Sex differences in brain structure and function are of substantial scientific interest because of sex-related susceptibility to psychiatric and neurological disorders. Neuroinflammation is a common denominator of many of these diseases, and thus microglia, as the brain's immunocompetent cells, have come into focus in sex-specific studies. Here, we show differences in the structure, function, and transcriptomic and proteomic profiles in microglia freshly isolated from male and female mouse brains. We show that male microglia are more frequent in specific brain areas, have a higher antigen-presenting capacity, and appear to have a higher potential to respond to stimuli such as ATP, reflected in higher baseline outward and inward currents and higher protein expression of purinergic receptors. Altogether, we provide a comprehensive resource to generate and validate hypotheses regarding brain sex differences.

Microglia are resident myeloid cells in the central nervous system (CNS) that control homeostasis and protect CNS from damage and infections. Microglia and peripheral myeloid cells accumulate and adapt tumor supporting roles in human glioblastomas that show prevalence in men. Cell heterogeneity and functional phenotypes of myeloid subpopulations in gliomas remain elusive. Here we show single-cell RNA sequencing (scRNA-seq) of CD11b+ myeloid cells in naïve and GL261 glioma-bearing mice that reveal distinct profiles of microglia, infiltrating monocytes/macrophages and CNS border-associated macrophages. We demonstrate an unforeseen molecular heterogeneity among myeloid cells in naïve and glioma-bearing brains, validate selected marker proteins and show distinct spatial distribution of identified subsets in experimental gliomas. We find higher expression of MHCII encoding genes in glioma-activated male microglia, which was corroborated in bulk and scRNA-seq data from human diffuse gliomas. Our data suggest that sex-specific gene expression in glioma-activated microglia may be relevant to the incidence and outcomes of glioma patients.

13Brain resident and infiltrating innate immune cells adapt a tumor-supportive phenotype in the 14 glioma microenvironment. Flow cytometry analysis supported by a single-cell RNA 15 sequencing study of human gliomas indicate considerable cell type heterogeneity. It remains 16 disputable whether microglia and infiltrating macrophages have the same or distinct roles in 17 supporting glioma progression. Here, we performed single-cell transcriptomics analyses of 18 CD11b+ cells sorted from murine syngeneic gliomas, indicating distinct activity of microglia, 19 infiltrating monocytes/macrophages and CNS border-associated macrophages. Our results 20 demonstrate a previously immeasurable scale of molecular heterogeneity in the innate 21 immune response in gliomas. We identified genes differentially expressed in activated 22 microglia from glioma-bearing mice of different sex, and profound overexpression of the 23 MHCII genes by male microglial cells, which we also observed in bulk human glioma 24 samples. Sex-specific gene expression in microglia in the glioma microenvironment may be 25 relevant to sex differences in incidence and outcomes of glioblastoma patients. 26 27 3 28 Introduction 29 Infiltrating immune system cells represent an abundant non-malignant component of the 30tumor microenvironment (TME). These cells play a pivotal role in tumor progression and 31 modulation of tumor responses to therapy 1 . A high number of macrophages within TME have 32 been associated with a poor prognosis in many cancers because those tumor-educated cells 33 suppress anti-tumor immunity, stimulate angiogenesis, and promote tumor invasion 2 . 34The central nervous system (CNS) is equipped with resident innate immune cells-microglia 35 and CNS border-associated macrophages (BAMs)-consisting of perivascular, meningeal, 36 and the choroid plexus macrophages. Those cells migrate to the CNS early in the prenatal 37 life and maintain a long-lasting population. In malignant gliomas, besides activation of local 38 microglia, circulating monocytes invade the brain from the periphery and differentiate within 39 the tumor; therefore, microglia and infiltrating monocytes/macrophages are commonly 40 referred to as glioma-associated macrophages (GAMs), due to the shortage 41 of immunocytochemical markers allowing their reliable identification 3 . Transcriptome profiling 42 of bulk CD11b+ cells isolated from human glioblastomas (GBMs) and rodent gliomas showed 43 a mixture of protumorigenic and antitumorigenic phenotypes and did not reveal consistent 44 markers and pathways 4-6 . Recent reports have demonstrated that GAMs consist of diverse 45 cell populations with likely distinct roles in tumor progression 7-10 . Dissecting the TME 46 composition and functional heterogeneity of tumor-infiltrating immune cells would extend the 47 understanding of the glioma immune microenvironment and allow to modulate functions of 48 distinct subpopulations for therapeutic benefits. 49Sex differences in incidence, transcriptomes, and patient outcomes in the adul...

Aim: Alterations of microglia, the brain-resident macrophages, are associated with numerous brain pathologies. Genetic manipulation of microglia in diseases using small interfering RNA (siRNA) is hampered by the lack of safe and efficient siRNA delivery methods. We assessed the amphiphilic dendrimer (AD) for functional siRNA delivery and gene knockdown in primary microglia. Materials & methods: We characterized the ability of AD to form nanoparticles with siRNA, and studied their size, surface potential, cell uptake and gene silencing in rodent microglia. Results: AD effectively delivered siRNA to primary microglia and decreased target gene and protein expression, leading to transcriptomic changes without affecting basal microglial functions. Conclusion: The dendrimer AD promises to be an innocuous carrier for siRNA delivery into microglia.

