Abstract 146: Studying the effect of compressive solid stress on macrophage phenotype and function in the context of glioblastoma

Abstract Glioblastoma (GBM) is one of the most aggressive forms of brain cancer, with an overall survival of less than two years. One barrier to therapeutic efficacy is the extremely immunosuppressive GBM tumor microenvironment (TME). Myeloid cells, such as macrophages, accumulate in GBM tumors and can comprise up to half of the tumor bulk. Macrophage phenotype typically ranges from pro-inflammatory (M1-like) to anti-inflammatory (M2-like). In GBM, they adopt a pathological, pro-tumor phenotype that thwarts anti-tumor immune responses. The GBM TME also features solid stress, which arises as the tumor expands and presses against the surrounding tissue. As residents of the tumor, macrophages therefore experience chronic compression. Here, we utilize 2D and 3D models to interrogate the macrophage response to and contribution to solid stress as seen in GBM. Murine macrophages were seeded in a cell culture insert and compression was applied with a 3D printed weight. An agarose cushion between the cells and the weight served as a media reservoir and prevented direct contact between the cells and the rigid weight. We measured the resulting changes in metabolic state, gene expression, nitric oxide production, phagocytosis, and influence on GBM cell proliferation. Using fluorescence lifetime imaging, we found that compression caused an increase in average cell fluorescence lifetime, indicating a shift towards increased oxidative phosphorylation. This is consistent with an M2-like, or anti-inflammatory phenotype. However, compressed macrophages also produced significantly more nitric oxide, a hallmark of pro-inflammatory macrophage function. Compressed macrophages upregulated both nitric oxide synthase and arginase-1, markers of canonically pro- and anti-inflammatory phenotype, respectively. They also had increased phagocytosis of microbeads. These results suggest that chronic compression results in a more complex phenotype than is represented on the traditional pro- and anti-inflammatory axis. Macrophages can also generate de novo solid stress in a confining 3-D hydrogel, with stress generation and phenotype responsive to physical and biochemical cues. This indicates that there may be reciprocal regulation between solid stress and macrophage phenotype in GBM, representing a promising therapeutic target to alleviate pathological solid stress and immunosuppression. Macrophages in GBM tumors also display elements of classical pro- and anti-inflammatory phenotypes. These results suggest that mechanical compression alone, independent from tumor-derived biochemical cues, may contribute to this pathological phenotype. Future work will elucidate targetable pathways involved in the macrophage response to chronic solid stress, with the aim of normalizing the tumor immune microenvironment to improve the efficacy of the natural or engineered anti-tumor immune response. Citation Format: Alice Burchett, Hao Chen, Scott Howard, Meenal Datta. Studying the effect of compressive solid stress on macrophage phenotype and function in the context of glioblastoma [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 146.