Extended Data Fig. 9: The combination of lenvatinib and gefitinib suppresses tumour growth without toxicity in immunocompetent mouse models in vivo.

a, Western blot analysis of total EGFR levels in mouse liver tissues and mouse liver cancer cells (Hepa1-6). Representative of three independent experiments. b, Hepa1-6 cells were treated with lenvatinib, the EGFR inhibitor gefitinib or their combination at the indicated concentrations. The cells were fixed and stained after 7 days. Representative of three independent experiments. c, After intrahepatic inoculation of Hepa1-6 cells in C57BL/6 mice, mice were treated with vehicle, lenvatinib (4 mg kg⁻¹), gefitinib (80 mg kg⁻¹) or lenvatinib (4 mg kg⁻¹) plus gefitinib (80 mg kg⁻¹) for 2 weeks. Tumour weight was measured. Data are mean ± s.e.m. n = 6 mice per group. P values were determined by two-sided unpaired Student’s t-test. d, Body weights of mice in the Hepa1-6 orthotopic models with the aforementioned treatments were assessed. n = 6 mice per group. Data are mean ± s.e.m. e, Schematic of gene delivery by hydrodynamic tail vein injection (HDTVi) of the Myc proto-oncogene transposon system and a CRISPR–Cas9 vector targeting the Trp53 tumour suppressor, which was used to induce HCC 2–3 weeks after hydrodynamic tail vein injections. f, Western blot analysis of total EGFR levels in mouse liver tissues and mouse liver cancer cells (Myc^OETrp53^KO). Representative of three independent experiments. g, Myc^OETrp53^KO mouse liver cancer cells were treated with lenvatinib, EGFR inhibitor gefitinib or their combination at the indicated concentrations, respectively. The cells were fixed and stained after 5 days. Representative of three independent experiments. h, Survival curve generated from mice bearing Myc^OETrp53^KO tumours, treated with vehicle (n = 6; median survival of 11 days), lenvatinib (4 mg kg⁻¹; n = 9; median survival of 23 days), gefitinib (80 mg kg⁻¹; n = 6; median survival of 13.5 days) or lenvatinib plus gefitinib (n = 9; median survival of 31 days). P values were determined by two-sided log-rank test. i, Body weights of mice in the Myc^OETrp53^KO mouse liver cancer models with the aforementioned treatments were assessed. n = 6–9 mice per group. Data are mean ± s.e.m. j, k, Mice bearing Myc^OETrp53^KO tumours treated with vehicle, lenvatinib (4 mg kg⁻¹), gefitinib (80 mg kg⁻¹) or a combination of both drugs were killed at the end point after treatment. Tumours were dissociated as single-cell suspensions, and flow cytometry analyses were performed to determine the content of tumour-associated lymphoid cells (NK cells, CD8⁺ T cells, CD4⁺ T cells and regulatory T cells (Tregs)) and myeloid cells (monocytes, neutrophils, tumour-associated macrophages (TAMs) and dendritic cells (DCs)). Data are mean ± s.e.m. P values were determined by two-sided unpaired Student’s t-test. Sample sizes are given in the Methods

Source data.