Ethics statement
Fresh tumour samples were obtained from patients undergoing surgery at Institut Curie and Paul Brousse hospitals. All patients provided written informed consent for use of tumour samples. The study was approved by institutional regulatory boards (DATA190160). All cLip-1 in vivo experiments were performed in compliance with the German Animal Welfare Law and have been approved by the Institutional Committee on Animal Experimentation and the Government of Upper Bavaria (approved no. ROB-55.2-2532.Vet_02-18-13).All intranodal injection mouse experiments complied with all relevant ethical regulations and were performed according to protocols approved by the Institutional Animal Care and Use Committee at Harvard T.H. Chan School of Public Health (protocol IS00003460). For mouse lymph and blood collection, animal experiments were performed in accordance with the European Community guiding in the care and use of animals.Animal experiments were performed in agreement with the French Guidelines for animal handling and approved by local ethics committee (Agreement no. 16487-2018082108541206 v3).
Lead contact.Further information and requests for resources and reagents should be directed to the lead contact Raphaël Rodriguez ([email protected]).
Materials availability.Please contact the lead author Raphaël Rodriguez for in-house reagents, which can be made available under a material transfer agreement with Institut Curie.
Data availability. Lipidomics data are presented in Supplementary Tables 1-5.
Chemical synthesis.Starting materials were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Anhydrous solvents were obtained by passing the degassed solvents through molecular sieves and activated alumina columns. Reactions were monitored by thin layer chromatography using aluminium plates coated with silica gel or neutral aluminium oxide neutral from Merck (60 F254). TLC plates were visualised by UV or by treatment with a ninhydrin, CAM or potassium permanganate solutions and heating. Reaction products were purified by flash column chromatography on silica gel 60 (230-400 mesh, Macherey Nagel) or aluminium oxide (activated neutral, Sigma-Aldrich), by Combiflash Rf, or by preparative HPLC Quaternary Gradient 2545 equipped with a Photodiode Array detector (Waters) fitted with a reverse phase column (XBridge BEH C18 OBD Prep column 5 μm 30 × 150 mm). NMR spectroscopy was performed on Bruker 300, 400 or 500 MHz instruments. Spectra were run in methanol-d4, dimethylsulfoxide-d6, methylene chloride-d2 or chloroform-d, at 298 K. 1H chemical shifts δ are expressed in ppm using the residual non-deuterated solvent as internal standard and the coupling constants J are specified in Hz. The following abbreviations are used: bs, broad singlet; s, singlet; d, doublet; dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet of triplets; dq, doublet of quadruplets; q, quadruplet; t, triplet; quint., quintet; m, multiplet. 13C chemical shifts δ are expressed in ppm using the residual non-deuterated solvent as internal standard. The purity of final compounds was determined to be >98% by UPLC-MS. Low-resolution mass spectra (LRMS) were recorded on a Waters Acquity H-class equipped with a Photodiode array detector and SQ Detector 2 (UPLC-MS) fitted with a reverse phase column (Acquity UPLC BEH C18 1.7 μm, 2.1x50 mm). HRMS were recorded on a Thermo Scientific Q-Exactive Plus equipped with a Robotic TriVersa NanoMate Advion. Procedures for the synthesis of small molecules are detailed in the Supplementary Information.
NMR of Lip-1-iron(III) complexes: 1H NMR spectra were recorded on a 500 MHz Bruker spectrometer at 310 K, and chemical shifts δ are expressed in ppm using the residual non-deuterated solvent signals as internal standard. General procedure: From 0 to 1 equivalent of FeCl3 (Alfa Aesar, 12357, lot E23Z042), portions of 0.5 equivalent of a solution of FeCl3 in methanol-d4 were added up to a solution of 1 equivalent of liproxstatin-1 (Lip-1, Sigma-Aldrich, SμL1414, lot 0000152075) in methanol-d4 into an NMR tube and NMR spectra were recorded. Then a drop of trifluoroacetic acid (TFA, Sigma-Aldrich, T6508) or sodium deuteroxide (NaOD, Eurisotop, D076Y) was added. 0.94 mg of Lip-1 were dissolved in 600 µL of methanol-d4. From 0 to 1 equivalent of FeCl3, portions of 3.0 µL of 92 mM solution of FeCl3 were added.
Cyclic voltammetry. Cyclic voltammetry51 experiments were performed with a three-electrode cell. A saturated calomel electrode (SCE) was used as reference, a steady glassy carbon (GC) electrode of diameter 3 mm was selected as working electrode and a platinum wire as counter-electrode. All cyclic voltammograms were recorded at room temperature with a μ-autolab III from Metrohm using Nova software with a scan rate of 2 V/s. MeCN and MeOH were used in HPLC grade (Carlo Erba). For all experiments we used a 0.1 M nBu4NBF4 in MeCN (32.9 mg/μL stock solution). 1 mM FeCl3 solutions were prepared with 50 µL of 20 mM FeCl3 solution in milliQ water and 950 µL of MeCN. Then, portions of 10 µL (0.2 eq.) of 20 mM stock solution of Lip-1 or analogues (solubilised in MeCN or MeOH) were added until 1.0 eq. was reached. Above 1.0 eq., 50 µL (1.0 eq.) of 20 mM stock solution of the analogues were added. After each addition, the solution was stirred for a few seconds and voltammograms were recorded.
Complexation studies in solution. 100 mM stock solutions of DFO mesylate salt (Sigma-Aldrich, D9533), FeCl3 or Lip-1 were dissolved in water for DFO and FeCl3, and MeCN for Lip-1. For dilution of each compound, 15 µL of these stock solutions were added to 135 µL of MeCN to reach a concentration of 10 mM in each vial. For mixture of compounds, 15 µL of each compound were added to 120 µL of MeCN to reach a concentration of 10 mM. miliQ water and HPLC grade MeCN (Carlo Erba) were used.
Liposome preparation. Liposomal structures were prepared using the traditional lipid film hydration method: 100 μL of a stock solution (1mg/μL chloroform) of 18:1 (Δ9-cis) PC (DOPC, Avanti Polar Lipids) were dissolved in 400 μL of chloroform and transferred into a round-bottom flask. The organic solvent was removed under reduced pressure in a rotary evaporator for 15 min at 200 rpm at 37 ºC in a water bath. Afterwards, the lipid film was dried with a vacuum pump overnight. Then was hydrated with 1 μL of 0.1 mM sodium acetate buffer (pH 4.5) and vortexed every 5 min for 20 min. Liposomes were extruded by passing the suspension through 2 polycarbonate membranes (pore size 0.2 mm) 20 times.
