplementary Information.
Alkyne Labeling, Imaging, and Quantification. The day before the experiment, 200,000 293FT cells or 50,000 U2OS cells/well were seeded into a 12-well plate (Cat:150628, Thermo Fisher Scientific) that had one 12mm2 coverslip (Cat: YA0350, Solarbio) in each well. On the day of the experiment, cells were washed once with PBS, and then treated in DMEM + 10% FBS ± Free fatty acid-alkyne, including AA-alkyne (50 μM, Cat: 10538, Cayman), ALA-alkyne (50 μM, Cat: GC40546, GLPBIO), PA-alkyne (50 μM, Cat: GC40292, GLPBIO), OA-alkyne (50 μM, Cat: GC41646, GLPBIO), and for 10 h. After 10 h, cells were washed 3 times with 1x PBS, then fixed in 4%PFA in 1x PBS (30 min, RT). Cells were washed 3 times with 1x PBS, then permeabilized with 0.1% Triton X-100 in PBS (2 min, RT). Cells were washed 3 times with 1x PBS, then treated with the click reaction: 0.1 mM Azide-fluor 488 (Cat: 760765, Sigma-Aldrich), 1 mM cupric sulfate (CuSO4, Cat: LSK900131, Lige Science), 1 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP, Cat: abs44077528, Absin) in 1x PBS (1 h, RT) in a light-impermeable humid chamber. Cells were washed 5 times with 1x PBS, then blocked with PBS-BT (1x PBS, 3% BSA, 0.1% Triton X-100, 0.02% NaN3) (1 h, RT). PBS-BT was removed and replaced with a primary antibody mixture: Integrin β1(1:1000, Cat: ab179471, Abcam) in 1x PBS-BT (1 h, RT). Cells were washed 3 times with 1x PBS-BT, then treated with a secondary antibody mixture: Anti-Rabbit Alexa Fluor 594 (1:500, Cat: 8889S, Cell Signaling) in 1x PBS-BT (1 h, RT). Cells were washed 3 times with 1x PBS-BT, then treated with 100 ng/ml 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, Cat: D1306, Thermo Fisher Scientific) in 1x PBS (15 min, RT). Cells were washed 3 times with 1x PBS, then the coverslips were mounted onto a glass slide with 5 mL of ProLong Gold Antifade Mountant (Cat: P10144, Thermo Fisher Scientific). Cells were imaged by confocal microscopy (Carl Zeiss, Jena, Germany). Images acquired for the alkyne experiments were processed in ImageJ 1.48v and quantified as follows. A region of interest (ROI) was defined for each image based on red fluorescence (i.e. Alexa Fluor 594) intensity falling within an empirically defined range. Then, the green fluorescence (i.e. Azide-fluor 488) intensity within the ROI was quantified. Images were background corrected by quantifying the green fluorescence in cell-free areas from at least four images and subtracting to determine the final green fluorescence values.
Immunofluorescence (IF) staining. IF staining was carried out on cell chamber slide cultures (Thermo Fisher Scientific) and followed by the antibody: anti-LPCAT1 (1:1000, Cat: HPA022268, Sigma-Aldrich) and Integrin β1(1:1000, Cat: ab179471, Abcam) antibodies. The secondary antibody was Anti-Rabbit Alexa Fluor 594 (1:500, Cat: 8889S, Cell Signaling) and Anti-Mouse Alexa Fluor 488 (1:500, Cat: 4408S, Cell Signaling). Then cells were mounted with Antifade Mountant with DAPI (Thermo Fisher Scientific). Cells were imaged by confocal microscopy (Carl Zeiss, Jena, Germany), and images acquired for the experiments were processed and analyzed in ImageJ 1.48v (described above).
Lipid peroxidation was assessed by Liperfluo staining. For flow cytometry experiments, the day before the experiment, 800,000 293FT cells or 200,000 U2OS cells, 200,000 HepG2 cells, or 200,000 SNU449 cells /well were seeded into 6-well plates (Cat:150628, Thermo Fisher Scientific), then treated with the compounds indicated in the relevant figure captions. On the day of the experiment, cells were incubated with Liperfluo (20 μM, Cat: L248, DojinDo) for 30 min at 37°C before they were harvested by trypsinization. Subsequently, cells were resuspended in fresh PBS strained and analyzed using the 524 nm laser of a flow cytometer for excitation.
