Fig. 4: Silencing DDR1 suppresses the metastatic colonization of UM cells in liver.

a Experimental schematic of liver metastasis model. b DDR1 knockdown in Mel270-luc and Omm2.3-luc cells stably transduced with Scramble or shDDR1 lentivirus was confirmed by Western blotting. c, d Mel270-luc and Omm2.3-luc cells stably transduced with Scramble or shDDR1 were intrasplenically inoculated in NOG mice for 3–4 weeks (n = 5 per group). Liver metastasis was analyzed by luciferase-based bioluminescence imaging system. Representative images (c) and quantitative analysis (d) of photon flux on day 21 are shown. Data are shown as the mean ± SD (n = 5). e, f The mice were sacrificed to count metastatic nodules on liver surface. Representative images (e) and quantitative analysis of liver surface nodules (f) are shown. Data are shown as the mean ± SD (n = 5). g Quantitative analysis of micrometastases in H&E staining liver sections from Scramble and shDDR1 mice. Data are shown as the mean ± SD (n = 3). **P < 0.01, Student’s t test for results in (d, f, g). h Mel270 cells transduced with lentiviral vector (pTSB) or construct encoding human STAT3 cDNA (pTSB-STAT3) were exposed to 10 μM 7rh for 24 h, and subjected to western blotting analysis with the indicated antibodies. i DDR1 knockdown and STAT3 overexpression was confirmed by western blotting in Mel270 cells stably expressed shDDR1 with or without STAT3 overexpression. j Experimental schematic diagram showing the location of STAT3-binding sites of SOX2 regulatory region. P1, P2 represent STAT3-binding sites at the SOX2 gene promoter. P3 was used as negative control and located in intronic region. ISG15 as a known non-target gene of STAT3 served as a negative control (top). ChIP-PCR analysis for STAT3 occupancy at the SOX2 gene promoter in Mel270 cells stably expressed shDDR1 with or without STAT3 overexpression (bottom). Data are shown as the mean ± SD (n = 3). k Enforced expression of STAT3 attenuated the 7rh-mediated decrease in percentage of ALDH⁺ cells in UM cells. Mel270 cells stably expressed with STAT3 were treated with or without 10 μM 7rh for 24 h, and then subjected to ALDH⁺ cells analysis by flow cytometry. Quantitive analysis of ALDH⁺ cells from three independent experiments is shown. Data are shown as the mean ± SD (n = 3). l Overexpression of STAT3 reversed the 7rh-mediated decrease in melanosphere growth and serially-replating capacity in UM. Mel270 cells stably expressed with STAT3 were treated with or without 10 μM 7rh for 24 h, and then subjected to melanosphere-formation assay. Data are shown as the mean ± SD (n = 3). m–q Mel270-luc cells stably transduced with lentiviral vector (pTSB) or constructs encoding human STAT3 cDNA (pTSB-STAT3) underwent intrasplenic injection in NOG mice, the mice were then administrated with vehicle (ddH2O:DMSO:EtOH:Cremophor EL = 90:2:4:4) or 7rh (25 mg/kg in vehicle, orally) every day for 21 days (n = 5 per group). Liver metastasis was analyzed by luciferase-based bioluminescence imaging and liver surface nodules were counted. Representative images were taken on day 21 post injection of cells (m) and quantitative analysis of bioluminescence intensity (n) are shown. Data are shown as the mean ± SD (n = 5). o Representative bright field images of liver surface and quantitative analysis of surface metastatic nodules on liver are shown. Data are shown as the mean ± SD (n = 5). p, q Microscopic observation of H&E staining in liver paraffin section from each group verified metastastic nodules. Data are shown as the mean ± SD (n = 3). Scale bar: 500 µm (40×), 200 µm (100×). *P < 0.05; ***P < 0.001, one-way ANOVA, post hoc comparisons, Tukey’s test for results in (j, k, l, n, o and q)