Exosomal miR‐6733‐5p mediates cross‐talk between glioblastoma stem cells and macrophages and promotes glioblastoma multiform progression synergistically

Abstract Aim Exosomal miRNAs derived from glioblastoma stem cells (GSCs) are important mediators of immunosuppressive microenvironment formation in glioblastoma multiform (GBM), especially in M2‐like polarization of tumor‐associated macrophages (TAMs). However, the exact mechanisms by which GSCs‐derived exosomes (GSCs‐exo) facilitate the remodeling of the immunosuppressive microenvironment of GBM have not been elucidated. Methods Transmission electron microscopy (TME) and nanoparticle tracking analysis (NTA) were applied to verify the existence of GSCs‐derived exosomes. Sphere formation assays, flow cytometry, and tumor xenograft transplantation assays were performed to identify the exact roles of exosomal miR‐6733‐5p. Then, the mechanisms of miR‐6733‐5p and its downstream target gene regulating crosstalk between GSCs cells and M2 macrophages were further investigated. Results GSCs‐derived exosomal miR‐6733‐5p induce macrophage M2 polarization of TAMs by positively targeting IGF2BP3 to activate the AKT signaling pathway, which further facilitates the self‐renewal and stemness of GSCs. Conclusion GSCs secrete miR‐6733‐5p‐rich exosomes to induce M2‐like polarization of macrophages, as well as enhance GSCs stemness and promote malignant behaviors of GBM through IGF2BP3 activated AKT pathway. Targeting GSCs exosomal miR‐6733‐5p may provide a potential new strategy against GBM.


| INTRODUC TI ON
Glioblastoma multiforme (GBM) accounts for 82% of malignant brain tumors and actually remains the most common and lethal form of cancer in the central nervous system. 1 GBM are prone to recurrence and the 5-year survival rate is less than 5%, even after surgery, chemotherapy, radiation, and immunotherapy. 2 Glioblastoma stem cells (GSCs) contribute to treatment resistance and have the ability to repopulate the tumor causing recurrence. 3Hence, an understanding of the specific functions of GSCs as well as the mechanisms of remodeling tumor microenvironment will help to develop a more effective therapeutic strategy.
Tumor-associated macrophages (TAMs), play vital roles in constructing immuno-suppressive tumor microenvironment, are enriched in GBMs, and promote tumor growth. 4High TAM infiltration has been shown to predict a poor prognosis in variety of malignancies, including GBMs. 5,6Several markers such as MHCII, CD80, and iNOS have been identified as M1 subtype TAM that can prevent tumor progression, while other markers including CD163, IL-10, and Arg1 are regarded as M2 subtype TAM that can promote GBM progression. 7Recent studies suggested that both TAMs and GSCs are localized and maintained in tumor perivascular niches, 8 and GSCs play a critical role in promoting TAM recruitment resulting in GBM development. 9Moreover, M2 polarization of TAMs and tissue remodeling of GSCs have been reported to be increased in recurrent GBMs after treatment. 10The cross-talking between TAMs and GSCs plays an important role in the constructing of highly immunosuppressive GBM microenvironment. 11However, the underlying molecular mechanisms between their cross-talk have not yet been fully elucidated.
3][14] Exosomal miRNAs from tumor cells have been found to regulate essential aspects of cancer, including angiogenesis, immunosuppression, and metastasis by remodeling the phenotype and function of recipient cells. 15,168][19] However, GSCs exosomal miRNAs mediated remodeling on TAMs, and the relevant regulation mechanism have not been extensively investigated.
In the current studies, we investigated the mutual interactions between GSCs and macrophages by co-culture transwell system and further evaluated whether GSC-derived exosomes (GSCs-exo) induce M2 macrophage polarization via secretion of specific miRNA, which may serve as a potential novel target against GBMs.

| Clinical specimens
Clinical GBM tissue specimens were collected from patients who underwent surgical removal and were diagnosed with GBM according to World Health Organization (WHO) pathological criteria at the Department of Neurosurgery, the Second Affiliated Hospital of Soochow University.The pathological diagnosis of GBM was independently achieved by two senior and experienced pathologists.
Ethical approval was obtained from the Second Affiliated Hospital of Soochow University (Approval number 2022148).Informed consent was obtained from all subjects.