Lipid oxidation in vitro. Control experiment: 200 μL of the liposome solution were added into an Eppendorf tube and heated at 37 ºC with agitation at 800 rpm. Then 5 μL of an aq. solution of Fe(OTf)2 (1.4 mg/1.5 μL) and 13 μL of 0.1 mM acetate buffer (pH 4.5) were added. At t = 0 min 13 μL of an aq. solution of H2O2 (10 μL H2O2 (30%)/1 μL) were added. Fentomycin experiment: 200 μL of the liposome solution were added into an Eppendorf tube and heated at 37 ºC with 800 rpm. Then 13 μL of a solution 1 mM of fentomycin in DMSO and 5 μL of an aq. solution of Fe(OTf)2 (1.4 mg/1.5 μL) were added. At t = 0 min 13 μL of an aq. solution of H2O2 (10 μL H2O2 (30%)/1 μL) were added. The kinetic process of DOPC oxidation was recorded with a QExactive mass spectrometer (Thermo Fisher Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). Samples were injected at 0.5 h, 1 h, 2 h, 3 h, 4 h, 7 h and 24 h reaction time.
Antibodies.Antibodies are annotated below as follows. WB, western blot; FCy, flow cytometry; FM, fluorescence microscopy. Hu, used for human samples. Ms, used for mouse samples. Dilutions are indicated. Any antibody validation by manufacturer is indicated and can be found on the manufacturers’ websites. Our antibody validation knockdown (KD) and/or KO strategies as described here for relevant antibodies is indicated. Primary antibodies: 4-Hydroxynonenal (4-HNE) (Abcam, ab48506, lot 1062274-2, FM, 1:200, Hu), ACSL4 (Santa Cruz Biotechnology, sc-271800, clone A-5, lot I1222, WB 1:1000, Hu, siRNA validated by us), AIFM2/FSP1 (Merck, MABC1638, clone 6D8-11, lot Q3745998, WB, 1:500, Hu, siRNA validated by us), Catalase (Cell Signaling, 12980T, clone D4P7B, lot 3, FM, 1:200, Hu), CD3-BV510 (BioLegend, 317332, clone OKT3, lot B263750, FCy, 1:100, Hu), CD31-PE-Cy7 (BioLegend, 303118, clone WM59, lot B276836, FCy, 1:100, Hu), CD31-BV605 (BioLegend, 303122, clone WM59, lot 331683, FCy, 1:100, Hu), CD31-BV605 (BioLegend, 102427, clone 390, lot B375532, FCy, 1:100, Ms), CD44-AF647 (Novus Biologicals, NB500-481AF647, clone MEM-263, lot D145771, FCy, 1:100, Hu), CD44-AF647 (BioLegend, 103018, clone IM7, lot B317762, FCy, 1:100, Ms), CD45-BV785 (BioLegend, 304048, clone HI30, lot B339809, FCy, 1:100, Hu), CD45-BV510 (BioLegend, 368526, clone 2D1, lot B373428, FCy, 1:100, Hu), CD45-BV510 (BioLegend, 103138, clone 30-F11, lot B386738, FCy, 1:100, Ms), CD163-PerCP/Cyanine5.5 (BioLegend, 326512, clone RM3/1, lot B291202, FCy, 1:100, Hu), Cytochrome c (Cell Signaling, 12963S, clone 6H2.B4, lot 2, FM, 1:200, Hu), EEA1 (Abcam, ab70521, clone 1G11, lot GR315680-1, FM, 1:200, Hu, validated in ICC/IF by manufacturer), FAP-AF700 (R&D Systems, FAB3715N, clone 427819, lot AEVI020011, FCy, 1:100, Hu), FAP-AF750 (Novus Biologicals, FAP3715S, lot 1718688, FCy, 1:100, Hu), Ferritin (Abcam, ab75973, clone EPR300AY, lot 10136442-29, WB, 1:1000, Hu, validated in WB by manufacturer), GPX4 (Abcam, ab125066, clone EPNCIR144, lot GR3369574-4, WB, 1:2000, Hu, KO validated by manufacturer), HSL (Cell Signaling, 18381T, clone D6W5S, lot 1, WB, 1:1000, Hu), IRP2 (Novus Biologicals, NB 100-1798, lot D-3, WB, 1:1000, Hu, validated by manufacturer), Lamp1 (Cell Signaling, 9091S, clone D2D11, lot 7, FM, 1:200, Hu), Lamp1 (Santa Cruz Biotechnology, sc-20011, FM, 1:100), Lamp2 (Abcam, ab25631, clone H4B4, FM, 1:200, Hu), PDIA3 (Sigma-Aldrich, AMAB90988, clone CL2444, lot 02879, FM, 1:200, Hu), MHCII-APC/Cyanine7 (BioLegend, 107628, clone M5/114.12.2, lot B370049, FCy, 1:100, Ms), Rcas1 (Cell Signaling, 12290S, clone D2B6N, lot 1, FM, 1:200, Hu), SLC7A11/xCT (Cell Signaling, 12691S, clone D2M7A, lot 5, WB, 1:1000, Hu, siRNA validated by us), TfR1-APC-AF750 (Beckman Coulter, A89313, clone YDJ1.2.2, lot 200060, FCy, 1:100, Hu), TfR1-PE (BioLegend, 334106, clone CY1G4, lot B364886, FCy, 1:100, Hu), γ-tubulin (Sigma-Aldrich, T5326, clone GTU-88, lot 0000140390, WB, 1:1000, Hu, validation by manufacturer). Secondary antibodies: Alexa Fluor 647 anti-mouse (Abcam, ab150115, tissue labelling, 1:500, Ms), Alexa Fluor 647 anti-mouse (Invitrogen, A21237, lot 1485202, FM, 1:1000, Hu), Alexa Fluor 647 anti-rabbit (Invitrogen, A21246, lot 2714437, FM, 1:1000, Hu), donkey anti-rabbit IgG-h+l HRP-conjugated (Bethyl Laboratories, A120-108P, lot 13, WB, 1:10000, Hu), goat anti-mouse IgG-h+l HRP-conjugated (Bethyl Laboratories, A90-116P, lot 39, WB, 1:10000, Hu), goat anti-rat IgG-h+l HRP-conjugated (Invitrogen, 31470, WB, 1:10000, Hu).
