1. ADAM8 knockdown profoundly decreases orthotopic tumor formation, CTC spread and metastasis A–DStable ADAM8 KD (shA8) clones were characterized in vitro.
ADAM8 knockdown profoundly decreases orthotopic tumor formation, CTC spread and metastasis A–DStable ADAM8 KD (shA8) clones were characterized in vitro. ADAM8 expression in WCEs from two shA8 clones (shA8‐17 and shA8‐20) were compared with two shCtrl clones (shCtrl‐3 and shCtrl‐5) using WB (LSBio antibody) (A). Clones were grown in 2D‐cultures for 48 h and subjected to an ATP assay. NS: non significant, Student's t‐test, n = 3 (B). Migration assays were performed as described in Fig 3B. *P = 6.8E‐14, Student's t‐test, n = 3 (C). Matrigel outgrowth assays were performed as in Fig 3D. Experiments were done twice with similar results. Bar: 100 μm (D). E–HMDA‐MB‐231 derived shCtrl‐3 and shA8‐20 cells were injected into the MFP of female mice (n = 7/group). Tumor volume was measured twice a week (mean ± s.e.m.). *P = 1.4E‐6, **P = 9.8E‐8, #P = 3.0E‐5, ##P = 9.7E‐7, †P = 1.7E‐5, ‡P = 5.1E‐6, §P = 2.0E‐7, Student's t‐test (E). At the end of the experiment, tumors were photographed and weighed (mean ± s.e.m.). *P = 5.6E‐8, Student's t‐test. Bar: 1 cm (F). Blood was collected by cardiac puncture and GFP‐positive CTCs were detected by flow cytometry. Average CTC count ± s.d. is given per μl of blood. *P = 0.006, Student's t‐test (G). Presence of brain metastases was examined by fluorescent microscopy (n = 6/group). Representative photographs are shown. Bars: 1 mm (H). IADAM8 expression in distant metastases of breast cancer patients (n = 56) was analyzed by immunohistochemistry. Representative pictures and percentages of ADAM8‐positive samples are presented.
Mathilde Romagnoli et al. EMBO Mol Med. 2014;6:
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