The NK cells phenotype and function in resected non small cell lung cancer: differences between squamous and adenocarcinoma.

The tumor microenvironment has come to light as a key player in carcinogenesis and progression. Tumors affect many host cell types, in particular immune cells. The immune system appears to select particularly fit tumor cells in the process of immuno-­editing, while the tumor cells influence the polarization of immune cells towards phenotypes that favor tumor growth and vascularization. Here we investigated the phenotype of tumor infiltrating natural killer (NK) cells, focusing on angiogenesis associated cytokines and activities in patient-­derived material from non-­small cell lung cancer (NSCLC). Samples from the tumor and adjacent normal issues, as well as peripheral blood and lung samples as well as from non-­oncologic patients with bullous emphysema were collected and rapidly processed to obtain single cell suspensions. Flow cytometry (FC) analyses were performed to evaluate specific markers (CD3, CD56, CD16) to identify NK cell subsets. We observed that in the NSCLC samples, the CD56+CD16-­ NK phenotype, associated with cytokine production, predominated in the tumor samples while the CD56dimCD16+ cytotoxic phenotype dominated in the adjacent normal tissues and in lung tissue derived from non-­oncologic patients. This was independent of tumor histotype and smoking status. We examined the angiogenic potential of tumor infiltrating NK cells by intracellular staining for production of VEGF, PlGF, IL-­8 (CXCL8), IFNand other markers. The CD56+CD16-­ subset was clearly associated with production of angiogenic cytokines in all samples. However, patients with squamous carcinoma histotypes showed remarkably and significantly higher production of angiogenic factors in tumor infiltrating, adjacent tissue and especially in peripheral blood CD56+CD16-­ NK cells than patients with adenocarcinomas. Following surgical intervention, these levels were reduced in disease-free patients. Moreover, supernatants derived from the tumor infiltrating CD56+CD16-­ NK cells were able to induce endothelial cell chemotaxis and formation of capillary-­like structures in vitro; this was particularly evident for NK cells isolated from squamous cell carcinomas. Our data suggest that squamous NSCLC tumors have a significant systemic effect on NK cells, enhancing angiogenic cytokine production in a manner dependent on the presence of disease. NK cells appear to participate in tumor neovascularization and could represent a peripheral marker for disease progression, angiogenesis and response to therapies in some tumor subsets.