Fig. 6

TGF-β1 secreted by UM cells activates microenvironmental HSCs to release collagen type Iα which in turn ligates cell surface DDR1 on UM cells. a Expression of TGF-β1 was analyzed by qRT-PCR assay in UM cells and ARPE-19 cells. Data are shown as the mean ± SD (n = 3). b ELISA analysis of TGF-β1 in the CM of UM cells and ARPE-19 cells is shown. Data are shown as the mean ± SD (n = 3). c–e qRT-PCR analysis of collagen Iα1 (c), collagen Iα2 (d) and collagen IV (e) in HSCs after treated with recombinant TGF-β1 (10 ng/mL) for indicated time periods or CM isolated from 92.1 and Mel270 cells for 6 h, then replaced with fresh HSC medium for 24 h. Data are shown as the mean ± SD (n = 3). f, g HSCs were cultured in the presence of 10 ng/mL recombinant TGF-β1 at different time points or the CM derived from 92.1 and Mel270 cells for 6 h, then replaced with fresh HSC medium for 24 h, The cells were pelleted by centrifugation to extract whole-cell lysates for western blotting analysis (f) and the corresponding supernatants were analyzed levels of pro-collagen I using ELISA assay (g). Data are shown as the mean ± SD (n = 3). h, i HSCs were treated with recombinant human TGF-β1 (10 ng/mL), or UM-derived CM in the presence or absence of neutralizing anti-TGF-β1 antibody or SB525334 (TGFβRI inhibitor) for 6 h, then replaced with fresh HSC medium for another 24 h. The collagen type Iα in the resultant medium was determined by ELISA assay (h). The resultant medium or HSC-derived CM was added into the culture of 92.1 and Mel270 cells for 12 h, and then subjected to western blotting analysis of DDR1 and its signaling molecules (i). Data are shown as the mean ± SD (n = 3). j–n After 5 × 10⁵ Mel270-luc cells were intrasplenically inoculated, the NOG mice were administered with vehicle (ddH2O:DMSO:EtOH:Cremophor EL = 90:2:4:4), 7rh (25 mg/kg, orally), SB525334 (30 mg/kg/day, i.p.) alone or combination 7rh (25 mg/kg, orally) with SB525334 (30 mg/kg/day, i.p.) every day for 21 days (n = 5 per group). j Representative images of luciferase signals on day 21 after treatment with vehicle, 7rh, SB525334 alone or combination 7rh with SB525334 (left). Quantitative analysis of photon flux for hepatic metastases in NOG mice was performed every week (right). Data are shown as the mean ± SD (n = 5). The mice were sacrificed to count metastatic nodules on liver surface. Representative images (k) and quantitative analysis (l) of liver nodules are shown. Data are shown as the mean ± SD (n = 5). m, n Metastasis nodules in paraffin sections of liver tissue from each group were identified by H&E staining. Scale bar: 500 µm (40×), 200 µm (100×). Data are shown as the mean ± SD (n = 3). o Collagens were detected by Masson trichrome staining in paraffin sections of liver tissue. Scale bar: 200 µm (100×). p A proposed working model is shown. UM cells secrete TGF-β1 which induces quiescent hepatic stellate cells (qHSCs) into activated HSCs (aHSCs) which secretes collagen type I in turn activates DDR1, strengthening survival through upregulating STAT3-dependent Mcl-1, strengthening stemness via upregulating STAT3-dependent SOX2, and clonogenicity in cancer cells and ultimately promotes liver colonization and metastasis in UM. *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA, post hoc comparisons, Tukey’s test for results in (a–e, g, h, j, l and n)