Fig. 5 | Signal Transduction and Targeted Therapy

Fig. 5

From: Activation of transmembrane receptor tyrosine kinase DDR1-STAT3 cascade by extracellular matrix remodeling promotes liver metastatic colonization in uveal melanoma

Fig. 5

UM cells educate hepatic stellate cells (HSCs) to secret collagen I. a Schema of mono-cultured HSCs or co-cultured HSCs with UM cells. I, mono-cultured HSCs with HSC-specific culture medium; II, mono-cultured HSCs with RPMI 1640 culture medium; III, directly co-culture HSCs mixed 1:1 with UM cells for 24 h; IV, HSCs were co-cultured with UM cells in Transwell systems for 24 h; V, UM cells-derived conditioned medium (CM) were added to culture starved HSCs for 6 h and replaced with HSCs specific medium for another 24 h. b, c CM were isolated from HSCs grown in mono-culture or co-cultured with 92.1 or Mel270 cells as indicated approaches (a), and then added into the culture of HSCs for 24 h, the marker of activated-HSCs α-SMA was determined by immunofluorescence assay (b) and western blotting (c). Scale bar: 10 µm. d DDR1 is required for melanosphere-formation in UM cells stimulated by HSCs-derived CM. 92.1 and Mel270 cells stably expressing Scramble or shDDR1 were cultured with HSCs-derived CM and then subjected to melanosphere assay. The medium of UM cells was added to culture starved HSCs for 6 h and replaced with HSCs specific medium for another 24 h, which collected as HSCs-derived CM. Data are shown as the mean ± SD (n = 3). e The CM from HSCs that were educated by UM cells activated DDR1 kinase in UM cells. The HSCs-derived CM was added into the culture of 92.1 and Mel270 cells for different times, and then phosphorylation of DDR1 and its downstream signal phosphorylation of STAT3 were analyzed by western blotting. f ELISA evaluation of the collagen type Iα was performed in various CM collected from different culture systems as illustrated in (a). Data are shown as the mean ± SD (n = 3). g HSCs were infected with lentiviral Scramble or specific shRNA targeting collagen type I, and verified by western blotting (top). The level of collagen type Iα in the CM harvested from HSCs that were stably transduced with lentiviral Scramble or shCol I were measured by ELISA assay (bottom). Data are shown as the mean ± SD (n = 3). h The medium of 92.1 or Mel270 cells was added to culture starved HSCs expressing Scramble or two shRNAs against Collagen I for 6 h and replaced with HSCs specific medium for another 24 h, which collected as Scramble HSC CM, shCol I #1 HSC CM, and shCol I #2 HSC CM, respectively. The indicated HSC-derived CM and recombinant collagen type I (10 μg/mL) were used for melanosphere assay in 92.1 or Mel270 cells. Data are shown as the mean ± SD (n = 3). i Western blotting was conducted after 92.1 or Mel270 cells incubation with recombinant collagen type I (20 μg/mL) or Scramble HSC CM, shCol I #1 HSC CM, and shCol I #2 HSC CM as illustrated in (h) for 24 h. j After UM cells treated with 20 μg/mL collagen I for different durations, the phosphorylation of DDR1 and STAT3 were examined by western blotting. k 92.1 and Mel270 cells stably transduced lentiviral Scramble or shDDR1 were cultured in the absence or presence of recombinant human collagen type I (10 μg/mL) to evaluate melanospheres formation. Data are shown as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA, post hoc comparisons, Tukey’s test for results in (d, f, g, h and k)

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