A proteomic approach to better understand the role of human neutrophil peptides in the NSCLC microenvironment

Aim: Although treatment of lung cancer, one of the deadliest diseases worldwide, with immune checkpoint inhibitors (ICIs) has shown promising outcomes, these survival outcomes are only observed in a relatively small subset of lung cancer patients. In a previous study, we elucidated that the presence of human neutrophil peptide (HNP) 1, 2 and 3 in non-small cell lung cancerous (NSCLC) biopsies is associated with a clinical response towards treatment with PD-1/PD- L1 immune checkpoint inhibitors. Furthermore, HNP1 has shown in vitro an immuneactivated function towards lung cancer cells, but the specific role of HNP1 in (lung) cancer is still unknown. The aim of this study was to provide a better understanding of HNP1 in an NSCLC microenvironment. Methods: To gain better insights into the role of HNP1 on cancer growth and to unravel immune responses, in vitro (SILAC-labelled) A549/PBMC (from three healthy donors) cocultures were set up and treated/not treated with HNP1. After 5 days, both secretome and cellular analysis using mass spectrometry were performed on these cocultures. After protein identification in all different tested conditions, pathway analyses (MetaCoreTM) were performed to investigate the biological significance of HNP1 stimulation on the A549/PBMC cocultures. The proteomic outcomes were confirmed by multiplex ELISA for a proinflammatory cytokine panel (TNF-a, IL-1 ss, IL-2, IL-4, IL-6, IL-12p70 and IL-18). Results: A number of biological pathways and process networks were observed to be upregulated after treatment of the coculture with HNP1. HNP1 stimulation leads to an increase in pathways and proteins stimulating chemotaxis (including plasmin signaling, leucocyte recruitment, CCL2 and CXCL8 expression), proinflammatory cytokine secretion (including IL-1 ss, IL-6 and TNF-a), dendritic cell (DC) maturation, phagocytosis and antigen presentation, leading to a more efficient adaptive anti-tumoral immunity. Conclusion: These results enhance our understanding of the role of HNP1 in the tumor microenvironment and suggest that HNP1 may be able to induce tumor necrosis by inducing prostimulatory immune responses.