Abstract P1-17-07: Preclinical characterization of neratinib in a blood-brain barrier co-culture model: Therapeutic implications for breast cancers with brain metastases
Authors: Martin TA , Lalani AS , Avogadri Connors F , Bryce RP , Jiang WG 
Published: February 2018

Abstract
Background: Breast cancer patients who develop brain metastases have poor prognosis/short overall survival. Human epidermal growth factor receptor 2 (HER2) +ive breast cancer have an increased propensity for brain metastases. Currently, these metastases have limited therapeutic options. The endothelial cells forming the blood-brain barrier (BBB) are highly specialized to allow precise control over substances that enter/leave the brain via the tight junction. Targeted agents that are efficacious/ able to penetrate the BBB are urgently needed. Neratinib is an orally available tyrosine kinase inhibitor that irreversibly binds and inhibits EGFR, HER2/HER4 receptor tyrosine kinases and has previously been shown to reduce the onset/delay symptomatic progression of CNS metastases in patients with advanced HER2-positive breast cancers with brain metastases (Awada 2016 JAMA Oncol; Freedman ASCO 2017 Ab 1005). Using a BBB model, this study aimed to determine if neratinib is able to pass through an endothelial barrier and demonstrate activity against human breast cancer cells lines. We also compared brain and vascular endothelial cells.
Methods: The BBB was represented by an in vitro transwell model using TY09 brain endothelial and HECV venous human cell lines in co-culture with a range of HER2+ive (MDA-MB-361;BT-474) or HER2-ive (MDA-MB-231;BT-549) human breast cancer cell lines. Effects on barrier function (trans-endothelial resistance/paracellular permeability) were assessed. Dose response effects on two brain endothelial cell lines (TY09/CMECD3) by neratinib were determined using ECIS (electric cell impedence sensing).
Results: Initially, we compared the effects of neratinib on a single brain endothelial cell line (TY09) in comparison to a venous endothelial cell line (HECV) in the presence/absence of EGF (20pg). We used temozolomide (TMZ) (40pmol) as a control treatment. Barrier function, assessed using trans-endothelial resistance and paracellular permeability revealed that neratinib had little effect on resistance over 1-72 hrs. When co-cultured with HER2+ive breast cancer cells, resistance was decreased at 1 hr which then returned to control levels (p&lt;0.05). There was little change following treatment to the HER2-negative cells. A similar effect was also observed with TMZ. There was also a marked effect in HECV cells, but this was mostly observed in those treated with TMZ, regardless of HER2 status of the breast cancer cells in co-culture (p&lt;0.04). Paracellular permeability was increased by 0.5 hr when using 10kDa (p&lt;0.05) but not 40kDa dextran. Neratinib reduced the resistance of both TY09 and CMECD3 cells in a dose-dependent manner, with the same effect observed in cell attachment (p&lt;0.01) and motility (0.05).
Conclusions: In our BBB co-culture model, neratinib was able to pass through the barrier created by both brain and venous endothelial cells and reduce the growth of HER2+ive breast cancer cells over 72 hrs. In addition, neratinib caused marked changes in barrier function of the brain endothelial cells with concurrent effects on cell behaviour. These data demonstrate that neratinib may be an attractive therapeutic for HER2-positive breast cancers with brain metastases.
Citation Format: Martin TA, Lalani AS, Avogadri Connors F, Bryce RP, Jiang WG. Preclinical characterization of neratinib in a blood-brain barrier co-culture model: Therapeutic implications for breast cancers with brain metastases [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-17-07.