Dm1 ch3

The HER2-positive breast cancer cell line BT-474 (catalog number: HTB-20) was used in this study. This cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). We previously established a trastuzumab-resistant cell line, BT-474-R, by treating BT-474 with increasing doses of trastuzumab (from 0.1 μg/mL to 40 μg/mL) for 10 months [8 (link)]. BT-474-R was additionally treated with a starting dose of 0.1 μg/mL, which was determined by the IC₁₀ values and generated by continuous exposure to increasing doses of T-DM1 up to 40 μg/mL for 12 months. The cells were exposed to T-DM1 until they were damaged at the 30% confluence of the dish, and they were then passaged when they reached 80% confluence in a drug-free state. When the cells reached 80% confluence, the drug was exposed again. The cells were repeatedly treated with the same concentration of T-DM1 until almost all of the cells survived the treatment. The cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum and maintained under 5% CO₂ at 37 °C. Trastuzumab and T-DM1 were purchased from Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan), dasatinib was purchased from the Bristol-Myers Squibb Company (New York, NY, USA) and DM1-CH_3, which is the methyl form of DM1, from Toronto Research Chemicals (Toronto, ON, Canada).

T-DM1, TRAS, and PER were provided by Chugai Pharmaceutical Co., Ltd. Human immunoglobulin G (HuIgG) was purchased from MP Biomedicals, LLC. TRAS and HuIgG were dissolved in distilled water. All agents were diluted with saline for in vivo experiments and with culture medium for in vitro experiments. CAPE, obtained from Chugai Pharmaceutical Co., Ltd., was suspended in 40 mmol/L citrate buffer (pH 6.0) containing 5% gum Arabic as the vehicle. DM1-CH₃ was purchased from Toronto Research Chemicals.