Matrigel

The invasion of cancer cells was performed by a 24-well transwell unit with Matrigel™ (Greiner Bio-One, Frickenhausen, Germany) coated on the upper side of polycarbonate filters into 8 μm filter pore size transwell inserts. The lower well was injected with 800 µl medium containing 10% FBS, without or with indicated concentrations of BPIQ. 1 × 10⁵ H1299 cells was resuspended in 200 µl of serum-free medium were seeded onto a transwell insert and allowed to invade for 16 h. Non-invaded cells on the upper part of the membrane were removed. Cells on the bottom surface of the filters were fixed with 4% paraformaldehyde, stained with Giemsa (Merck), and counted under a microscope. Each experiment was done in triplicate, and the results from three independent experiments were expressed as mean ± SD.

For co‐culture, medium contained neurospheres was centrifuged at 100 × g for 5 min and dissociated the obtained neurospheres to single cell using ACCUTASE. About 80%–90% confluent BMECs were trypsinized (with 0.02% EDTA) and centrifuged at 150 × g for 5 min and resuspension with serum‐free culture medium. NSCs and BMECs were mixed and resuspension with Matrigel (BD Biosciences, growth factor reduced) at a density ratio of 1:10. The final density of BMECs was 2.0 × 10⁶·ml⁻¹, and the final density of NSCs was 2.0 × 10⁵·ml⁻¹. The 100 μl cell/Matrigel mixture was then evenly spread on the bottom of the confocal dishes (NEST, 15 mm) or cell culture inserts (Greiner Bio‐One, 24‐well), and then, placed in an incubator to solidify. After 1 h, 50 μl mixture of NSCs/Matrigel was evenly spread on the surface of the solidified gels, which was further placed in the incubator for 2 h, and then, the culture medium was added. BMECs‐NSCs co‐culture system was grown in DMEM/F12: Neurobasal medium 1:1, 2% B27 (without vitamin A), 20 ng·ml⁻¹ rh EGF, 10 ng ml⁻¹ rh bFGF and the medium was supplemented with 1% FBS. After 3 days, B27 in the medium was replaced with B27 containing vitamin A. The BMEC‐NSC co‐cultures or control BMEC or NSC monocultures were analyzed following 3 or 7 days of culture, using live cell microscopy and immunofluorescence staining.

In this platform, 4 × 10⁵ RTskin01 fibroblasts were resuspended in 125 µL of a Matrigel^® solution diluted 1:8 in serum-free L-15 medium and layered upon the ThinCert inserts with 3 µm pores (Greiner Bio-One, Kremsmunster, Austria, cat. no. 665631), the same used in the MM platform. After 1 h incubation at 20 °C for allowing polymerization, L-15 medium, supplemented with 10% FCS, was added in both apical (AP) and basolateral (BL) compartments. After 1 week, 2.5 × 10⁵ RTpiMI cells/cm² were seeded on the apical surface.