Fig. 5
Neoadjuvant neratinib prolongs survival and inhibits metastasis more potently than late intervention. a Kaplan-Meier survival analysis of control (vehicle) and neratinib-treated (60 mg/kg) mice. TBCP-1 cells were inoculated into the left cardiac ventricle and the mice treated daily by oral gavage starting 2 days after tumour cell inoculation. Control, n = 9; neratinib, n = 10. *p = 0.0424. b Incidence of mice with visible brain metastases was determined by screening of three H&E step-sections/brain (100 μm apart) from the control and neratinib-treated groups. n/s, not significant. c–h Orthotopic tumour-bearing mice (8/group) were treated daily with vehicle (control) or neratinib (60 mg/kg) by oral gavage for up to 3 weeks, starting when tumours reached 100 mm3 and surgically resected on day 25, as described in the “Methods” section. c Tumour growth rate. Data show mean ± SEM. ****p < 0.0001. Arrows show start/end of treatment. d Tumour weight at resection. Each dot represents one mouse. Data show mean tumour weight ± SEM. ****p < 0.0001. e Image of primary tumours from control and neratinib-treated mice. f Kaplan-Meier survival analysis. ***p < 0.00001. g Incidence of soft tissue macro-metastases at endpoint in vehicle control and neratinib-treated mice. h Ex vivo fluorescence imaging (mCherry) of the brains at endpoint. Macro-metastases (mCherry+ve) are delineated by a dotted line. i Incidence of mice with visible brain micro-/macro-metastases (left panel) was determined by screening of three H&E step-sections/brain (100 μm apart) from control and neratinib-treated mice (n = 8) and further confirmed by cytokeratin IHC staining. *p < 0.05. Metastatic burden in the lung and bone (combine matched femur and spine burden) (middle panels) was determined by genomic qPCR of the mCherry marker gene (tumour cells) relative to vimentin (tumour + host cells) as described in the “Methods” section. Data are expressed as relative tumour burden (RTB) and show one point per mouse and mean burden (horizontal bar) ± SEM (n = 8/group). *p < 0.05, **p < 0.01, ***p < 0.001. Spleen weight in naïve (n = 3), vehicle control (n = 8) and neratinib-treated (n = 8) mice were measured at endpoint. Each point represents one mouse, and data show mean spleen weight ± SEM