Functional Contribution of Cancer-Associated Fibroblasts in Glioblastoma

Abstract The abundance and biological contribution of cancer associated fibroblasts (CAFs) in glioblastoma are poorly understood. Here, we applied single-cell RNA sequencing and spatial transcriptomics analyses to identify and characterize CAFs in human glioblastoma tumors and then performed functional enrichment analysis and in vitro assays to investigate their interactions with malignant glioblastoma cells. We found that CAF abundance was significantly correlated with tumor grade, poor clinical outcome, and activation of extracellular matrix remodeling, using three large databases containing bulk RNA-sequencing data and clinical information. Proteomic analysis of the CAFs and their secretome revealed fibronectin (FN1) as a strong candidate mediating CAF functions. This was validated using in vitro cellular models, which demonstrated that CAF conditioned media and recombinant FN1 could facilitate the migration and invasion of glioblastoma cells. In addition, we showed that CAFs were more abundant in the mesenchymal-like state (or subtype) than in other states of glioblastomas, while cell lines resembling the proneural-state responded to the CAF signaling better in terms of the migratory and invasive phenotypes. Investigating the in-situ expression of gene markers specifically associated with CAFs and mesenchymal malignant cells further indicated that CAFs were enriched in the perinecrotic and pseudopalisading zones of human tumors, where mesenchymal-like glioblastoma cells co-resided and thus likely interacted. Overall, this study characterized the molecular features and functional impacts of CAFs in glioblastoma, alluding to a novel cell-to-cell interaction axis mediated by CAFs in the glioblastoma microenvironment.