Extracellular matrix protein 1 confers resistance toward trastuzumab. (A) Cells were seeded with Matrigel and treated with trastuzumab (Ttzm; 20 μg/ml) and recombinant human ECM1 (rhECM1; 200 ng/ml). The number of colonies 20 μm or greater in diameter was counted at 12 days (*P < 0.05, **P < 0.005). WT, Wild type. (B) At 24 hours after cell seeding, each cell line was treated with anti-ECM1 antibody (5 μg/ml) and Ttzm (20 μg/ml) in fresh medium. After a further 48 hours, cell viability was analyzed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (*P < 0.05, **P < 0.005, ***P < 0.0005). (C) BT-474 vector and ECM1 cells and BT-474 Ttzm-resistant (TR) control (Cont) short-hairpin (shRNA; shC) and ECM1 shRNA (shE) cells were passaged by subcutaneous injection into the lower flank of each mouse. When the tumor size increased up to 250 mm3, Ttzm at 20 mg/kg was administered to each mouse by intraperitoneal injection twice per week (n = 5 or 6 for each group). (D) Circulating levels of ECM1 in serum from TR breast cancer patients were assessed by enzyme-linked immunosorbent assay (left) and Western blot analysis and compared with (right) corresponding data for Ttzm-responsive patients (*P < 0.05).