Tmic-12. complement signaling in hypoxic niches of the glioma microenvironment

Abstract Glioblastoma is the most common and aggressive primary brain tumor in adults. Despite treatment through surgery, irradiation and chemotherapy, all patients suffer recurrence of treatment-resistant tumors. Previous studies from our lab showed that astrocytes become reactive and generate tumor-supportive conditions for glioma cells, when exposed to hypoxia or irradiation. Reactive astrocytes are known to upregulate proteins of the complement system, especially complement component 3 (C3), in several neurological disorders. However, it remains unexplored how these complement proteins are expressed in stromal astrocytes in glioblastoma. Tissue sections from a glioma mouse model showed presence of C3 in the invasive front, and in hypoxic, and perivascular spaces of the tumors, where there is an abundance of astrocytes. C3 expression co-localized with Nestin and CD44, two markers of mesenchymal and/or stem-like glioma cells. Primary human astrocytes grown in hypoxic conditions upregulated C3 as well as other proteins associated with a more extensive infiltrative phenotype of glioblastoma. In general, a strong correlation between hypoxia (195 genes) and complement (200 genes) gene signatures were found in TCGA GBM dataset (R= 0.82, p-value= 0). Single-cell sequencing data from primary GBM tumors showed a subpopulation of highly C3-expressing astrocytes, which were enriched for cellular pathways comprising epithelial mesenchymal transition (EMT), TNF-alpha, complement, hypoxia, and interferon signaling. Human glioma cell lines from proneural, classical and mesenchymal subtypes all showed increased CAIX and GLUT1 expression grown under lower oxygen tensions, while two out of three subtypes showed upregulation of stemness markers (OCT4 and NANOG1) as well as C3 and C3aR. Overall, our data indicate a strong link between hypoxia and complement expression in the brain tumor microenvironment, where a local expression of complement proteins could possibly lead to tumor promoting signaling, which will be further explored.