Microglia Induce PDGFRB Expression in Glioma Cells to Enhance Their Migratory Capacity

Abstract

High-grade gliomas (HGGs) are the most aggressive and invasive primary brain tumors. The platelet-derived growth factor (PDGF) signaling pathway drives HGG progression, and enhanced expression of PDGF receptors (PDGFRs) is a well-established aberration in a subset of glioblastomas (GBMs). PDGFRA is expressed in glioma cells, whereas PDGFRB is mostly restricted to the glioma-associated stroma. Here we show that the spatial location of TAMMs correlates with the expansion of a subset of tumor cells that have acquired expression of PDGFRB in both mouse and human low-grade glioma and HCGs. Furthermore, M2-polarized microglia but not bone marrow (BM)-derived macrophages (BMDMs) induced PDGFRB expression in glioma cells and stimulated their migratory capacity. These findings illustrate a heterotypic cross-talk between microglia and glioma cells that may enhance the migratory and invasive capacity of the latter by inducing PDGFRB.

Keywords: Cancer; Immunology; Molecular Mechanism of Behavior; Pathophysiology.

Graphical abstract

Figure 1

PDGFB-Driven Gliomas in the N/tv-a;Arf^−/− Mouse Model Display Necrosis and Decreased Vessel Perfusion (A) Images display tumor sections immunostained with H&E. Scale bars, 200 μm. (B) Glioma sections were immunostained for OLIG2 (green) and GFAP (red). Scale bars, 50 μm. (C–G) (C) Images display tumors perfused with fluorescein isothiocyanate-conjugated lectin (green) and immunostained for the endothelial marker CD31 (blue). Graphs display (D) vessel area, (E) vessel density, (F) area of perfused vessels, and (G) % number of perfused vessels. (n = 3–5). Scale bars, 100 μm. Statistical analysis: one-way ANOVA was used; *p < 0.05, **p < 0.01, ***p < 0.001; * indicates significance compared with grade II-like tumors; ^#p < 0.05; ^# indicates significance between grade III- and grade IV-like tumors.

Figure 2

PDGFB-Driven Gliomas in the N/tv-a;Arf^−/− Mouse Model Display Hypoxia and Vessel Hyperproliferation (A and B) (A) Glioma sections were immunostained for CD31 (blue), fluorescein isothiocyanate (FITC)-conjugated lectin (green), and the hypoxic marker pimonidazole (PIMO; red). (B) Graph shows morphometric analysis of hypoxic areas (n = 3–5). Scale bars, 100 μm. (C and D) (C) Glioma sections were immunostained for podocalyxin (green) and Ki67 (red). (D) Graph depicts quantification of podocalyxin⁺Ki67⁺ vessels (n = 3). Scale bars, 100 μm. (E and F) (E) Glioma sections were immunostained for CD31 (blue), FITC-conjugated lectin (green), and the pericyte marker α-SMA (red). (F) Graph displays quantification of α-SMA (n = 3–5). Scale bars, 100 μm. (G and H) (G) Glioma sections were immunostained for OLIG2 (blue), FITC-conjugated lectin (green), and α-SMA (red). (H) Graph shows analysis of α-SMA⁺ sheets associated with vessels. Scale bars, 100 (left panel) and 50 (right panel) μm. (I) Glioma sections were immunostained for podocalyxin (blue), microglia marker IBA1 (green), and α-SMA (red). Scale bars, 100 (left panel) and 50 μm (right panel). Statistical analysis: one-way ANOVA (A–F) and student's t test (G and H) were used: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; * indicates significance (A–F) compared with grade II-like tumors and (H) between α-SMA⁺ sheets associated and non-associated with vessels; ^#p < 0.05; ^# indicates significance between grade III- and grade IV-like tumors.

Figure 3

Glioma Cells Express Pericyte Markers in Mouse Gliomas (A–H) Mouse glioma sections were immunostained for (A) α-SMA (red) and the pericyte marker PDGFRB (green), (B) α-SMA (red) and the pericyte and oligodendrocyte marker NG2 (blue), (C) α-SMA (red) and the mesenchymal and stem cell marker CD44 (green), (D) α-SMA (red) and the GSC marker SOX2 (green), (E) α-SMA (red) and the oligodendrocyte marker OLIG2 (green), (F) α-SMA (red) and the astrocyte marker GFAP (green), and (G) α-SMA (red) and the HA tag expressed by PDGF-B-transformed cells (green). Left panels show α-SMA⁺ sheets, whereas right panels display round α-SMA⁺ cells. Scale bars, 100 (left panel) and 50 (right panel) μm. (H) Graph displays quantification of HA⁺, OLIG2⁺, SOX2⁺, and GFAP⁺ cells out of α-SMA⁺ cells (n = 3). Statistical analysis: one-way ANOVA was used.

