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
View: Text | PDF
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

×

Figure 4

GSCs mediate CAF invasion and proliferation via PDGF and TGF-β pathways.

Options: View larger image (or click on image) Download as PowerPoint
GSCs mediate CAF invasion and proliferation via PDGF and TGF-β pathways....
Compared with NM, CM from GBM6 stem cell–enriched neurospheres (A and B) attracted more GBMpt1CAFs in chemotaxis assays (n = 6/group; P < 0.001, t test) and (C) stimulated GBMpt5CAF proliferation (P < 0.001 at all time points; n = 5/group; t test). Scale bars: 20 μm. (D) We mapped the expression of receptors expressed by GBMpt1CAFs and GBMpt2CAFs (Supplemental Table 2) to that of their cognate ligands/agonists expressed by GBM6 neurospheres (37) based on a database of 491 known receptor-ligand interactions (67). Shown are cognate pairs coexpressed by GBM CAFs and GSCs for which FPKM of the ligand is greater than 0.05 and read counts of the receptor are greater than 10, which represented 189 GSC ligands with receptors expressed by CAFs. (E) Chemotaxis of GBMpt1CAFs toward GBM6 neurosphere CM was abrogated by neutralizing antibodies against PDGF (P < 0.001 at 5 and 10 μg/mL), but not TGF-β. TGF-β–neutralizing antibodies did not abrogate invasion at 2.5–10 μg/mL (P = 0.3–0.7). PDGF-neutralizing antibodies reduced the number of invading cells at 5 and 10 μg/mL (P < 0.001; n = 6/group). ANOVA with post hoc Tukey’s test. (F) PDGF-neutralizing antibodies minimally reduced and TGF-β antibodies did not alter GBM43 GSC CM-induced GBMpt5CAF proliferation (PDGF: P = 0.1–0.5 from 0–119 hours, P = 0.02–0.047 from 120–140 hours; TGF-β: P = 0.2–0.6 from 0–140 hours), while combining these antibodies reduced GBM43 GSC CM-induced GBMpt5CAF proliferation (P = 0.01–0.04 from 105–119 hours, P = 0.007–0.009 from 120–140 hours, both antibodies versus no antibodies in GBM43 GSC CM; P = 0.02–0.04 from 120–140 hours, both antibodies versus anti-PDGF; P = 0.01–0.04 from 100–119 hours and P = 0.006–0.009 from 120–140 hours, both antibodies versus anti–TGF-β). HER2 antibodies exerted no effect (P = 0.6–0.8 from 0–140 hours) on GBM43 GSC CM-induced GBMpt5CAF proliferation. n = 5/group; ANOVA with post hoc Tukey’s test. ***P < 0.001.