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M2-Macrophage-Derived Exosomes Promote Meningioma Progression through TGF-β Signaling Pathway

Tumor-associated macrophages (TAMs) have been shown to be an essential component of the tumor microenvironment and facilitate the proliferation and invasion of a variety of malignancies. However, the contribution of TAMs to meningioma progression has not been characterized in detail. In this study, we aimed to discover a novel regulatory pathway by which exosome-mediated M2-polarized macrophages participate in meningioma tumorigenesis and progression. Methods. First, the distribution and functional phenotype of macrophages in meningioma tissues were assessed by immunohistochemistry. Macrophage-derived exosomes (MDEs) were characterized, and further cell coculture experiments were performed to explore the effects of M2-MDEs on the proliferation, migration, and invasion of meningioma cells. RNA sequencing was used to analyze the transcriptomic signatures in meningioma cells treated with M2-MDEs. Three-dimensional tumorspheres and xenograft tumor models were used to evaluate the effects of M2-MDEs on meningioma tumorigenesis and development. Results. We found that M2 macrophages were enriched in meningioma tissue. Coculture with meningioma cells induced the M2 polarization of macrophages. We also found that M2-MDEs were able to significantly promote cell proliferation, cell migration, cell invasion, and tumorigenesis in meningiomas. Bioinformatic analysis suggested that the TGF-β pathway was activated in meningioma cells treated with M2-MDEs. Functional experiments demonstrated that blocking the TGF-β signaling pathway could effectively reverse the tumor-promotive effects mediated by M2-MDEs. Conclusions. Overall, our study showed that M2-MDEs promoted meningioma development and invasion by activating the TGF-β signaling pathway. Targeting exosome-mediated intercellular communication in the tumor microenvironment may be a novel therapeutic strategy for meningioma patients.


Introduction
As one of the most common primary tumors of the central nervous system, meningiomas arise from the arachnoid tissue surrounding the brain [1,2]. Meningiomas can be defined as grade I (benign meningioma), grade II (atypical meningioma), and grade III (malignant meningioma) based on the grading criteria of the World Health Organization (WHO) [3]. The histopathological features of most meningi-omas are benign (grade I), and patients with these meningiomas usually have an ideal prognosis with surgery [4]. Atypical and anaplastic meningiomas often exhibit biological characteristics of malignant tumors such as abnormal proliferative activity and aggressive growth patterns, which pose challenges for the treatment of meningiomas [5][6][7]. However, the molecular mechanisms of meningioma tumorigenesis, especially the role of the tumor microenvironment in its progression, are still underexplored.

Materials and Methods
2.1. Cell Culture and Treatment. Human meningioma cell lines (IOMM-Lee and CH157-MN) and human mononuclear macrophages THP-1 were bought from ATCC. IOMM-Lee and CH157-MN cells were passaged in DMEM medium supplemented with 10% exosome-free fetal bovine serum (FBS). THP-1 cells were cultured in RPMI 1640 medium supplemented with 10% exosome-free FBS. The medium was changed every 48 hours. THP-1 cells were induced to differentiate into M0 macrophages by treatment with 100 ng/mL of phorbol myristic acetate (PMA, Sigma) for 24 hours. To induce the M2 polarization of macrophages, 20 ng/mL IL-4 and 20 ng/ mL of IL-13 were used to treat M0 macrophages for 48 hours. Transwell chambers (0.4 μm, Corning) were used for coculturing meningioma cells (lower chamber) and macrophages (upper chamber). For the culture of 3D tumorspheres, 1000 meningioma cells were cultured in U-shaped microplates (Thermo Scientific) with a low-adhesion surface for 7 days.
2.2. Immunohistochemistry (IHC) and Immunofluorescence (IF) Staining. The study was approved by the Ethics Committee of the West China Hospital of Sichuan University. All the meningioma samples were obtained from West China Hospital. Paraffin sections were deparaffinized and rehydrated for antigen retrieval according to standard IHC and IF protocols [28]. Then, 3% H 2 O 2 was used to block endogenous peroxidase, followed by the blocking of tissue sections. After overnight incubation with the primary antibodies at 4°C, the tissue sections were counterstained with hematoxylin after DAB staining. For IF staining, Alexa Fluor® 555-conjugated secondary antibody was used to incubate sections, and the nuclear chromatin is marked by DAPI staining. Sections were observed under an upright microscope, and image data were collected. The specific primary antibody information is listed in Supplementary Information Table S1.

