GoKawiilThe key reagents and resources required to replicate our study are listed in Supplementary Table 2.
Mouse colony management
Animal experimentation was carried out in accordance with the Animals (Scientific Procedures) Act 1986 (UK) and with the approval of the Cancer Research UK Cambridge Institute Animal Welfare and Ethical Review Body: the maximum approved tumour burden was 10% body weight, which was not exceeded. Animals were maintained using standard husbandry: mice were group-housed in Tecniplast GM500 IVC cages with a 12 h–12 h light–dark cycle (07:00–19:00) and ad libitum access to water, food (LabDiet 5058) and environmental enrichments.
The following mouse strains and species were used: M. musculus domesticus C3H/HeOuJ (C3H mice) and C57BL/6J (BL6), M. musculus castaneus CAST/EiJ (CAST) and M. caroli CAROLI/EiJ (CAROLI; Supplementary Table 2). For simplicity, these strains, subspecies and species are here referred to as ‘strains’.
Chemical model of hepatocarcinogenesis
We treated 15-day-old (P15) male mice of all strains with a single intraperitoneal injection of DEN (N0258, Sigma-Aldrich; 20 mg kg−1 body weight) diluted in 0.85% saline. This is a well-established tumour-induction protocol and therefore DEN treatment was not randomized or blinded. Injections were performed in a fixed-time interval between 08:00 and 09:00 to control for circadian effects in DEN metabolism. Liver tumour samples were collected from DEN-treated mice 25 weeks (C3H), 36 weeks (BL6), 38 weeks (CAST) or 78 weeks (CAROLI) after treatment; pilot data indicated that 100% of surviving DEN-treated mice would develop tumours by these timepoints. The time of tumour removal was recorded for DEN-exposed BL6 mice, with 5-min intervals added for each sequential tumour isolated (Extended Data Fig. 9a–e). Our existing cohort of 370 C3H tumours25 included multiple tumours per animal and multiple animals per litter. To capture this hierarchical structure across genetic backgrounds, we included a minimum of 50 tumours per strain.
Untreated control mice from each strain were assessed for the presence or absence of tumours at the same ages as DEN-induced tumours to assess inherent susceptibility to spontaneous tumours (Fig. 1a and Supplementary Table 3). In addition, spontaneous liver tumours were collected from untreated mice identified opportunistically as part of routine colony health surveillance (including one female mouse; Fig. 1b and Supplementary Table 3). All macroscopically identified tumours were isolated and processed in parallel for DNA and RNA extraction and histopathological examination. Additional tissues from untreated P15 mice (ear, tail and liver), untreated age-matched adult mice (liver only; C3H 27 weeks, BL6 38 weeks, CAST 40 weeks and CAROLI 80 weeks) and DEN-treated background (non-tumour) liver (BL6 38 weeks) were sampled for control experiments.
Tissue collection and processing
Liver tumours of sufficient size (2 mm or larger diameter) were bisected; one-half was flash frozen in liquid nitrogen and stored at −80 °C for DNA and RNA isolation, and the other half was processed for histology. Tissue samples for histology were fixed in 10% neutral-buffered formalin for 24 h, transferred to 70% ethanol, machine processed (Leica ASP300 Tissue Processo) and paraffin embedded. All formalin-fixed paraffin-embedded sections were 3 μm in thickness.
Histochemical staining
... continue reading