Gi254023x

Mice were purchased from the Laboratory Animal Center, Army Medical University (Chongqing, China). To establish mouse orthotopic models, MB428 and MB913 cells were injected into the cerebellum of 6-week-old NOD-SCID mice at 1 × 10⁵ cells per mouse (n = 7). The tumorigenesis and metastasis were monitored by bioluminescent imaging on Day 14, Day 28, and Day 42 using In Vivo Imaging System (IVIS) Spectrum (Perkin-Elmer, USA). The mouse survival was observed for up to all the animals dead. NOD-SCID mouse models established with MB428 cells as above were used for drug studies. On the 7th day after implantation, animals were intraperitoneally injected with Adam10 inhibitor, GI254023X (Selleck; formulated in 10% DMSO in 0.1 M carbonate buffer), 100 mg/kg/day, 5 days per week, for two weeks. Control groups were given identical volume of vehicle instead of GI254023X. Xenograft growth and metastasis were monitored by bioluminescent imaging using In Vivo Imaging System (IVIS) Spectrum (Perkin-Elmer, USA) at 7, 14, and 21 days. The animal experiments were approved by the Institutional Animal Care and Use Committee of Army Medical University (AMUWEC20201439).

Human ovarian cancer cell line A2780 were kindly gifted by the Cancer Research Institute, Zhejiang Cancer hospital (Hangzhou, China). Human ovarian cancer cell lines SKOV3 were purchased from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China). These cells were authenticated by DNA(STR) profiling by iCell Bioscience Inc (Shanghai, China). SKOV3 was cultured with McCoy 5 A, HEK293T cell was cultured with DMEM, IOSE80 AND A2780 was cultured with RMPI 1640 medium. Human peritoneal mesothelial cells were isolated from the tumor-free omentum of patients without malignancy according to the methods described in another study [17 (link)]. Primary peritoneal mesothelial cells were cultured on collagen-coated plates with RPMI-1640 media. All media were supplemented with 1% penicillin-streptomycin and 10% FBS and incubated in 37 °C humidified atmosphere with 5% CO2. Cell Proliferation and Toxicity Detection Kit (CCK-8, CK101-01, DIB Data Inventory Biotechnology) were used according to manufacturer’s instruction. ADAM10 inhibitor GI254023X (S8660) was purchased from Selleck. F-actin was visualized with Actin-Tracker Red-Rhodamine (C2207S, Beyotime).