Characterization of Lung Cancer Cell Lines

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.