In glioblastoma (GBM), tumor-associated macrophages (TAM) represent up to one half of the cells of the tumor mass, including both infiltrating macrophages and resident brain microglia. In an effort to delineate the temporal and spatial dynamics of TAM composition during gliomagenesis, we employed two genetically engineered mouse models where oncogenic drivers and fluorescent reporters were expressed coordinately under the control of the monocyte/microglia-selective Cx3cr1 or Ccr2 promoters, respectively. Using this approach, we demonstrated that CX3CR1LoCCR2Hi monocytes were recruited to the glioblastoma, where they transitioned to CX3CR1HiCCR2Lo macrophages and CX3CR1HiCCR2− microglia-like cells. Infiltrating macrophages/monocytes constituted ~85% of the total TAM population, with resident microglia accounting for the ~15% remaining. Bone marrow-derived infiltrating macrophages/monocytes were recruited to the tumor early during GBM initiation, where they localized preferentially to perivascular areas. In contrast, resident microglia were localized mainly to peritumoral regions. RNA-sequencing analyses revealed differential gene expression patterns unique to infiltrating and resident cells, suggesting unique functions for each TAM population. Notably, limiting monocyte infiltration via Ccl2 genetic ablation prolonged the survival of tumor-bearing mice. Our findings illuminate the unique composition and functions of infiltrating and resident myeloid cells in GBM, establishing a rationale to target infiltrating cells in this neoplasm.

Monocytes/macrophages have begun to emerge as key cellular modulators of brain homeostasis and central nervous system (CNS) disease. In the healthy brain, resident microglia are the predominant macrophage cell population; however, under conditions of blood-brain barrier leakage, peripheral monocytes/macrophages can infiltrate the brain and participate in CNS disease pathogenesis. Distinguishing these two populations is often challenging, owing to a paucity of universally accepted and reliable markers. To identify discriminatory marker sets for microglia and peripheral monocytes/macrophages, we employed a large meta-analytic approach using five published murine transcriptional datasets. Following hierarchical clustering, we filtered the top differentially expressed genes (DEGs) through a brain cell type-specific sequencing database, which led to the identification of eight microglia and eight peripheral monocyte/macrophage markers. We then validated their differential expression, leveraging a published single cell RNA sequencing dataset and quantitative RT-PCR using freshly isolated microglia and peripheral monocytes/macrophages from two different mouse strains. We further verified the translation of these DEGs at the protein level. As top microglia DEGs, we identified P2ry12 , Tmem119, Slc2a5 and Fcrls , whereas Emilin2 , Gda, Hp and Sell emerged as the best DEGs for identifying peripheral monocytes/macrophages. Lastly, we evaluated their utility in discriminating monocyte/macrophage populations in the setting of brain pathology (glioma), and found that these DEG sets distinguished glioma-associated microglia from macrophages in both RCAS and GL261 mouse models of glioblastoma. Taken together, this unbiased bioinformatic approach facilitated the discovery of a robust set of microglia and peripheral monocyte/macrophage expression markers to discriminate these monocyte populations in both health and disease. Electronic supplementary material The online version of this article (10.1186/s40478-019-0665-y) contains supplementary material, which is available to authorized users.

While carcinogenesis requires the acquisition of driver mutations in progenitor cells, tumor growth and progression is heavily influenced by the local microenvironment. Previous studies from our laboratory have employed Neurofibromatosis-1 (NF1) genetically engineered mice to characterize the role of stromal cells and signals to optic glioma formation and growth. Previously, we have shown that Nf1+/- microglia in the tumor microenvironment are critical cellular determinants of optic glioma proliferation. To define the role of microglia in tumor formation and maintenance further, we employed CD11b-TK mice, in which resident brain microglia (CD11b+, CD68+, Iba1+, CD45low cells) can be ablated at specific times following ganciclovir (GCV) administration. GCV-mediated microglia reduction reduced Nf1 optic glioma proliferation during both tumor maintenance and tumor development. We identified the developmental window during which microglia are increased in the Nf1+/- optic nerve and demonstrated that this accumulation reflected delayed microglia dispersion. The increase in microglia in the Nf1+/- optic nerve was associated with reduced expression of the chemokine receptor, CX3CR1, such that reduced Cx3cr1 expression in Cx3cr1-GFP heterozygous knockout mice led to a similar increase in optic nerve microglia. These results establish a critical role for microglia in the development and maintenance of Nf1 optic glioma.

The original publication of this article [1] contained 3 minor errors in Figs. 1, 3 and 5. In this correction article the updated figures are published. The figure captions describe the updated information in these figures.

While traditional models of carcinogenesis have largely focused on neoplastic cells, converging data have revealed the importance of non-neoplastic stromal cells in influencing tumor growth and progression. Leveraging a genetically-engineered mouse model of NF1-associated optic glioma, we now demonstrate that stromal microglia express the CX3CR1 chemokine receptor, such that reduced CX3CR1 expression decreases optic nerve microglia. Moreover, genetic reduction of Cx3cr1 expression in Nf1 optic glioma mice delays optic glioma formation. Coupled with previous findings demonstrating that microglia maintain optic glioma growth, these new findings provide a strong preclinical rationale for the development of future stroma-directed glioma therapies in children.