Cell culture studies

Primary HBMECs were purchased from ScienCell™ Research Laboratories and used at low passage number (<P5). Cells were cultured in ECM medium (ScienCell™1001) in 5% FBS and ECGS. Astrocytes were isolated from the brains of mouse pups at post-natal day 3 and cultured in low glucose DMEM medium from ThermoFisher (sh3002101). Normal Human Astrocytes were purchased from Lonza (CC-2565) and cultured in ABM basal media with supplemental growth factors (Lonza CC-4123). HBMECs were plated at 70% confluence and cultured in conditioned media from both astrocytes and tumor cells were collected at 72 hours for treatment. The growth factors and concentrations used for endothelial cell treatment were 0.1 ng/ml for TGFβ1 (R&D Systems, 240-B-002), 100 ng/ml for bFGF (Gibco), and 100 ng/ml for VEGFA (Biolegend 583702). The neutralizing antibodies used are anti-TGFβ (1–4 μg/ml, R&D Systems, MAB1835) and anti-bFGF (16 μg/ml, R&D Systems, AF-233). U0126 inhibitor (20 μM, Tocris) was used to inhibit Mek1/2-mediated phosphorylation of Erk in HBMEC cells. Concentrated lentivirus for pLOC-MFSD2a (Clone ID-PLOHS_100073371) and control pLOC were obtained from the MD Anderson Open Reading Frame core facility. HBMECs were infected at MOI 5. Cells overexpressing MFSD2a were lysed and levels of mRNA expression were measured by real time PCR. For the TGFβ1 ELISAs, 250,000 metastatic cells were plated in a 6 well plate and conditioned media was collected at 72 hours for treatment. The levels of TGFβ1 were measured using human TGFβ1 ELISA kits (R&D Systems) according the manufacturer’s protocol.