Tumor-derived exosomes deliver the tumor suppressor miR-3591-3p to induce M2 macrophage polarization and promote glioma progression

Abstract

Exosomes can selectively secrete harmful metabolic substances from cells to maintain cellular homeostasis, and complex crosstalk occurs between exosomes and tumor-associated macrophages (TAMs) in the glioma immune microenvironment. However, the precise mechanisms by which these exosome-encapsulated cargos create an immunosuppressive microenvironment remain unclear. Herein, we investigated the effect of glioma-derived exosomes (GDEs) on macrophage polarization and glioma progression. We performed sequencing analysis of cerebrospinal fluid (CSF) and tumor tissues from glioma patients to identify functional microRNAs (miRNAs). High levels of miR-3591-3p were found in CSF and GDEs but not in normal brain tissue or glial cells. Functionally, GDEs and miR-3591-3p significantly induced M2 macrophage polarization and increased the secretion of IL10 and TGFβ1, which in turn promoted glioma invasion and migration. Moreover, miR-3591-3p overexpression in glioma cell lines resulted in G2/M arrest and markedly increased apoptosis. Mechanistically, miR-3591-3p can directly target CBLB and MAPK1 in macrophages and glioma cells, respectively, and further activate the JAK2/PI3K/AKT/mTOR, JAK2/STAT3, and MAPK signaling pathways. In vivo experiments confirmed that macrophages lentivirally transduced with miR-3591-3p can significantly promote glioma progression. Thus, our study demonstrates that tumor-suppressive miR-3591-3p in glioma cells can be secreted via exosomes and target TAMs to induce the formation of an immunosuppressive microenvironment. Collectively, these findings provide new insights into the role of glioma exosomal miRNAs in mediating the establishment of an immunosuppressive tumor microenvironment and show that miR-3591-3p may be a valuable biomarker and that blocking the encapsulation of miR-3591-3p into exosomes may become a novel immunotherapeutic strategy for glioma.

Fig. 1. Glioma cell-derived exosomal miR-3591-3p promotes macrophage toward an M2-like phenotype.

A Venn diagram exhibiting the overlap of the miRNAs in exosomes from CSF of glioma patients (preoperation vs. postoperation) and tissues (glioma tissues vs. normal brain tissues). B Preoperative and postoperative expression levels of miR-3591-3p in the CSF of glioma patients (n = 32). C The expression levels in tumor tissues (n = 32) and normal brain tissues (n = 12). D qPCR analysis of the expression levels of miR-3591-3p in the exosomes of different glioma cell lines and one normal cell line. E qPCR analysis of the expression levels of miR-3591-3p in the exosomes and cell lines of U118MG and U251. F qPCR analysis of the expression of M2 markers (CD163, IL10, TGFβ1, Arg-1, and IL1ra) and M1 markers (TNFα and iNOS) in PMA-pretreated THP-1 cells cocultured with U118MG cells (GW4869, exosome secretion inhibitor). G Representative immunofluorescence image showing the internalization of PKH67-labeled exosomes (green) and Cy3-labeled miR-3591-3p (red) by PMA-treated THP-1 cells. (scale bar,10 um). H The expression levels of M2 markers (CD163, IL10, Arg-1, TGFβ1, and IL1ra) and M1 markers (TNFα and iNOS) were determined by qRT–PCR in PMA-pretreated THP-1 cells treated with U118MG exosomes. I ELISAs were used to quantify the expression of cytokines (IL10, TGFβ, TNFα) in PMA-treated THP-1 cells cocultured with exosomes derived from U118MG cells transfected with miR-3591-3p mimics. J Flow cytometry assay was applied to analyze CD11b⁺CD163⁺ macrophages treated by exosomes derived from U118MG cells, and quantification was performed. Data are shown as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 2. miR-3591-3p induced M2 macrophage polarization and promoted the progression of glioma cells in vitro and in vivo.

A qRT–PCR analysis revealed that miR-3591-3p mimics could promote M2 macrophage polarization. B The secreted levels of cytokines (IL10, TGFβ1, and TNFα) were measured by ELISA in PMA-treated macrophages, which were transfected with miR-3591-3p mimics. C Representative flow cytometry plots of CD11b⁺ CD163⁺ macrophages. D Invasion and migration capacity of U118MG cocultured with conditioned macrophages were tested using transwell assays (PMA-treated THP-1 cells were transfected with miR-3591-3p mimics). Representative images of invaded and migrated cells are shown (scale bar, 100 μm). E In vivo bioluminescent imaging assay of tumor burden in xenograft nude mice bearing U118MG with macrophages pretreated with LV-miR-3591-3p or LV-NC. Representative images and relative photon flux on day 7 and day 14 postimplantation are shown (n = 5 of each group). F Survival proportion Kaplan–Meier curves of animals in each study group (n = 10 of each group). G HE staining of representative tumor tissues from two groups (scale bar, 2.5 mm). H Representative images and quantification of the expression of Ki67, CD163, and TGFβ1 using immunohistochemistry (scale bar, 100 μm). Data are shown as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 3. CBLB is the direct target of miR-3591-3p in macrophages.

