Cell lines
The KP cells used here were established previously66. Atf4 and Lcn2 knockout cell lines were generated by transient transfection of PX458 (Addgene, 48138) expressing guides targeting either Atf4 or Lcn2. The list of sequences and primers is included in Supplementary Table 4. Single GFP-positive clones were selected, and gene of interest (GOI) loss was validated by western blot or ELISA. B16F10 cells were a gift from the I. Aifantis laboratory. Cells were cultured in RPMI with 10% fetal bovine serum (FBS) and gentamicin. mLCN2 and hLCN2 KP cells were selected and maintained in hygromycin-supplemented medium (800 μg ml−1). Commonly available cell lines have been authenticated using short tandem repeat (STR) profiling. All cells were mycoplasma-negative.
The mouse KPC7 pancreatic cancer cell line, derived from a tumour in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre mice, was used. This cell line is maintained on a C57BL/6 background.
Doxycycline-induced knockdown of Lcn2 was achieved by cloning hairpin targeting Lcn2 into the pLKO.1-TETON-Puro vector (Addgene plasmid 21915). In brief, hairpins were designed according to the Genetic Perturbation Platform (Broad Institute), and the annealed hairpin is ligated into the EcoRI+AgeI–digested backbone with Quick Ligase (NEB) at a 3:1 insert:vector molar ratio. Sequences and primers are listed in Supplementary Table 4. Vectors were transduced into cells through lentivirus and selected with puromycin (8 μg ml−1). Oligos were obtained from Integrated DNA Technologies.
Mouse models
All experiments were approved by the New York University (NYU) Institutional Animal Care and Use Committee (IA16-01627). KrasLSL-G12D/+;Trp53fl/fl;Rosa26LSL-Cas9-P2A-GFP/LSL-Cas9-P2A-GFP (KPC) mice have already been described55,66,67,68,69,70,71. Mice aged six to eight weeks of both sexes with the appropriate genotype were selected to begin tumour initiation studies with the bifunctional lentiviruses (pUSEC), which express Cre recombinase and sgRNAs for conditional CRISPR–Cas9-based loss of Atf4 (sgAtf4) or control (sgTom). The sgRNAs used are listed in Supplementary Table 4. For the experiments with the Lcn2-loss GEMM, we used a double-guide lentiviral system with a control vector carrying sgNeo-1 and sgNeo-2, and KPC mice with Lcn2 loss infected with lentivirus carrying sgLcn2-1 and sgLcn2-2 guides. The total lung area occupied by each tumour was measured on haematoxylin and eosin (H&E)-stained slides using NIS-Elements software (Nikon). Transplant experiments were performed using nude (JAX strain 002019), NOD scid gamma (NSG; JAX strain 005557) or C57BL/6J (JAX strain 000664) mice. Cells (100,000 cells in 100 μl PBS) were injected s.c. into each flank of the mouse. B16F10 melanoma cells (2 × 105) were injected s.c. into female mice aged four to six weeks. LLC cells (5 × 105) were injected s.c. into female mice aged four to six weeks. Tumours were measured by caliper, and tumour volume was calculated according to the formula 0.5 × length × width2. Tumours were not allowed to grow more than 2 cm in any linear measurement, and did not exceed that limit in any of the experiments performed. To generate orthotopic lung tumours, KP cells expressing luciferase–GFP were injected intravenously (100,000 cells in 100 μl PBS) into male C57BL/6J (JAX strain 000664) mice, and tumour burden was measured by bioluminescence (Perkin Elmer IVIS Spectrum in vivo imaging system, d-luciferin, Perkin Elmer 122799). Data were analysed using Living Image software. Sample sizes are provided in the figure legends. The minimum sample size was predetermined on the basis of the stochastic variability expected in the individual model, and taking into consideration the difference in the expected effect magnitude. When possible, a higher number of mice was used to reduce waste and increase confidence. Precise numbers are provided in the figure legends.
