Extended Data Fig. 10: Pro-senescence treatment combined with an mTOR inhibitor suppresses tumour growth in p53-deficient, immunocompetent somatic mouse models of HCC.

a, Schematic of hydrodynamic-tail-vein gene delivery of the Myc proto-oncogene transposon system and a CRISPR–Cas9 vector targeting either Trp53 or the Pten tumour suppressor, used to induce models ofHCC two to three weeks after HDTVi. b, Quantification of SA-β-gal staining performed on frozen sections from mouse models of Myc^OE;Pten^KO or Myc^OE;Trp53^KO HCC, 14 days after treatment with vehicle or XL413 monotherapy (results from Myc^OE;Trp53^KO HCC are also shown in Fig. 4f). For analyses of Myc^OE;Pten^KO tumours, vehicle-treated, n = 9 biologically independent nodules from 3 mice; XL413-treated, n = 16 biologically independent nodules from 3 mice. For analyses of Myc^OE;Trp53^KO tumours, vehicle-treated, n = 41 biologically independent nodules from 7 mice; XL413-treated, n = 81 biologically independent nodules from 11 mice. Graph shows the mean ± s.e.m. of the number of SA-β-gal⁺ cells per tumour nodule per mm². Statistics were calculated by two-sided unpaired Student’s t-test. c, Trial design to evaluate the efficacy of the pro-senescence treatment combined with an mTOR inhibitor in mice bearing Myc^OE;Trp53^KO HCC. Mice were monitored by weekly MRI after HDTVi, and enrolled into treatments with vehicle, XL413 (100 mg kg⁻¹, daily gavage), AZD8055 (20 mg kg⁻¹, daily gavage) or XL413 + AZD8055 combination at the first signs of tumour development (revealed by MRI). Drugs were administered six days per week, and mice were killed when they became symptomatic. Immunohistochemical analyses confirmed MYC expression and p53 knockout in endpoint Myc^OE;Trp53^KO HCC. d, Longitudinal individual-body-weight curves from mice bearing Myc^OE;Trp53^KO tumours, treated with the combination of XL413 + AZD8055. e, Individual tumour-growth curves from mice treated with vehicle, XL413, AZD8055 or a combination of both drugs were calculated on the basis of MRI images from mice bearing Myc^OE;Trp53^KO tumours. f, Volumes of Myc^OE;Trp53^KO tumours from mice bearing HCC, treated with vehicle (n = 5, as shown in Fig. 4c), sorafenib (n = 4) or XL413 + AZD8055 (n = 6) at day 0 and day 14. Graphs show mean ± s.e.m., analysed with two-sided unpaired Student’s t-test. g, h, Representative images of SA-β-gal (g) and p16 (h) staining performed on frozen and paraffin-embedded sections, respectively, from mice bearing Myc^OE;Trp53^KO tumours, treated with the indicated drugs and killed at the intermediate time point (14–16 days in time-matched treated cohorts). Quantifications are shown in Fig. 4f, g. Scale bar, 50 μm. i, Mice bearing Myc^OE;Trp53^KO tumours, treated with vehicle, XL413, AZD8055 or a combination of both drugs were killed at the indicated time point after treatment. Tumours were dissociated as single-cell suspensions, and flow cytometry analyses were performed to determine the content of tumour-associated macrophages (CD45⁺ CD11b⁺Ly6C⁻Ly6G⁻), CD8 T cells (CD45⁺CD3⁺CD19⁻NK1.1⁻CD8⁺) and CD4 T cells (CD45⁺CD3⁺CD19⁻NK1.1⁻CD4⁺) relative to total CD45⁺ leucocytes. Cell proliferation (Ki67⁺) was determined within CD8 T cells and CD4 T cell populations. Graphs show mean ± s.e.m., analysed with two-sided unpaired Student’s t-test. Sample sizes are given in Methods. j, Mice bearing Myc^OE;Trp53^KO HCC were treated with XL413 (n = 20) or XL413 + AZD8055 combination (n = 8) for 14 days. Among the XL413-treated mice, a subset (n = 10) was killed at 14 days after treatment, concomitantly with the group treated with the combination of drugs. The rest of the XL413-treated mice (n = 10) underwent withdrawal of XL413 for 4 days. The absolute number of senescent cells per tumour nodule were visualized by SA-β-gal staining, performed on frozen sections and quantified for each treatment group (XL413-treated, n = 60 biologically independent nodules from 10 mice; XL413-withdrawn, n = 63 biologically independent nodules from 10 mice; XL413 + AZD8055-treated, n = 57 biologically independent nodules from 7 mice). Graphs show mean ± s.e.m. analysed with two-sided unpaired Student’s t-test. Data in c are representative of three independent biological experiments.

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