A Venn diagram showing the two predicted miR-3591-3p target genes in three databases (down in mRNA_seq, FC > 1.5, p <0.05; Targetscan; miRDB). B The expression of the two predicted genes was detected by qPCR analysis. C Relative luciferase activities of the CBLB luciferase reporters (WT and MUT) were performed in macrophages. D qRT–PCR was performed for M2 markers (CD163, IL10, TGFβ1, Arg-1, and IL1ra) and M1 markers (TNFα, iNOS) in macrophages transfected with si-CBLB or si-NC. E The expression levels of cytokines (IL10, TGFβ, TNFα) in macrophage transfected with si-CBLB or si-NC were detected by ELISA. F Flow cytometry assay was used to analyze CD11b+CD163+macrophages transfected with si-CBLB or si-NC, and quantification was performed. G Transwell assay was applied to determine the invasion and migration of U118MG cells cocultured with macrophages transfected with si-NC or si-CBLB. Representative images (scale bar, 100 μm) and quantification are shown. Data are shown as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 4. miR-3591-3p promotes macrophage toward M2 phenotype via downregulation of CBLB expression and activation of the JAK2/PI3K/AKT/mTOR and JAK2/STAT3 signaling pathway.

A Macrophages were transfected with plasmids of CBLB following miR-3591-3p transfection, and the expression levels of M2 markers and M1 markers were detected by qRT–PCR. B The expression of cytokines (IL10, TGFβ1, and TNFα) were determined in macrophages, which were transfected with miR-3591-3p mimics and CBLB encoding plasmids. C The CD11b⁺CD163⁺ cells of macrophages were measured using flow cytometry. Representative images and quantification are shown. D Transwell assay was applied to determine the invasion and migration of U118MG cells cocultured with macrophages transfected with miR-3591-3p mimics and CBLB encoding plasmids. Representative images (scale bar, 100 μm) and quantification are shown. E Protein levels of JAK2/PI3K/AKT/mTOR and JAK2/STAT3 pathways were measured in macrophages cocultured with exosomes derived from glioma cells. F Protein levels of the two above pathways were detected following transfection of miR-3591-3p mimics or inhibitors in macrophages. G Proteins levels of the two above pathways were measured in macrophages transfected with si-CBLB or si-NC. H Western blot analysis of the two pathways protein levels after the indicated treatment. The intensity of protein bands was quantified by densitometry. Macrophages: PMA-pretreated THP-1 cells. Data are shown as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 5. miR-3591-3p suppresses tumor growth in vitro and in vivo by blocking cell cycle progression.

A Flow cytometry analysis of cell cycle phase is shown of U118MG cells transfected with miR-NC or miR-3591-3p mimics. B Western blots were used to detect the levels of G2/M-related proteins. C Representative flow cytometry plots of cell apoptosis and quantitative analysis are shown. D Western blot analysis of apoptosis-associated proteins. E In vivo bioluminescent imaging assay of tumor burden in xenograft nude mice bearing U118MG pretreated with LV-miR-3591-3p or LV-NC. Representative images and quantifications are shown (n = 5 of each group). F Kaplan–Meier survival curves for overall survival in different groups (n = 10 of each group). G Representative HE staining of tumor tissues are shown from the two groups (scale bar, 2.5 mm). H Representative pictures of immunohistochemical staining of Ki67 and Bax are shown (scale bar, 100 μm). Data are shown as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 6. MAPK1 is the direct downstream target of miR-3591-3p in glioma cells.

A Venn diagram showing the predicted miR-3591-3p target genes in three databases (down in mRNA_seq, FC > 1.5, p < 0.05; Targetscan; miRDB). B DO (Disease Ontology) enrichment analysis of differential expression genes in mRNA_seq. C GO and KEGG pathway enrichment analysis of the 75 overlapping genes. D, E The relative mRNA expression levels of the 7 MAPK signaling pathway-related genes were measured transfected with miR-mimics/NC in U118MG and U251 cell lines, respectively. F Relative luciferase activity was detected by dual-luciferase reporter assays. G Representative flow cytometry results of the cell cycle phase are shown in U118MG cells transfected with si-NC or si-MAPK1. H The expression of G2/M-related proteins was detected by western blot. I Representative flow cytometry results of apoptotic cells are shown in U118MG cells transfected with si-NC or si-MAPK1. J The expression of apoptosis-related proteins was detected by western blot in groups as indicated. K MAPK1 overexpressing plasmids were transfected following miR-3591-3p mimics transfection, and the cell cycle distributions were determined by flow cytometry in U118MG cells. L The expression of G2/M-related proteins was detected by western blot in groups as indicated. M MAPK1 overexpressing plasmids were transfected following miR-3591-3p mimics transfection, and the apoptotic cells were quantified by flow cytometry in U118MG cells. N Western blot assessment of the expressions of apoptosis-related proteins in groups as indicated. Data are shown as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001).

Fig. 7. miR-3591-3p suppresses tumor via MAPK signaling pathway.

A Western blot results for the expression of proteins in the MAPK signaling pathway in U118MG cells transfected with miR-NC, miR-3591-3p mimics, inhibitor-NC, and miR-3591-3p inhibitor. B Western blot results for the expression of proteins in the MAPK signaling pathway in U118MG cells transfected with si-NC or si-MAPK1. C MAPK1 overexpressing plasmids were transfected following miR-3591-3p mimics transfection, and the MAPK signaling pathway-related proteins were detected by western blot in U118MG cells. The intensity of protein bands was quantified by densitometry. D Schematic diagram depicting the key assumptions and conclusions. Briefly, miR-3591-3p was excreted by glioma cells via exosomes and then taken up by macrophages. In macrophages, miR-3591-3p promotes macrophage to M2 phenotype by inhibiting the activity of CBLB and then activating JAK2/PI3K/AKT/mTOR and STAT3 pathways. On the other hand, since miR-3591-3p plays a tumor suppressor role in glioma cells, when it is excreted, the inhibitory effect on MAPK1 is relieved and thus promoting glioma progression by activating the MAPK pathway. Data are shown as the mean ± SD of three independent experiments. (*p < 0.05; **p < 0.01; ***p < 0.001).