HER2-Driven EMT and T-DM1 Resistance

HMLE cells were a kind gift from Sendurai Mani (MD Anderson Cancer Center). These cells were constructed to stably express firefly luciferase via lentiviral transduction and selection with blasticidine. HER2-transformed HMLE cells (HME2) were constructed via lentiviral transduction of pBabe (addgene #40978) and stable selection using puromycin. Once transformed by HER2, HME2 cells are cultured in DMEM containing 10% FBS and 10 μg/ml of insulin. BT474 cells were obtained from the ATCC. HME2 and BT474 cells stably overexpressing FGFR1 were constructed by lentiviral transduction and stable selection using hygromycin as previously described^{18 (link)}. Trastuzumab and Trastuzumab emtansine (T-DM1) were obtained from Genentech through the material transfer agreement program. Where indicated, HME2 cells were treated with TGF-β1 (5 ng/ml) every 3 days for a period of 4 weeks to induce EMT. These EMT-induced HME2 cells were further treated with T-DM1 (250 ng/ml) every 3 days until resistant colonies emerged, these cells were pooled and cultured as the TDM1R population. Cells were validated for lack of mycoplasma contamination using the IDEXX Impact III testing on July 24, 2018.