The Role of Fibrinogen-Like Protein 2 on Immunosuppression and Malignant Progression in Glioma

Abstract

Background: Virtually all low-grade gliomas (LGGs) will progress to high-grade gliomas (HGGs), including glioblastoma, the most common malignant primary brain tumor in adults. A key regulator of immunosuppression, fibrinogen-like protein 2 (FGL2), may play an important role in the malignant transformation of LGG to HGG. We sought to determine the mechanism of FGL2 on tumor progression and to show that inhibiting FGL2 expression had a therapeutic effect.

Methods: We analyzed human gliomas that had progressed from low- to high-grade for FGL2 expression. We modeled FGL2 overexpression in an immunocompetent genetically engineered mouse model to determine its effect on tumor progression. Tumors and their associated microenvironments were analyzed for their immune cell infiltration. Mice were treated with an FGL2 antibody to determine a therapeutic effect. Statistical tests were two-sided.

Results: We identified increased expression of FGL2 in surgically resected tumors that progressed from low to high grade (n = 10). The Cancer Genome Atlas data showed that LGG cases with overexpression of FGL2 (n = 195) had statistically significantly shorter survival (median = 62.9 months) compared with cases with low expression (n = 325, median = 94.4 months, P < .001). In a murine glioma model, HGGs induced with FGL2 exhibited a mesenchymal phenotype and increased CD4+ forkhead box P3 (FoxP3)+ Treg cells, implicating immunosuppression as a mechanism for tumor progression. Macrophages in these tumors were skewed toward the immunosuppressive M2 phenotype. Depletion of Treg cells with anti-FGL2 statistically significantly prolonged survival in mice compared with controls (n = 11 per group, median survival = 90 days vs 62 days, P = .004), shifted the phenotype from mesenchymal HGG to proneural LGG, and decreased M2 macrophage skewing.

Conclusions: FGL2 facilitates glioma progression from low to high grade. Suppressing FGL2 expression holds therapeutic promise for halting malignant transformation in glioma.

Figure 1.

The influence of fibrinogen-like protein 2 (FGL2) on survival in low-grade glioma (LGG) and glioblastoma (GB). A) Kaplan-Meier curve showing overall survival of The Cancer Genome Atlas (TCGA) glioma (grades II–IV) cases stratified by over- or low-expression of FGL2 mRNA (n = 664, log-rank test, P < .001). Median survival in all TCGA patients with LGG or GB stratified by high or low FGL2 expression. B) Kaplan-Meier curve showing overall survival of TCGA patients with GB stratified by over- or low-expression of FGL2 mRNA (n = 160, log-rank test, P = .41). C) Kaplan-Meier curve showing overall survival of TCGA patients with LGG stratified by over- or low-expression of FGL2 (n = 520, log-rank test, P < .001). D) FGL2 expression in a representative human LGG before and after progression to high-grade glioma (HGG). Representative magnetic resonance images (fluid-attenuated inversion recovery and T1-weighted postcontrast images) at diagnosis and at the time of malignant progression from a patient are shown. The LGG demonstrates a paucity of FGL2 staining, whereas the subsequent HGG in the same patient demonstrates abundant FGL2 staining (magnification, 400×; scale bar = 50 μm). FLAIR = fluid-attenuated inversion recovery; GB = glioblastoma; LGG = low-grade glioma; MRI = magnetic resonance imaging.

Figure 2.

Fibrinogen-like protein 2 (FGL2) expression in glioblastoma stem cells and in human tumors. A) Immunoblot analysis of FGL2 expression in proneural (PN/P) and mesenchymal (MES/M) glioma stem cells (GSCs). Actin was used as the loading control. U87 was used as the positive control for FGL2 expression. B) Confirmation of FGL2 expression in GSCs using enzyme-linked immunosorbent assay (t test, two-sided, P = .04). Error bars = SD. C) Characterization of GSC lines for PN and MES signatures using RNA sequencing for Olig2 and CD44 markers. FGL2 = fibrinogen-like protein 2; MES/M = mesenchymal; PN/P = proneural.

Figure 3.