Cell culture. Dissociated human and mouse tumour cells and 4T1 cells were cultured in RPMI 1640 supplemented with GlutaMAX (Gibco, 61870010), 10% foetal bovine serum (FBS, Eurobio Scientific, CVFSVF00-01). HT-1080 cells were cultured in Dulbecco’s Modified Eagle Medium GlutaMAX (DMEM, Gibco, 61965059) supplemented with 10% FBS (Gibco, 10270-106) and penicillin/streptomycin (BioWhittaker/Lonza, DE17-602E). FC1242 and FC1245 murine pancreatic cancer cells, 4a cells and human pancreatic hMIA-2D cells were a generous gift from the Tuveson laboratory (CSHL) and were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Primary human pancreatic PDAC090T, PDAC053T, PDAC211T and PDAC030T cells were grown in serum-free ductal medium: DMEM/F12 supplemented with 0.61g/500μL nicotinamide (Sigma-Aldrich, 3376), 2.50g/ 500μL glucose (Sigma-Aldrich, G6152), 1:200 ITS+ (Corning, 354352), 1:20 Nu-serum IV (Corning, 355104), 100 ng/μL cholera toxin, 1 µM dexamethasone (Sigma-Aldrich, D4902), 50 nM 3,3’,5-triiodo-L-thyronine (Sigma-Aldrich, T6397) and penicillin/streptomycin.
Dissociation of human and murine tumour samples:Tumour samples were collected from patients after surgery. Tumour samples correspond to pancreatic ductal adenocarcinoma (PDAC), undifferentiated pleomorphic sarcoma (UPS), liposarcoma, angiosarcoma, epithelioid sarcoma and PDAC liver metastasis. Tumours were dissociated using the human tumour dissociation kit (Miltenyi, 130-095-929) according to the manufacturer’s protocol. In brief, tumours were cut into small pieces of 1-5 mm, put in presence of the enzyme mix in RPMI and dissociated on the gentleMACS Octo Dissociator with Heaters (Miltenyi) with the appropriate gentleMACS program (37C_h_TDK). Per tumour sample, a total volume of 9.4 μL of medium was used with the corresponding enzyme concentration according to the manufacturer’s protocol. Mouse tumour samples were dissociated using the mouse tumour dissociation kit (Miltenyi, 130-096-730) according to the manufacturer’s protocol. Tumours were cut into small pieces of 1-5 mm, put in presence of the enzyme mix in RPMI and dissociated on the gentleMACS Octo Dissociator with Heaters (Miltenyi) with the appropriate gentleMACS program (37C_m_TDK). Subsequently, the dissociated tumour suspension was applied to a MACS SmartStrainer (30 µm) (Miltenyi). Samples were diluted with 1× PBS (Phosphate-buffered saline) and centrifuged at 300× g. The cell pellet was resuspended in RPMI (10%FBS, penicillin/streptomycin) and cells were counted using an automated cell counter (Entek)
Establishment of xenograft derived primary cell cultures (XDPCC). XDPCC models were originally derived from PDX patient models. The PDX fragments designated for cell culture were processed in a biosafety chamber. After fine mincing they were treated with collagenase type V (Sigma-Aldrich, C9263) and trypsin/EDTA (Gibco, 25200-056) and were suspended in Dulbecco’s modified Eagle’s medium supplemented with 1% w/w penicillin/streptomycin and 10% FBS. After centrifugation, cells were resuspended in serum-free ductal media adapted from previous protocols52 at 37 °C in a 5% CO2 incubator. Amplified cells were stored in liquid nitrogen. Cells were weaned from antibiotics for more than 48 h before testing. This protocol was used to establish the cells designated as PDAC053T, PDAC090T, PDAC211T and PDAC030T.
Establishment of xenograft derived pancreatic organoids (XDPO). XDPO models were originally derived from PDX patient models. Xenografts were split into several small pieces and processed in a biosafety chamber and after a fine mincing they were treated with the tumour dissociation kit. Undigested pellets were digested with accutase at 37 °C for 30 min. The pancreatic tissue slurry was transferred into a tissue strainer 100 μm and was placed into 12-well plate coated with 150 μL GFR matrigel (Corning). The samples cultured with pancreatic organoid feeding media (POFM) consisted of advanced DMEM/F12 supplemented with 10 mM HEPES (Thermo Fisher Scientific, 15630056), 1× Glutamax (Thermo Fisher Scientific, 35050087), penicillin/streptomycin, 100 ng/μL animal-free recombinant human FGF10 (Thermo Fisher Scientific, 500-P151G-50UG), 50 ng/μL animal-free recombinant human EGF (Thermo Fisher Scientific, AF-100-15-1MG), 100 ng/μL recombinant human noggin (Biotechne, 6057-NG), Wnt3a-conditioned medium (30% v/v); RSPO1-conditioned medium (10% v/v), 10 nM human gastrin 1 (Sigma-Aldrich, SCP0152) 10 mM nicotinamide (Sigma-Aldrich, N0636), 1.25 mM N-acetylcysteine (Sigma-Aldrich, A9165), 1× B27 (Thermo Fisher Scientific, 17504001), 500 nM A83-01 (Tocris, 2939/10) and 10.5 μM Y27632 (Tocris, 1254/1). The plates were incubated at 37 °C in a 5% CO2 incubator, and the media were changed every 3 or 4 days. This procedure was used to generate the XDPOs PDAC009T, PDAC003T, PDAC117T and PDAC372T.
Chemosensitivity profiling of XDPO and XDPCC. For chemosensitivity profiling, XDPO were plated into 96-well plates and then subjected to incrementally increasing concentrations of drugs. Cell viability was measured 72 h after treatment using CellTiter-Glo 3D (Promega, G9683). Doubling times (DT) of XDPO viability for untreated control conditions were calculated on days 0 and 3. The ratio of day 3 over day 0 corresponds to the replication rate (RR) of the cells at 72 h. Doubling time was calculated with the formula 72 × 2/RR. Fluorescence and luminescence values were quantified using the plate reader Tristar LB941 (Berthold Technologies). Each experiment was performed at least 3 times with at least 3 replicates.
