Shanna M. Handgraaf

Tumor-associated macrophages (TAMs) and microglia (MG) are potent regulators of glioma development and progression. However, the dynamic alterations of distinct TAM populations during the course of therapeutic intervention, response, and recurrence have not yet been fully explored. Here, we investigated how radiotherapy changes the relative abundance and phenotypes of brain-resident MG and peripherally recruited monocyte-derived macrophages (MDMs) in glioblastoma. We identified radiation-specific, stage-dependent MG and MDM gene expression signatures in murine gliomas and confirmed altered expression of several genes and proteins in recurrent human glioblastoma. We found that targeting these TAM populations using a colony-stimulating factor-1 receptor (CSF-1R) inhibitor combined with radiotherapy substantially enhanced survival in preclinical models. Our findings reveal the dynamics and plasticity of distinct macrophage populations in the irradiated tumor microenvironment, which has translational relevance for enhancing the efficacy of standard-of-care treatment in gliomas.

Tumors growing in metabolically challenged environments, such as glioblastoma in the brain, are particularly reliant on crosstalk with their tumor microenvironment (TME) to satisfy their high energetic needs. To study the intricacies of this metabolic interplay, we interrogated the heterogeneity of the glioblastoma TME using single-cell and multi-omics analyses and identified metabolically rewired tumor-associated macrophage (TAM) subpopulations with pro-tumorigenic properties. These TAM subsets, termed lipid-laden macrophages (LLMs) to reflect their cholesterol accumulation, are epigenetically rewired, display immunosuppressive features, and are enriched in the aggressive mesenchymal glioblastoma subtype. Engulfment of cholesterol-rich myelin debris endows subsets of TAMs to acquire an LLM phenotype. Subsequently, LLMs directly transfer myelin-derived lipids to cancer cells in an LXR/Abca1-dependent manner, thereby fueling the heightened metabolic demands of mesenchymal glioblastoma. Our work provides an in-depth understanding of the immune-metabolic interplay during glioblastoma progression, thereby laying a framework to unveil targetable metabolic vulnerabilities in glioblastoma.

Abstract Glioblastomas are aggressive primary brain tumors with an inherent resistance to T cell-centric immunotherapy due to their low mutational burden and immunosuppressive tumor microenvironment. Here we report that fractionated radiotherapy of preclinical glioblastoma models induce a tenfold increase in T cell content. Orthogonally, spatial imaging mass cytometry shows T cell enrichment in human recurrent tumors compared with matched primary glioblastoma. In glioblastoma-bearing mice, α-PD-1 treatment applied at the peak of T cell infiltration post-radiotherapy results in a modest survival benefit compared with concurrent α-PD-1 administration. Following α-PD-1 therapy, CD103 + regulatory T cells (Tregs) with upregulated lipid metabolism accumulate in the tumor microenvironment, and restrain immune checkpoint blockade response by repressing CD8 + T cell activation. Treg targeting elicits tertiary lymphoid structure formation, enhances CD4 + and CD8 + T cell frequency and function and unleashes radio-immunotherapeutic efficacy. These results support the rational design of therapeutic regimens limiting the induction of immunosuppressive feedback pathways in the context of T cell immunotherapy in glioblastoma.