In vitro neutrophil assays to support immuno-oncology drug development

Neutrophils play important roles in cancer progression. The presence of tumor-associated neutrophils can promote tumor growth, invasion, angiogenesis, and the formation of metastasis. On the other hand, neutrophils have also been associated with multiple anti-cancer functions, such as killing of antibody-opsonized cancer cells, trogocytosis, or indirectly through the release of cytotoxic mediators. Thereby, Neutrophils represent a target of interest for the development of novel cancer immunotherapy approaches. Here, we report a variety of in vitro assays allowing to evaluate the putative impact of drug candidates on neutrophil functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), Neutrophil Extracellular Traps (NET) formation and the release of Reactive Oxygen Species (ROS) by flow cytometry and live-cell imaging. Furthermore, multiplex cytokine and peroxidase release analysis after activation and stimulation of neutrophils can be evaluated using ELISA or Luminex technology. Co-cultures of freshly isolated human neutrophils and fluorescent-labeled tumor cells can be used to screen the potency of candidate therapeutics to enhance anti-cancer neutrophil functions. Lastly, the toxicity of drug candidates on Neutrophils can be evaluated. Key factors to obtain reliable and reproducible results are the access to fresh blood as well as optimized protocols for purifying and culturing untouched neutrophils. Taken together, functional in vitro assay using fresh primary human Neutrophils provide cost-effective tools for the characterization and lead selection of drug candidates targeting Neutrophils.