Fig. 3

Creation of acquired trastuzumab-resistant cell models and characteristics of CXCR4 expression. To create cell models of acquired trastuzumab resistance, we continuously exposed BT474 and SKBR3 cells to trastuzumab (20 µg/ml) for at least 1 year. Tolerances to trastuzumab of primary cells (A) or the trastuzumab-resistant cells (B) were tested in 3D Matrigel culture. Photographs were taken on day 13. Expression of CXCR4 and HER2 was evaluated in BTRT (C) and SKRT (E) cells. Quantitative analysis of the density was performed using AlphaView SA software (D, F). CXCR4 expression in BTRT (G) and SKRT (H) cells was verified with immunofluorescent staining (green) and examined under a confocal microscope (see “Materials and methods” section). I Dynamic expression of CXCR4 with cell cycle progression in BTRT cells was detected by flow cytometry. J BTRT cells received BrdU pulse. Immunofluorescent staining for CXCR4, BrdU, and 7-AAD was performed and followed by flow cytometry analysis (“Materials and methods” section). The correlation between CXCR4 and 7-AAD or BrdU was analyzed using Pearson r coefficients