RNA Extraction and Quantitative
Real-Time PCR (qPCR). Nucleic acids in the samples were extracted using TRIzol Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. Briefly, dishes with 1 × 10 6 cells were washed three times with PBS. 1 mL of TRIzol Reagent was used to lyse cells and release RNA. High-speed centrifugation was performed after mixing TRIzol with ethanol, and the mRNA was distributed in the upper aqueous phase. Collect mRNA using an adsorption column and dissolve it in 50 μl RNase-free water. The 260/280 absorbance ratio (A260/A280) was used to assess RNA purity. A Prime-Script™ RT reagent Kit (TAKARA, Kusatsu, Japan) was used for the reverse transcription of mRNA. qPCR experiments were performed using the TB Green® Premix Ex Taq™ II kit (TAKARA). The amplification program was as follows: 95°C for 1 min, 40 cycles of 95°C for 5 s, and 60°C for 20 s. ACTB was used as the housekeeping gene to evaluate the relative expression level of the target gene. The PCR primers are listed in Supplementary Information Table S2. 2.4. Western Blot. Total protein from cells and tissues was extracted using RIPA lysis buffer (Beyotime). Cytoplasmic and nuclear proteins were extracted by sequentially disrupting the cell membrane and nuclear membrane through an osmotic pressure gradient using the Nuclear/Cytoplasmic Protein Extraction Kit (Beyotime). The protein extraction 2 Journal of Immunology Research solutions were quantified using the BCA kit (Solarbio) and then thermally denatured using a metal bath. Proteins of different mass sizes were separated by vertical SDS-PAGE and then transferred to PVDF membranes. The PVDF membranes were incubated in blocking solution for 1 hour and then incubated with primary antibodies, followed by chemiluminescent detection using the ECL method. The specific primary antibody information is listed in Supplementary Information Table S1.
2.5. Exosome Isolation and Analysis. The exosomes secreted by macrophages were isolated using gradient centrifugation [29]. Briefly, macrophages were cultured for 48 hours in exosome-free medium. Then, the macrophage culture medium was collected and centrifuged at 1000 × g for 10 min, 3000 × g for 30 min, 6000 × g for 40 min, and 12,000 × g for 2 hours to remove the cell debris. 2.14. Statistical Analysis. The GraphPad Prism software (v8.0, USA) was used for statistical analysis. Student's t-test and one-way ANOVA were used to calculate the p values for differences between groups. When p < 0:05, differences were considered statistically significant.

Macrophages Are Enriched in Meningioma Tissue, and
Coculture with Meningioma Cells Promoted M2 Polarization of Macrophages. To identify the infiltration and functional characteristics of macrophages in meningiomas and adjacent normal meningeal tissue, we performed IHC staining and analyzed the expression of CD68, CD163, and CD206. The IHC results show that macrophages were abundantly infiltrated in tumors relative to meningeal tissues (Figure 1(a)). Interestingly, CD163 and CD206 are ubiquitously expressed in these infiltrating macrophages, especially in high-grade meningiomas, suggesting that they may be induced to become TAMs with an M2 phenotype in the TME. Previous studies have provided evidence that the paracrine secretion of tumor in various malignant tissues is an inducer of the tumor-promoting phenotypic transformation of macrophages [30,31]. In vitro, THP-1 was induced to differentiate into M0 macrophages by coculture with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) for 24 hours. In order to explore the paracrine effect of meningioma cells on macrophage polarization, we constructed a noncontact tumor-macrophage coculture system by using Transwell chambers. We assessed the expression levels of polarization markers in macrophages after 48 hours of coculture with meningioma cell lines. As shown, macrophages cocultured with IOMM-Lee and CH157-MN cells had higher mRNA levels of CD163, CD206, IL-10, and ARG1 as detected by qPCR assay (Figure 1(b)). Similar results were detected in western blot ( Figure 1(c)). At the same time, we detected greater IL-10 secretion by macrophages cocultured with meningioma cells by ELISA (Figure 1(d)). These results indicate that macrophages are enriched in meningioma tissue and exhibit M2-prone polarization during coculture with tumor cells.

Characterization of Macrophage-Derived Exosomes and
Internalization. THP-1 was induced to differentiate into M0 macrophages in the presence of 100 ng/mL PMA for 24 hours and subsequently cocultured with IL-4 and IL-13 to induce M2-phenotype polarization. Macrophage-derived exosomes (MDEs) were collected by ultracentrifugation. The detection results of transmission electron microscopy (TEM) and the nanoparticle tracking analyzer indicated that MDEs exhibited morphological characteristics typical of exosomes ( Figure S1A, B). Western blotting showed that CD81, TSG101, and CD9 were highly enriched in M2-MDEs and M0-MDEs, all of which are positive markers of exosomes ( Figure S1C). And calnexin, an intracellular calcium-binding protein considered to be a negative marker for exosomes, was absent or underexpressed in isolated MDEs. To examine whether meningioma cells could take up MDEs, we incubated them with PKH67-labeled MDEs. Green fluorescence was observed outside the DAPI-labeled nuclei, which suggested that M0-MDEs and M2-MDEs could be phagocytosed by tumor cells ( Figure S1D).

