Abstract 173: Studying the interaction between macrophages and solid stress in novel models of the tumor immune microenvironment

Abstract

Abstract Glioblastoma (GBM) is one of the most aggressive forms of cancer, resisting conventional and immune-based therapies and typically killing patients within two years of diagnosis. This is largely due to the immunosuppressive GBM tumor microenvironment (TME), mediated in large part by tumor-resident macrophages. Macrophages’ role as immune regulators, as well as their sheer quantity in brain tumors, makes them attractive therapeutic targets. The GBM TME also features mechanical abnormalities. As a tumor grows, it expands into the surrounding brain, generating compressive solid stress. Macrophages can contribute to up to half of tumor mass and are known to respond to mechanical cues, allowing them to both contribute to and respond to solid stress, but this relationship has not yet been explored experimentally. To isolate the contribution of macrophages on solid stress, we incorporated RAW264.7 murine macrophages into agarose gels with varying stiffnesses. Over time, spheroids form as the cells proliferate. Because cells cannot alter agarose, cell aggregates must generate stress as they grow and displace the surrounding gel matrix. The resulting agarose-embedded spheroids were stained with calcein AM to observe spheroid viability and morphology. Using confocal microscopy and image processing, we obtained the final spheroid geometry and imported it into COMSOL to model the solid stress resulting from spheroid formation. Our initial experiments show that macrophages have distinct growth patterns dependent on the stiffness of their surrounding gel. At larger sizes, the spheroids adopt an ellipsoid shape and generate a distinct stress field compared to spheroidal shapes. To stimulate the solid stress that macrophages experience within a growing mass, we applied a custom weight to macrophages grown on a porous membrane. This applies 0.15 kPa of compressive solid stress, corresponding to the stress measured in murine glioma models. After 24 hours of compression, macrophages upregulate expression of both nitric oxide synthase and arginase-1, canonical M1 and M2 markers, respectively. This indicates that compression causes a more complex phenotype than can be described by the traditional M1/M2 axis, something that has been observed in tumor-associated macrophages. We also found an apparent difference in the fluorescence lifetime of compressed and uncompressed macrophages, indicating that compression alters macrophage metabolism. Both the spheroid and compression culture models are unique in the context of macrophages. This work will elucidate key pathways involved in myeloid “immunomechanics” and inform strategies to target the innate immune system in cancers such as GBM, as well as other immunological diseases. Citation Format: Alice Burchett, Saeed Siri, Hao Chen, Scott Howard, Meenal Datta. Studying the interaction between macrophages and solid stress in novel models of the tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 173.