In vivo tumour studies

All studies were performed under an animal research ethics project license that was approved by the UK Home Office and in accordance with institutional welfare guidelines.

Kras^LSL-G12D/+;Trp53^fl/fl mice (KP) were sourced from the Mouse Models of Human Cancer Consortium and maintained on a pure C57BL/6 background. Kras^LSL-G12D/+; Trp53^fl/fl;Rosa26^A3Bi mice (KPA) and Kras^LSL-G12D/+Trp53^fl/fl;Rosa26^A3Bi;Rag1^-/- mice (KPAR) were generated by breeding KP mice with Rosa26^A3Bi mice and Rag1^-/- mice (see Supplementary Methods for development and validation of the Rosa26::LSL-A3Bi model). Tumours were induced by intratracheal intubation of 1x10⁶ adenovirus expressing Cre recombinase as previously described (13). Tumour volume was assessed via micro-CT scanning.

For the urethane-induced models, tumours were induced by 3 intra-peritoneal injections of 1mg/g of urethane over the period of a week. Three weeks following urethane first injection, APOBEC3Bi was induced by 3 doses of 100mg/g tamoxifen over a period of a week in Rosa26^{A3Bi/CreER(t2)} mice (UrA3Bi-CreER). Tumour volume was assessed via micro-CT scanning.

All transplantation animal experiments were carried out using 8-12-week C57BL/6 mice. For subcutaneous studies, 1.5x10⁵ KPAR1.3 or KPAR1.3^G12C cells and 5x10⁵ KPB6^G12C cells (1:1 mix with Matrigel) were injected subcutaneously into the flank. Tumour volume was measured twice weekly and calculated using the formula 0.5 x [Length x Width²]. Mice were euthanised when the average tumour dimensions exceeded 1.5 mm. For re-challenge experiments, tumour-free mice were injected subcutaneously into the opposite flank with 1.5x10⁵ KPAR1.3 tumour cells. For orthotopic studies, 1.5x10⁵ KPAR1.3 cells, 1x10⁵ KPAR1.3^G12C and KPB6 cells were injected intravenously into the tail-vein. Mice were euthanised when the humane endpoint of 15% weight loss was reached.

For treatments, 200μg anti-PD-1 (clone RMP1-14, BioXcell) and 200μg anti-CTLA-4 (clone 9H10, BioXcell), or their respective IgG controls, were administered per mouse via intraperitoneal injection twice weekly for a maximum of three weeks. Anti-PD-L1 (clone 10F.9G2, BioXcell) or the respective IgG control were administered at 10 mg/kg via intraperitoneal injection twice weekly, for two weeks.

AZ-8037 or vehicle (10% Pluronic-F127) was administered 5 days per week via oral gavage at 100mg/kg. Mice were randomised into groups and treatments initiated once tumours reached an average volume of 150mm³ for subcutaneous studies or were detectable by micro-CT for orthotopic experiments.