Fibrillar extracellular matrix produced by pericyte-like cells facilitates glioma cell dissemination

Abstract

Gliomagenesis induces profound changes in the composition of the extracellular matrix (ECM) of the brain. In this study, we identified a cellular population responsible for the increased deposition of collagen I and fibronectin in glioblastoma. Elevated levels of the fibrillar proteins collagen I and fibronectin were associated with the expression of fibroblast activation protein (FAP), which is predominantly found in pericyte-like cells in glioblastoma. FAP+ pericyte-like cells were present in regions rich in collagen I and fibronectin in biopsy material and produced substantially more collagen I and fibronectin in vitro compared to other cell types found in the GBM microenvironment. Using mass spectrometry, we demonstrated that 3D matrices produced by FAP+ pericyte-like cells are rich in collagen I and fibronectin and contain several basement membrane proteins. This expression pattern differed markedly from glioma cells. Finally, we have shown that ECM produced by FAP+ pericyte-like cells enhances the migration of glioma cells including glioma stem-like cells, promotes their adhesion, and activates focal adhesion kinase (FAK) signaling. Taken together, our findings establish FAP+ pericyte-like cells as crucial producers of a complex ECM rich in collagen I and fibronectin, facilitating the dissemination of glioma cells through FAK activation.

Keywords: collagen type I; extracellular matrix proteins; fibronectin; glioblastoma; pericytes; proteomics.

FIGURE 1

Expression of COLI genes and FN1 in GBM is associated with high expression of FAP. (A) TCGA database‐based bioinformatic analysis of COL1A1, COL1A2, and FN1 expression in control, non‐tumorous brain tissue (n = 10) and GBMs with high (n = 119, upper tercile) and low (n = 119, lower tercile) expression of FAP. *p <0.05 **p <0.01, ***p <0.001, Kruskal–Wallis test. (B) Expression of COL1A1, COL1A2, and FN1 in distinct regions of GBM. Box–10th to 90th percentile, whiskers—min to max values, dots—original data, line—mean, ***p <0.001, ANOVA, Tukey's multiple comparison test. (C) Relative expression of COL1A1, COL1A2, and FN1 in vascular regions of GBM with high (n = 17, upper tercile) and low (n = 17, lower tercile) expression of FAP. Box—10th to 90th percentile, line—mean, whiskers—min‐max values, dots—raw data, ***p <0.001, Mann–Whitney U test.

FIGURE 2

COLI and FN1 are expressed by FAP+ pericyte‐like cells in perivascular areas in GBM. A) COLI and FN1 concentration determined by ELISA in PRE (n = 7), LowFAP (n = 15), and HighFAP (n = 15) GBMs. Box–10th to 90th percentile, whiskers–min to max values, dots–original data, line–mean, *p <0.05 **p <0.01, ***p <0.001, Kruskal–Wallis test. (B) Representative immunofluorescence images of COLI and FN1 in PRE, LowFAP, and HighFAP GBMs. (C) Percentage of area covered by COLI and FN1 in PRE (n = 3), LowFAP (n = 11), and HighFAP (n = 11) GBMs. Box—10th to 90th percentile, line—mean, whiskers—min‐max values, dots—raw data, ***p < 0.0001, Kruskal–Wallis test. (D) A representative image of perivascularly localized collagen I (COLI) in close vicinity of FAP expressing cells; expression of von Willebrand factor (vWf) was used to identify endothelial cells.

FIGURE 3

COLI and FN are expressed by FAP+ pericyte‐like cells in vitro. (A) A schema of experimental design. Created in Biorender.com. (B) Immunocytochemical detection and (C) quantification by ELISA of COLI and FN in cell types present in GBM. HBVP—human brain vascular pericytes, pEC—primary endothelial cells derived from GBM, BC1/2M2—primary M2 macrophages derived from monocytes, GSC—glioma stem‐like cells. Results are presented as mean ± SD from four parallel cell culture replicates, each measured in triplicate.

FIGURE 4

FAP+ pericyte‐like cells from human glioblastomas produce an ECM rich in collagen I, fibronectin, and essential proteins of basement membrane. (A) A schema of in vitro production of cell‐derived 3D matrices. Created in Biorender.com. (B) Immunodetection of COLI and FN1 in cell‐derived 3D matrices produced by FAP+ pericyte‐like cells (80A) and (C) U87 glioma cells. Representative confocal microscopy images (maximum intensity projection) and a 3D reconstruction. (D) Mass spectrometry analysis of matrisome proteins in cell‐derived 3D matrices produced by FAP+ pericyte‐like cells (80A, 46A), HBVP (human brain vascular pericytes), and U87 glioma cells. Principal component analysis (PCA), two‐dimension representation according to PC1 and PC2 based on the levels of 82 identified matrisome proteins. (E) Relative abundance of COL1A1, COL1A2, and FN1 in cell‐derived 3D matrices. Results are presented as mean ± SD from four replicates for each cell culture, ***p <0.001, ANOVA, Tukey's post hoc test. (F) Heatmap of relative protein abundance of basement membrane proteins in cell‐derived 3D matrices produced by FAP+ pericyte‐like cells (80A, 46A), HBVP, and U87 cells. Columns represent cell lines (quadruplicates) and rows represent expressed proteins as quantified by LC–MS/MS. Color in each tile represents the scaled log2 transformed abundance value.

FIGURE 5

Extracellular matrix produced by FAP+ pericyte‐like cells enhances glioma cell migration. (A) Haptotaxis of U251 and U87 cells through inserts coated with ECM produced by FAP+ pericyte‐like cells (39A, 46A, 49A) and HBVP, respectively. Data in each experiment were normalized to the migration of glioma cells on uncoated inserts. (B) Representative images of migrating U251 and U87 glioma cells. (C) Haptotaxis of glioma stem‐like cells NCH364 and NCH407 through inserts coated with ECM produced by FAP+ pericyte‐like cells (80A, 46A) and HBVP. Results are presented as a number of migrating cells per visual field (10× objective). (D) Representative images of migrating NCH364 and NCH407 glioma stem‐like cells. Box—10th to 90th percentile, line—mean, whiskers—min‐max values, dots—raw data. Results of two independent experiments performed in quadruplicates, **p <0.01, ***p <0.001 compared to the corresponding control, Kruskal–Wallis test.

FIGURE 6

Extracellular matrix produced by FAP+ pericyte‐like cells induces glioma cell adhesion and activates FAK signaling. (A) Representative phase contrast images and immunodetection of phospho FAK [pY397] in glioma stem‐like cells NCH364 and NCH407 seeded on an uncoated surface (control) or on a surface coated with ECM produced by FAP+ pericyte‐like cells. (B) Western blot detection of phospho FAK [pY397] and total FAK in NCH364 and NCH407 18 h after seeding on an uncoated surface (control) or on a surface coated with ECM produced by FAP+ pericyte‐like cells (86A, 90A, 95A). Ratio between total protein (stain‐free method)‐normalized phospho FAK [pY397] and total FAK was used to assess the activation of FAK signaling. The experiment was performed in three biological replicates. Box—min and max, line—median, dots—raw data.