Fig. 4. Function-blocking humanized anti-ITGA5 monoclonal antibody M200 specifically inhibits breast cancer cell adhesion and spreading to fibronectin and blocks MDA-B02 cell colonization in the bone marrow.

A MDA-B02 cells treated or not treated with increasing concentrations of M200 (50, 150, and 250 μg/ml) were allowed to adhere for 1 h to human fibronectin, type I collagen, or vitronectin. Attached cells were then fixed, stained, and counted under microscope. Data are expressed as mean ± SEM of three separate experiments. ***p < 0.0001. B Representative images of paxillin immunofluorescent labeling of focal adhesion contacts (black spots at the edge of the plasma membrane) in MDA-MB-231 and MDA-B02 cells treated with a control IgG or M200 (250 μg/ml) that attached and spread to fibronectin, type I collagen, or vitronectin. C Effect of increasing concentrations of control IgG or M200 (50, 150, and 250 μg/ml) on MDA-B02 cell migration through 8-µm diameter pore-size inserts coated with fibronectin. Data are the mean ± SEM of three separate experiments. **p < 0.001; ***p < 0.0001. D Top-left panel: schematic representation of the treatment protocol. MDA-B02 cells were inoculated intra-arterially to Balb/c nude mice. Animals received a treatment with a control IgG or M200 (15 mg/kg) every other day, starting 1 day before tumor cell inoculation. Seven days after tumor cell inoculation, animals were culled, and the bone marrow collected for tumor cell colony assays. Bottom panel: representative images of tumor cell colonies in the bone marrow from animals treated with M200 or the control IgG.