Abstract PS4-06-22: Niche-specific clustering of immune-suppressive cell populations and signals in breast cancer bone metastases

Abstract Breast cancer cells frequently metastasize to bone and home to the osteogenic niche (bone surface), where they are thought to lay dormant until reactivated. Our knowledge of how tumor cells are impacted by the bone microenvironment is limited, as the spatial integrity of bone is often compromised. We hypothesized that myeloid populations infiltrate the osteogenic niche and sustain tumor burden by creating an immunosuppressive environment. We performed Digital Spatial Profiling (DSP) of bone metastases from 6-week-old female C57Bl/6 mice challenged with E0771 mouse mammary carcinoma cells by intracardiac injection (n=3/group) and evaluated tumor and immune cells residing in the marrow or endosteum. Mice were treated with α-PD-1 every 3-4 days to activate T cells and create a pro-inflammatory microenvironment. Upon sacrifice, formalin-fixed bone tissue sections were decalcified, paraffin-embedded, sectioned, and stained with syto13, pan-cytokeratin (PanCK), and CD45 as morphological markers to identify nuclei, tumor, and immune cells respectively. Antibodies for 70+ proteins (e.g., immune activation, cell death, proliferation, and cell stress), were then hybridized to the bone tissue slides, and regions of interest (ROIs) were selected along the osteogenic niche (within 100µm of the bone surface) and the marrow region (>100µm from the bone surface). Each ROI (n=5-6/bone) was segmented into two areas of interest (AOIs), PanCK or CD45 positive staining, and antibody reads were collected in each segment using Nanostring GeoMx/nCounter instruments and normalized to GAPDH and Histone H3 housekeeping proteins. Within the CD45+ segments (n=9 AOIs), we observed increased CD163 (p<0.0043) and FOXP3 (p<0.0385) expression in the osteogenic niche compared to the marrow niche, suggesting immune-suppressive M2-like macrophages and T regulatory cells (Tregs) may cluster in the osteogenic niche. Moreover, immune checkpoint proteins PD-1 (p<0.0202) and PD-L1 (p<0.0253), which are also immune-suppressive, were enriched in the osteogenic niche compared to the marrow in the CD45+ segments. In PanCK+ segments, we observed elevated p38 (p<0.0012), MEK1 (p<0.0280), pan-RAS (p<0.0411), and phospho-ERK1/2 (p<0.0177) proteins in the marrow niche compared to the osteogenic niche, suggesting that oncogenic pathways are enriched in breast cancer cells in the marrow. These data suggest that while the osteogenic niche is immune suppressive and may therefore provide an environment conducive to breast cancer immune evasion, breast cancer cells may preferentially proliferate within the marrow niche, away from the osteogenic niche/bone surface. Citation Format: D. Grant, G. Joseph, M. Searcy, R. Johnson. Niche-specific clustering of immune-suppressive cell populations and signals in breast cancer bone metastases [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS4-06-22.