| Cell lines and cell culture
Human glioma stem cell lines GSC11 and GSC23 (M.D. Anderson Cancer Center) were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco) containing 20 ng/mL basic fibroblast growth factor (bFGF) (Gibco) and 20 ng/mL epidermal growth factor (EGF) (Gibco).THP-1 cell line (ATCC) was cultured in RPMI 1640 (Gibco) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen), then incubated with 100 ng/mL phorbol-12-myristate-13-acetate (PMA; Sigma-Aldrich) for 24-48 h to induce resting macrophages (M0).Primary monocyte-derived macrophages were prepared from fresh blood from healthy volunteers.The study was approved by the Research Ethics Committee of the Second Affiliated Hospital of Soochow University, and written informed consent was obtained from all participants.Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation using Ficoll-Paque (TBDScience LTS1077) at 2000 g for 25 min using minimum acceleration and no brake.PBMC fractions were washed in sterile PBS after lysing erythrocytes (CWBIO, CW0613) and plated to select for adherent cells.Non-adherent cells were washed away after 6 h and the remaining cells incubated in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated pooled human serum (Sigma) and 40 ng/mL macrophage colony-stimulating factor (M-CSF, PeproTech, 300-25).Medium changed every 3 days until the cells differentiated into macrophages by 7 days.All cells were maintained in a humidified chamber containing 5% CO 2 at 37°C.

| Cell transfection
The miR-6733-5p mimics/inhibitors and the IGF2BP3 small interfering RNAs (GenePharma, Shanghai, China) were transiently transfected using siRNA-mate (GenePharma) according to the manufacturer's instructions.Sequences information is shown in Table S2.
The RT-qPCR assay was performed with three technical replicates, and at least three independent biological replicates were performed and showed similar results.

| Transwell assay
Transwell inserts (8.0 μm, Corning) were used to evaluate the migration ability of macrophages (THP1 + PMA) cells.The upper chambers contained 300 μL of 2% FBS-containing RPMI-1640 medium, while the lower chambers contained 600 μL of 10% FBS-containing RPMI-1640 medium.After 48 h, cells in the upper chamber were wiped and the cells that migrated were captured with a microscope (AMG EVOS).

| Isolation and purification of exosomes
The culture supernatant was collected from GSCs (GSC11 or GSC23) culture in DMEM/F12 medium containing bFGF and EGF for 4 days, NHA-derived exosomes were harvested from NHAs culture medium (DMEM supplemented with 10% exosome-depleted FBS) for 2 days under 5% CO 2 at 37°C.Briefly, the collected culture medium was centrifuged at 2000 g for 30 min, then 12,000 g for 45 min to remove cell debris and large vesicles.For exosome purification, the supernatant was ultracentrifuged at 100,000 g for 70 min at 4°C to collect the pellet, then was resuspended in 50-100 μL PBS for the subsequent studies.The concentration of exosomes was detected using a BCA Protein Assay (Beyotime).For exosome addition, the culture medium of recipient cells was supplemented with purified exosomes at 20 μg/mL unless otherwise specified.

| Electron microscopy and nanoparticle tracking analysis
Exosomes to be examined by TEM were applied to assess the morphology of exosomes.In brief, exosomes (10 μg) fixed with 4% formaldehyde were placed on copper grids, washed with filtered PBS, and stained with uranyl acetate solution.After 24 h of incubation, samples were analyzed with TEM (Hitachi HT-7700).Besides, the size distribution and concentration were detected by NTA (ZetaView PMX 110, Particle Metrix).

| Engulf of exosomes by macrophages
Purified exosomes were collected and labeled with PKH26 Red Fluorescent membrane linker dye (Sigma-Aldrich) according to the manufacturer's instructions.THP-1 cells were seeded in eight-well chamber slides (5000 cells/well) and pretreated with PMA for 24 h.Then, 10 μg exosomes were incubated with PKH26 dye at room temperature for 5 min.After centrifuged at 10,000 g for 30 min at 4°C, the labeled exosome pellets were resuspended and added to THP-1 derived macrophages for exosomes uptake studies.After incubation for 8 h at 37°C, cells were fixed, stained with DAPI (Invitrogen), and examined by confocal microscope (Zeiss).

| ELISA
Cell culture medium was collected 48 h after the indicated treatment.Secretion of IL-10 and TNFα was examined using ELISA Kit (Biolengend, 430607, 430207) according to the manufacturer's instructions.

