Oligomer-Aβ42 suppress glioma progression via potentiating phagocytosis of microglia

Abstract

Aims: Glioma is characterized by an immunosuppressed environment and a poor prognosis. The accumulation of Amyloid β (Aβ) leads to an active environment during the early stages of Alzheimer's disease (AD). Aβ is also present in glioma tissues; however, the biological and translational implications of Aβ in glioma are elusive.

Methods: Immunohistochemical (IHC) staining, Kaplan-Meier (KM) survival analysis and Cox regression analysis on a cohort of 79 patients from our institution were performed to investigate the association between Aβ and the malignancy of glioma. Subsequently, the potential of oligomer-Aβ42 (OAβ42) to inhibit glioma growth was investigated in vivo and in vitro. Immunofluorescence staining and phagocytosis assays were performed to evaluate the activation of microglia. Finally, RNA-seq was utilized to identify the predominant signaling involved in this process and in vitro studies were performed to validate them.

Results: A positive correlation between Aβ and a favorable prognosis was observed in glioma. Furthermore, OAβ42 suppressed glioma growth by enhancing the phagocytic activity of microglia. Insulin-like growth factor 1 (IGF-1) secreted by OAβ42-activated microglia was essential in the engulfment process.

Conclusion: Our study proved an anti-glioma effect of Aβ, and microglia could serve as a cellular target for treating glioma with OAβ42.

Keywords: IGF-1; OAβ42; glioma; microglia; phagocytosis.

FIGURE 1

The clinical significance of Aβ in glioma patients. (A) Human glioma tissues were collected for IHC staining of Aβ. White bars represent 100 μm, and black bar represent 20 μm. Each grade of glioma tissues was repeated fourth, and the results were obtained and analyzed by experienced pathologists; (B) Quantification of DAB signal intensity was performed and represented in graphs, the non‐parametric equivalent with the Kruskal‐Wallis test was used; (C) Survival analysis of Aβ in 79 glioma patients' cohort, the Kaplan–Meier survival analysis with Log Rank test was applied. (D) Survival analysis of Aβ in 62 GBM patients' cohort, the Kaplan–Meier survival analysis with Log Rank test was conducted. Columns indicate median [95% CI]; n.s., not significant; p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

OAβ42 suppresses glioma growth in vivo. (A) schematic diagram of OAβ42 treatment in GL261‐inoculated C57 allogeneic model; (B) Representative (In vivo Imaging System) IVIS images of glioma‐inoculated mice treated with vehicle or OAβ42 in early (2 weeks) and late stage (4 weeks); (C) Quantification of tumor volumes in two groups of mice recorded in IVIS, data was analyzed by the non‐parametric equivalent with Mann–Whitney U‐Test; (D) Survival analysis of glioma‐inoculated mice along with vehicle or OAβ42 treatment, the Kaplan–Meier survival analysis with Breslow test was applied; (E, F) Representative images of Ki‐67 staining and quantification of Ki‐67 staining. White bars represent 100 μm, the Student's t‐test was conducted for the data analysis. Columns indicate median [95% CI]; n.s., not significant; p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

OAβ42 active microglia and re‐modulate the tumor microenvironment. (A) Representative images of the immune microenvironment of Glioma‐bearing mice treated with vehicle or OAβ42. White bars represent 100 μm; (B–E) Quantification of the immune cells in the two groups, the Student's t‐test was conducted for the data analysis. CD3 represented T cells (B), GFAP represented astrocytes (C), IBA1 represented microglia (D), and CD68 represented activated microglia (E). Columns indicate median [95% CI]; n.s., not significant; p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

OAβ42 potentiates phagocytosis of microglia. (A) Representative confocal images of microglia that engulf glioma cells after OAβ42 stimulation, White bars represent 20 μm; (B) Representative images of microglia morphology with vehicle treatment or OAβ42, White bars represent 100 μm; (C) Quantification of activation ratio by the ratio of CD68 to IBA1 positive microglia, the Student's t‐test was conducted for the data analysis. Columns indicate median [95% CI]; n.s., not significant; p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

OAβ42 enhances the phagocytosis capacity of microglia in vitro. (A) Dynamic process captured with ImageXpress microscope 10 h subsequent to the incubation of vehicle, low concentrate or high concentrate OAβ42. GL261 cells were labeled with GFP and microglia without labeling at the top 3 panels. White dotted circles indicate the hotspots of cell interactions. 2 zoomed‐in panels of the co‐culture system containing GFP‐labeled GL261 cells and CellTracke Red CMTPX stained microglia were presented subsequently. White bars represent 100 μm; (B) Quantification for glioma cell inhibition in the co‐culture system, the two‐way ANOVA with Dunnett's multiple comparison test was conducted to data analysis. (C) Quantification for live cell phagocytosis assay in the co‐culture system, the one‐way ANOVA test with least significance difference post hoc comparison was performed to data analysis. (D) Representative images of microglia engulf beads subsequent to the incubation of vehicle, low concentrate, or high concentrate OAβ42. White bars represent 100 μm; (E) Quantification of beads phagocytosis assay, the one‐way ANOVA test with least significance difference post hoc comparison was performed to data analysis; (F) Representative images of microglia engulfed beads with vehicle or OAβ42 treatment. Microglia were labeled with CellMaskTM Green Actin Tracking Stain (1:1000) before beads were enrolled. The red arrow indicates the beads engulfed in the cell body, and the yellow arrow indicates the beads contacted by the cell processes. White bars represent 20 μm. Columns indicate median [95% CI]; n.s., not significant; p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

IGF1 is essential for the OAβ42 induced microglia phagocytosis. (A) The Gene Ontology (GO) and functional pathway analysis of coding genes associated with positive correlation coefficients in TAMs treated with OAβ42, p‐value ≤0.05. The x‐axis is the ‐log 10 p value; (B) GSEA of TAMs concerning genes upregulated in the exposure of OAβ42. The analysis demonstrates a positive correlation (q < 0.1, Enrichment Score = 0.15) between many genes upregulated in phagocytosis and AD; (C) PCA of the transcriptome of glioma condition medium pre‐treated BV2 (TAMs) treated with OAβ42 compared to vehicle. Volcano plot shows gene expression differences between microglia treated with OAβ42 and vehicle. Log 2 gene ratios are plotted against negative log 10 p values; (D) qPCR reveals IGF‐1 upregulated in OAβ42 stimulated microglia, the non‐parametric equivalent with Mann–Whitney U‐Test was performed to data analysis; (E) Western blot of IGF‐1 signaling related proteins of microglia treated with vehicle, OAβ42, and OAβ42+ IGFBP3; (F) IGFBP3 decreased the phagocytosis of microglia treated with OAβ42. White bars represent 100 μm; (G) Quantification of the phagocytosis assay, the one‐way ANOVA test with least significance difference post hoc comparison was performed to data analysis. Columns indicate median [95% CI]; n.s., not significant; p ≥ 0.05; *p < 0.05; **p < 0.01; ***p < 0.001.