Non-small cell lung cancer (NSCLC) affects millions of patients each year worldwide. Existing therapies include epidermal growth factor receptor (EGFR) inhibition using small molecules or antibodies with good efficacy. Unfortunately, intrinsic and acquired resistance to EGFR therapy remains a persistent complication for disease treatment. A greater understanding of the role of EGFR in NSCLC etiology is crucial to improving patient outcomes. In this study, the role of EGFR in tumor angiogenesis was examined in H292 NSCLC cells under the pretense that confluent cells would exhibit a more angiogenic and growth-centered phenotype. Indeed, confluent H292 cells potentiated endothelial cell angiogenesis in co-culture models in an EGFR-dependent manner. While confluent H292 cells did not exhibit any change in EGFR protein expression, EGFR localization to the extracellular membrane was increased. EGFR membrane localization coincided with a comparable potentiation of maximal EGFR phosphorylation and was followed by a 3-fold increase in vascular endothelial growth factor A (VEGF-A) production as compared to subconfluent cells. EGFR-mediated VEGF-A production was determined to be dependent on signal transducer and activator of transcription 3 (STAT3) activation and not phosphoinositide 3-kinase (PI3K) signaling. These results identify unique cell density dependent phenotypes within a monoclonal NSCLC cell line and provide a potential mechanism of resistance to anti-EGFR therapy in metastatic NSCLC.

ST2L is a transmembrane receptor that belongs to the IL-1 receptor family. The receptor is expressed on various cell types including Th2 cells, mast cells, basophils, growth-activated fibroblasts, and vascular endothelial cells. ST2L activation by its ligand IL-33 has been implicated in Th2-mediated immunity, inflammation, and allergic responses in vivo. Inhibition of ST2L activity can attenuate Th2-dominated immune responses such as lung eosinophilia, airway hyper-responsiveness, and arthritis in animal models. Here we report the generation and in vitro characterization of a panel of rat anti-mouse ST2L monoclonal antibodies. We demonstrate that the antibodies specifically bind to recombinant receptor protein and that a subset of the binders inhibits mouse ST2L activity in multiple in vitro assays. Four of the identified anti-mouse ST2L antibodies were shown to prevent IL-33 from binding to ST2L, down-regulate IL-33-induced NF-κB signaling, and neutralize the ability of IL-33 to stimulate mouse Th2 cell proliferation. The characterized monoclonal antibodies are important tools that will be used to study mouse ST2L receptor functionality in vivo.