Abstract B061: Defining Early Glial Responses in Breast Cancer Brain Metastasis

Abstract Background: Early detection of the footprints of brain metastasis in breast cancer remains an unresolved clinical challenge. Because metastatic sites often lack identifiable signs until the blood-brain barrier (BBB) is compromised, there is a critical need for reliable techniques to detect brain metastasis early. We hypothesized that early changes within the tumor microenvironment, particularly the responsive behaviors of glial cells, might serve as diagnostic indicators of metastatic initiation, preceding observable BBB disruption. Here, we devised an experimental system capable of elucidating primary tumor–glia interactions in vivo. Methods: Breast cancer cells were injected via the tail vein. Tumor development and brain metastatic events were analyzed in 3D using light-sheet and confocal microscopy after tissue clearing. Immunofluorescence staining for Iba1, TMEM119, CD68, and GFAP was used to evaluate the two main glial cell populations, microglia and astrocytes. A 70kDa fluorescently labeled dextran tracer was used to assess the integrity of the BBB. Lesion volume, glial cell distribution, and BBB permeability were quantified using 3D image reconstruction and spatial fluorescence analysis. Results: Within 4 days, small metastatic deposits were detectable predominantly in the cerebral cortex, while no hemorrhagic BBB disruption was confirmed. In these early stages, microglial activation was detectable, as indicated by a transition of Iba1-positive microglia from a ramified to an amoeboid morphology, characterized by reduced process complexity and increased CD68 expression, even in the absence of dextran leakage. Astrocytes also accumulated in peritumoral regions, forming an early astrocytic boundary around nascent metastatic foci. As metastatic progression continued, a pronounced BBB disruption associated with hemorrhagic leakage developed. These findings indicate that microglial activation and astrocytic accumulation arise during the window preceding overt BBB breakdown. Conclusions: This high-resolution in vivo platform demonstrates that distinct microglial activation and astrocytic accumulation appear before overt, hemorrhage-associated BBB disruption during early breast cancer brain colonization. These early glial responses may represent biologically meaningful changes that precede clinically detectable barrier breakdown, supporting their further investigation as potential early indicators of metastatic initiation. Citation Format: Naoki Hama, Alysha Ho, Gargi Chitre, Hunter Dickens, Yoshiko Nagaoka-Kamata, Masakazu Kamata. Defining Early Glial Responses in Breast Cancer Brain Metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B061.