Modeling Blood–Brain Barrier Efflux Transport Using a Breast Cancer Resistance Protein Overexpression Cell Line

Background: The blood–brain barrier (BBB) separates the circulation from the central nervous system (CNS) and serves to maintain brain homeostasis. The BBB comprises highly specialized brain endothelial cells (BECs) with unique properties that allow the BBB to maintain strict regulation of molecules entering and exiting the CNS. These characteristics include tight junctions, low endocytosis rates, and efflux and nutrient transporters. Breast cancer resistance protein (BCRP) is an efflux transporter found at the BBB that plays a key role in protecting the CNS. Together with other efflux transporters, BCRP contributes to multidrug-resistant cancers and difficulty delivering drugs and therapeutics to the brain and other organs. Methods: Using the hCMEC/D3 line, we utilized BCRP substrate rosuvastatin to effectively select for cells expressing high amounts of BCRP, thus generating hCMEC/D3-BCRP. To assess protein abundance, we utilized flow cytometry and confirmed expression via qPCR. To investigate BCRP efflux function in evolved hCMEC/D3-BCRP, we performed substrate accumulation assays with BCRP and P-gp substrates. Results: We found hCMEC/D3-BCRP had increased BCRP abundance and expression relative to parent hCMEC/D3. We also observed an increase in BCRP function via substrate accumulation of two BCRP substrates compared to parent hCMEC/D3. Conclusions: BCRP serves a protective role within the BBB and is a major hurdle in drug delivery. We generated a BCRP overexpression BEC cell line (hCMEC/D3-BCRP) under the influence of endogenous promoters. This cell line can be used to further investigate the role of BCRP in BECs and utilized in efflux transport studies.