For confocal imaging experiments, the day before the experiment, 200,000 293FT cells or 50,000 U2OS cells/well were seeded into a 12-well plate that had one 12mm2 coverslip in each well. On the day of the experiment, cells were incubated with Liperfluo (20 μM, Cat: L248, DojinDo), Dil (15 μM, Cat: 42364, Sigma), and Hoechst (10 μM, Cat: 62249, Sigma-Aldrich) for 30 min at 37°C. Subsequently, cells were washed twice with PBS. Then, cells were imaged by confocal microscopy (Carl Zeiss, Jena, Germany), and images acquired for the experiments were processed and analyzed in ImageJ 1.48v (described above).
Cell viability assay. Cells were measured using a Cell Counting Kit-8 (CCK-8, Cat: CK04, Dojindo). In brief, cells were seeded onto 96-well plates at a density of 5 × 103 per well. The next day, cells were treated with the compounds indicated in the relevant figure captions, including ferroptosis inducer. Subsequently, cells were exposed to 10 μl CCK-8 reagent (100 μl medium per well) for 1 h at 37 °C, 5% CO2 in an incubator. The absorbance at a wavelength of 450 nm was determined using a Spectro fluorimeter (SpectraMax i3X, Molecular Devices).
Cell Death Assay. Cell death was measured using a flow cytometer after propidium iodide (PI) staining. The day before the experiment, 2× 105 cells were seeded into 6-well plates, then treated with the compounds indicated in the relevant figure captions. On the day of the experiment, cells were incubated with PI (5 μM, Cat: KGA108, KeyGen) for 15 min at 37°C before they were harvested by trypsinization. Subsequently, cells were resuspended in fresh PBS strained, and analyzed using a flow cytometer for excitation.
Analysis of cellular ROS. Intracellular ROS levels were examined using Fluoro-metric Intracellular ROS Kit (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 5 × 103 cells were cultured in a 96-well plate, and then cell-permeable oxidative fluorescent dye 2’,7’ dichlorodihydrofluorescein diacetate (DCFHDA) was added to wells and incubated for 1 h at 37 °C. ROS levels were quantified by measuring fluorescence intensity at excitation and emission wavelength of 490 and 525 nm, respectively using a Spectro fluorimeter (SpectraMax i3X, Molecular Devices).
Analysis of cellular Fe2+ amount. The day before the experiment, 800,000 293FT cells or 200,000 U2OS cells/well were seeded into 6-well plates (Cat:150628, Thermo Fisher Scientific), then treated with the compounds indicated in the relevant figure captions. On the day of the experiment, cells were incubated with Ferro-Orange (1 μM, Cat: F374, DojinDo) for 30 min at 37°C before they were harvested by trypsinization. Subsequently, cells were resuspended in fresh PBS strained and analyzed using the 543 nm laser of a flow cytometer for excitation.
Analysis of cellular Glutathione. The GSH-Glo™ Glutathione Assay Kit (Promega, Madison, WI, USA) was used to determine the reduced GSH content in indicated cells. In brief, cells were seeded onto 96-well plates at a density of 5 × 103 per well. The next day, cells were treated with the compounds indicated in the relevant figure captions. Cells were prepared for the measurement of glutathione using the GSH-Glo™ Glutathione Assay Kit according to the manufacturer’s protocol. The luciferase signal was determined using a Spectro fluorimeter (SpectraMax i3X, Molecular Devices).
Immunohistochemistry (IHC). IHC analysis was performed to determine altered protein expression in paraffin-embedded HCC tissues with anti-LPCAT1 (1:500, Cat: HPA022268, Sigma-Aldrich) and anti-MDA (1:100, Cat: JAI-MMD-030N, Adipogenic), anti-4-HNE (1:200, Cat: ab46545, Abcam), anti-Cleaved-caspase 3 (1:400, Cat: #9661S, Cell signaling technology) antibodies overnight at 4 °C. The degree of immunostaining of formalin-fixed, paraffin-embedded sections was reviewed and scored separately by two independent pathologists uninformed of the histopathological features and patient data of the samples. The scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The scores given by the two independent pathologists were combined into a mean score for further comparative evaluation. Tumor cell proportions were scored as follows: 0, no positive tumor cells; 1, < 10% positive tumor cells; 2, 10–35% positive tumor cells; 3, 35–75% positive tumor cells; 4, > 75% positive tumor cells. Staining intensity was graded according to the following standard: 1, no staining; 2, weak staining (light yellow); 3, moderate staining (yellow-brown); 4, strong staining (brown). The staining index (SI) was calculated as the product of the staining intensity score and the proportion of positive tumor cells. Using this method of assessment, we evaluated protein expression in benign esophageal epithelial and malignant lesions by determining the SI, with possible scores of 0, 2, 3, 4, 6, 8, 9, 12, and 16. Samples with a SI ≥ 8 were determined as high expression and samples with a SI < 8 were determined as low expression. Cutoff values were determined based on a measure of heterogeneity using the log-rank test concerning overall survival.
