Hepatocyte growth factor protects triple-negative breast cancer cells from gefitinib treatments. (A) SUM102 and SUM149 cells were plated at 35,000 cells/well of a six-well plate on day 0. Under normal growth conditions, 50 ng/ml hepatocyte growth factor (HGF) and/or 0.5 μM gefitinib was added to the cells on day 1 and subsequently every other day. Cells were counted on days 1, 4, and 8 using a Coulter Counter. (B) SUM102 and SUM149 cells under normal growth conditions were treated with increasing concentrations of gefitinib in the presence or absence of 50 ng/ml HGF for 7 days. Cells were trypsinized and replated at 4,000 cells/35-mm dish and grown under normal growth conditions for 7 days. Colonies were stained with crystal violet and counted using a cell counter. The percentage of surviving cells was calculated by dividing the number of colonies on the treated plate by the number of colonies on the untreated plate. (C) Under normal growth conditions, SUM102 and SUM149 cells were plated in a layer of agar sandwiched between two layers of agar of different percentage. Every other day for 3 weeks, 50 ng/ml HGF and/or 0.5 μM gefitinib were added to the top layer in a final volume of 300 μl. Colonies were stained with p-iodonitrol and counted using a cell counter. Each experiment was performed in triplicate at least three times. Student's t test was used to determine significance between gefitinib-treated and gefitinib + HGF-treated cells. *P < 0.05. UT, untreated.