Extended Data Fig. 1: Justification for and validation of the genetics and genetic induction approach employed in the suite of GEMMs.
From: Human-correlated genetic models identify precision therapy for liver cancer
(a) Dose finding for clonal induction using AAV.TBG.Cre in a R26-LSL-tdTomato reporter mouse model. Experimental scheme for b-d. (b-d) Decreasing doses of AAV.TBG.Cre lead to decreased recombination of the LSL and thus less RFP+ hepatocytes (b-c) Quantification of RFP+ hepatocytes by sex and dose. n = 4 mice throughout except n = 3 for 5×109 female (single sample excluded due to inconsistent RFP staining). Data shown as mean ± s.e.m.. GC = genomic copies. (d) Representative images of immunofluorescent staining demonstrating exclusive and dose-dependent targeting of hepatocytes by AAV.TBG.Cre. Individual Channels for the zone 3 marker glutamine synthetase (GS, yellow), HNF4a (magenta), RFP (green), DAPI (blue). Scale bar equals 100 µm. (e) AAV.TBG.Cre sex-dependent clonal induction variation over time. Experimental scheme for f-i. (f) Quantification of RFP+ hepatocytes using GS shows clonal induction within zone 3 and outside zone 3 but no significant zonal expansion over time using a dose of 6.4*108 GC/mouse. n = 5 (male d3 + d7, female d3 + d7), 8 (female d5), 9 (male d5). Data shown as mean ± s.e.m. Two-way ANOVA with Tukey correction (g) Male mice recombine at a higher rate than female mice after induction with 6.4*108 GC/mouse with no additional residual recombination from 5 to 7 days post induction. n = 5 (male d3 + d7, female d3 + d7), 8 (female d5), 9 (male d5). Data shown as mean ± s.e.m., Kruskal-Wallis test with Dunn’s correction (h) Representative images of Cre-driven recombination rates in males and females on d3, d5, and d7 post induction; GS (green), HNF4a (magenta), RFP (yellow) and DAPI (blue). Scale bar equals 50 µm. (i) Summary of mouse cohorts used in j-l. (j) A lower induction rate in females leads to a lower tumour burden compared to males with the same mutational background. n = 28 (Cohort 5) and 19 (Cohort 6) mice. Data shown as mean ± s.e.m. Unpaired two tailed t-test. (k) Lower tumour burden due to a lower induction rate causes a prolonged survival in female mice compared to males with the same mutational background. n = 53 (male, Cohort 5) and 22 (female, Cohort 6) mice. Log rank test. (l) Mutational burden and induction dose influence tumour penetrance and survival outcomes. n = 11 (Cohort 1), 8 (Cohort 2), 14 (Cohort 3), 9 (Cohort 4), 53 (Cohort 5 – same data as k), 3 (Cohort 7). Log rank test. All panels: GC = genomic copies. Please note that individual cohort survival data shown for Cohort 5 and 6 are also shown in Extended Data Fig. 2a to allow direct comparison with data in that figure. (m) Analysis of the TCGA PanCancer dataset shows odds ratio for co-occurrence and mutual exclusivity of modelled HCC driver genes. n = 353. One-sided Fisher Exact Test, */**/*** denote p ≤ 0.05/0.01/0.001 respectively. (n) Mutual exclusivity of drivers is fluid and can change depending on tumour stage as shown for CTNNB1 and TP53; reanalysis of data from Nault et al.86. n = 73 (BCLC 0), 404 (BCLC A), 157 (BCLC B), 101 (BCLC C). One-sided Fisher Exact Test. (o) Genotyping of bulk end stage tumour in representative cohorts. Genotyping by cohort and allelic recombination within individual mice (bars) – representative mice are aligned between alleles within cohorts. Comparison is made to known colony genotype testing presence/absence of allelic recombination to prediction displayed as false/true positives/negatives. Recombination accuracy denotes true positive + true negative/positive + negative. Cdkn1a and Cdkn2a were constitutive knockouts in the relevant colonies.
