The tumor microenvironment (TME) is a dynamic ecosystem surrounding a tumor. The TME consists of various components such as cancer cells, stromal tissue, immune cells and the extracellular matrix (ECM). In particular, the interaction between cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) is a key regulator of immunosuppression in the TME, subsequently contributing to tumor progression, resistance and metastasis. CAFs stimulate TAMs to differentiate into a M2-like macrophage phenotype, supporting immune evasion and tumor growth. It has been found that reactive oxygen species (ROS) produced by NADPH oxidase (NOX) enzymes are involved in process. ROS have been reported to stimulate CAF activity and TAM differentiation, but the role of NOX in the interaction between CAF and TAM has not yet been elucidated. Herein, we aimed to investigate the role of NOX2 in M2 polarization of macrophages, especially CAF-mediated TAM differentiation, and evaluate the therapeutic potential of targeting NOX2 in the TME. In this study, we utilized THP-1 monocytic cell lines and human pancreatic CAFs (pCAFs) to examine the functional significance of NOX2. NOX2 was predominantly expressed in THP-1 cells compared to other NOX isozymes when polarized into M2-like macrophages and knockdown of NOX2 effectively inhibited M2-like polarization of THP-1-derived macrophages. In addition, knockdown of NOX2 significantly reduced the secretion of M-CSF by pCAFs. These findings suggest that NOX2 plays a crucial role in CAF-mediated M2 differentiation of macrophages. Based on these findings, we confirmed the effects of NOX2 inhibition using com-19, a selective NOX inhibiting molecule. Com-19 modulated M2 polarization of macrophages and downregulated NOX2 mRNA levels in the cells. Moreover, com-19 also significantly reduced ROS production and the secretion of cytokines/chemokines in macrophages under M-CSF-induced conditions. Importantly, the results also demonstrated that com-19 inhibited pCAF-induced M2-like polarization of THP-1. Taken together, we confirmed that CAFs and TAMs interact closely and NOX2 inhibition could be a potential therapeutic target for CAF- and TAM-mediated immunosuppression, especially for M2-polarization of macrophages in the TME. Furthermore, com-19 can provide a novel approach to overcome immunosuppressive barriers by interrupting their interaction in the TME, offering the potential for more effective combination therapies in cancer treatment.Citation Format:Hye ji Jang, Jihyun Um, Eun Sil Lee, Sung Hwan Moon, Soo Jin Lee. NOX2 plays a key role in M2 polarization of macrophages associated with cancer-associated fibroblast in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2238.