The Novel Protein, Olfactomedin‐like 3, Alters Microglia Biology and May Contribute to Glioma Progression

Abstract

Despite decades of research, glioblastoma multiforme (GBM) remains a uniformly lethal brain tumor. Transforming growth factor-, in concert with glioma associated microglia (GAMS), promotes GBM growth and invasion. Although TGF- has a significant role in GBM progression, failed clinical trials suggest a complex role in GBM pathogenesis similar to non-CNS cancer. Thus, an improved understanding of the TGF- /GAM axis is critical to refine the therapeutic approach toward precise molecular targets. Olfactomedin-like 3 (OLFML3), a novel secreted glycoprotein, is elevated 9-fold in GBM. Although poorly studied within the context of GBM, OLFML3 contributes to non-CNS tumor progression via increased angiogenesis and cell growth pathways. Importantly, Olfml3 has been identified in mice as a direct target gene of TGF- in microglia. Therefore,we hypothesized that microglia-derived Olfml3 polarizes microglia cells toward a pro-tumorigenic phenotype and promotes tumor cell growth kinetics. We first demonstrated that Olfml3is a direct target gene of all three TGF- isoforms in microglia, but not a mouse glioma cell line (GL261). Using CRISPR/Cas9, we generated an Olfml3-knockout (Olfml3-/-) microglial cell line. Loss of Olfml3 altered microglial production of several pro-tumorigenic molecules. Most notably, secretion of key pro-tumorigenic molecules GM-CSF, IL-6, and CSF-1, was attenuated in Olfml3-/- microglia. Additionally, loss of Olfml3 attenuated microglia chemotaxis. Importantly, the effects of OLFML3 were non-cell autonomous. Following 48-hour treatment with increasing concentrations of rOLFML3, we observed a linear increase in migration of GL261. However, only a single concentration of rOLFML3 increased GL261 invasion, suggesting a concentration-dependent cellular response. In conclusion, Olfml3 is a direct target gene of TGF- in microglia, but not in glioma cells, suggesting that increased OLFML3 in human GBM may be microglia-derived. OLFML3 supports glioma progression via pro-tumorigenic microglia gene expression and promotion of glioma cell malignant phenotype. Further studies aim to elucidate microglia derived OLFML3 and its role in GBM progression in vivo.