GoKawiilMice
Sftpc–CreERT2 (028054), R26R–Confetti (013731), Pdgfrα–CreERT2 (032770), R26R–iDTR (007900), NOD/Scid Il2rg null Tg (NSG: 005557) and Ai6/RCL–ZsGreen (007906) animals were obtained from The Jackson Laboratory. AregloxP/loxP animals were kindly provided by M. Clatworthy from the University of Cambridge. Red2Kras mice were generated in-house as previously described22. CCR2–CreERT2 mice were kindly provided by B. Becher (University of Zurich)58. All transgenic mouse strains were maintained on a C57BL or C57BL/6Brd-Tyr 597 c-Brd mixed background. Mouse studies in the UK were approved under UK Home Office Project Licences PC7F8AE82 and PP3176550, and experiments in the US and Korea were approved by the Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee (protocol no. 24-04-003) and Gwangju Institute of Science and Technology (GIST) Institutional Animal Care and Use Committee (protocol no. GIST-2022-043). All procedures complied with institutional and national guidelines. The mice were housed under specific pathogen-free conditions at the Gurdon Institute (University of Cambridge), MSKCC and GIST on a 12-h light/dark cycle with food and water provided ad libitum.
Both male and female mice aged 6–15 weeks were used. Experiments were randomized where feasible. Blinding was not performed, as treatment effects on tumour volume were readily distinguishable between groups. Humane end points were defined as a single tumour exceeding 2 cm in diameter, a tumour burden exceeding 10% of body mass or multiple tumours with a cumulative volume greater than 3,000 mm3. As this study focused on early tumour development, these limits were not approached or exceeded in any experiment.
Mouse procedures
Tamoxifen administration
Tamoxifen (Sigma; T5648) was dissolved in corn oil (Sigma; C8267) at 20 mg ml−1. Aliquots were heated to 50 °C and vortexed before administration. Animals were weighed and received tamoxifen by oral gavage. They received either a single dose (0.1 mg g−1 body weight) for clonal analysis or two to four doses (0.2 mg g−1 body weight) administered every other day. Tissue collection time points are specified in the relevant figures and detailed in Methods.
MRTX1133 administration
Red2Kras animals received two doses of tamoxifen through oral gavage (0.2 mg g−1 body weight) every other day to induce KrasG12D expression. At 4 weeks post-induction, the mice received freshly prepared MRTX1133 (MedChemExpress; HY-134813) through intraperitoneal injection at 15 mg kg−1 twice daily for 10 days. The stock solution was prepared in DMSO and diluted in 40% polyethylene glycol 300 (PEG300; MedChemExpress; HY-Y0873), 5% Tween-80 (MedChemExpress; HY-Y1891) and 45% phosphate-buffered saline (PBS) for injection, as recommended by the manufacturer.
Gefitinib administration
Red2Kras animals received two doses of tamoxifen through oral gavage (0.2 mg g−1 body weight) every other day to induce KrasG12D expression. Four days after the final tamoxifen dose, the mice received freshly prepared gefitinib (80 mg kg−1 in 50 μl of DMSO) or DMSO (vehicle control) through intraperitoneal injection every 4 days for 20 days.
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