Trastuzumab

Human breast cancer cell lines SKBR3 and BT474 were purchased from Shanghai Academy of life Science (Shanghai, China) and cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) harboring with 15% fetal bovine serum (FBS) and 0.1 IU/mL insulin at 37°C with 5% CO₂. Trastuzumab-resistant breast cancer cells, named SKBR3-TR and BT474-TR, were established by continuously exposing parental cells to increasing concentration of Trastuzumab (Sigma, St. Louis, MO, USA) for more than 6 months until cells displayed resistance to Trastuzumab. Trastuzumab-resistant cells were maintained in the same media supplemented with 3 μg/mL Trastuzumab.

Viability assays were performed to determine the antiproliferative effects of trastuzumab, pertuzumab and T-DM1 (all from Roche, Basel, Switzerland), using the Cell Proliferation Reagent WST-1 (Abcam, Cambridge, United Kingdom) and following the manufacturer’s instructions. Briefly, cell lines were seeded in 96-well plates to obtain a confluency of 90%, after 24 h (5 × 10³ cells/well for CAT-MT and FMCp, 15 × 10³ cells/well for FMCm and 10 × 10³ cells/wells for SKBR-3), and then exposed to increasing concentrations of each antibody (Table 1), with the control wells left unexposed. Phosphate buffered saline (PBS; Corning) was used as a vehicle for mAbs and ADC. After 72 h of exposure, the WST-1 reagent (Abcam) was added, followed by an incubation period of 4 h, at 37 °C, and absorbance was measured at 440 nm using a plate reader (FLUOStar Optima, BMG LabTech, GmbH, Ortenberg, Germany). Triplicate wells were used to determine each data point and three independent experiments were performed.
For the combined assays: trastuzumab plus pertuzumab, trastuzumab plus lapatinib (Sigma-Aldrich, Darmstadt, Germany) and pertuzumab plus lapatinib, a similar methodology was used, testing concentrations that covers different cytotoxic responses (Table 2).