Peshoff, Mekenzie

Abstract Introduction Microglia are the resident immune cells of the central nervous system, classically studied for neuroinflammation. A specialized subset, vessel-associated microglia (VAM), whose somata directly contact capillaries, constitutes ∼30% of microglia in the naïve brain. Only recently have VAM been recognized as regulators of brain circulation, influencing blood—brain barrier (BBB) integrity and cerebral blood flow, yet their role in pathological vascular remodeling remains unclear. To address this gap, we used glioma models in which peritumoral capillaries undergo extensive vascular remodeling and BBB dysfunction, providing an in vivo platform to probe capillary vulnerability and vascular stability. Methods We combined pharmacological microglial ablation via CSF1R inhibitors with longitudinal in vivo two-photon imaging to visualize microglia—capillary interactions. Behavioral testing, survival analysis, T2-weighted MRI, and immunostaining were performed in parallel. Results Over half of the microglia-depleted mice developed acute, lethal paralysis by 15 days, whereas control tumor-bearing mice remained asymptomatic and survived 30 days. MRI revealed large intracranial hemorrhages and severe edema, and two-photon imaging showed nodular, abnormal vasculature at paralysis onset. The peritumoral region contained dense capillary networks with a marked increase in VAMs (>60%). Genetic lineage tracing confirmed these cells were microglia, exhibiting elongated, vessel-wrapping morphology, also observed following single-vessel photothrombosis and consistent with a response to capillary injury. Conclusion Our findings identify VAMs as critical neurovascular stabilizers that preserve peritumoral capillary integrity and prevent lethal hemorrhage in glioma, highlighting an essential immune role in brain vascular homeostasis. Notably, intracranial hemorrhage has repeatedly emerged across clinical trials of CSF1R inhibitors, raising important safety considerations for CSF1R-targeted therapies in the diseased brain. Funding Source AHA postdoc fellowship (26POST1560008) (J.Z.) Topic Categories Neuroimmunology (NEUR)

The discovery of genes encoding the volume-regulated anion channel (VRAC) has enabled detailed exploration of its cell type–specific roles in the brain. LRRC8A (SWELL1) is the essential VRAC subunit. We observed seizure-induced, subunit-specific changes in microglial VRAC expression and investigated its function using conditional KO (cKO) of LRRC8A in microglia. SWELL1 cKO mice exhibited a male-specific increase in kainate-induced seizure severity, yet showed paradoxical neuroprotection against seizure-associated neuronal loss. Mechanistically, SWELL1 deletion led to a cell-autonomous reduction in microglial density and decreased release of VRAC-permeable neuroactive metabolites, including taurine, GABA, and glutamate in culture. Additionally, impaired phagocytic kinetics and reduced lysosomal biogenesis contributed to the observed neuroprotection. These findings reveal potentially novel roles for microglial VRAC in regulating seizure outcomes and microglia-neuron interactions.

Abstract Triggering receptor expressed on myeloid cells 2 (TREM2) is a critical modulator of phagocytosis, inflammation, and survival for microglia and other macrophages. In neurodegenerative disorders, microglia expressing TREM2 play a protective role at distinct phases of disease progression, enabling uptake of toxic protein aggregates and dead neurons. TREM2 is also highly expressed on tumor-associated macrophages in a variety of cancers, promoting an immunosuppressive microenvironment. We and others have previously investigated the role of TREM2 in gliomas with confounding results, suggesting TREM2 could be pro-tumorigenic or anti-tumorigenic depending on experimental context (Peshoff, et al. Neuro Oncol. and Zheng, et al. Neuro Oncol.) In this study, we aim to resolve these findings by investigating the role of TREM2 across progression of gliomas with unique genetic drivers using immunocompetent mouse models. We employ syngeneic glioma stem cell lines generated via retroviral vectors expressing a dominant negative p53 along with amplification of human PDGFB or oncogenic HRAS G12V to model proneural and mesenchymal glioblastomas, respectfully. In contrast to our previous results demonstrating enhanced GL261 and CT2A growth in Trem2-/- mice, we demonstrate that Trem2 deficiency results in decreased tumor volume and improved survival when mice are challenged with Ras-driven, but not PDGFB-driven, gliomas. Furthermore, Trem2-/- mice display a more immunostimulatory microenvironment with increased T cell infiltration to mesenchymal-like tumors. We employ an in vitro co-culture system of primary mouse microglia with glioma stem cells to understand how microglia sense and respond to different genetic subtypes. We find that TREM2-mediated processes including microglial phagocytosis, chemotaxis, and survival are impacted by glioma genetic drivers. Together, these results indicate discrepancies in the literature regarding the function of TREM2 in gliomas may be explained by tumor genetics behind the cell lines used, which can provide a framework for understanding subtypes of gliomas that could respond to TREM2-modulating therapy.

Abstract Aberrant vasculature is a hallmark of glioblastoma (GBM), contributing to both blood–brain barrier (BBB) dysfunction and frequent intracranial hemorrhage. Myeloid cells, including microglia and macrophages, are known to co-opt tumor vasculature during tumor progression, but the nature and consequences of their physical interactions with blood vessels require clearer definition. To address this, we characterize vessel-associated myeloid cells in murine gliomas with high spatial and temporal resolution, leveraging in vivo two-photon imaging, MRI, and transgenic mouse lines. To investigate the role of microglia in glioma vasculature, we pretreated glioma-bearing mice with CSF1R inhibitors (PLX3397 & PLX5622) to ablate microglia prior to tumor inoculation. Strikingly, over half of the microglia-depleted mice developed acute, lethal paralysis between days 11–15, whereas control tumor-bearing mice remained asymptomatic. MRI revealed extensive intracranial hemorrhage and edema, and two-photon imaging showed nodular, abnormal vasculature. This vascular dysfunction led to a dramatic reduction in median survival—from 30 days in control glioma-bearing mice to 15 days in PLX-treated mice with gliomas. To identify the protective microglial population involved, we examined vascular–myeloid interactions. We found that the peritumoral region harbored dense capillary networks supporting tumor expansion, along with a marked increase in myeloid cells with somas directly contacting blood vessels. Using TMEM119CreER/+:R26tdTomato/+ mice, we confirmed these vessel-associated cells as brain-resident microglia, not infiltrating macrophages. Quantitatively, ~60% of peritumoral microglia were vessel-associated, compared to ~30% in naïve or contralateral hemispheres. These microglia displayed elongated somata and processes aligned with vessels or wrapping around vessels, which is a phenotype also observed following single-vessel photothrombosis, suggesting a response to impaired blood flow and BBB leakage. Our findings uncover a critical neurovascular role for vessel-associated microglia in maintaining vascular integrity during glioma progression and highlight the importance of preserving, reprogramming, and further investigating this population in GBM therapies.