Skull-Derived Myeloid Cells Augment T Cell Responses and Suppress Glioblastoma Growth

Abstract

INTRODUCTION: Recent studies have implicated skull marrow as a functional myeloid reservoir in CNS pathology. However, its contribution to malignancy remains unexplored. METHODS: Fluorescently labeled calvarial flaps were transplanted onto syngeneic glioblastoma-bearing mice, followed by flow cytometric profiling of tumor-infiltrative leukocytes. To assay the functional relevance of SDMCs, tumor-bearing C57BL/6 mice were treated with preemptive calvarial ablation (13 Gy irradiation), or conversely intracalvarial AMD3100 to augment myeloid egress from marrow niches. Tumor burden was monitored via survival and bioluminescent imaging. RESULTS: 72 h following CMFDA-stained calvarial transplantation into tumor-bearing Balb/C mice, 40.3% of tumor-infiltrative myeloid cells were fluorescent and thus skull-derived; transplantation in an alternative longitudinal model (GFP+ flaps, C57BL/6) validated that SDMCs were robust and viable at 3 weeks post-operatively. As MHCII+ cells were disproportionately enriched among SDMCs (87.9% vs 67.3%, p = 0.012), we interrogated the immunogenicity of these cells through targeted ablation. Skull-ablated mice had reduced infiltration by antigen-presenting cells than controls (cDCs: 1.1% vs 4.5%, p = 0.002; macrophages: 14.4% vs 36.6%, p < 0.001) and 6-fold greater necrosis (67.7% vs 13.5% apoptotic, p < 0.001), suggesting an impaired ability to mount a controlled, adaptive immune response. The stimulatory role of SDMCs was further demonstrated via intracalvarial AMD3100 administration, which through its promotion of myeloid precursor egress (0.26% vs 0.02%, p < 0.001) and cDC2 infiltration (10.4% vs 3.0%, p = 0.003) yielded robust CD4+ T cell activation and effector memory differentiation. These differences were clinically meaningful, as skull irradiation accelerated tumor growth and attenuated survival compared to controls, while AMD3100 treatment accomplished the opposite (median OS: 17d vs 22d, p < 0.001; 26.5d vs 22d, p = 0.006). CONCLUSIONS: The preferential polarization of SDMCs into immunostimulatory APCs warrants the continued exploration of local marrow-targeting agents (e.g., AMD3100) as therapeutic modalities.