Background Glioma-associated microglia/macrophage (GAM) markedly influence glioma progression. Under the influence of transforming growth factor beta (TGFB), GAM are polarized toward a tumor-supportive phenotype. However, neither therapeutic targeting of GAM recruitment, nor TGF Beta (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. Further, 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 up-regulation of the canonical TGFB signaling axis. Moreover, TGFB1 enhanced 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 investigation of GAM-associated therapeutic strategies for human malignant glioma.

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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.

BackgroundNo definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF‐H) is a promising biomarker for nervous system diseases.Hypothesis/ObjectiveCerebrospinal fluid (CSF) and serum pNF‐H is a detectable biological marker for diagnosis of canine DM.AnimalsFifty‐three DM‐affected, 27 neurologically normal, 7 asymptomatic at‐risk, and 12 DM mimic dogs.MethodsArchived CSF and serum pNF‐H concentrations were determined by a commercially available ELISA. A receiver‐operating characteristic (ROC) curve was generated with CSF values.ResultsCompared 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.Conclusions and Clinical Importance 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.