Cell death and viability assays. Cell death assay with Annexin-V (A) & Propidium Iodide (PI): PDAC-053T cells were seeded on 6-well plates at the density of 2× 105 cells/well. Baf-A1 (75 nM) was added 7 h prior to the experiment. RSL3 (Sigma-Aldrich, SμL2234, 0.1, 0.5, 2, 10 μM) was added along with Lip-1 (1 µM), cLip-1 (1 µM), metcLip-1 (1 µM) or alcLip-1 (1 µM) on the following day, and after 24 h, the media was recovered and cells were trypsinised. Cells were harvested, pelleted along with the media recovered, washed with 1× PBS, and 100 μL of 1x Annexin-V binding buffer containing Annexin-V and PI according to the manufacturer’s protocol (Annexin-V flow cytometry kit, Thermo Fisher Scientific, V13242). 1× PBS buffer containing 10% FBS and EDTA (0.1% v/v) was added for flow cytometry. Flow cytometry was run on an AttuneTM NxT Flow Cytometer and analysed on FlowJo. Lactate dehydrogenase release was measured using a Cytotoxicity detection kit (Sigma-Aldrich, 11644793001) according to the manufacturer’s protocol in a 96-well format. Cell viability was assessed using a CellTiterGlo 2.0 (Promega, G9241) or CellTiter blue (Promega, G8081) kit according to the manufacturer’s protocol in a 96-well format. In brief, 4000 HT-1080 cells were seeded per well in clear-bottom and darkened 96-well plates (Greiner, 655090, lot E23063EG) 24 h prior to the experiment. Cells were then pre-treated for 2 h with Lip-1 (10 µM), cLip-1 (10 µM), ferrostatin-1 (Fer-1, SμL0583, 10 µM), deferoxamine (DFO, Sigma-Aldrich, D9533, 100 µM), deferasirox (DFX, Cayman chemical, 16753, 10 µM), deferiprone (Def, Sigma-Aldrich, Y0001976, 100 µM), tocopherol (Toc, Sigma-Aldrich, PHR1031, 100 µM), Vitamin K3 (Sigma-Aldrich, M5625-25G, 10 µM), Z-VAD-FMK (Enzo Life Sciences, ALX-260-020-M005, 50 µM) or Necrostatin-1 (Nec-1, Sigma-Adrich, N9037, 20 µM). Subsequently fentomycin (10 µM, 6 h) was added. Samples were processed detailed in the manufacturer’s protocols and data recorded on a SpectraMax ID3 plate reader (Molecular Devices). For standard-of-care cell survival measurements, cells were plated at 2000 cells per well 24 h prior to the experiment. Cells were incubated with serial dilutions of fentomycin, irinotecan (Sigma-Aldrich, I1406), 5-FU (Alfa Aesar, A13456-06) or oxaliplatin (Biotechne, 2623) for 72 h. Cell viability was assessed using a CellTiter blue assay and data recorded on a SpectraMax ID3 plate reader (Molecular Devices). For MTT assays, cells were plated in a 96-well plate, incubated for 24 h, and then pre-treated with Lip-1 (1uM) for 10 min prior to treatment with vehicle control or fentomycin for 24 h. After 24 h, media was carefully removed and 50 μL of serum-free media and 50 μL of MTT solution (Cayman Chemical, 21795) were added per well. The plate was incubated at 37 °C for 3 h. After incubation, the solution was removed and 100 μL of DMSO was added per well. The plate was covered and shaken on an orbital shaker for 15 min prior to reading absorbance on a plate reader (OD=590). For RSL3 treatment, Pfa1 cells were seeded onto 96-well plates (2000 cells per well) and cultured overnight. The following day, cells were cotreated with RSL3 (500 nM) and indicated small molecules in serial dilution. For 4-OH-TAM treatment, Pfa1 cells were seeded onto 96-well plates (500 cells per well) with 4-OH-TAM (1 μM) and a dilution series of the indicated compounds. After 24 h (for RSL3) or 72 h (for 4-OH TAM) incubation, cell viability was assessed using resazurin as a viability indicator. Fluorescence intensity was measured at Ex/Em = 540/590 nm using a SpectraMax iD5 microplate reader with SoftMax Pro v7 software (Molecular Devices) after 4 h of incubation in standard cell culture medium containing 0.004% resazurin.
cLip-1 treatment in vivo. Mice were kept under standard conditions with water and food ad libitum and in a controlled environment (22 ± 2 °C, 55 ± 5% humidity, 12 h light/dark cycle). For animal studies, C57BL6/J mice were randomised into separate cages. 12 to 24-week-old sex-matched mice were used for all experiments. For the survival cohort study, Rosa26-CreERT2;Gpx4f/f mice were intraperitoneally treated with tamoxifen (Sigma-Aldrich, T2859, 2 mg/day dissolved in Myglyol 812) on day 0 and day 1 for deletion of Gpx4 in the whole body except in the brain4. From day 2, cLip-1 (10 mg/kg/day dissolved in 1× PBS containing 20% PEG400 and 5% Solutol HS15) or vehicle was intraperitoneally administered to the mice each day until the completion of the survival study. For histochemistry analyses with click chemistry, Rosa26-CreERT2;Gpx4f/f mice treated with intraperitoneal tamoxifen injection (2 mg/day on day 0 and day 1) were used. On day 7, cLip-1 (10 and 100 mg/kg) or vehicle was intraperitoneally injected to the mice. One h after the injection, mice were euthanised and the kidney and liver samples were collected.
