Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects

TLDR

Glioblastoma was long thought to lack cancer‑associated fibroblasts because brain tissue does not normally contain fibroblasts. The study isolated CAF‑like cells from glioblastoma specimens by serial trypsinization and single‑cell RNA‑seq, identifying them by absence of CNVs and high expression of nine CAF markers, and mapped ligand‑receptor interactions that link GSCs to CAFs via PDGF/TGF‑β and CAFs to GSC enrichment via osteopontin/HGF. These CAFs were found in 12 patient tumors, differentiated from immature to mature subtypes, localized near GSCs, endothelial cells, and M2 macrophages, attracted and enriched GSCs, induced M2 polarization through EDA‑fibronectin/TLR4, and promoted tumor growth in xenografts, establishing glioblastoma CAFs as a novel therapeutic target.

Abstract

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-β as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.