Immune cells accumulating in the microenvironment of malignant tumors are tumor educated and contribute to its growth, progression, and evasion of antitumor immune responses. Glioblastoma (GBM), the common and most malignant primary brain tumor in adults, shows considerable accumulation of resident microglia and peripheral macrophages, and their polarization into tumor-supporting cells. There are controversies regarding a functional phenotype of glioma-associated microglia/macrophages (GAMs) due to a lack of consistent markers. Previous categorization of GAM polarization toward the M2 phenotype has been found inaccurate because of oversimplification of highly complex and heterogeneous responses. In this study, we characterized functional responses and gene expression in mouse and human microglial cultures exposed to fresh conditioned media [glioma-conditioned medium (GCM)] from human U87 and LN18 glioma cells. Functional analyses revealed mutual communication reflected by strong stimulation of glioma invasion by microglial cells and increased microglial phagocytosis after GCM treatment. To define transcriptomic markers of GCM-activated microglia, we performed selected and global gene expression analyses of stimulated microglial cells. We found activated pathways associated with immune evasion and TGF signaling. We performed computational comparison of the expression patterns of GAMs from human GBMs and rodent experimental gliomas to select genes consistently changed in different datasets. The analyses of marker genes in GAMs from different experimental models and clinical samples revealed only a small set of common genes, which reflects variegated responses in clinical and experimental settings. Tgm2 and Gpnmb were the only two genes common in the analyzed data sets. We discuss potential sources of the observed differences and stress a great need for definitive elucidation of a functional state of GAMs.

Glioblastomas (GBM) are the common and aggressive primary brain tumors that are incurable by conventional therapies. Immunotherapy with immune checkpoint inhibitors is not effective in GBM patients due to the highly immunosuppressive tumor microenvironment (TME) restraining the infiltration and activation of cytotoxic T cells. Clinical and experimental studies showed the upregulation of expression of the arginase 1 and 2 (ARG1 and ARG2, respectively) in murine and human GBMs. The elevated arginase activity leads to the depletion of L-arginine, an amino-acid required for the proliferation of T lymphocytes and natural killer cells. Inhibition of ARG1/2 in the TME may unblock T cell proliferation and activate effective antitumor responses. To explore the antitumor potential of ARG1/2 inhibition, we analyzed bulk and single-cell RNA sequencing (scRNA-seq) data from human and murine gliomas. We found the upregulation of ARG1/2 expression in GBMs, both in tumor cells and in tumor infiltrating microglia and monocytes/macrophages. We employed selective arginase inhibitors to evaluate if ARG1/2 inhibition in vitro and in vivo exerts the antitumor effects. A novel, selective ARG1/2 inhibitor - OAT-1746 blocked microglia-dependent invasion of U87-MG and LN18 glioma cells in a Matrigel invasion assay better than reference compounds, without affecting the cell viability. OAT-1746 effectively crossed the blood brain barrier in mice and increased arginine levels in the brains of GL261 glioma bearing mice. We evaluated its antitumor efficacy against GL261 intracranial gliomas as a monotherapy and in combination with the PD-1 inhibition. The oral treatment with OAT-1746 did not affect the immune composition of TME, it induced profound transcriptomic changes in CD11b+ cells immunosorted from tumor-bearing brains as demonstrated by RNA sequencing analyses. Treatment with OAT-1746 modified the TME resulting in reduced glioma growth and increased antitumor effects of the anti-PD-1 antibody. Our findings provide the evidence that inhibition of ARG1/2 activity in tumor cells and myeloid cells in the TME unblocks antitumor responses in myeloid cells and NK cells, and improves the efficacy of the PD-1 inhibition.

Microglia are the resident immune cells of the central nervous system (CNS) that have distinct ontogeny from other tissue macrophages and play a pivotal role in health and disease. Microglia rapidly react to the changes in their microenvironment. This plasticity is attributed to the ability of microglia to adapt a context-specific phenotype. Numerous gene expression profiling studies of immunosorted CNS immune cells did not permit a clear dissection of their phenotypes, particularly in diseases when peripheral cells of the immune system come to play. Only recent advances in single-cell technologies allowed studying microglia at high resolution and revealed a spectrum of discrete states both under homeostatic and pathological conditions. Single-cell technologies such as single-cell RNA sequencing (scRNA-seq) and mass cytometry (Cytometry by Time-Of-Flight, CyTOF) enabled determining entire transcriptomes or the simultaneous quantification of >30 cellular parameters of thousands of individual cells. Single-cell omics studies demonstrated the unforeseen heterogeneity of microglia and immune infiltrates in brain pathologies: neurodegenerative disorders, stroke, depression, and brain tumors. We summarize the findings from those studies and the current state of knowledge of functional diversity of microglia under physiological and pathological conditions. A precise definition of microglia functions and phenotypes may be essential to design future immune-modulating therapies.

Single-cell technologies allow precise identification of tumor composition at the single‑cell level, providing high-resolution insights into the intratumoral heterogeneity and transcriptional activity of cells in the tumor microenvironment (TME) that previous approaches failed to capture. Malignant gliomas, the most common primary brain tumors in adults, are genetically heterogeneous and their TME consists of various stromal and immune cells playing an important role in tumor progression and responses to therapies. Previous gene expression or immunocytochemical studies of immune cells infiltrating TME of malignant gliomas failed to dissect their functional phenotypes. Single-cell RNA sequencing (scRNA-seq) and cytometry by time-of-flight (CyTOF) are powerful techniques allowing quantification of whole transcriptomes or >30 protein targets in individual cells. Both methods provide unprecedented resolution of TME. We summarize the findings from these studies and the current state of knowledge of a functional diversity of immune infiltrates in malignant gliomas with different genetic alterations. A precise definition of functional phenotypes of myeloid and lymphoid cells might be essential for designing effective immunotherapies. Single-cell omics studies have identified crucial cell subpopulations and signaling pathways that promote tumor progression, influence patient survival or make tumors vulnerable to immunotherapy. We anticipate that the widespread usage of single-cell omics would allow rational design of oncoimmunotherapeutics.