Tumor Microenvironment and Immune Escape in the Time Course of Glioblastoma

Abstract

Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with a malignant prognosis. GBM is characterized by high cellular heterogeneity and its progression relies on the interaction with the central nervous system, which ensures the immune-escape and tumor promotion. This interplay induces metabolic, (epi)-genetic and molecular rewiring in both domains. In the present study, we aim to characterize the time-related changes in the GBM landscape, using a syngeneic mouse model of primary GBM. GL261 glioma cells were injected in the right striatum of immuno-competent C57Bl/6 mice and animals were sacrificed after 7, 14, and 21 days (7D, 14D, 21D). The tumor development was assessed through 3D tomographic imaging and brains were processed for immunohistochemistry, immunofluorescence, and western blotting. A human transcriptomic database was inquired to support the translational value of the experimental data. Our results showed the dynamic of the tumor progression, being established as a bulk at 14D and surrounded by a dense scar of reactive astrocytes. The GBM growth was paralleled by the impairment in the microglial/macrophagic recruitment and antigen-presenting functions, while the invasive phase was characterized by changes in the extracellular matrix, as shown by the analysis of tenascin C and metalloproteinase-9. The present study emphasizes the role of the molecular changes in the microenvironment during the GBM progression, fostering the development of novel multi-targeted, time-dependent therapies in an experimental model similar to the human disease.

Keywords: Astrocytes; FIB-2; Glioma; MHCII; MMP-9; Macrophages; Microglia; Neuroinflammation; Spatio-temporal heterogeneity.

Fig. 1

Tumor monitoring at 21D. A C57Bl/6 mice with vehicle (SHAM) or tumor cells on day 21 (21D). Mice were intravenously injected with 10 nmol of 2-DG-750 probe or vehicle 750 dye and imaged at 21D FMT 1000 Imaging System (Perkin Elmer). The SHAM mouse showed no specific targeting. Bioluminescence imaging showed. B Macroscopic photographs of a Sham and two scanned 21D tumor-bearing brains. The GBM development was differently oriented from the original tumor cells injection site

Fig. 2

Glioblastoma time-course. A ki67 Immunofluorescence in the primary site of injection (right striatum) at different time points. Ki67⁺ cells increase at 14D. Scale bar 20 μm. B GBM tumor bulk at 14D and 21D stained by cresyl violet acetate. The tumor tissue appears as necrotic. Massive infiltration occurs at 21D. Scale bar 50 μm. C 2D reconstruction of immuno-stained tumor-bearing slices at 14D and 21D for GFAP. GFAP⁺ astrocytes surround the tumor mass. A secondary mass (white square) is evident in the contralateral hemisphere at 21D. Scale bar 100 μm

Fig. 3

Astrocytes dynamic during GBM progression. A GBM-associated astrocytes expressing GFAP increase with GBM progression. Scale bar 50 μm. B Quantification of peritumoral astrocytes immunostained for GFAP. The results are expressed as proportional area (One-way Anova for multiple comparisons, post-hoc Holm-Sidak correction, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for comparisons vs. SHAM; ^#p ≤ 0.05; ^##p ≤ 0.01; ^###p ≤ 0.001 for comparisons between the stages of GBM progression). C Morphological changes in representative high-magnified GFAP-expressing tumor-associated astrocytes. Scale bar 50 μm. D GBM-associated astrocytes expressing GFAP in the contralateral (left) hemisphere under SHAM condtion and at 21D. Scale bar 50 μm. The secondary tumor mass is indicated with an asterisk. E Quantification of peritumoral astrocytes immunostained for GFAP in the contralateral hemisphere. The results are expressed as proportional area. (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, Student’s t test)

Fig. 4

Extra-cellular matrix remodeling during GBM progression. A TN-C immunohistochemistry in the peritumoral region. TN-C peak is evident at 14D. Brain sections were counterstained with cresyl violet acetate (Nissl staining). Scale bar 20 μm. B Quantification of peritumoral TNC. The results are expressed as a measure of optical density (Kruskal–Wallis test was used for non-parametric analysis followed by Tukey’s test for pairwise multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for comparisons vs. SHAM; ^#p ≤ 0.05; ^##p ≤ 0.01; ^###p ≤ 0.001 for comparisons between the stages of GBM progression). C Western blot of right hemisphere lysate showed differential expression for FIB-2, pro-MMP9, and MMP9 during GBM progression. Samples derive from the same experiment; gels/blots were processed in parallel. D–F Quantification of FIB-2, MMP-9, pro-MMP9 western blot bands relative to the total protein content stained with Ponceau-S. FIB-2 and MMP-9 upregulation was prominent in the earlier stages of GBM development. The expression of MMP-9 was in equilibrium with its precursor pro-MMP-9(One-way Anova for multiple comparisons, post-hoc Holm-Sidak corrections. Kruskal–Wallis test was used for non-parametric analysis followed by Tukey’s test for pairwise multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for comparisons vs SHAM; ^#p ≤ 0.05; ^##p ≤ 0.01; ^###p ≤ 0.001 for comparisons between the stages of GBM progression)

