Relationship of extracellular matrix protein 1 level to breast cancer cell proliferation. (A) Each cell line was treated with 200 ng/ml recombinant human extracellular matrix protein 1 (rhECM1) or 5 μg/ml anti-ECM1 antibodies, and cells were counted with a hemocytometer over the course of 3 days (*P < 0.05, **P < 0.005). Cont, Control; shC, Control short-hairpin RNA; shE/shECM1, Short-hairpin extracellular matrix protein 1 RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) Cell cycles were analyzed by flow cytometry (*P < 0.05, **P < 0.005). (C) Each cell line was passaged by subcutaneous injection into the lower flank of each mouse. Tumor volume was measured every 3 days (n = 5 or 6 for each group). (D) Kaplan-Meier analyses for recurrence-free survival, overall survival and distant metastasis-free survival in the HER2-positive breast cancer patients. Data were obtained from the Kaplan-Meier Plotter Breast Cancer database . Overall survival data from breast cancer patients were stratified by ECM1 levels in HER2-positive patient sera (n = 27).