Isolation of blood, serum and lymphatic fluid from mice. Balb/C mice (25-week-old adult female mice) were purchased from Charles River and grow in CRCM animal core facility. Mice were housed under sterile conditions with sterilised food and water provided ad libitum and maintained on a 12 h light/dark cycle.Mice were not subjected to any procedures prior to the lymph, blood and serum samples collection. Lymph sample collection:Thirty min before the beginning of the experiment, buprenorphine (Buprecare), an analgesic, was administered by intraperitoneal injection (0.5 mg/kg). Mice were euthanised by intraperitoneal injection of a ketamine/xylazine combination (ketamine 100 mg/kg (Imalgène)/xylazine 10 mg/kg (Rompum); 20 µl/g). After cutaneous and peritoneal incisions, the lymph has been collected in the intestinal lymph trunk53 with a glass capillary. The lymph collection was placed in cryotubes, frozen at -20 °C and stocked at -80 °C. Blood and serum sample collection: After lymph collection, we performed a terminal cardiac puncture (23G needle with 1 to 2 μL syringe) with thoracotomy to collect a large volume of blood without anticoagulants. 100 to 200 µL of blood sample were placed in a vial, frozen at -20 °C and stocked at -80 °C. The rest of the blood collection was left to stand at room temperature for 30 min, then centrifuged at 2000× g for 15 min. The supernatant (serum) was collected in a vial, frozen at -20 °C and stored at -80 °C.
Intranodal murine metastasis models and fentomycin treatment. Murine breast cancer cells (4T1) were transplanted into 6 to 8-week-old female Balb/c mice (syngeneic with the 4T1 model). To perform injections into lymph nodes, the lymphatics were first traced by injecting 2% Evans Blue dye (Sigma-Aldrich, E2129,) into the foot pedal 5 min before performing intranodal injections. After injecting Evans Blue dye, the mice were anesthetised using isoflurane and a small (5–10 mm) incision was made in the region of the right popliteal lymph node. The lymph node was located based on Evans Blue staining, immobilised with forceps, and 20000 cells (Experiment 1) to 10000 cells (Experiments 2 and 3) suspended in 1× PBS were injected in a volume of 10 μL into the popliteal lymph node using a 27 G Hamilton syringe. Injection into the lymph node was confirmed by visible swelling of the lymph node. The incision was closed using surgical glue (3M VetBond Tissue Adhesive, 1469SB,) and the mice were closely monitored for signs of pain or distress. Once tumours were palpable in at least 75% of the mice (~1 week after injection), 10 μL of volume of fentomycin (0.003 mg per animal every-other-day) of vehicle delivered intralymphatically into the tumour-bearing lymph node every other day until the experimental endpoint. Intranodal tumour diameters were measured thrice weekly with calipers until any tumour in the mouse cohort reached 2.0 cm in its largest diameter which was the pre-determined experimental endpoint for these experiments. At that point, all mice in the cohort were killed, per approved protocol, for analysis of intranodal tumour diameter, tumour mass and mouse. Tumour samples were frozen in 10% DMSO in FBS (1 °C/ min until -80 °C) for subsequent cellular analyses. No formal randomisation techniques were used. However, animals were allocated randoμLy to treatment groups and specimens were processed in an arbitrary order. For all experiments, the maximum permitted tumour diameter was 2.0 cm and this limit was not exceeded in any experiment. For all experiments, mice were kept on a normal Chow diet and fed ad-libitum.
Fluorescence microscopy. Cells were plated on coverslips and treated as indicated. BODIPY 665/676 (Thermo Fisher Scientific, B3932, 10 µM, 45 min), LysoTracker Deep red (Thermo Fisher Scientific, L12492, 100 nM, 45 min), SQSS (in-house, 50 nM, 24 h) and 1-Red (in-house, 100 nM, 1 h before fixation) were added to live cells before fixation. For fentomycin and marmycin A treatments, cells were treated at the indicated temperature with 1 µM compound for 1 h. BacMam transduced cells (see transduction section) were treated with BODIPY 665/676 16 h after transduction. Cells were then washed three times with 1× PBS, fixed with 2% paraformaldehyde in 1× PBS for 12 min and then washed three times with 1× PBS. For antibody staining, cells were then permeabilised with 0.1% Triton X-100 in 1× PBS for 5 min and washed three times with 1× PBS. Subsequently, cells were blocked in 2% bovine serum albumin/BSA, 0.2% Tween-20/1× PBS (blocking buffer) for 20 min at room temperature. Cells were incubated with the relevant antibody in blocking buffer for 1 h at room temperature, washed three times with 1× PBS and were incubated with secondary antibodies for 1 h. Finally, coverslips were washed three times with 1× PBS and mounted using VECTASHIELD containing DAPI (Vector Laboratories, H-1200-10). BODIPY 665/676 treated cells were fixed using ice cold reagents and placed at 4 °C immediately after mounting on cover slips and imaged immediately. Fluorescence images were acquired using a Deltavision real-time microscope (Applied Precision) or a thunder microscope (Leica). 40×/1.4NA, 60×/1.4NA and 100×/1.4NA objectives were used for acquisitions and all images were acquired as z-stacks. Images were deconvoluted with SoftWorx (Ratio conservative - 15 iterations, Applied Precision) and processed with FIJI 2.0.0-rc-69/1.52n. Images were taken in black and white and colouring was applied with FIJI. Fluorescence intensity is displayed as arbitrary units (AU) and is not comparable between different panels. Colocalisation quantification was calculated using FIJI 2.0.0-rc-69/1.52n. Nuclei were detected using DAPI or Hoechst fluorescence as indicated.
