Fetal bovine serum (fbs)

GEC were seeded at 1 × 10⁴ cells/cm² at 37 °C and 5% CO₂ in medium supplemented with 0.1% VEGF, 0.1% ECGS, 0.1% Heparin, 0.1% EGF, 0.1% Hydrocortisone, 1% L-Glutamine, 1% antibiotic-antimycotic solution, 5% FBS (CellBiologics). GEC were overstimulated with recombinant VEGF (Gibco, ThermoFisher Scientific) at 100ng/ml. Simultaneously, AFSC (ration 1:1, on transwell inserts, Corning) or EVs and EV^Flt1−/− (10,000:1 ratio, on culture media) were co-cultured with GEC under the same growth conditions. Experiments were terminated at 24 hours and culture media and cells were collected for analysis. Integration of EVs into GEC was performed by applying EVs with green and red fluorescence tags derived from AFSC^GFP co-labeled with CM-DiI as previously reported^{12 (link)} to GEC overnight. Integration of EV into GEC was assessed by inverted fluorescence microscope (Leica DMI6000 B). Experiments were repeated in triplicate.

Primary adult rat lung microvascular endothelial cells (EC) were purchased from Cell Biologics Inc (Cell Biologics Inc., Chicago, IL) and maintained in basal medium supplemented with fetal bovine serum (FBS), epidermal growth factor, L-glutamine, and an antibiotic-antimycotic solution per manufacturer’s recommendations. Cells were analyzed between passage numbers 8 and 12 and for each experiment; within each experiment cells were matched by passage and lot numbers. 293A cell line was purchased from Life Technologies (Grand Island, NY) and was maintained according to manufacturer’s recommendations in media supplemented with 10% FBS (Corning Cellgro, Manassas, VA) and penicillin-streptomycin (Life Technologies, Grand Island, NY). Plasmids encoding hemagglutinin A (HA)-tagged CDK5 and the dominant negative CDK5 mutant (D144N) were purchased from Addgene (Cambridge, MA) [11 (link)]. On-Target plus siRNA targeting rat CDK5, p35 (CDK5r1), and non-targeting control siRNA were purchased from Dharmacon (Lafayette, CO, USA. Transfection of EC with duplex RNAs was carried out using Geneporter B reagent (Genelantis, San Diego, CA, USA) according to the manufacturer’s recommendations as detailed previously [12 (link)]. Lipofectamine 2000 reagent (Life technologies,Carlsbad, CA) was used for transfection of plasmid DNA per manufacturer’s recommendations.

Primary mBECs were purchased from Cell Biologics and grown in endothelial cell medium supplemented with growth factors and 5% FBS (Cell Biologics). Stealth RNAi duplexes targeting Rab7a were purchased from Invitrogen. Stealth RNAi negative control Med CG Duplex #3 (Invitrogen) was used as negative control in all experiments. 12.5 nM of siRNA was transfected using Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions.

A549, NCI-H1975 (H1975 throughout the text), and HOP62 lung cancer cells were grown as previously described [60 (link),61 (link),62 (link)]. Human primary lung microvascular endothelial cells (HPLMECs) and human umbilical vein endothelial cells (HUVECs) from Cell Biologics were grown in human endothelial cell medium with the addition of VEGF, heparin, EGF, FGF, hydrocortisone, L-glutamine, antibiotic–antimycotic Solution, and FBS according to the manufacturer’s instructions (Cell Biologics, Chicago, IL, USA). To generate A549 cells stably expressing FPR1 or TLR7 shRNA, we used pools of five constructs (shRNA FPR1-Qiagen, Valencia, CA, USA; shRNA TLR7-Origene, Rockville, MD, USA) containing 21-mer short hairpin RNAs (shRNA) directed to various coding regions of each target gene. Conditioned media from NSCLC cells (CM) were collected 18 h after incubation in 1% serum containing media or cell activation with specific stimuli.