| Sphere-formation assay
GSCs were cocultured with GSC-exo activated macrophages for 2 days.GSCs were dispersed into single cell suspension and seeded 1000 GSCs into each well of the 96-well plate with 100 μL DMEM/ F12 medium containing bFGF and EGF.
The brains of xenograft mice were fixed with 4% PFA and embedded in paraffin.Then, 5 μm slices were cut by a microtome (Leica) and deparaffinized, dehydrated, and incubated in heat-mediated antigen retrieval.Subsequently, the endogenous catalase was eliminated with 3% H 2 O 2 -methanol, and tissue slices were incubated with indicated primary antibodies against Ki67 (1:500, Servicebio, GB121141), IMP3 (1:500; abcam, ab179807) and F4/80 (1:2000, Sanying, 28463-1-AP) at 4°C overnight.After washing with PBS, sections were incubated with biotinylated secondary antibodies at room temperature for 1 h.They were then incubated with peroxidase solution for 30 min and then the sections were stained with DAB reagent and counterstained with hematoxylin.The images of each section were taken and analyzed under an optical microscope.
In HE staining, the paraffin-embedded sections were sequentially deparaffinized, dehydrated, stained by hematoxylin, differentiated by the addition of hydrochloric ethanol, backed to blue with ammonia water, and stained with eosin.Then, the sections were dehydrated with gradient alcohol, cleared with xylene, and sealed with neutral resin.Finally, tissue HE staining photos were acquired under an optical microscope.

| Statistical analysis
All statistical analysis were conducted with GraphPad Prism 9.0 (GraphPad Software).Results are presented as means ± SD standard deviation on three independent experiments.The normality of the data distribution was analyzed by the Shapiro-Wilk test.Student's t test was performed to analyze the statistical difference between two groups, and analysis of variance (ANOVA) was applied to evaluate the differences between multiple groups.The p-value <0.05 was considered statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).p-Value >0.05 was considered not significant and was denoted by "NS."

| TAMs were accumulated in GBM and can be induced transformation toward M2 polarization via GSCs
To investigate the population of M2-like TAMs in surgical specimens of GBM patients, immunofluorescence staining of M2-like TAMs makers CD163 and CD206 was performed, which disclosed that CD163 and CD206 expression elevated higher in tumor tissue in comparison to para-tumor tissue (Figure 1A).Further investigations focus on exploring the relationships between M2-like TAMs signature and clinic prognosis of GBM with Kaplan-Meier analysis, which demonstrated that higher M2-like TAMs infiltration indicated poorer survival of GBM patients (Figure 1B; Figure S1A).M2-like TAMs infiltration and accumulation can be observed in GBM, which was in accordance with the previous studies. 5,6 vitro differentiation of THP-1 cells into macrophages was achieved with addition of PMA (100 ng/mL), which was verified by both adherent cells morphological changes (Figure S1B) and increased expression of the cell surface markers CD11b and CD68 (Figure 1C).To explore whether GSCs were involved in promoting the infiltration of M2-like TAMs in the GBM microenvironment, the key stemness biomarkers were examined (Figure S1C,D) and a coculture model was established based on transwell chambers in vitro.The transwell migration assay showed that conditioned media (CM) of GSC11 and GSC23 promoted the migration of M0 macrophages differentiated by THP-1, compared to the CM of normal human astrocytes (NHAs) group (Figure 1D,E).To further explore whether GSCs mediated the M2 polarization of macrophages, M0 macrophages derived from THP-1 cells were cocultured with GSCs in a transwell system (Figure S1E).Two days later, the levels of macrophage-associated phenotypic markers were detected by RT-qPCR, the level of M2 macrophages markers (CD163, Arg1, and IL-10) in macrophages cocultured with GSCs (GSC11 or GSC23 cells) significantly increased, while one of the M1 markers CD80 slightly decreased, and another M1 marker iNOS kept stable, compared with the naive resting macrophages (Mφ) group (Figure 1F).