Transmission electron microscopy. TEM analysis was performed by the High-Resolution Electron microscopy facility at the instrument center of Zhongshan School of Medical, Sun Yat-Sen University. Samples were fixed with a solution containing 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3), then washed in 0.1 M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid, postfixed with 1% buffered osmium and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. The samples were polymerized in a 60 °C oven for approximately 3 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica UC6), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a Tecnai G2 SpiritTwin transmission electron microscope at an accelerating voltage of 80 kV. Digital images were obtained using the GATAN 832.10W System.
RNA-seq analysis. The total RNA was extracted and purified using the Trizol (Life Technologies) reagent according to the manufacturer’s instructions. RNA quantitation and quality control were performed by Bioanalyzer 2100 (Agilent Technologies). Construction of stranded RNA-seq libraries for high-throughput sequencing was done on Illumina HiSeq X Ten following the manufacturer’s protocol. RNA-seq reads were mapped to the reference genome of Illumina Ensembl genome GRCh37 using HISAT2 version 2.2.9. Mapped reads were summarized for each gene using htseq-count version 0.11.2. Differential expression analysis was implemented using DEGseq version 1.36.1. P-value was calculated by Student’s t-test.
Lipidomic analyses. Metabolite extraction. 1*107 cells were collected, and 1ml 0.9% normal saline was added to clean the cells twice until the cell medium was colorless. 800ul pre-cooled methanol (pre-cooled overnight in -80℃ refrigerator) and 320ul ice water were added to the cells, and the mixture was homogenized by shaking for 10s. The cell metabolites were scraped with a cell spatula and absorbed into a 1.5ml centrifuge tube. 800 μl of pre-cooled chloroform was added to the tube and centrifuged at 4℃ for 15min at 15000 r.c.f. 700 μl of the lower liquid was placed in a new 1.5ml centrifuge tube and dried with nitrogen at room temperature. 120 μl of redissolved solvent (chloroform: methanol: water =6:3:0.5, V/V/V) was added to each layer of the dry sample, followed by voraciously shaking for 5min, centrifugation at 15000 r.c.f for 5min at room temperature, and 100 μl of supernatant was taken into 2mL mass spectrometry vial with internal intubation for machine analysis.
Liquid chromatography with tandem mass spectrometry analysis. The liquid chromatography method was based on the QExactive LC (Thermo Fisher) system, where Ultimate 3000 LC (Thermo Fisher) was used for the liquid phase portion. Metabolomics liquid mass spectrometry was performed on the Hyperil Gold C18 column (100 x 2.1mm, 1.9 μm; Thermo Fisher), the column temperature was 40℃, the flow rate was 0.30 ml/min, and the injection volume was 1 μl. Mobile phase A consisted of 40% water, 60% acetonitrile, and 10 mM ammonium formate, 0.1% formic acid. Mobile phase B consisted of 10% acetonitrile and 90% isopropanol, and 10 mM ammonium formate, 0.1% formic acid. The analysis was carried out with the following elution gradient: 0–6 min, 100%-50% phase A, 0%-50% phase B; 6–30 min, 50%-0% phase A, 50%-100% phase B; 30-32 min, 0% phase A, 100% phase B; and 32–32.1 min, 0%-100% phase A, 100%-0% phase B; and 32.1–36 min, 100% phase A, 0% phase B. The mass spectrometry method was established in QExactive (Thermo Fisher) system. The lipidomics mass spectrometry method was performed in Full Scan/Data-Depend MS2 Top10 analysis mode and positive and negative spectrum analysis in ESI mode. Spray voltage +3500V/ -3000V, the sheath gas is 40 arb, the auxiliary gas is 10 arb, the ion transfer tube temperature is 320℃, the auxiliary gas heating temperature is 350℃; The first-level Full scan resolution is 70000, and the mass range is 100-1500m/z. Data depend ms2 has a secondary resolution of 17500, top 10, the impact energy of 20, 40, and mass range of 70-1500m/z.
Data pre-processing and annotation. The data obtained from the above lipidomics method were imported into Lipid Search 4.1 software for Lipid identification and queue analysis. The full MS/Data depend MS2 data collected by the QExactive instrument was selected to use the daughter ion mode for lipid identification. The accurate mass deviation threshold of parent ion and daughter ion was set to 5ppm, and isotope matching was performed to display the ions with fragment matching score above 2. The retention time window threshold was set to 1min. Positive mode selects +H peak, +Na peak, +NH4 peak addition, and negative mode select -H peak, +HCOO peak addition, and sum for database retrieval. The retrieval results select and identifies the ions with grade A, B, and C for queue analysis to obtain the lipid profile information and the relevant data results can be derived.
