(B) imaging of the accumulation of Flamma 675 NIR dye-labeled NLN, NEW, or control peptide in the tumors and other organs isolated from mice 6 h after peptide injection. binding to CD44v6-high cells was inhibited by the knockdown of CD44v6 gene expression and competition with an anti-CD44v6 antibody. A pull-down assay with biotin-labeled peptides enriched CD44v6 from cell lysates. NLN and NEW induced CD44v6 internalization and inhibited hepatocyte growth factor-induced c-Met internalization, c-Met and Erk phosphorylation, and cell migration and invasion. In mice harboring tumors, intravenously administered NLN and NEW homed to the tumors and inhibited metastasis to the lungs. When combined with crizotinib, a c-Met inhibitor, treatment with each peptide inhibited metastatic growth more efficiently than each peptide or crizotinib alone. In addition, KLAKLAKKLAKLAK pro-apoptotic peptide guided by NLN (NLN-KLA) or NEW (NEW-KLA) killed tumor cells and inhibited tumor growth and metastasis. No significant systemic side effects were observed after treatments. Conclusions: These results suggest that NLN and NEW are promising metastasis-inhibiting peptide therapeutics and targeting moieties for CD44v6-expressing metastases. whole-body and fluorescence imaging of tumor homing of peptides Mice for animal experiments were purchased from Orient Bio Inc. (Seongnam, Korea) and Mouse monoclonal to IL-1a managed in conformance with the Guidelines of the Institutional Animal Care and Use Committee of Kyungpook National University (permission no. 2014-1-121). A total of 1 1 106 MDA-MB231 cells were subcutaneously injected into the right flank of each 6-week-old female BALB/c nude mouse. When the tumor reached a volume of approximately 100 mm3, the mouse was injected intravenously with Flamma 675 NIR fluorescence dye-labeled peptides (1 mg/kg body weight). Whole-body fluorescence imaging was performed under inhalational anesthesia using an IVIS imaging system (Perkin Elmer). After imaging, each mouse was euthanized, the tumor and control organs (liver, kidney, spleen, heart, and lung) were isolated, and images were obtained using the IVIS imaging system. Anti-tumor therapy using experimental tumor metastasis model A mouse model of lung metastasis of breast cancer was prepared by injecting 1 106 MDA-MB231-luc cells into 6-week-old female BALB/c nude mice via tail vein. To monitor the localization of the tumor cells Clorprenaline HCl in the lungs, the mice were injected intraperitoneally with D-luciferin at a dose of 150 mg/kg body weight and subjected to a whole-body bioluminescence imaging using IVIS Clorprenaline HCl imaging system (Perkin Elmer) after a Clorprenaline HCl 10-min resting period. Mice (= 10 per group) were randomly assigned to groups based on the luminescence intensity. At 1 h after tumor cell injection, tumor-bearing mice received intravenous injections of CD44v6-binding peptides through the tail vein (14.2 mg/kg body weight, thrice weekly for 3 weeks) alone or in combination with orally administered crizotinib in 5% dimethyl sulfoxide (DMSO) (25 mg/kg of Clorprenaline HCl body weight, twice weekly for 3 weeks) as previously described 28, 29. Metastatic tumor growth after treatments was monitored by measuring the total photon flux (quantity of photons/second) in the whole body using the IVIS imaging system. The body weights of mice and tumor ulceration were monitored throughout the treatment period. At the end of the treatment period, half of the mice (= 5 per group) were utilized for the collection of blood, sacrificed, the lungs were harvested and weighed, and the numbers of metastatic tumor nodules in the lungs were counted. The remaining mice (= 5 per group) were maintained until death to determine the survival rates. For the analysis of hematological parameters, 1 mL of blood was collected Clorprenaline HCl from each mouse and separated into 500 L aliquots used to prepare serum and plasma. Serum was obtained by centrifuging clotted blood at 4 C twice, followed by filtration (pore size: 0.22 m). Plasma was obtained by centrifuging ethylenediaminetetraacetic acid-treated samples. Hematological parameters and liver and kidney function markers were measured by DGMIF (Daegu, Korea). Anti-tumor therapy using spontaneous tumor metastasis model 4T1-luc cells (1 106 cells) were orthotopically inoculated into the mammary excess fat pads in 6-week-old female BALB/c mice. Panc-1 cells (1.