| GSCs induced M2 polarization of macrophages in exosome-dependent manner
The exosomes from GSCs or NHAs were isolated by ultracentrifugation.TEM verified that the harvested exosomes were vesicle-like structures with size ranged from 30 to 100 nm (Figure 2A).Particle size analysis of the exosomes further confirmed that these particles distributed mainly varied in 30-150 nm (Figure 2B), which contained their specific markers (HSP70, TSG101, and CD9) but negative for endoplasmic reticulum marker Calnexin (Figure 2C).To evaluate whether macrophages can engulf GSCs-derived exosomes, PKH26 (red fluorescence)-labeled GSCs exosomes were cocultured with macrophages, which disclosed that the unstained macrophages internalized the PKH26-labeled exosomes obviously under confocal microscopic observation (Figure S1H).
To verify whether GSCs-exo can regulate M2 macrophage polarization, macrophages were treated with PBS, NHA-exo, or GSCsexo, respectively.When compared with the control M0 macrophages treated with PBS, the expression of M2 markers significantly increased in the M0 treated with GSCs-exo, while the expression of M1 markers reduced or remained unchanged.When compared with the macrophages treated by the NHA-derived exosomes, the expression of relevant macrophage markers had no obvious changes (Figure 2D).
A total of 7 days after isolation from healthy donor whole blood and in vitro cultured in RPMI 1640 culture medium, human PBMCs-derived macrophages underwent morphological changes as well after addition of GSCs exosomes (Figure S1I).Flow cytometry analysis showed that CD163 expression in PBMCs-induced macrophages treated with GSCs-exo also increased (Figure 2H,I).In summary, these data indicated that GSCs can polarize macrophages toward M2 polarization through regulation by GSCs secreted exosomes.

| MiR-6733-5p was highly expressed in GSCs and enhanced the self-renewal and stemness of GSCs
To reveal the potential exosomal miRNAs involved in GBM development, bioinformatic analysis on expression profiles of miRNAs in serum of GBM patients and healthy donors was predicted with the latest two miRNA-Seq datasets from Gene Expression Omnibus database (GEO) (GSE139031, GSE113740).Besides, miRNA sequencing for exosomes derived from GSCs and NHAs was performed and the intersect miRNAs of the abovementioned four groups were shown in a Venn diagram (Figure 3A), and we finally chose miR-6733-5p for further study (Figure 3B).The RT-qPCR results showed that miR-6733-5p was the highest expression in GSCs and higher than glioma cells (U251, LN229, SNB19, and SF295) (Figure 3C).
Compared with the peri-tumor tissue, miR-6733-5p level elevated obviously in the GBM tissue (Figure 3D), indicating that aberrantly high expression of miR-6733-5p may be associated with GBM development.Sphere-formation assay showed that sphere-forming ability significantly elevated after miR-6733-5p overexpression and decreased after miR-6733-5p knockdown both in GSC11 and GSC23 cells (Figure 3E,F).Flow cytometry analysis revealed that the CD133 and OCT4 expression significantly elevated after miR-6733-5p overexpression, and decreased after miR-6733-5p knockdown both in GSC11 and GSC23 cells (Figure 3G-J).These data suggested that miR-6733-5p was highly expressed in GSCs and enhanced the self-renewal and stemness of GSCs.

| MiR-6733-5p highly expressed in GSCs-exo can be transferred to macrophages
Mounting evidence indicated that miRNAs play a vital role in exosome mediate intercellular communications. 21In current studies, we disclosed that high level of miR-6733-5p in GSCs-exo and can be engulf actively by macrophages.Macrophages incubated with GSCs-exo expressed higher miR-6733-5p than those incubated with NHA exosomes or PBS only (Figure 4A).The role of miR-6733-5p on polarization of M2 macrophages was investigated, which showed that the expression of M2 markers increased after addition of miR-6733-5p mimics in macrophages derived from THP-1 and decreased by miR-6733-5p inhibitors (Figure 4B,C; Figure S2A).ELISA results showed that miR-6733-5p mimics promoted the secretion of IL-10 and inhibited the secretion of TNFα, while miR-6733-5p inhibitors decreased the secretion of IL-10 without changing the secretion of TNFα (Figure 4D,E; Figure S2B).Flow cytometry analysis showed that the proportion of CD11b + CD163 + double positive THP-1 derived macrophages increased by miR-6733-5p mimics and decreased by miR-6733-5p inhibitors (Figure 4F-H).Consistent with this finding, the proportion of CD11b + CD163 + double positive macrophages (PBMCs) also increased (Figure 4I).Taken together, these data suggest that GSCs-derived exosomal miR-6733-5p contributed to M2 polarization of macrophages in vitro.

