An in vitro migration assay was performed as described in Materials and Methods. migration and invasion. The MMP-induced FNIII14 exposure was also shown to be functional in the migration and invasion of highly metastatic mouse breast cancer cell collection 4T1. Overexpression and knockdown experiments of eEF1A in Mum2B cells revealed that this migration and invasion were dependent on the membrane levels of eEF1A. experiments using tumor xenograft mouse models derived from Mum2B and 4T1 cell lines showed that this anti-FNIII14 Ab has a significant PF6-AM anti-metastatic effect. Thus, these results provide novel insights into the regulation of malignancy cell migration and invasion and suggest promising targets for anti-metastasis strategies. [30]. The gelatinolytic bands were quantified with image J software. eEF1A knockdown and overexpression Reverse transfection was performed for transient knockdown and overexpression of human eEF1A (GenBank Accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001958″,”term_id”:”1732746393″NM_001958). For eEF1A knockdown, cells were transfected with Sigma ultra siPerfect unfavorable control (Merck-Sigma-Aldrich) or eEF1A siRNA using Lipofectamin RNAiMAX (Thermo Fisher Scientific, Waltham, MA) according to the manufacturers instructions. The following eEF1A siRNAs were used: sense, 5-AGGAGAAGACCCACAUCAATT-3; antisense, 5-UUGAUGUGGGUCUUCUCCUTG-3 (Hs_EEF1A2_6 FlexiTube siRNA; Qiagen). For eEF1A overexpression, human eEF1A cDNA was amplified by PCR using following primers: forward, 5-CCAGCCCCTCACACTCCCAG-3; reverse, 5-TACCTCCGCATTTGTAGATGA-3. Cells were transfected with vacant pCMV vector or pCMV vector encoding a HA-tagged eEF1A using Opti-MEM medium (Thermo Fisher Scientific) and FuGENE HD transfection reagent (Promega, Madison, WI) according to the manufacturers instructions. These cells were used for subsequent experiments 48 hours after transfection. In vivo metastasis assay KSN/SLC mice (6 weeks aged) and BALB/c mice (5 weeks aged) were obtained from Japan SLC (Shizuoka, Japan). All animals were cared for in accordance with the guidelines set out by Tokyo University or college of Science. Animal protocols were approved by the Animal Care Committee of Tokyo University or college of Science. For the evaluation of melanoma metastasis, Mum2B cells (2 106 PF6-AM cells/mouse) suspended in serum-free RPMI-1640 made up of fibronectin (1 g/mL) was subcutaneously injected into the right hind footpads of 7-week-old male KSN/SLC mice. Immediately after the implantation, 3 mice each was randomly assigned to 2 groups, control group received normal rabbit IgG (100 g/mouse) pHZ-1 or treated group received anti-FNIII14 Ab (100 g IgG/mouse). The antibodies dissolved in PBS were injected intraperitoneally to the mice on days 10, 15, 20 and 25 after the implantation. Tumor sizes at the implanted site were measured once every five days after implantation. On day 30 after the implantation, the mice were sacrificed. The lungs and subiliac lymph nodes were excised and subjected to the evaluation of metastasis by detecting human genomic DNA in these tissues, as follows. Purified genomic DNA from your lungs and subiliac lymph nodes was isolated with the GenElute Mammalian Genomic DNA Miniprep Kit (Merck-Sigma-Ardrich) accordance to the manufacturers instructions. The following primers were used to amplify the human protein tyrosine phosphatase receptor type C genomic DNA (experiments were positively correlated with their invasive potential, which has been confirmed by experiments using a xenograft model [36]. Both the migratory and invasive capabilities of highly invasive Mum2B cells were much higher than those of low invasive Mum2C cells (Physique 1A and ?and1B1B). Open in a separate windows Physique 1 Cryptic anti-adhesive site FNIII14 stimulates melanoma migration and invasion. A. Comparison of in vitro migratory capability between Mum2B and Mum2C cells. An in vitro migration PF6-AM assay was performed as explained in Materials and Methods. Representative phase contrast images (left, scale bar = 100 m) and migration distances (right) are shown at 0 and 6 hours after scratching. B. Comparison of in vitro invasive capability between Mum2B and Mum2C cells. An in vitro invasion assay was performed as explained in Materials and Methods. Representative phase contrast images (top, grid = 100 m squares) and the numbers of cells invaded (bottom) are shown at 6-hour (Mum2B) or 24-hour (Mum2C) culture. C. Inactivation of 1-integrin by de-adhesive peptide FNIII14 in Mum2B cells. Mum2B cells were incubated with MnCl2 (1 mM) in the presence or absence of peptide FNIII14 (50 g/mL). Control is usually untreated cells. Activation status of 1-integrin was evaluated by circulation cytometry using an anti-1-integrin monoclonal antibody, AG89, which recognizes the active 1-integrin conformation-specific epitope. Representative data of three individual experiments is usually shown. D. Detection of the de-adhesive site FNIII14 in uncovered state within the fibronectin substrate. Mum2B cells were subjected to adhesion assay in the presence or absence of normal rabbit IgG (Control IgG, 12.5 g/mL) or anti-FNIII14 Ab (Ani-FNIII14, 12.5 g IgG/mL). E. Suppression of Mum2B cell migration.