Cell lines
KP LUAD cell lines were obtained from the laboratory of T. Jacks. KP, Fsp1KO cell lines were generated by transient transfection of PX458 (Addgene #48138) expressing an sgRNA targeting Fsp1. Single GFP-positive clones were selected and FSP1 loss was validated by western blot. The 16645 cell line was developed from KrasG12D Stk11−/− GEMM as previously described52. KPC7 cells were obtained from the laboratory of D. Simeone. All cell lines were maintained in DMEM or RPMI 160 (Corning) supplemented with 10% FBS (Sigma Aldrich) and gentamicin (Invitrogen) and were tested for mycoplasma regularly (PlasmoTest, InvivoGen). All mouse cell lines were authenticated by PCR genotyping. All human cell lines used were purchased from ATCC and were authenticated by short-tandem-repeat profiling. Genetic manipulation of cell lines was performed via lentiviral transduction of plasmids, detailed in next section, followed by either puromycin (7 μg ml−1) or hygromycin (800 μg ml−1) selection for 1 week.
Cloning/lentivirus generation
CRISPR–Cas9-mediated knockout of target genes was achieved by cloning sgRNAs into pLenti-USEC or lentiCRISPRv2-puro vectors, as previously described31. In short, backbones were digested with Esp3I (New England Biosciences) and purified with a gel extraction kit (QIAGEN). sgRNAs were designed using CRISPick (Broad Institute), obtained from Integrated DNA Technologies (Coralville), annealed, phosphorylated, Esp3I-digested, and ligated into the purified digested backbones using Quick Ligase (New England Biosciences). Double sgRNA ultramers were designed and generated as previously described38. In brief, ultramers were Gibson-assembled to digested pDonor_sU6 (Addgene #69351), Esp3I-digested, and ligated to purified digested backbones. Guide RNA sequences used to make gene knockouts can be found in Supplementary Data 4. GPX4, mouse FSP1 and human FSP1 expression plasmids were generated using Gibson assembly of the respective cDNA into pLenti-v2-filler.
Lentivirus was generated by co-transfection of HEK293 cells with a viral vector and packaging plasmids psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) using PEI transfection reagent. Cell supernatant containing lentivirus was collected 72 h after transfection and filtered through 0.45-µm PVDF filters. For in vivo experiments, lentivirus was concentrated by ultracentrifugation at 25,000 rpm for 2 h at 4 °C. The viral pellet was resuspended in PBS and stored at −80 °C until use. Viral titre was quantified with the use of a Cre-dependent GreenGo reporter cell line. For in vitro experiments, medium containing virus was collected, filtered, and added directly to recipient cells with polybrene at 8 µg ml−1 for 48 h before selection.
Mouse models
All mouse experiments described in this study were approved by the NYU Institutional Animal Care and Use Committee (IACUC). Mice were housed according to IACUC guidelines in ventilated caging in a specific pathogen-free animal facility. For all mouse studies, ≥4 mice were used for each experimental condition. KrasLSL-G12D/+; Tp53fl/fl; Rosa26LSL-Cas9/LSL-Cas9 (KPC GEMMs) mice were bred as previously described31,32,33,34,35. C57BL/6 J (JAX strain 000664) mice with the appropriate genotype, aged 8 to 12 weeks were randomly selected to begin tumour initiation studies with pUSEC lentivirus. Care was taken to ensure each experimental arm had an equal number of male and female mice. Mice were intratracheally infected with lentiviruses as described and monitored until experimental endpoint. Tumour burden was quantified by H&E staining and analysed using QuPath software as a measurement of total tumour area/total lung lobe area. All quantifications were done with investigator blinded to the respective sample genotypes. All transplantation experiments were performed using nude (JAX strain 002019), NOD SCID Gamma (NSG; JAX strain 005557 F), C57BL/6 J Fsp1-knockout (Conrad group16) or C57BL/6 J wild-type (JAX strain mice aged 8 to 12 weeks old). For mouse cell xenograft experiments, 100,000 cells in 100 µl of phosphate-buffered saline (PBS) was injected subcutaneously into each flank of the mouse. For the xenograft studies in Fig. 3 the number of cells injected per flank and whether they were injected with 50:50 PBS and Matrigel (Corning) are indicated as follows: H2009 (2 million cells + Matrigel), H1299 (1 million cells), PC9 (1 million cells), H1975 (2 million cells + Matrigel), A549 (1 million cells), 16645 (500,000 cells), KPC7 (250,000 cells). All human cell line xenograft experiments were carried out in male NSG mice unless specified. For the PDX experiment, tumours were implanted subcutaneously in male NSG mice as previously described34. Tumours were measured with callipers, and volume was calculated based on 0.5 × length × width2. The maximum tumour diameter permitted by our IACUC protocol was 2 cm, and this was not exceeded in any experiment. For orthotopic lung tumour experiments, 100,000 luciferase-expressing cells in 200 μl of PBS were injected intravenously into tail vein of male mice unless specified in the legend. Tumour growth was measured by bioluminescence (PerkinElmer IVIS Spectrum In Vivo Imaging System; D-luciferin, PerkinElmer 122799). Data were analysed using Living Image software.
Antioxidant and drug treatments
For LIP1 treatment, mice were dosed with 10 mg kg−1 LIP1 (BOC Sciences) or vehicle (2% DMSO + 40% PEG300 + 2% Tween 80 in sterile H 2 O) by intraperitoneal injection for frequency and duration indicated in figure schematics. For icFSP1 treatment, mice were dosed with 50 mg kg−1 icFSP1 (WuXi LabNetwork) or vehicle (45% PEG300 in sterile PBS) by intraperitoneal injection twice daily. High (TD.2108412) and low (TD.210841) irradiated vitamin E diets were obtained from Inotivco and provided ad libitum for length of time indicated in figure legends. In all experiments, mice were randomly assigned to treatment group.
Cell clonogenic and viability assays
... continue reading