| GSCs exosomal miR-6733-5p targeted IGF2BP3 and induced M2 macrophages polarization by regulating AKT signaling pathway
To explore the function of IGF2BP3 in macrophages, overexpression efficiency in macrophages was verified by Western blot (Figure 5I; For IGF2BP3 is an activated regulator of AKT pathway, 25 and AKT signaling pathway is involved in M2 polarization, 26 it is reasonable to be speculated that exosomal miR-6733-5p can activate AKT signaling pathway by regulating IGF2BP3 expression.Western blot results showed that the expression of phosphorylated AKT in macrophages treated with exosomes derived from GSC11 cells increased obviously, and can be abolished by miR-6733-5p inhibitors (Figure 6E; Figure S2H).In addition, the effects of IGF2BP3 knockdown can be partially attenuated by miR-6733-5p overexpression (Figure 6F; Figure S2I).Collectively, these results suggested that GSCs-derived miR-6733-5p regulated IGF2BP3 expression to activate AKT signaling in macrophages, thus polarizing macrophages toward the M2 phenotype.
To elucidate whether overexpressing IGF2BP3 in macrophages promote GSCs maintenance, the sphere-formation assay and flow cytometry assay were performed, which demonstrated that macrophages overexpressing IGF2BP3 significantly increased the efficiency of sphere-forming ability and stemness (Figure 8A,B).