For the PDAC orthotopic model experiments, 8- to 12-week-old female C57BL/6 mice were used. Syngeneic KPC7 cells, with or without Lcn2 knockout (Lcn2KO), were orthotopically implanted (200,000 cells per mouse) into the mice, and tumour growth was monitored weekly using ultrasound. Five weeks after implantation, the mice were euthanized, primary tumours were collected and the tissues were formalin-fixed for further IHC analysis.
In addition, KPC7 cells were orthotopically implanted (200,000 cells per mouse) into C57BL/6 mice. When the tumour size reached 100 mm3, the mice were randomized into two groups to receive either a control vehicle (Ctr) (n = 10) or anti-mLCN2 (n = 10) treatment by intraperitoneal (i.p.) injection. Pancreatic tumour burden was quantified by ultrasound imaging before and after the drug treatment. After the treatment, the mice were euthanized, and tumour tissues were collected for IHC studies. All experiments were approved by the NYU Institutional Animal Care and Use Committee (IA16-01627).
CRISPR screen using a library of sgRNAs targeting ATF4-controlled genes
More information on library cloning is provided in a previous report23. In brief, the oligo pool from a reconstituted oligonucleotide library synthesis (OLS) library was single-amplified with barcoded primers and inserted into an Esp3I-digested pUSEPB (U6-sgRNA-EFS-Puro-P2A-TagBFP) vector72. The screen included a total of 3,240 sgRNAs targeting 470 ATF4-controlled genes, 25 essential genes and 279 unique non-targeting Ctr sgRNAs (Supplementary Table 1). KP cells with stable expression of Cas9–blasticidin were generated by introducing the lentivector pLX_311-Cas9, which expresses blasticidin resistance from the SV40 promoter and Cas9 from the EF1a promoter, as described73. For the in vitro screen, at least 3.3 × 106 cells were maintained throughout the experiment to ensure a representation of at least 1,000×. A total of 6 × 107 cells were transduced with the sgRNA library in 10-cm dishes at a multiplicity of infection of 0.3, then selected with 10 μg ml−1 puromycin for 30 h. After this, the medium was refreshed for 24 h for medium without puromycin, after which cells were collected, counted and used for screening in vitro and in vivo. A fraction of cells representing 1,000× coverage were pelleted and stored at −80 °C (t0 population). Cells were passaged every 2–3 days, and 3.3 × 106 cells were plated after each passage. After 10 population doublings (t10), 3.3 × 106 cells were pelleted and stored at −80 °C. For the in vivo screen, the ATF4 CRISPR library was divided into two sub-libraries, containing 1,626 and 1,614 sgRNAs, respectively. KP cells were infected and selected in a similar manner as for the in vitro screen, and 1.6 × 106 were injected s.c. into the flanks of C57BL/6J (n = 4) and NSG (n = 4) mice. Twelve days after s.c. transplantation, tumours were resected and genomic DNA was isolated using lysis buffer (50 mM Tris, 50 mM EDTA, 1% SDS, pH 8.0) supplemented with 30 μl of 20 mg ml−1 proteinase K (QIAGEN, 19131) that was added to tissue (up to 200 mg) or cells (up to 30 million cells) and incubated at 60 °C overnight. This was followed by phenol:chloroform:isoamyl alcohol (25:24:1) phase separation, isopropanol precipitation and ethanol wash. Five micrograms of in vitro and 10 μg of in vivo samples’ genomic DNA were used for screen deconvolution with NEB Q5 High-Fidelity 2X Master Mix (M0492L). Integrated sgRNAs were amplified by PCR to attach sequencing adapters and barcodes, as described previously23. PCR products of around 248 bp in length were size-selected on 2% agarose and purified using the QIAquick Gel Extraction Kit (QIAGEN, 28704). Samples were sequenced on an Illumina NextSeq 500 (75-nt single-end reads) at NYU Genome Technology Center. sgRNA dropout results were identified using the MAGeCK algorithm74.
... continue reading