Small molecule labelling using click chemistry. In-cell click labelling: Cells on coverslips were treated as indicated with cDFO (in-house, 100 µM, 15 min), cLip-1 (in-house, 1 µM or 10 µM, 1 h), metcLip-1 (in-house, 10 µM, 1 h), alcLip-1 (in-house, 10 µM, 1 h), then fixed and permeabilised as indicated in the fluorescence microscopy paragraph. LysoTracker Deep Red was added to live cells for 45 min before fixation. The click reaction cocktail was prepared using the Click-iT EdU Imaging kit (Invitrogen, C10337) according to the manufacturer’s protocol. In a typical experiment we mixed 50 μL of 10× Click-iT reaction buffer with 20 μL of CuSO4 solution, 1 μL Alexa-Fluor-azide, 50 μL reaction buffer additive (sodium ascorbate (Asc)) and 379 µL ultrapure water to reach a final volume of 500 μL. Coverslips were incubated with the click reaction cocktail in the dark at room temperature for 30 min, then washed three times with 1× PBS. Immunofluorescence was then performed as described in the fluorescence microscopy section. Click labelling in tissues: The kidney and liver tissue samples collected from the cLip-1-treated mice were fixed in 4% paraformaldehyde in 1× PBS overnight at 4 °C. Fixed tissues were incubated in 10% sucrose in 1× PBS for 30 min and then incubated in 20% sucrose in 1× PBS at 4 °C for 4 h, followed by embedding in OCT mounting compound (TissueTek, Sakura) on dry ice and storage at -80 °C. Frozen tissues were cut into 5-µm-thick sections using a Cryostat Microm HM 560 (Thermo Fisher Scientific) at -30 °C. Tissue sections were postfixed with 1% paraformaldehyde in 1× PBS for 10 min and subsequently incubated with 100% acetone for 10 min at -20 °C. Sections were incubated in blocking solution (1× PBS containing 3% BSA and 0.2% Triton X-100) for 30 min. The specimens were labelled using a click reaction as described above. For co-staining with a lysosome marker, the click reaction labelled specimens were incubated with anti-Lamp1 antibody diluted in 1× PBS containing 10% normal goat serum overnight at 4 °C. The next day, sections were incubated with secondary antibody in 1× PBS containing 1% BSA and 0.3% Triton X-100 for 2 h at room temperature. Nuclei were visualised with Hoechst 33342 staining, and slides were mounted in Aqua/PolyMount (Polysciences). Images were acquired using a fluorescence microscope (DS-Qi2, Nikon) or confocal microscope (LSM880, Carl Zeiss) and the corresponding appropriate filter sets for fluorophores.
Western blotting. Cells were treated as indicated and then washed with 1× PBS. Proteins were solubilised in 2× LaemμLi buffer containing benzonase (VWR, 70664-3, 1:100). Extracts were incubated at 37 °C for 1 h, heated at 94 °C for 10 min and quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). Protein lysates were resolved by SDS-PAGE electrophoresis (Invitrogen sure-lock system and Nu-PAGE 4-12% Bis-Tris precast gels). In a typical experiment, 10-20 µg of total protein extract were loaded per lane in 2× LaemμLi buffer containing bromophenol blue. On each gel a size marker was run: 3 µL PageRuler (Thermo Fisher Scientific, 26616) or 3 µL PageRuler plus (Thermo Fisher Scientific, 26620) and 17 µL 2× LaemμLi buffer). Proteins were then transferred onto nitrocellulose membranes (Amersham Protran 0.45 μm) using a Trans-Blot SD semi-dry electrophoretic transfer cell (Bio-Rad) using 1× NuPage transfer buffer (Invitrogen, NP00061) with 10% methanol. Membranes were blocked with 5% non-fat skimmed milk powder (Régilait) in 0.1% Tween-20/1× PBS for 20 min. Membranes were cut at the appropriate marker size to allow for the probing of several antibodies on the same membrane. Blots were then probed with the relevant primary antibodies in 5% BSA, 0.1% Tween-20/1× PBS or in 5% non-fat skimmed milk powder in 0.1% Tween-20/1× PBS at 4 °C overnight with gentle motion in a hand-sealed transparent plastic bag. Membranes were washed with 0.1% Tween-20/1× PBS three times and incubated with horseradish peroxidase conjugated secondary antibodies (Jackson Laboratories) in 5% non-fat skimmed milk powder, 0.1% Tween-20/1× PBS for 1 h at room temperature and washed three times with 0.1% Tween-20/1× PBS. Antigens were detected using the SuperSignal West Pico PLUS (Thermo Fisher Scientific, 34580) and SuperSignal West Femto (Thermo Fisher Scientific, 34096) chemiluminescent detection kits. Signals were recorded using a Fusion Solo S Imaging System (Vilber). γ-Tubulin served as loading control on the same gels. Band quantifications were performed with FIJI 2.0.0-rc-69/1.52n using pixel intensity normalised against the signal of γ-tubulin. All full scans of blots are displayed in the Supplementary Information.
Transduction. PDAC053T cells were seeded at 200000 cells per well 24 h prior to the experiment in 1 μL of medium. Cells were then transduced with CellLight Lysosomes-GFP, BacMam 2.0 (Thermo Fisher Scientific, C10596), CellLight ER-GFP, BacMam 2.0 (Thermo Fisher Scientific, C10590), CellLight Mitochondria-GFP, BacMam 2.0 (Thermo Fisher Scientific, C10600) according to the manufacturer’s procedure. In brief, 70 µL of BacMam reagent was added to the medium and mixed. Cells were incubated for 16 h.
