Meningiomas are the most common primary intracranial tumors. Treatments for patients with meningiomas are limited to surgery and radiotherapy, and systemic therapies remain ineffective or experimental. Resistance to radiotherapy is common in high-grade meningiomas and the cell types and signaling mechanisms that drive meningioma tumorigenesis and resistance to radiotherapy are incompletely understood. Here, we report that NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy and find that perivascular NOTCH3+ stem cells are conserved across meningiomas from humans, dogs, and mice. Integrating single-cell transcriptomics with lineage tracing and imaging approaches in genetically engineered mouse models and xenografts, we show NOTCH3 drives tumor-initiating capacity, cell proliferation, angiogenesis, and resistance to radiotherapy to increase meningioma growth and reduce survival. To translate these findings to patients, we show that an antibody stabilizing the extracellular negative regulatory region of NOTCH3 blocks meningioma tumorigenesis and sensitizes meningiomas to radiotherapy, reducing tumor growth and improving survival. Significance: There are no effective systemic therapies to treat meningiomas, and meningioma stem cells are poorly understood. Here, we report perivascular NOTCH3+ stem cells to drive meningioma tumorigenesis and resistance to radiotherapy. Our results identify a conserved mechanism and a therapeutic vulnerability to treat meningiomas that are resistant to standard interventions.

Background: Glioma-associated microglia/macrophages (GAMs) markedly influence glioma progression. Under the influence of transforming growth factor beta (TGFB), GAMs are polarized toward a tumor-supportive phenotype. However, neither therapeutic targeting of GAM recruitment nor TGFB signaling demonstrated efficacy in glioma patients despite efficacy in preclinical models, underscoring the need for a comprehensive understanding of the TGFB/GAM axis. Spontaneously occurring canine gliomas share many features with human glioma and provide a complementary translational animal model for further study. Given the importance of GAM and TGFB in human glioma, the aims of this study were to further define the GAM-associated molecular profile and the relevance of TGFB signaling in canine glioma that may serve as the basis for future translational studies. Methods: GAM morphometry, levels of GAM-associated molecules, and the canonical TGFB signaling axis were compared in archived samples of canine astrocytomas versus normal canine brain. Furthermore, the effect of TGFB on the malignant phenotype of canine astrocytoma cells was evaluated. Results: GAMs diffusely infiltrated canine astrocytomas. GAM density was increased in high-grade tumors that correlated with a pro-tumorigenic molecular signature and upregulation of the canonical TGFB signaling axis. Moreover, TGFB1 enhanced the migration of canine astrocytoma cells in vitro. Conclusions: Canine astrocytomas share a similar GAM-associated immune landscape with human adult glioma. Our data also support a contributing role for TGFB1 signaling in the malignant phenotype of canine astrocytoma. These data further support naturally occurring canine glioma as a valid model for the investigation of GAM-associated therapeutic strategies for human malignant glioma.

No definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF-H) is a promising biomarker for nervous system diseases. Cerebrospinal fluid (CSF) and serum pNF-H is a detectable biological marker for diagnosis of canine DM. Fifty-three DM-affected, 27 neurologically normal, 7 asymptomatic at-risk, and 12 DM mimic dogs. Archived CSF and serum pNF-H concentrations were determined by a commercially available ELISA. A receiver-operating characteristic (ROC) curve was generated with CSF values. Compared with old control dogs, median CSF pNF-H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4–9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8–29.6; P < .05) versus DM stage 2 36.8 ng/mL (IQR 22.9–51.2; P < .0001) versus DM stage 3 25.2 ng/mL (IQR 20.2–61.8; P < .001) versus DM stage 4 38.0 ng/mL (IQR 11.6–59.9; P < .01). Degenerative myelopathy stage 1 dogs had increased median CSF pNF-H concentrations compared with asymptomatic, at-risk dogs (3.4 ng/mL [IQR 1.5–10.9; P < .01]) and DM mimics (6.6 ng/mL [IQR 3.0–12.3; P < .01]). CSF pNF-H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09–90.64%) and 93.6% specific (CI 78.58–99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92–0.9974). No differences in serum pNF-H concentration were found between control and DM-affected dogs. pNF-H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies.

Purpose: Spontaneously occurring glioma in pet dogs is increasingly recognized as a valuable translational model for human glioblastoma. Canine high-grade glioma and human glioblastomas share many molecular similarities, including the accumulation of immunosuppressive regulatory T cells (Tregs) that inhibit anti-tumor immune responses. Identifying in dog mechanisms responsible for Treg recruitment may afford to target the cellular population driving immunosuppression, the results providing a rationale for translational clinical studies in human patients. Our group has previously identified C-C motif chemokine 2 (CCL2) as a glioma-derived T-reg chemoattractant acting on chemokine receptor 4 (CCR4) in a murine orthotopic glioma model. Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma. Methods: We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine. Results: We established a flow cytometry gating strategy for identifying and isolating FOXP3⁺ Tregs in dogs. The canine CD4 + CD25^high T-cell population was highly enriched in FOXP3 and CCR4 expression, indicating they are bona fide Tregs. Canine Treg migration was enhanced by CCL2 or by glioma cell line-derived supernatant. Blockade of the CCL2-CCR4 axis significantly reduced migration of canine Tregs. CCL2 mRNA was expressed in all glioma cell lines, and expression increased when exposed to Tregs but not CD4 + helper T-cells. Conclusion: Our study validates CCL2-CCR4 as a bi-directional Treg-glioma immunosuppressive and tumor-promoting axis in canine high-grade glioma.

Under the influence of transforming growth factor-beta (TGFβ), glioma-associated microglia produce molecules that promote glioma growth and invasion. Olfactomedin-like 3 (Olfml3), a novel, secreted glycoprotein, is known to promote several non-CNS cancers. While it is a direct TGFβ1 target gene in microglia, the role of microglia-derived OLFML3 in glioma progression is unknown. Here, we tested the hypotheses that microglial Olfml3 is integral to the pro-tumorigenic glioma-associated microglia phenotype and promotes glioma cell malignancy. Using an Olfml3 knockout microglial cell line (N9), we demonstrated that Olfml3 is a direct target gene of all TGFβ isoforms in murine microglia. Moreover, loss of Olfml3 attenuated TGFβ-induced restraint on microglial immune function and production of cytokines that are critical in promoting glioma cell malignancy. Importantly, microglia-derived OLFML3 directly contributes to glioma cell malignancy through increased migration and invasion. While exposure to conditioned medium (CM) from isogenic control microglia pre-treated with TGFβ increased mouse glioma cell (GL261) migration and invasion, this effect was abolished with exposure to CM from TGFβ-treated Olfml3^-/- microglia. Taken together, our data suggest that Olfml3 may serve as a gatekeeper for TGFβ-induced microglial gene expression, thereby promoting the pro-tumorigenic microglia phenotype and glioma cell malignancy.