Figure 1.

A. The effect of trastuzumab on the viability of the BT-474 (parental, sensitive) cell line for 72 h remains constant as of 10 µg/ml. The horizontal axis shows the different concentrations of trastuzumab (0.01 to 10 µg/ml, nonlinear scale) used to test the line BT-474. The values of cell growth are represented on the vertical axis and show that IC50 remains constant at concentrations greater than 10 µg/ml. B. Generation of acquired resistance to trastuzumab in cell lines BT-474, SK-BR-3, AU-565, and EFM-192A. Monthly cell count (average of a minimum of 3 replica) to assess the generation of resistance in the cell lines after sustained exposure to trastuzumab (10 µg/ml, first month; 30 days’ pause; 15 µg/ml, later). In all cases resistant cells (dark gray boxes) showed a higher growth rate in the presence of the drug than the sensitive cells (pattern fill). Every trastuzumab-treated condition was compared to its corresponding non-treated one (light gray bars). C. Phase contrast images showing cultured monolayers of parental and trastuzumab-resistant cells for every BCCL. Morphological characteristics did not differ between sensitive and resistant cells of the same line. D. BT-474.R cells were resistant to trastuzumab in vivo. Female SCID/beige mice were injected with BT-474 (round markers) or BT-474.R cells (square markers) into the subcutaneous space. Once tumor volumes reached approximately 100 mm³, five mice per group were randomized to either IgGĸ 10 mg/kg (open markers) or trastuzumab 10 mg/kg (filled markers). Overall, significantly higher growth rates of BT-474.R cells were detected between initial and final days of treatment with trastuzumab, as compared to BT-474 cells (P < 0.001). Points: mean tumor volume of five mice per group; bars: SD.