The effect of fibrinogen-like protein 2 (FGL2) overexpression on malignant progression in vivo. A) Kaplan-Meier analysis demonstrating symptom-free survival in Ntv-a mice from the replication-competent ASLV long terminal repeat with a splice (RCAS) platelet-derived growth factor subunit B (PDGFB), RCAS-PDGFB+FGL2, or RCAS-FGL2 groups. B and C) CD44 and Olig2 expression in RCAS-PDGFB and RCAS-PDGFB+FGL2 LGGs and HGGs. Immunohistochemical staining of representative tumor sections (magnification, 400×; scale bar = 50 μm). Left) Scatter plot demonstrating differences in CD44 and Olig2 expression between the two groups (t test, two-sided). FGL2 = fibrinogen-like protein 2; HGG = high-grade glioma; LGG = low-grade glioma; PDGF = platelet-derived growth factor; PDGFB = platelet-derived growth factor subunit B.

Figure 4.

The effect of fibrinogen-like protein 2 (FGL2) depletion on glioma formation in GL261 intracranial tumors. A) FGL2 mRNA expression in mock and FGL2-KO GL261 cells. Glyceraldehyde 3-phosphate dehydrogenase was used for normalization. B) Whole-mount photograph showing tumor occurrence in GL261-FGL2-knockout (GL261-FGL2KO) mice and the mock GL261 mice. FGL2 = fibrinogen-like protein 2; KO = knockout.

Figure 5.

The influence of fibrinogen-like protein 2 (FGL2) expression on the recruitment of immune cells to the tumor microenvironment. A) Forkhead box P3 (FoxP3) expression in replication-competent ASLV long terminal repeat with a splice (RCAS) platelet-derived growth factor subunit B (PDGFB) and RCAS-PDGFB+FGL2 tumors. Scatter plot demonstrating the difference in FoxP3 expression between the two groups (magnification, 400×; scale bar = 50 μm) among LGGs and HGGs from each injection set. B) Immunosuppressive CD4⁺FoxP3⁺ cells in the tumor microenvironment were analyzed by fluorescence-activated cell sorting at different time points (10, 20, 40, 60, and 80 days). Data are presented as the mean ± standard deviation (n = 5 mice). An unpaired t test was used to calculate the two-sided P values. C) Kaplan-Meier curve demonstrating symptom-free survival in Ntv-a mice injected with replication-competent ASLV long terminal repeat with a splice (RCAS) platelet-derived growth factor subunit B (PDGFB)+FGL2 and treated with anti-CD25 antibody or rat immunoglobulin G (IgG) control starting three weeks after inoculation (n = 10 mice). D) Immunohistochemical staining of FoxP3 expression between treated and control groups (magnification, 400×; scale bar = 50 μm). E) Flow analysis of the depletion of CD4⁺FoxP3⁺ cells in the CD25 antibody–treated tumor compared with IgG control tumors (n = 5 mice). An unpaired t test was used to calculate the two-sided P values. FGL2 = fibrinogen-like protein 2; HGG = high-grade glioma; LGG = low-grade glioma; PDGF = platelet-derived growth factor; PDGFB = platelet-derived growth factor subunit B.

Figure 6.

The effect of fibrinogen-like protein 2 (FGL2) inhibition on overall survival, macrophage polarization, and Treg infiltration. A) Kaplan-Meier curve demonstrating symptom-free survival in Ntv-a mice injected with replication-competent ASLV long terminal repeat with a splice (RCAS) platelet-derived growth factor subunit B (PDGFB)+FGL2 and treated with anti-FGL2 antibody (FGL2-Ab) or immunoglobulin G (IgG) control starting three weeks after inoculation (n = 11 mice). B) CD44 and Olig2 expression in representative IgG control– and FGL2-Ab–treated tumors (magnification, 400×; scale bar = 50 μm). C) Immunosuppressive CD4⁺forkhead box P3 (FoxP3)⁺ cells in the tumor microenvironment were analyzed by fluorescence-activated cell sorting after 20 days of treatment with anti-FGL2 antibody or IgG antibody (control). Data are presented as the mean ± standard deviation (n = 4 mice). An unpaired t test was used to calculate the two-sided P values. D) Expression of Arginase 1⁺/Iba1⁺ cells in the RCAS-PDGFB+FGL2 tumors treated with IgG or anti-FGL2 antibody. Representative immunofluorescence staining for Arginase 1 (red) and Iba1 (green) macrophages (magnification, 400×; scale bar = 50 μm). FGL2 = fibrinogen-like protein 2; HGG = high-grade glioma; LGG = low-grade glioma.