| DISCUSS ION
Considerable attention has been focused on the significance of tumor environment (TME) on tumor progression, a complex community that includes cancer cells, immune-inflammatory cells, and diverse stromal cells and variety of regulating factors. 4Targeting M2 TAMs in TME is considered as an potential effective therapeutic strategy against cancer. 27Our previous studies have found that GBM could promote glioma-associated macrophage infiltration and M2 polarization. 28Exosomes are membrane-enclosed extracellular vesicles carrying biomolecules that include proteins and nucleic acids, especially miRNAs. 16In the current studies, we explored the effects and underlying mechanisms of cosstalk between GSCs and macrophages on M2 polarization, which disclosed that GSCs-derived exosomal miR-6733-5p can induce M2 macrophage polarization, and polarized M2 macrophages promote F I G U R E 2 Glioblastoma stem cells (GSCs)-exo induces M2 macrophage polarization.(A) Representative transmission electron microscopy (TEM) images of GSC11, GSC23, and NHA cell-secreted exosomes (scale bar, 100 nm).(B) Nanoparticle tracking the size distribution of exosomes released by GSC11, GSC23, and NHA.(C) Western blot analysis was performed to detect typical exosomal biomarkers (HSP70, TSG101, CD9, and Calnexin) in exosomes derived from NHA, GSC11, and GSC23 cell lines.(D) RT-qPCR was applied using primers for M2 markers (CD163, Arg1, and IL-10) and M1 markers (CD80 and iNOS) in PMA-pretreated THP-1 cells treated with PBS, NHA-exo, GSC11exo, GSC23-exo.(E) Macrophages were treated with PBS, NHA-exo, GSC11-exo, GSC23-exo, and the supernatants cultured for 48 h were examined to determine the secretion of IL-10 and TNFα via ELISA.(F, G) Flow cytometry was used to examine the M2 macrophages (CD11b + CD163 + ) expression of PMA-pretreated THP-1 cells treated with PBS, NHA-exo, GSC11-exo, GSC23-exo, and quantification was performed.(H, I) Flow cytometry was used to detect the M2 macrophages (CD11b + CD163 + ) expression of M-CSF-pretreated PBMC cells treated with PBS, NHA-exo, GSC11-exo, GSC23-exo, and quantification was performed.Data depict the mean ± standard deviation and are representative of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).Exosomes secreted by various tumor cells or stromal cells can influence tumor progression by transfer of bioactive molecules among different cell types in the TME. 29,30Recently, accumulating studies have shown that tumor cells derived exosomes were involved in remodeling of immune suppressive microenvironment through crosstalk between tumor cells and tumor-associated stromal cells. 15Tumor cells-derived exosomes can induce exhaustion of natural killer cells and induce resistance to immunocheckpoint blocking therapy. 31,32Besides, tumorderived exosomes can also induce pro-tumor activation of neutrophils to facilitate tumorigenesis. 33,34Besides, our current studies demonstrated that GSCs can induce the polarization of macrophages toward M2 phenotypes via delivering GSCs exosomes.
MicroRNAs (miRNAs) are a class of small, noncoding RNAs that regulate the translation of target genes at posttranscription level.
The abnormal expression of miRNAs and dysregulation of miRNA regulators are involved in the progression of many malignancies. 35e biological function of miR-6733-5p in tumor development has not yet been elucidated previously.Our investigations disclosed that miR-6733-5p was significantly enriched in GSCs, GSCs exosomes, and GBM tumor surgical specimen of patients, and further discovered that it can be delivered intracellularly via engulf of exosomes by macrophages.Besides, we demonstrated that intracellular miR-6733-5p can bind with IGF2BP3 to induce M2 macrophage polarization via regulating the AKT pathway.
IGF2BP3, together with IGF2BP1 and IGF2BP2, are members of RNA-binding protein family that belongs to mRNA-binding proteins, which influence the cytoplasmic fate of mRNAs through localization, stability, and translation. 36,37Recent studies have showed that IGF2BP3 played key roles in GBM maintenance and promoting the emergence of M2-subtype macrophages, highlighting its vital roles on development of gliomas. 38,39However, the associated mechanisms of IGF2BP3 in gliomas have never been fully elucidated.
Our data disclosed that miR-6733-5p can bind to the 5′-UTR of IGF2BP3 and promote its expression, furthermore, overexpression of IGF2BP3, modulated by miR-6733-5p, resulted in activation of AKT signaling, indicating that miR-6733-5p played crucial roles on promoting M2 macrophage polarization by regulating the IGF2BP3/ AKT pathway.
TAMs are responsible for various tumor-promoting activities associated with GBM development, progression, and resistance to therapies. 40,41In this investigation, we found that GSCs-derived exosomal miR-6733-5p induced the alternative M2 macrophage activation, which help to promote the self-renewal and stemness of GSCs both ex vivo and in vivo, indicating that exosomes are important regulators of GBM microenvironment remodeling and act as an important messenger that mediated the cross-talk between GSCs and relevant stromal cells.
In summary, we demonstrated that GSCs-derived exosomal miR-6733-5p can induce M2 macrophage polarization by activation of the IGF2BP3/AKT pathway, and remodeled macrophages can further promote the self-renewal and stemness of GSCs.Our findings shed light on the mechanisms of GSCs exosomal miR-6733-5p in the development of GBM immuno-microenvironment and highlight this pathway as a potential therapeutic target against gliomas.However, one common issue associated with immunodeficiency mouse F I G U R E 4 Glioblastoma stem cells (GSCs)-derived exosomal miR-6733-5p induces M2 macrophage polarization.(A) Macrophages were incubated with GSC11-exo or HNA-exo for 48 h.The miR-6733-5p expression levels in macrophages were determined using RT-qPCR.(B, C) THP1 (Mφ) cells transfected with mimics NC or miR-6733-5p mimics or incubated with GSC11-exo and transfected with NC inhibitors or miR-6733-5p inhibitors.RT-qPCR was adopted to detect the levels of M2 and M1 markers in THP1 (Mφ) cells.(D, E) Macrophage transfected with mimics NC or miR-6733-5p mimics or treated with GSC23-exo and transfected with NC inhibitors or miR-6733-5p inhibitors, and the supernatants of 48-h cultures were used to determine the secretion of IL-10 and TNFα via ELISA.(F-I) Flow cytometry and quantification were performed to analyze the proportion of CD11b + CD163 + macrophages transfected with mimics NC or miR-6733-5p mimics or incubated with GSC11-exo or GSC23-exo and transfected with NC inhibitors or miR-6733-5p inhibitors.Data depict the mean ± standard deviation and are representative of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).models in glioma research fields is the lack of T lymphocytes, which limits the interpretation of immune related experimental data.Our experimental design can be improved if applying more powerful in vivo characterization methods to further elucidate the role of macrophages in GSCs-microenvironment.Recent studies have already reported that molecular magnetic resonance imaging (mMRI) may be useful for enhancing the feasibility and accuracy of in vivo glioma studies. 42,43Using molecular MRI could help visualize biological processes at the molecular level in immunodeficiency mouse models, thereby improving future studies.