Flow cytometry. Cells were washed with ice-cold 1× PBS. For antibody staining, cells were incubated with Fc block (Human TruStain FcX, Biolegend, 422302, 1:20) for 15 min, then incubated with antibodies in ice cold 10% FBS, 1× PBS, 2 mM EDTA for 20 min at 4 °C and then washed with 1× PBS and resuspended in 10% FBS, 1× PBS, 2 mM EDTA before analysis using a flow cytometer. For BODIPY-C11 581/591 staining, live cells were incubated with BODIPY-C11 581/591 (Thermo Fisher Scientific, D3861, 1 µM, 45 min) before fixation. RPE probe flow cytometry: PDAC-053T cells were seeded in 6-well plates at the density of 2× 105 cells/well. On the following day, compounds were added as indicated in the figure for 1 h; ferric ammonium citrate (FAC, Sigma-Aldrich, F5879, 100 µg/μL), Lip-1 (10 µM), hydroxychloroquine (HCQ, Sigma-Aldrich, H0915, 100 µM), Bafilomycin A1 (Baf-A1, Sigma-Aldrich, B1793, 75 nM). After 30 min of treatment with compounds, RPE probe (in-house, 40 µM) was added for 30 min. The media was removed and cells were washed with 1× PBS once before trypsinisation. Cells were harvested, pelleted, washed with 1× PBS and finally 250 µL of 1× PBS buffer containing 10% FBS and EDTA (0.1% v/v), was added for flow cytometry. Data were recorded on a BD LSR Fortessa X-20. BODIPY-C11 581/591 flow cytometry on PDAC-053T cells: PDAC-053T cells were seeded in 6-well plates at the density of 2× 105 cells/well. On the next day, cells were treated with Baf-A1 (75 nM) and HCQ (10 µM) for 7 h before adding RSL3 (200 nM). Then after 16 h, cells were treated with BODIPY-C11 581/591 (4 µM) for an additional 1 h. The media was removed and cells were washed with 1× PBS twice before trypsinisation. Cells were harvested, pelleted, washed with 1× PBS and finally 250 µL of 1× PBS buffer containing 10% FBS and EDTA (0.1% v/v), was added for flow cytometry. Data were recorded on an AttuneTM NxT Flow Cytometer (Thermo Fisher Scientific). For flow cytometry analyses of lysosomal GSH and lysosomal hydroxyl radicals: Cells were incubated with SQSS (100 nM, 24 h) or 1-Red. HT-1080 cells were incubated with RSL3 (1 µM), μL210 (10 µM, Sigma-Aldrich, SμL0521), FIN56 (5 µM, Sigma-Adrich, SμL1740), Buthionine sulfoximine (BSO, 10 µM, Sigma-Aldrich, B2515) or erastin (10 µM, Sigma-Aldrich, 329600) for the indicated time points. Data were recorded on an AttuneTM NxT Flow Cytometer (Thermo Fisher Scientific). For flow cytometry analyses of lysosomal iron content typically 200000 dissociated human tissue cells were incubated in media (RPMI 1610, 10% FBS, penicillin/streptomycin) with RhoNox-M (in-house, 1 mM, 1 h). Lysosomal iron content of human tumour samples and healthy adjacent tissues was analysed with the following antibody and stain panel: DAPI (0.1 µg/μL), CD3-BV510 (BioLegend, 317332), CD31-PE-Cy7 (BioLegend, 303118), CD44-AF647 (Novus Biologicals, NB500-481AF647), CD45-BV785 (BioLegend, 304048), CD163-PerCP/Cyanine5.5 (BioLegend, 326512), FAP-AF700 (R&D Systems, FAB3715N), TfR1-APC-AF750 (Beckman Coulter, A89313). The live tumour cells corresponded to DAPIneg/CD45neg/CD31neg/FAPneg cells. Data were recorded on a BD LSRFortessa X-20. For flow cytometry analyses of CD44 in fentomycin treated tumour samples, typically 200000 dissociated tumour cells were incubated in media (RPMI 1610, 10% FBS, penicillin/streptomycin) with 1 µM fentomycin for 24 h. Cells were pre-treated with 1 µM Lip-1, 1 µM cLip-1, 100 µM Toc or 100 µM Def for 2 h. Toc was kept pure under inert atmosphere and a fresh stock solution was prepared throughout the study before each experiment. The following antibody and stain panel was used for subsequent flow analysis: SYTOX blue (Thermo Fisher Scientific, S34857, 1 µM), CD31-BV605 (BioLegend, 303122), CD45-BV510 (BioLegend, 368526, lot B373428), CD44-AF647 (Novus Biologicals, NB500-481AF647), TfR1-PE (BioLegend, 334106), FAP-AF750 (Novus Biologicals, FAP3715S). The live tumour cells corresponded to SYTOX blueneg/CD45neg/CD31neg/FAPneg cells. Data were recorded on an AttuneTM NxT Flow Cytometer (Thermo Fisher Scientific). For flow cytometry analyses of CD44 levels in mouse 4T1 tumours, typically 200000 dissociated tumour cells were used per condition. Freshly dissociated cells were stained using the following antibody and stain panel: SYTOX blue (Thermo Fisher Scientific, S34857, 1 µM), CD31-BV605 (BioLegend, 102427), CD44-AF647 (BioLegend, 103018), CD45-BV510 (BioLegend, 103138), MHCII-APC/Cyanine7 (BioLegend, 107628). The live tumour cells corresponded to SYTOX blueneg/CD45neg/CD31neg/MCH IIpos cells. Data were recorded on an AttuneTM NxT Flow Cytometer (Thermo Fisher Scientific). All data were analysed with FlowJo software v. 10.10.0.
Fluorescence-activated cell sorting. Sorting of human cells was performed using the following antibodies: CD31-PE-Cy7 (BioLegend, 303118), CD44-AF647 (Novus Biologicals, NB500-481AF647), CD45-BV785 (BioLegend, 304048). The sorted cells corresponded to CD45neg/CD31neg/CD44pos cells and CD45neg/CD31neg/CD44neg cells and were isolated on a FACSAria Fusion (BD). ICP-MS experiments were conducted in CD44pos and CD44neg tumour cells as described in the ICP-MS section. Sorting of murine cells was performed using the following antibodies: CD44-AF647 (Biolegend, 103018), MHCII-APC/Cyanine7 (BioLegend, 107628). The sorted cells corresponded to MHCIIpos/CD44pos cells and MHCIIpos/CD44neg cells. Sorted cells were centrifuged at 300× g and cell pellets were processed for subsequent applications.
Inductively coupled plasma mass spectrometry (ICP-MS). Glass vials equipped with teflon septa were cleaned with nitric acid 65% (VWR, Suprapur, 1.00441.0250), washed with ultrapure water (Sigma-Aldrich, 1012620500) and dried. Cells were harvested and washed twice with 1× PBS. Cells were then counted using an automated cell counter (Entek) and transferred in 200 µL 1× PBS or ultrapure water to the cleaned glass vials. The same volume of 1× PBS or ultrapure water was transferred into separate vials for the background subtraction, at least in duplicate per experiment. For tissue samples, a small piece of about 1 mm3 was transferred into a clean pre-weighed vial. Samples were lyophilised using a freeze dryer (CHRIST, 2-4 LDplus). Vials with tissue samples were weighed subsequently to determine the tissue dry weight. Samples were then mixed with nitric acid 65% and heated at 80 °C overnight in the same glass vials closed with a lid carrying a teflon septum. Samples were then cooled to room temperature and diluted with ultrapure water to a final concentration of 0.475 N nitric acid and transferred to metal-free centrifuge vials (VWR, 89049-172) for subsequent mass spectrometry analyses. Amounts of metals were measured using an Agilent 7900 ICP-QMS in low-resolution mode, taking natural isotope distribution into account. Sample introduction was achieved with a micro-nebulizer (MicroMist, 0.2 μL/min) through a Scott spray chamber. Isotopes were measured using a collision-reaction interface with helium gas (5 μL/min) to remove polyatomic interferences. Scandium and indium internal standards were injected after inline mixing with the samples to control the absence of signal drift and matrix effects. A mix of certified standards was measured at concentrations spanning those of the samples to convert count measurements to concentrations in the solution. Values were normalised against cell number or tissue dry weight.
