Hbmec

The NSCLC cell line A549 was obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Non-fetal-derived human brain microvascular endothelial cells (HBMECs) were purchased from Bioleaf Biotechnology (Shanghai, China). A549 and HBMECs were grown in Roswell Park Memorial Institute-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum at 37 °C with 5% CO₂. The lnc-MMP2-2 overexpression and silencing lentivirus and their control lentivirus were packaged by Genomeditech (Shanghai, China). EPB41L5-targeting siRNA and scramble control siRNA were obtained from Ribobio (Guangzhou, China). The EPB41L5-targeting sequences were as follows: siRNA#1, 5′-GGATCACGATTTA GATATA-3′; siRNA#2, 5′-GTCCTGAACTTGTCTCAGA-3′; siRNA#3: 5′-CGACTATTTTGGTCTGAG A-3′. The overexpression plasmid (pcDNA3.1-EPB41L5) and the empty vector (pcDNA3.1) were obtained from Genomeditech (Shanghai, China). miR-1207-5p antagomir, antagomir control, and miR-1207-5p agomir and agomir control were purchased from Ribobio (Guangzhou, China). Cell transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. For exosome transfection, lnc-MMP2-2 Smart Silencer (RiboBio Co, Ltd., Guangzhou, China) were loaded in exosomes using the Exo-Fect Exosome Transfection Kit.

Human brain microvascular endothelial cells (HBMECs), obtained from Cell Systems (ACBRI 376, Kirkland, WA, USA), were cultured in endothelial cell medium (#1001, ScienCell Research Laboratories, Carlsbad, CA, USA). Inducible gene expression in HBMECs was achieved through lentiviral infection. Lentiviruses were generated in HEK293T cells with the assistance of two other plasmids, pMD2.G (#12259, Addgene) and psPAX2 (#12260, Addgene). For siRNA-mediated knockdown experiments, siRNAs were introduced into HBMECs using standard electroporation techniques (Neon Transfection System, Invitrogen, Carlsbad, CA, USA). Autophagy was induced by treating HBMECs with Earle’s Balanced Salt Solution (EBSS) for 2 h, followed by LC3 puncta or p62 turnover assays. All chemicals, including pepstatin/E-64D, MG132, and FAK inhibitor 14, were purchased from Cayman Chemical.

hBMECs were purchased from ScienCell Research Laboratories (Catalog #1000). HEK293T cells (ATCC® CRL-3216TM) and THP-1 cells (ATCC® TIB-202™) were purchased from American Type Culture Collection. The hBMECs and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, No. c1995500bt) supplemented with 10% fetal bovine serum (Gibco, No.10091148); the THP-1 cells were cultured in RPMI 1640 (Gibco, No. c11875500bt) medium with 10% fetal bovine serum (Gibco, No.10091148). To differentiate THP-1 cells into MΦs, cells were stimulated with 5 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, 79346-1MG) in RPMI complete medium for 72 h and rested in PMA-free RPMI-1640 medium without FBS for an additional 48 h. Further details regarding cell survival assay, microscopy observation, construction of derivative hBMECs, protocol of inhibitor usage, CRISPR screen, and endothelium monolayer model construction assay are provided in SI Appendix, Materials and Methods.

To assess the transmigratory capacity of immune cells across the BBB, an established cell migration assay was performed as described in detail before (14 (link)).
Shortly, on a monolayer of HBMECs (PELOBiotech GmbH, Planegg, Germany), thawed PBMCs (5 × 10⁵ cells per transwell) were added to the HBMEC monolayer in the upper compartment (3 × 10⁴ HBMECs per transwell) and the lower compartment was filled with transmigration medium (RPMI + 2% B27, Gibco™ life technologies, Carlsbad, CA, USA). After incubation for 6 h at 37°C flow count fluorospheres (Beckmann Coulter) were added for quantification of the migrated cells in the lower compartment, followed by cell surface staining and flow cytometry analysis. A well containing 5 × 10⁵ thawed PBMCs in 600 µl of RPMI + 2% B27 served as an in vitro control. The percentage of migrated cells for each cell type was calculated after the following equation:
$\text{migrated cells (\%) = }\frac{\begin{array}{l}\text{absolute number\hspace{0.17em}of\hspace{0.17em}cells\hspace{0.17em}\hspace{0.17em}in\hspace{0.17em}\hspace{0.17em}}\\ \text{\hspace{0.17em}lower\hspace{0.17em}\hspace{0.17em}compartment *\hspace{0.17em}}100\end{array}}{\text{absolute\hspace{0.17em}\hspace{0.17em}number of cells in vitro}}$