| CON CLUS IONS
We discovered that M2-like TAMs infiltration and accumulation can be observed in GBM.GSCs can induce macrophage M2 polarization of TAMs, which further facilitates the self-renewal and stemness of GSCs.Given that exosomes have been shown to transport miRNAs to alter cellular functions, we disclosed that miR-6733-5p was enriched in GSCs, GSCs-derived exosomes, and surgical specimen of

Female
BALB/c nude mice (4 weeks old, 15-20 g) were randomly divided into four groups.For the subcutaneous xenografts, 5 × 10 6 GSC23 cells were mixed with 5 × 10 5 conditioned macrophages stimulated by PBS, GSCs-exo, or miR-6733-5p mimics transfection, respectively, at a ratio of 10:1 ratio were injected subcutaneously into the right flank of each mouse, respectively.The mice were sacrificed 4 weeks after tumor inoculation.The tumors were obtained by surgery and the tumor volume and weight were measured and calculated.For the orthotopic xenografts, 5 × 10 5 GSC23 cells were mixed with 5 × 10 4 conditioned macrophages stimulated by PBS, GSCs-exo, or miR-6733-5p mimics transfection, respectively, at a ratio of 10:1 ratio orthotopically xenografted by intracerebral injection into the right cerebral cortex of the mouse at a depth of 3.5 mm.Mice were euthanized when neurological symptoms were observed.Then, the whole brain was harvested, PFA-fixed, paraffin-embedded, and sectioned coronally from anterior to posterior.All the animal experiments were approved by the Institutional Animal Care and Use Committee of Second Affiliated Hospital of Soochow University.

Flow
cytometry analysis further verified that the double positive expression rate of the M0 marker CD11b and M2 marker CD163 elevated obviously after mutual interactions with GSC11 or GSC23 cells in vitro (Figure 1G,H).For exosomes are important vehicles for intercellular communications, 20 whether GSCs exosomes were involved in mediating the M2 polarization of macrophages needs further investigations.Addition of GW4869 resulted in inhibition of GSCs exosomes secretion, RT-qPCR disclosed the level of M2 markers (CD163, Arg1, and IL-10) in macrophages decreased simultaneously (Figure S1F,G), flow cytometric analysis showed that the proportion of CD11b + CD163 + double positive macrophages significantly decreased after inhibition of GSCs exosomes production (Figure 1I,J), which implied the possibility that GSCs induced M2 polarization of macrophages in an exosome-dependent manner.

FIGURE 4
FIGURE 4 Legend on next page )-derived exosomal miR-6733-5p directly targets IGF2BP3 in macrophages.(A, B) Schematic diagram of the wild-type and mutated-type binding site between miR-6733-5p and the IGF2BP3 3/5′UTR.(C, D) Luciferase reporter assays in HEK 293T cells with transfection of wild-type or mutant IGF2BP3 3/5′-UTR as well as either miR-6733-5p mimics or miR-6733-5p inhibitors were performed.Luciferase activity was normalized by the ratio of firefly and renilla luciferase signals and determined 48 h after transfection.(E-G) Comparison of IGF2BP3 expression in macrophages either treated with GSC11-derived exosomes, or transfected with miR-6733-5p mimics/inhibitors, or transfected with si-IGF2BP3, respectively, by Western blot.(H) The RT-qPCR results showed that knocked down IGF2BP3 with small interfering RNAs significantly decreased the expression of IGF2BP3.(I, J) Comparison of IGF2BP3 expression in macrophages either transfected with miR-6733-5p mimics, or transfected with si-IGF2BP3, or transfected with ov-IGF2BP3, respectively, by Western blot.(K) Macrophages were transfected with plasmids overexpressing nonsense sequence or full-length IGF2BP3 cDNA.RT-qPCR was adopted to detect the levels of M2 (CD163, Arg1, and IL-10) and M1 markers (CD80 and iNOS) in THP1 (Mφ) cells.(L) Macrophages were transfected with plasmids overexpressing nonsense sequence or full-length IGF2BP3 cDNA.The supernatants of cultures were used to determine the secretion of IL-10 and TNFα via ELISA.(M, N) Flow cytometry was applied to measure CD11b + CD163 + macrophages transfected with plasmids overexpressing nonsense sequence or full-length IGF2BP3 cDNA, and quantification was performed.Data depict the mean ± standard deviation and are representative of three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).