Increasing evidence suggests that macrophages critically shape brain homeostasis and disease.However, while the pivotal role of parenchymal microglia has gradually emerged, other brain-resident myeloid cells remain elusive. By dissecting border regions and combining single-cell RNA sequencing with high-dimensional cytometry, bulk RNA-sequencing, fate-mapping and microscopy, we reveal the remarkable diversity of non-parenchymal brain macrophages. Border-associated macrophages or BAMs residing in the dura mater, subdural meninges and choroid plexus consisted of distinct subsets that exhibited tissue-specific transcriptional signatures and underwent strong compositional changes during postnatal development. The gene regulatory networks of BAMs were identified and fundamentally differed from those of microglia. Importantly, we identified a unique non-homeostatic microglia-like population residing on the apical surface of the choroid plexus epithelium. Niche accessibility drove BAM ontogeny and determined whether embryonic macrophages were progressively replaced by bone marrow progenitors. Together, our work provides important insights into the biology of brain macrophages and offers a solid framework for future investigations.

Glioblastomas are aggressive primary brain cancers that recur as therapy-resistant tumors. Myeloid cells control glioblastoma malignancy, but their dynamics during disease progression remain poorly understood. Here, we employed single-cell RNA sequencing and CITE-Seq to map the glioblastoma immune landscape in newly diagnosed and recurrent patients and in mouse tumors. This revealed a large and diverse myeloid compartment, with dendritic cell and macrophage populations that were conserved across species and were dynamic across disease stages. Tumor-associated macrophages (TAMs) consisted of microglia-or monocyte-derived populations, with both exhibiting additional heterogeneity, including subsets with conserved lipid and hypoxic signatures. Microglia-and monocytederived TAMs (Mo-TAMs) were self-renewing populations that competed for space and could be depleted via CSF1R blockade. Microglia-derived TAMs were predominant in newly diagnosed tumors but were outnumbered by Mo-TAMs upon recurrence, especially in hypoxic tumor environments. Our results unravel the glioblastoma myeloid landscape and provide a framework for future therapeutic interventions.

Cancer immunotherapy by immune checkpoint blockade has proven its great potential by saving the lives of a proportion of late stage patients with immunogenic tumor types. However, even in these sensitive tumor types, the majority of patients do not sufficiently respond to the therapy. Furthermore, other tumor types, including glioblastoma, remain largely refractory. The glioblastoma immune microenvironment is recognized as highly immunosuppressive, posing a major hurdle for inducing immune-mediated destruction of cancer cells. Scattered information is available about the presence and activity of immunosuppressive or immunostimulatory cell types in glioblastoma tumors, including tumor-associated macrophages, tumor-infiltrating dendritic cells and regulatory T cells. These cell types are heterogeneous at the level of ontogeny, spatial distribution and functionality within the tumor immune compartment, providing insight in the complex cellular and molecular interplay that determines the immune refractory state in glioblastoma. This knowledge may also yield next generation molecular targets for therapeutic intervention.

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