GoKawiilMice and in vivo studies
KrasLSL-G12D/+Trp53flox/flox mice (designated as KP)60 were maintained on a mixed C57BL/6-129/Sv genetic background. Lung tumours were induced in young KP (2–3 months old; referred to as KP-Young) and old KP (18–19 months old, referred to as KP-Old) mice through intratracheal instillation with Lenti-Cre as described in ref. 61 or with Ad5mSPC-Cre viral particles (PFU 2 × 107; University of Iowa; VVC-Berns-1168) under general anaesthesia as described in ref. 10.
NXG (NOD Xenograft Gamma) mice (NOD-Prkdcscid-IL2rgTm1/Rj) were obtained from Janvier Laboratories and were 6–10 weeks old at the start of the experiments. All transplantation studies were conducted using age-matched cohorts of mice. For subcutaneous implantations, a total of 2.5 × 105 of green fluorescent protein (GFP)-luciferase-expressing versions of the indicated cells suspended in PBS were subcutaneously injected into the lower right and lower left flanks of NXG mice. Tumours were measured every other day by callipers and the tumour volume was calculated using the formula volume = (length × width2)/2. At no point during the study did any tumour exceed the permitted maximal measurements of 2 cm × 2 cm or endpoint criteria.
For intravenous injections, a total of 5 × 104 of the indicated cells were injected into the lateral tail vein of NXG mice. All transplantation experiments were performed in young immunodeficient NXG hosts to minimize confounding by host age or immunity.
For CB-839 studies, mice were randomized and subjected to treatment with either 200 mg kg−1 body weight CB-839 (no. HY-12248, MedChemExpress) or vehicle. Treatments were administered twice a day every other day following either the tumour-establishment phase (subcutaneous implantations) or within 6 h after the bloodstream injection of cells. The vehicle control consisted of 25% (w/v) 2-hydroxypropyl-β-cyclodextrin in 10 mM citrate buffer (pH 2.0).
For doxycycline-induced Atf4 silencing, 1 mg ml−1 of doxycycline (no. HY-N0565B, MedChemExpress) was administered in the drinking water supplemented with 5% sucrose. For ISRIB (no. HY-12495, MedChemExpress) studies, indicated cells were pretreated 3 times a day for 7 days with either ISRIB (2 µM) or vehicle (dimethylsulfoxide, DMSO) and then injected into mice. ISRIB was administered at 10 mg kg−1 twice daily by intraperitoneal injection or vehicle alone (45% saline, 50% PEG 400, 5% DMSO).
Same-sex mice were housed in individually ventilated cages, under a 12–12 h light–dark cycle, with ambient temperature (15–21 °C) and humidity control (45–70% relative humidity), enrichment material and ab libitum rodent chow and water. All mouse experiments described in this study were approved by the Research Animal Ethics Committee in Gothenburg (2071/19; 2077/19 and 6057/24).
IVIS imaging
Mice were injected intraperitoneally with 30 mg ml−1 of d-luciferin (no. 15225733, Fisher Scientific) and organs were imaged ex vivo 10 min after injection. Luminescence was quantified as constant, or as total flux (p s−1). Analysis was performed with Living Image v.4.7.4 software, maintaining the region of interest over the tissues at a constant size. The radiance (p s−1 cm−2 sr−1) from the region of interest was normalized against the background radiance.
Histology and IHC analyses
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