Abstract Introduction and rationale: Immune checkpoint blockade therapy (ICB) is a cutting-edge treatment strategy modulating T cell activity and has emerged at the forefront of non-small-cell lung cancer (NSCLC) management, yet most patients do not durably respond. Studies have shown that innate immune cells could create an immune-suppressive tumor microenvironment (TME) thus, resulting in resistance to ICB. Therefore, to design an effective therapeutic strategy, it is imperative to consider the diverse immune cells and their complex interactome. In noncancerous conditions, pro-inflammatory neutrophils release extracellular traps or NETs that interfere with monocyte differentiation to macrophages. However, their interaction and its effect in mediating immune evasion in NSCLCs is poorly understood. Methods: To study the role of neutrophils in macrophage behaviour, freshly isolated monocytes were differentiated into pro-tumorigenic M2-like macrophages and polarized with or without NETs. These NETs-educated macrophages (NeMacs) were phenotypically characterized by flow cytometry. Functionally, their role in the TME was investigated using a Tumor-Immune-Microenvironment on-chip (TIMEoC) platform. Here, immune cells such as donor matched T cells or neutropils were loaded into a microfluidic channel magnetically attached to active NSCLC spheroids embedded in a collagenous matrix containing NeMacs. T-cell or neutrophil migration and tumor cytotoxicity in the presence of NeMacs were monitored on TIMEoC. Results: In the presence of NETs, macrophages presented an immunosuppresive phenotype with significantly reduced expression of CD80, a key co-stimulatory receptor for T cell activation. NeMacs recruited significantly more neutrophils to the tumor than M2-like macrophages. In contrast, NeMacs retarded activated CD8+ T cell migration to the TME and significantly impacted their cytotoxic abilities observed by caspase 3/7 expression. The presence of NeMacs also affected ICB efficacy in vitro. Conclusion: Therefore, NETs could reprogram macrophages to create an immune-suppressive TME. This study was able to unveil previously unexplored relationship between NETs and macrophages in NSCLCs which could help identify prospective targets that could augment the current standard of care. Citation Format: Simrit Safarulla, Meghan De Meo, Roni Rayes, Jade Canape, Arvind Chandrasekaran, Betty Giannias, France Bourdeau, Pierre-Olivier Fiset, Jonathan Spicer. Neutrophil extracellular traps reprogram macrophages to an immunosuppressive phenotype in non-small-cell lung cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Mechanisms of Cancer Immunity and Cancer-related Autoimmunity; 2025 Sep 24-27; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(9 Suppl):Abstract nr A017.
Safarulla et al. (Wed,) studied this question.