Subcutaneous xenograft model. All of the animal procedures were approved by the Sun Yat-sen University Animal Care Committee. Informed consent was obtained from the patients and the study was compliant with all relevant ethical regulations regarding research involving human participants. Five-week-old male BALB/c nude mice were obtained from Beijing Vital River (Beijing, China). In brief, PDX tumors in cold DMEM were minced into fragments 1–2 mm3 in volume. Then each PDX tumor fragment was subcutaneously inoculated into the dorsal flank of an BALB/c nude mice. When the tumors reached 50–100 mm3 in volume, the mice were assigned randomly into different treatment groups described for cell line-derived xenografts. The tumor volume was measured every 3 days until the endpoint and calculated according to the equation volume = length × width2 × 1/2. The doxycycline was dissolved and administered in mouse drinking water at concentrations of 0.5-8mg/ml. Imidazole ketone erastin (IKE) (40 mg/kg) and Ferrostatin-1 (5 mg/kg) were intraperitoneal injected. Animals were killed when the xenograft tumor size reached 1,500 mm3. No mouse exhibited severe loss of body weight (>15%) or evidence of infections or wounds.
Orthotopic cell line-derived xenograft (CDX) model. All of the animal procedures were approved by the Sun Yat-sen University Animal Care Committee. Five-week-old male BALB/c nude mice were obtained from Beijing Vital River (Beijing, China). In brief, mice were injected with 5 × 105 indicated cells expressing luciferase in the liver of mice, respectively (n = 6 per group). Orthotopic xenograft bioluminescence was examined every 3 days. When the bioluminescence reached 1.0 × 106p/s/cm2/sr, doxycycline was dissolved and administered in mouse drinking water at concentrations of 0.5-8mg/ml for 10 weeks. The tumors were observed and assessed weekly in mice injected with D-luciferin (75 mg/kg) by bioluminescence imaging using the IVIS Spectrum In Vivo Imager. After 11 weeks, mice-bearing HCC were executed. Experiments associated with animals comply with the Laboratory Animal Care and Use Guide and the Institutional Research Ethics Committee has approved these experiments.
Patient-derived xenograft (PDX) model. All of the animal procedures were approved by the Sun Yat-sen University Animal Care Committee. Informed consent was obtained from the patients and the study was compliant with all relevant ethical regulations regarding research involving human participants. All of the BALB/c nude mice were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and were housed in a barrier facility on a 12-h light/dark cycle. The fragments of human HCC tissues were orthotopic implantation into the liver of 5-week-old mice. The mice were divided into four treatment groups: (1) AAV-Scr + vehicle, (2) AAV-si-LPCAT1 + vehicle, (3) AAV-Scr + IKE, and (4) AAV-si-LPCAT1 + IKE. After 3 weeks, mice-bearing HCC were locally injected with AAV-Scr or AAV-si-LPCAT1 and intraperitoneal injected with vehicle or Imidazole ketone erastin (IKE) (40 mg/kg, once every other day). After 11 weeks, mice-bearing HCC were executed. Experiments associated with animals comply with the Laboratory Animal Care and Use Guide and the Institutional Research Ethics Committee has approved these experiments.
Patient information and tissue specimens. The clinical tissues of fresh pancreatic cancer and liver cancer used in this study, which complied with all relevant ethical regulations for work with human participants, were from the Sun Yat-sen University Cancer Center and the Third Affiliated Hospital of Sun Yat-sen University. The study protocols were approved by the Institutional Research Ethics Committee of Sun Yat-sen University for the use of these clinical materials for research purposes. All Patients’ samples were obtained according to the Declaration of Helsinki and each patient signed written informed consent for all the procedures.
Statistics and reproducibility. All data were presented as the mean ± standard deviation (SD). n represents the number of independent experiments performed on different mice or different batches of cells or different clinical tissues. Statistical analysis was performed using the student’s two-tailed t-test and one-way analysis of variance (ANOVA). Bivariate correlations between study variables were calculated by Spearman’s rank correlation coefficients. Survival curves were plotted by the Kaplan–Meier method and compared by the log-rank test. The significance of various variables for survival was analyzed by univariate and multivariate Cox regression analyses. p-values of 0.05 or less were considered statistically significant. Statistical analysis was performed using the GraphPad Prism 8.0.1 and SPSS 19.0 statistical software. Representation of the p-values was *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant (p > 0.05).