Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects
Saket Jain, Jonathan W. Rick, Rushikesh S. Joshi, Angad Beniwal, Jordan Spatz, Sabraj Gill, Alexander Chih-Chieh Chang, Nikita Choudhary, Alan T. Nguyen, Sweta Sudhir, Eric J. Chalif, Jia-Shu Chen, Ankush Chandra, Alexander F. Haddad, Harsh Wadhwa, Sumedh S. Shah, Serah Choi, Josie L. Hayes, Lin Wang, Garima Yagnik, Joseph F. Costello, Aaron Diaz, Dieter Henrik Heiland, Manish K. Aghi
Saket Jain, Jonathan W. Rick, Rushikesh S. Joshi, Angad Beniwal, Jordan Spatz, Sabraj Gill, Alexander Chih-Chieh Chang, Nikita Choudhary, Alan T. Nguyen, Sweta Sudhir, Eric J. Chalif, Jia-Shu Chen, Ankush Chandra, Alexander F. Haddad, Harsh Wadhwa, Sumedh S. Shah, Serah Choi, Josie L. Hayes, Lin Wang, Garima Yagnik, Joseph F. Costello, Aaron Diaz, Dieter Henrik Heiland, Manish K. Aghi
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Research Article Oncology

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

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.

Authors

Saket Jain, Jonathan W. Rick, Rushikesh S. Joshi, Angad Beniwal, Jordan Spatz, Sabraj Gill, Alexander Chih-Chieh Chang, Nikita Choudhary, Alan T. Nguyen, Sweta Sudhir, Eric J. Chalif, Jia-Shu Chen, Ankush Chandra, Alexander F. Haddad, Harsh Wadhwa, Sumedh S. Shah, Serah Choi, Josie L. Hayes, Lin Wang, Garima Yagnik, Joseph F. Costello, Aaron Diaz, Dieter Henrik Heiland, Manish K. Aghi

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Figure 6

Regional variation of CAF localization in GBM.

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Regional variation of CAF localization in GBM. (A) Schematic showing whe...
(A) Schematic showing where site-directed biopsies from patient GBMs were taken. (B) qPCR revealed that SVZ GBM had 22-fold increased expression of EDA (P < 0.001), 22-fold increased total FN expression (P < 0.001), and 5-fold increased EDB expression (P = 0.02) compared with the tumor core (n = 3/group ANOVA with post hoc Tukey’s test). (C) Immunofluorescence confirmed elevated EDA (green) and total FN (red) in SVZ GBM compared with the tumor core. Scale bars: 30 μm. (D) Flow cytometry for CAF marker α-SMA reveals elevation in the SVZ compared with the tumor core (n = 3 paired specimens; P = 0.02 paired t test). (E) Immunofluorescence revealed no PDGFR-α or EDA staining in the SVZ of a GBM patient whose tumor did not involve the SVZ. Original magnification, ×100. Scale bar: 30 μm. (F) Total and EDA FN expression by qPCR was elevated in SVZ GBM but virtually undetectable in tumor-free SVZ from epilepsy surgery (P < 0.001; t test; n = 3). *P < 0.05; ***P < 0.001.