Extended Data Fig. 4: Recombinant AREG mimics tumour cell-derived AREG phenotype in vivo.
From: Radiation-induced amphiregulin drives tumour metastasis
a, H&E staining of lungs 7 days post i.v. injection of 1 × 105 LLCAR+ and LLCAR− tumour cells. Black arrows indicate metastatic lesions. b, Number of lung metastases (left), and mean size of five largest metastases per lung area (right), n = 4 mice per group. Unpaired two-sided t-test. c, H&E staining of lungs 14 days post i.v. injection of 1 × 105 LLCAR+ and LLCAR− tumour cells. Black arrows indicate metastatic lesions. d, Number of lung metastases (left), and mean size of five largest metastases per lung area (right), n = 4 mice per group. Unpaired two-sided t-test. e, Number of metastases per lung (left) and mean size of largest metastases per lung area after ± intravenous (i.v.) recombinant AREG (rAR) treatment (right), n = 5 in LLCAR+ + rAR group, n = 4 mice in all other groups. One-way ANOVA, Šídák’s test. f, Growth curve of LLC flank tumours ± 20 Gy RT or complete surgical resection of the flank tumour on day 11, 14, and 18, n = 4 mice in 20 Gy, n = 5 in all other groups. g, AREG concentration in serum of tumour-bearing mice five days after 20 Gy RT or complete surgical resection of the flank tumour, measured by ELISA, n = 3 mice in 20 Gy, n = 5 in all other groups. One-way ANOVA, Šídák’s test. h, Number of lung metastases (left - One-way ANOVA, Šídák’s test), and mean size of the five largest metastases per lung area (right – unpaired two-sided t-test), n = 4 mice in 20 Gy, n = 5 in all other groups. i, H&E staining. Black arrows indicate metastatic lesions. j, Dot plot of Gene Set Enrichment Analysis (GSEA) comparing upregulated Hallmark pathways in RNA-seq of LLCAR+ + 4 Gy. k, Volcano plot of up- and downregulated genes in LLCAR+ cells + 4 Gy RT. Significantly upregulated differentially-expressed genes (DEGs) involved in type-I-IFN pathway are coloured in red. DEGs determined by Wald test, Benjamini-Hochberg FDR correction (DESeq2). l, Relative expression of Areg in LLCAR+ cells after interferon β (IFNβ) treatment, measured by RT-qPCR. One-way ANOVA, Dunnett’s test. n = 3 technical repeats. m, AREG concentration in LLCAR+ cells after IFNβ treatment, measured by ELISA. Kruskal-Wallis, Dunn’s test. n = 3 technical repeats. n, Relative expression of Areg in LLC cells post-RT and siSTAT2 treatment, measured by RT-qPCR. One-way ANOVA, Šídák’s test. n = 3 technical repeats. o, AREG concentration in LLCAR+ cells after 4 Gy RT and siSTAT2 treatment, measured by ELISA. One-way ANOVA, Šídák’s test. n = 3 technical repeats. Data are shown as mean ± SEM; box plots show median (Q2), Q1–Q3, and whiskers (min–max).
