Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects
Saket Jain, … , Dieter Henrik Heiland, Manish K. Aghi
Saket Jain, … , Dieter Henrik Heiland, Manish K. Aghi
Published March 1, 2023
Citation Information: J Clin Invest. 2023;133(5):e147087. https://doi.org/10.1172/JCI147087.
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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 2

Identification of CAFs in GBM by scRNA-Seq of patient GBMs.

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Identification of CAFs in GBM by scRNA-Seq of patient GBMs. (A–D) scRNA-...
(A–D) scRNA-Seq results from 12 patient GBMs (30, 31) were analyzed using mutual nearest neighbor horizontal integration followed by SNN clustering. (A) Optimal number of clusters was determined by the cluster stability score (upper right) resulting in 18 robust cell clusters. While most stromal cells clustered away from tumor cells, some stromal cells clustered close to tumor cells. (B) Green cells were tumor cells based on CNV analysis, while red cells were stromal. (C) CAF probability scores based on exclusive gene signatures and defined exclusion criteria were computed (left side). CAFs exhibited no CNV alterations (upper right) and were identified in each of the 12 patients (lower right). (D) Presence of early versus late-stage CAF subtypes was evaluated in cells with high CAF probability scores, with late-stage CAFs predominating over early stage CAFs in these 12 patients. (E–H) Deconvolution of spatially resolved transcriptomics was performed. (E) Surface plots obtained from 6 × 6 mm tissue samples revealing that CAFs (left) spatially correlated with the MES and astrocyte-like (AC-like) GBM cell signatures (30). Two examples of low overlap (top) and high overlap (bottom) are demonstrated. (F) Spatial correlation between CAFs and mes-GBM cells was significant (P < 0.001, Pearson’s R2 = 0.79). (G and H) Line diagrams show the spatial relationship between CAFs and other cell types or states (tumor subtypes). The x axis represents the relative distance to CAFs. The y axis shows the cell type/state probability of a particular gene set or spotlight probability. The spatial distance of CAFs to different cell types or states was computed based on ranked cell-type probability. If high cell probability values are displayed at a short distance (dist) from CAFs, the likelihood of a spatial relationship is high, as occurred for (G) mes- and AC-GBM cells and M2 TAMs and (H) CD44+ GSCs and CD34+ endothelial cells.