Abstract 1605: Extracellular vesicles from metastatic non-small cell lung cancer induce the angiogenic switch in natural killer cells
Authors: Gallazzi Matteo , Fortunato Orazio , Pastorino Ugo , Sozzi Gabriella , Noonan Douglas , Albini Adriana , Bruno Antonino 
Published: September 2020

Abstract
Lung cancer is the leading cause of cancer-related death worldwide and the only curative treatment remains surgery. Recurrence rates in resected patients remain high: from 30% for stage I to 80% for stage III. We previously demonstrated the role of 24 microRNAs in the pathogenesis and aggressiveness of lung tumors. Natural Killer (NK) cells, cellular mediators involved in tumor immunosurveillance are functionally compromised in cancer patients. We discovered that tumor infiltrating NK cells (TINKs) in non-small cell lung cancer (NSCLC) acquire the proangiogenic CD56brightCD16-VEGF+PlGF+ phenotype and functions and that TGFβ is involved the NK cell angiogenic switch. Tumor cells can release large amounts of extracellular vesicles (EVs), that have been reported to be endowed with potent immunosuppressive activities, also in NK cells. Here, we investigate the abilities of EVs from NSCLC cell lines and patients to induce a pro-angiogenic polarization in NK cells.
EVs were isolated from conditioned media of NSCLC primary tumor (A549, LT73) and metastatic (H460) cell lines, and from plasma of NSCLC metastatic patients or heavy smoker individuals (EV-donor), by ultra-centrifugation and their purity were confirmed by transmission electron microscopy (TEM), Nanosight and western blot. NK cells, FACS sorted from healthy controls were exposed for 72 hours to 15 μg of EVs, then characterized for surface antigen expression, by multicolour flow cytometry. miRNA-profiling was performed on NK cells polarized for 72 hours with 15 μg of EVs isolated from A549 or LT73 NSCLC cells lines, or from plasma of patients with metastatic NSCLC or heavy smoker individuals.
NanoSight analysis revealed that the EVs-Patients and EVs-Donors had a similar median size and morphology. We found that EVs from NSCLC cell lines and NSCLC patients induce the CD56brightCD9+CD49a+ NK cell phenotype, together with upregulation of CXCR4 expression and decreased levels of the NKG2D activation marker. Interestingly, the metastatic H460 cell line exhibited stronger NK cell polarization effects as compared to those from A549 and LT73 cells. Strong pro-angiogenic NK cell polarization was also observed in cytolytic NKs, exposed to EVs from metastatic NSCLC patients, as compared to those exposed to EVs-donors Finally, miRNA profiling on NK cells polarized with EVs, showed up-regulation of miR-133a and miR-221 suggesting a possible role of these miRNAs in NK angiogenic switch.
Our study demonstrates that NSCLC EVs can support lung cancer progression acting on the NK cell angiogenic switch.
Citation Format: Matteo Gallazzi, Orazio Fortunato, Ugo Pastorino, Gabriella Sozzi, Douglas Noonan, Adriana Albini, Antonino Bruno. Extracellular vesicles from metastatic non-small cell lung cancer induce the angiogenic switch in natural killer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1605.