Figure 4

Spatial Location of α-SMA⁺SOX2⁺, α-SMA⁺OLIG2⁺, and α-SMA⁺PDGFRB⁺ GCs in Mouse Gliomas (A–C) Mouse glioma sections were immunostained for (A) OLIG2 (blue), α-SMA (red), and podocalyxin (green); (B) SOX2 (blue), α-SMA (red), and podocalyxin (green); and (C) PDGFRB (blue), α-SMA (red), and podocalyxin (green). Scale bars, 100 μm. (D) Mouse grade IV-like glioma sections were immunostained for HA tag (expressed by PDGFB-transformed cells; blue), α-SMA (red), and IBA1 (green). Scale bars, 50 μm. (E) Mouse grade IV-like glioma sections were immunostained for HA tag (blue), PDGFRB (red), and IBA1 (green). Scale bars, 50 μm. See also Figure S1.

Figure 5

Glioma Cells Express PDGFRB and Correlate with IBA1⁺ TAMMs in Mouse and Human Gliomas (A–E) (A) Mouse glioma sections were immunostained for H&E, HA tag (green/blue), PDGFRB (red), and IBA1 (green). Graphs display (B) number of PDGFRB⁺HA⁺ cells out of HA⁺ cells, (C) area of IBA1⁺ cells, (D) Pearson's correlation between PDGFRB⁺ cells versus area of IBA1, and (E) number of PDGFRB⁺HA⁺ cells in contact with IBA1⁺ cells (n = 3–5). Scale bars 200 (left panel), 50 (middle panel), and 20 (far right panel) μm. (F–J) (F) Human glioma sections were immunostained for IDH1-R132H or GFAP (blue), PDGFRB (red), and IBA1 (green). Graphs depict (G) number of PDGFRB⁺IDH1-R132H⁺ cells or PDGFRB⁺GFAP⁺ cells out of IDH1-R132H⁺ or GFAP⁺ cells, (H) area of IBA1⁺ cells, (I) Pearson's correlation between PDGFRB⁺ cells versus area of IBA1, and (J) number of PDGFRB⁺IDH1-R132H⁺ or PDGFRB⁺GFAP⁺ cells in contact with IBA1⁺ cells (n = 5–6). Scale bars, 50 (left panel) and 20 (far right panel) μm. Statistical analysis: one-way ANOVA (B, C, E, G, H, and J) and Pearson's correlation (D and I) were used: *p < 0.05, ***p < 0.001, ****p < 0.0001; * indicates significance compared with grade II-like tumors; ^###p < 0.001, ^####p < 0.0001; ^# indicates significance between grade III- and grade IV-like tumors. See also Figure S2.

Figure 6

TAMMs Induce Mouse Glioma Cells to Express PDGFRB (A) Microglia were polarized to either an M1 or M2 phenotype. Graph shows gene expression levels of cxcl9, cxcl10, cxcl11, il-6, il-1b, arginase1, mrc1, and ym1 analyzed by qRT-PCR (n = 3). (B–F) GFP⁺GSCs were co-cultured with M1- or M2-polarized microglia. Graphs depict qRT-PCR analysis of gene expression levels of flow-sorted tumor cells for (B) pdgfrb, (C) α-sma, (D) pdgfra, (E) fgfr and igf-1r, and (F) egfr and hgfr (n = 3). (G) GFP⁺GSCs were cultured with M0 (not polarized)-, M1- or M2-like microglia conditioned media. Graph displays gene expression levels for α-sma quantified by quantitative RT-PCR (n = 3). (H) GFP⁺GSCs were co-cultured with M1- or M2-polarized BMDMs. Graph shows qRT-PCR analysis of gene expression levels of flow-sorted tumor cells for α-sma (n = 3). (I–K) GFP⁺GSCs were co-cultured with M1- or M2-polarized microglia. Graphs depict quantitative RT-PCR analysis of gene expression levels of flow-sorted tumor cells for (I) nestin, olig2, and sox2; (J) cnp, ng2, and hes1; and (K) gfap (n = 3). (L and M) GFP⁺GSCs were co-cultured with M1- or M2-polarized microglia, and migrated tumor cells were stained for the OLIG2 marker. Graphs display (L) migrated tumor cells (n = 3) and (M) migrated tumor cells with isotype control or neutralizing PDGFRB antibody (n = 3). Statistical analysis: Student's t test (A) and one-way ANOVA (B–M) were used: *p < 0.05, **p < 0.01, ***p < 0.001; * indicates significance (B–L) compared with tumor cells alone, and (M) compared with tumor cells without PDGFRB blockage; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001; ^# indicates significance between (A) M1-like and M2-like microglia and (B–L) tumor cells co-cultured with M1 microglia compared with M2 microglia and (M) tumor cells co-cultured with M2-like microglia without PDGFRB blockage compared with PDGFRB blockage. All data represent one out of three independent experiments and are presented as the mean +SD. See also Table S1.