Figure 4

Met phosphorylation and DNA synthesis in triple-negative breast cancer cells in the presence of gefitinib. Co-culture with hepatocyte growth factor (HGF)-expressing fibroblasts increases Met phosphorylation and DNA synthesis in triple-negative breast cancer cells in the presence of gefitinib. (A) SUM102 cells were labeled with CytoTracker green and were co-cultured with RMF-HGF cells at different ratios. Lysates were analyzed for phosphorylation of Met by immunoblotting. β-actin and total Met protein expression were used as controls. Numbers below the immunoblots represent relative densitometry measurements of the ratio of pMet to total Met. (B) RMF-HGF fibroblasts were plated on coverslips. SUM102 and SUM149 cells were labeled with CytoTracker green and plated on top of the RMF-HGF fibroblasts at the indicated ratios. Then 1.0 μM gefitinib (gef) was added to the cells for 24 hours. SUM102 cells were incubated with BrdU for 18 hours and SUM149 cells were incubated with BrdU for 4 hours. Cells were fixed and stained using anti-BrdU-Alexa-Fluor-595. SUM102 and SUM149 cells labeled green were counted as BrdU-positive or BrdU-negative. Student's t test was used to determine statistical significance between gefitinib-treated cells with or without co-cultures. *SUM102, P = 0.0471; SUM149, P = 0.0028. UT, untreated; 0/10, fibroblast to tumor cell ratio; 10/10, fibroblast to tumor cell ratio.