**3. Conclusions**

During the last decades, it became more clear that antimicrobial peptides (AMPs) are not restricted in characteristics as being antibacterial, but that AMPs are also serving as a first-line of defense against fungi, viruses, and even tumor cells. Furthermore, these small cationic molecules have been shown to exhibit potent immune regulatory activities, including chemoattraction, activation and differentiation of leukocytes and monocytes to initiate and further enhance adaptive immunity. Combined, we believe that AMPs may hold great potential to be used as lead for new (co-)therapeutic agents.

With proteomic approaches, more in particular mass spectrometry, it is feasible to identify (cyclic) AMPs in their active form after their corresponding proteolysis. While emphasis has been put strongly on HNP identification in a tumorimmunology-related context, it is also important to stress out that mass spectrometric analyses allows for the detection and identification of native AMPs in different specimens, even when present at low concentrations. These unbiased analyses can lead to the detection and identification of new AMPs, important for future drug development.

In this way, human neutrophil peptide (HNP) 1, 2 and 3 were identified in a non-small cell lung cancer (NSCLC) related context, of which the presence have been shown to be discriminative between a responding and a nonresponding NSCLC patient towards anti-PD-(L)1 immunotherapeutics. Although the biological activity of HNPs is well described against bacteria and viruses, little is known about their antitumor characteristics. Some studies suggest a direct antitumoral activity

of HNP1-3, while it has also been proven that these HNPs do not show a direct cytolytic activity towards NSCLC, but a reduced NSCLC proliferation was observed in the presence of HNP and peripheral blood mononuclear cells (PBMC) *in vitro.* In the latter case, increasement of IFN-γ was observed, referring a cell-mediated immunity. The question raised by these findings is; what is the mechanism of HNP-mediated immunity? Do the HNPs attack the NSCLC cells directly through interaction with the negatively charged phosphatidylserine on the outer leaflet of the tumor cells? This may result in tumor-associated antigen release responsible for activation of a proper immune response. Also, it is increasingly evident that these HNPs are responsible for an enhanced antigen-specific immune response by activating dendritic cells: upon HNP stimulation, dendritic cells are recruited to the tumor site, followed by maturation and differentiation of these dendritic cells, leading to an enhanced adaptive immune response. However, the exact HNPdriven mechanisms are still largely unknown. Further investigation will reveal if HNPs could be a promising approach in future anticancer therapeutics, possibly in synergy with immunotherapy.