Lipidomics. For comparison of ferroptosis inducers, HT1080 cells were treated with fentomycin (1 µM), erastin (10 µM), RSL3 (100 nM) or iFSP1 (10 µM, Sigma-Aldrich, SμL2749). For co-treatment with ferroptosis inhibitors, HT1080 cells were pre-treated with Toc (100 µM), Def (100 µM) or Lip-1 (10 µM) for 2 h, and then co-treated with fentomycin (1 µM) for 24 h. Dissociated human tumour samples were pre-treated with 100 µM Toc and then co-treated with 1 µM fentomycin for 24 h. Dissociated mouse tumour samples were counted and processed directly after dissociation. Cells were subsequently washed with 1× PBS and then with 150 mM ammonium bicarbonate. Cells were then resuspended in 150 mM ammonium bicarbonate and centrifuged at 300× g for 5 min. The supernatant was removed and cells were resuspended in 1 μL of 150 mM ammonium bicarbonate. The solutions were centrifuged at 12000 rpm for 10 min and the supernatant was removed. 200 µL of 150 mM sodium bicarbonate was added to the pellet and samples were flash frozen in liquid nitrogen. Lipidomics analysis was performed on the same day for all technical and biological replicates for a given dataset. For lipidomics analysis, the 200 µL cell lysates were spiked with 1.4 μL of internal standard lipid mixture containing 300 pmol of PC 17:0-17:0, 50 pmol of PE 17:0-17:0, 50 pmol of PI 16:0-16:0, 50 pmol of PS 17:0-17:0, and 30 pmol of PA 17:0-17:0, 30 pmol of LPC 12:0, 30 pmol of LPE 17:1, 30 pmol of LPS 17:1 and 30 pmol of LPA 17:0 and subjected to lipid extraction at 4 °C as previously described54. Briefly, the sample was extracted with 1 μL of chloroform:methanol (10:1) for 2 h at 4 °C with vigorous shaking in a thermomixer (1000 rpm). The lower organic phase was collected and dried in a Speedvac vacuum concentrator. The remaining aqueous phase was re-extracted with 1 μL of chloroform:methanol (2:1) for 1 h at the same temperature and shaking conditions. The lower organic phase was collected and evaporated in a SpeedVac vacuum concentrator. Lipid extracts were dissolved in 100 μL of infusion mixture consisting of 7.5 mM ammonium acetate dissolved in propanol:chloroform:methanol [4:1:2 (v/v)]. Samples were analysed by direct infusion in a QExactive mass spectrometer (Thermo Fisher Scientific) equipped with a TriVersa NanoMate ion source (Advion Biosciences). 5 µL of sample were infused with gas pressure and voltage set to 1.25 psi and 0.95 kV, respectively. PC and PCOx were detected in the 10:1 extract, by positive ion mode FTMS as protonated adducts by scanning m/z= 580–1000 Da, at Rm/z=200=280 000 with lock mass activated at a common background (m/z=680.48022) for 30 seconds. Every scan is the average of 2 micro-scans, automatic gain control (AGC) was set to 106 and maximum ion injection time (IT) was set to 50 ms. PE, PEO, PEOx and LPE were detected as deprotonated adducts and LPC were detected as acetate adducts in the 10:1 extract, by negative ion mode FTMS by scanning m/z= 420–1050 Da, at Rm/z=200=280 000 with lock mass activated at a common background (m/z=529.46262) for 30 seconds. Every scan is the average of 2 micro-scans, automatic gain control (AGC) was set to 106 and maximum ion injection time (IT) was set to 50 ms. The following abbreviations are used: PC = phosphatidylcholine, PE = phosphatidylethanolamine, PS = phosphatidylserine, PI = phosphatidylinositol, ox = oxidised. LPC = lysophosphatidylcholine, LPE = lysophosphatidylethanolamine, LPS = lysophosphatidylserine, LPI = lysophosphatidylinositol. PI, PIOx, PS, PSOx, LPI, and LPS were detected in the 2:1 extract, by negative ion mode FTMS as deprotonated ions by scanning m/z= 400–1100 Da, at Rm/z=200=280 000 with lock mass activated at a common background (m/z=529.46262) for 30 seconds. Every scan is the average of 2 micro-scans, automatic gain control (AGC) was set to 106 and maximum ion IT was set to 50 ms. All data was acquired in centroid mode. All lipidomics data were analysed with the lipid identification software, LipidXplorer (http://genomebiology.com/2011/12/1/R8). Tolerance for MS and identification was set to 2 ppm. Data post-processing and normalisation to internal standards were done in Excel (Microsoft).
Glycerol quantification. HT1080 cells were treated with 1 or 2 µM fentomycin for 24 h. Glycerol was quantified using the Glycerol-glo assay (Promega, J3150) according to the manufacturer’s protocol. In brief, the assay was performed in a 96-well plate and 4000 cells were plated per well 24 h prior to the experiment. A standard curve was prepared for each biological experiment and three technical replicates were performed for each condition and each biological replicate. Luminescence signals were recorded using a SpectraMax ID3 plate reader (Molecular Devices). Data were exported and analysed using Excel (Microsoft) and PRISM software.
Software for illustrations.Illustrations were created using FIJI 2.0.0-rc-69/1.52n, Prism 10.0.3 and Adobe Illustrator 26.0.2 and biorender.com. Biorender.com was used for Fig. 2b and Fig. 4.
Quantification, statistical analysis and reproducibility.Results are presented as mean values ± standard error of the mean (s.e.m.) or standard deviation (s.d.) as indicated. Box plots: boxes represent interquartile range and median, and whiskers indicate the minimum and maximum values. Prism 10.0.3 software was used to calculate P values using a two-sided Mann-Whitney test, two-sided unpaired t-test, Kruskal-Wallis test with Dunn’s post-test, 2-way ANOVA or Mantel-Cox log-rank test as indicated. Prism 10.0.3 software was used to generate graphical representations of quantifications unless stated otherwise. Exact P values are indicated in the figures. Sample sizes (n) are indicated in the figure legends.
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