Additional file 3: of Neoadjuvant neratinib promotes ferroptosis and inhibits brain metastasis in a novel syngeneic model of spontaneous HER2+ve breast cancer metastasis

Figure S3. Determination of neratinib IC50 and pro-ferroptotic activity in mouse and human breast cancer lines and schematic of neratinib treatment protocols. (A) Sensitivity of mouse (left panel) and human (middle panel) breast cancer cell lines to neratinib, and IC50 values were determined in short-term (72 h) assays as described in the “Methods” section. Expression of EGFR and HER2 in human lines (right panel) was examined by standard western blotting. The bottom panels show response to neratinib or RSL3 (0.5 μM) treatment in the presence or absence of liproxstatin-1 (2 μM) in the indicated lines. Neratinib was used at 800 nM (67NR), 2.5 μM (4T1.2), 5 μM (MCF-7), 2 nM (BT474) and 500 nM (MDA-MB-231HM). Data show mean ± SD three independent experiment (n = 3) done in triplicate wells. **p < 0.01, ****p < 0.001; ns, not significant. (B) Metastatic setting. TBCP-1 cells (5 × 105/100 μl saline) were inoculated into the left cardiac ventricle. Daily treatment by oral gavage commenced 2 days post-inoculation and continued for up to 3 weeks. Mice were sacrificed individually when showing signs of advanced metastatic disease. (C) Neoadjuvant setting. TBCP-1 cells (1 × 106/20 μl) were inoculated orthotopically and daily treatment by oral gavage commenced when tumours reached 100 mm3 (~ 1 week). Treatment continued for up to 3 weeks or until tumours were resected when they reached 0.4–0.5 cm3 (~ 3 weeks). Mice were sacrificed individually when showing signs of advanced metastatic disease. (TIF 13948 kb)