Fig. 5

Microglia/macrophages dynamic during GBM progression. A Immunohistochemistry for Iba1 showed a bland reaction by microglia/macrophages in the site of the primary GL261 cells injection. A massive infiltration is evident at 21D. Scale bar 50 μm. B Quantification of peritumoral Iba1-expressing cells in the peritumoral region, right hemisphere. The results are expressed as proportional area (Kruskal–Wallis test was used for non-parametric analysis followed by Tukey’s test for pairwise multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for comparisons vs. SHAM; ^#p ≤ 0.05; ^##p ≤ 0.01; ^###p ≤ 0.001 for comparisons between the stages of GBM progression). C Immunohistochemistry for Iba1 showed an increase of Iba1⁺ cells at 21D compared to the SHAM group in the contralateral hemisphere. D Quantification of peritumoral Iba1-expressing cells in the peritumoral region, contralateral hemisphere. The results are expressed as proportional area (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001, Student’s t test). E 2D reconstruction of tumor-bearing section immunostained for Iba1 at 21D. Scale bar 100 μm. The morphology of Iba1 expressing cells at the tumor border is shown in detail. Scale bar 50 μm. The tumor bulk is indicated with an asterisk*

Fig. 6

Microglia reaction appears inhibited during GBM progression. A Western blot analysis of right hemisphere lysate revealed differential expression for microglia specific marker TMEM119 during GBM progression. Samples derive from the same experiment; gels/blots were processed in parallel. B Quantification of TMEM119 Western blot bands relative to the β-actin content used as the internal loading control. The expression of TMEM119 does not differ from the SHAM group at 7D but is downregulated at 21D (One-way Anova for multiple comparisons, post-hoc Holm-Sidak corrections. Kruskal–Wallis test was used for non-parametric analysis followed by Tukey’s test for pairwise multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for comparisons vs. SHAM; ^#p ≤ 0.05; ^##p ≤ 0.01; ^###p ≤ 0.001 for comparisons between the stages of GBM progression). C Western blot of contralateral hemisphere lysate showed differential expression for microglia specific marker TMEM119 during GBM progression. Samples derive from the same experiment; gels/blots were processed in parallel. D Quantification of TMEM119 western blot bands relative to the β-actin content from the contralateral hemisphere. The expression of TMEM119 is downregulated at 21D compared to the SHAM group in the contralateral hemisphere (One-way Anova for multiple comparisons, post-hoc Holm-Sidak corrections. Kruskal–Wallis test was used for non-parametric analysis followed by Tukey’s test for pairwise multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for comparisons vs SHAM; ^#p ≤ 0.05; ^##p ≤ 0.01; ^###p ≤ 0.001 for comparisons between the stages of GBM progression)

Fig. 7

Antigen-presenting function and microglia/macrophage-associated inflammation are compromised in the initiation of the GBM. A Western blot analysis of right (top) and left (bottom) hemisphere lysate revealed differential expression for MHCII and CCL2 during GBM progression. Samples derive from the same experiment; gels/blots were processed in parallel. B Quantification of CCL2 right hemisphere western blot bands relative to the β-actin content used as the internal loading control (One-way Anova for multiple comparisons, post-hoc Holm-Sidak corrections. Kruskal–Wallis test was used for non-parametric analysis followed by Tukey’s test for pairwise multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for comparisons vs SHAM; ^#p ≤ 0.05; ^##p ≤ 0.01; ^###p ≤ 0.001 for comparisons between the stages of GBM progression). C, D Quantification of MHCII western blot bands relative to the β-actin content used as internal loading control from the right and left hemisphere, respectively (One-way Anova for multiple comparisons, post-hoc Holm-Sidak corrections. Kruskal–Wallis test was used for non-parametric analysis followed by Tukey’s test for pairwise multiple comparisons. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 for comparisons vs SHAM; ^#p ≤ 0.05; ^##p ≤ 0.01; ^###p ≤ 0.001 for comparisons between the stages of GBM progression)