MMP-1 functionally contributes to HEp3-hi/diss intravasation and metastasis. MMP-1 functionally contributes to HEp3-hi/diss intravasation and metastasis.

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MMP-1 functionally contributes to HEp3-hi/diss intravasation and metastasis. MMP-1 functionally contributes to HEp3-hi/diss intravasation and metastasis. A, long-term downregulation of MMP-1 expression in HEp3-hi/diss cells by siRNA treatment. Western blot analysis for the levels of MMP-1 produced by HEp3-hi/diss cells treated with control siRNA or MMP-1–specific siRNA constructs. Serum-free conditioned medium was harvested at the indicated time points after siRNA treatments. Sample of recombinant MMP-1 (rMMP-1), containing both proenzyme and activated enzyme species, is shown on the left along with positions of molecular weight markers. B, rescue of HEp3-hi/diss intravasation and metastasis by exogenous MMP-1. HEp3-hi/diss cells treated with control and MMP-1–specific siRNAs were grafted on the CAM of day 10 embryos. On day 1 and 3 after cell grafting, a fraction of primary tumors developing from MMP-1–deficient cells were topically supplemented with 100 ng of human recombinant MMP-1, alone or premixed with 0.5 mmol/L GM6001. Tumor weights and levels of CAM intravasation and liver metastasis were determined on day 5 after cell grafting. Data from individual embryos were analyzed against the mean of control group (100%) in 3 independent experiments, each using from 6 to 11 embryos per treatment. Bars are mean ± SEM. *, P < 0.05; ***, P < 0.001, respectively; two-tailed Student t test. C, supplementation of exogenous MMP-1 induces intravasation and metastasis of HEp3-lo/diss variant. On day 1 and 3 after grafting of HEp3-lo/diss cells on the CAM, the developing tumors were treated topically with 100 ng of human recombinant MMP-1. On day 5, primary tumors were excised and weighed (left) and intravasation to the distal CAM (middle) and metastasis to the liver (right) were quantified by Alu-qPCR. Data from individual embryos were analyzed against the mean of control group (100%) in 3 independent experiments, each using from 7 to 8 embryos per treatment. Bars are mean ± SEM. *, P < 0.05; **, P < 0.01; two-tailed Student t test. Anna Juncker-Jensen et al. Cancer Res 2013;73:4196-4211 ©2013 by American Association for Cancer Research