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

Saket Jain(University of California, San Francisco), Jonathan W. Rick(University of California, San Francisco), Rushikesh S. Joshi(University of California, San Francisco), Angad Beniwal(University of California, San Francisco), Jordan Spatz(University of California, San Francisco), Sabraj Gill(University of California, San Francisco), Alexander Chih-Chieh Chang(University of California, San Francisco), Nikita Choudhary(University of California, San Francisco), Alan Nguyen(University of California, San Francisco), Sweta Sudhir(University of California, San Francisco), Eric Chalif(University of California, San Francisco), Jia‐Shu Chen(University of California, San Francisco), Ankush Chandra(University of California, San Francisco), Alexander F. Haddad(University of California, San Francisco), Harsh Wadhwa(University of California, San Francisco), Sumedh S. Shah(University of California, San Francisco), Serah Choi(University of California, San Francisco), Josie Hayes(University of California, San Francisco), Lin Wang(University of California, San Francisco), Garima Yagnik(University of California, San Francisco), J Costello(University of California, San Francisco), Aarón Díaz(University of California, San Francisco), Dieter Henrik Heiland(University of Freiburg), Manish K. Aghi(University of California, San Francisco)
Journal of Clinical Investigation
February 28, 2023
10.1172/jci147087
Cited by 183Open Access
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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.

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