3.3.
M2-Macrophage-Derived Exosomes Promote Meningioma Cell Proliferation and Inhibit Apoptosis. Previous studies suggested an important role for TAM-derived exosomes in tumor proliferation and antiapoptotic processes. To further investigate the role of M2-MDEs in meningioma progression, we coincubated meningioma cells with M0-MDEs and M2-MDEs separately for 48 h and then detected the proliferative activity and apoptosis levels of the tumor cells. As shown in Figure 2(a), the CCK-8 results indicate that, compared with the control and M0-MDEs group, meningioma cells cocultured with M2-MDEs exhibited higher proliferative activity, while M0-MDEs failed to promote tumor cell proliferative activity. Similarly, a higher proportion of EdU + tumor cells was identified in the M2-MDE coculture group by flow cytometry, suggesting that M2-MDEs, but not M0-MDEs, can promote tumor cell mitosis (Figure 2(b)). The expression of apoptosis-related proteins in meningioma cells was further examined by western blotting. As shown in Figure 2(c), the expression of the antiapoptotic protein Bcl2 was significantly increased in tumor cells after M2-MDE treatment, while the expression of the proapoptotic protein Bax was downregulated. The expression of the apoptotic marker cleaved-caspase-3 was downregulated in meningioma cells after M2-MDE treatment but did not change significantly in the M0-MDE group. Furthermore, we detected the fraction of Annexin V/PI-positive cells by flow cytometry to characterize early and late apoptotic cell death (Figure 2(d)). The results showed that the proportion of apoptotic cells was significantly reduced in both IOMM-Lee and CH157-MN after 48 hours of treatment with M2-MDEs. Overall, according to our results, coculture with M2-MDEs can specifically promote the proliferative viability and reduce the level of apoptosis of meningioma cells.

M2-MDEs Promote Metastasis of Meningioma Cells.
Next, we tried to delve into the effect of M2-MDEs on the migration and invasion ability of meningioma cells. The wound healing assay was used to quantify meningioma cell mobility. Meningioma cells cocultured with M2-MDEs exhibited increased mobility compared to the control and M0-MDE groups (Figure 3(a)). Transwell assays were subsequently performed, and M2-MDEs induced a significant increase in the number of tumor cells migrating across the membrane to the lower chamber (Figure 3(b)). In addition, we preincubated Matrigel on the membrane of the upper Transwell chamber and then examined the tumor cell invasiveness. The results indicate that M2-MDE-treated meningioma cells exhibited a stronger invasive ability (Figure 3(b)). It is well known that EMT is highly correlated with tumor invasion and metastasis, and transformation to a mesenchymal-like phenotype confers enhanced motility of   Journal of Immunology Research tumor cells. Therefore, we suspected that M2-MDEs might have an impact on the EMT process and related signaling pathways in meningioma cells. To assess whether coculture with M2-MDEs induces EMT processes in meningioma cells, the expressions of canonical EMT markers were detected. As shown in Figure 3(c), the protein expression levels of mesenchymal markers slug, snail, vimentin, and N-cadherin in meningioma cells were significantly increased after treatment with M2-MDEs for 48 h, relative to the control and M0-MDE groups. In short, our results suggest that coculture with M2-MDEs increases the migratory and invasive abilities of meningioma cells.

TGF-β Pathway Is Activated in M2-MDE-Treated
Meningioma Cells. To explore the molecular mechanism by which M2-MDE regulates the proliferative activity and invasive ability of meningioma cells, we performed RNA-seq to explore the transcriptome differences in meningioma cells cocultured with M0-MDEs and M2-MDEs. Differential gene analysis and pathway enrichment analysis revealed potential differences in biological functions in the tumor cells (Figures 4(a)-4(c)). We noticed that genes such as TGFBI, SNAI1, MMP2, TGFB1, TGFB2, IGFBP7, and LTBP2 were upregulated by the treatment of M2-MDEs in meningioma cells and may contribute to tumor invasion and proliferation behavior. In particular, GO enrichment analysis suggested that multiple TGF-β signaling-related pathways including "Regulation of TGF-beta activation" and "Regulation of smad protein complex assembly" were activated in meningioma cells cocultured with M2-MDEs, which was similar to the trend of the results of KEGG analysis. Previous studies have not reported the functional characterization for the TGF-β signaling in macrophage-tumor communication in the meningioma microenvironment. Therefore, we hypothesized that the TGF-β pathway might be closely related to the protumor biological effects induced by M2-MDEs, and we validated the RNA-seq conclusions at the mRNA level. The results of qPCR suggested that the expression of TGF-β1 was upregulated by the treatment of M2-MDEs, which supported the above results (Figure 4(d)). Furthermore, we analyzed the protein expression levels of key molecules of the TGF-β pathway in meningioma cells by western blotting (Figure 4(e)). The results showed that coculture with M2-MDEs increased the phosphorylation level of Smad2/3 in meningioma cells. As classical downstream transcription factors of the TGF-β pathway, translocation of phosphorylated Smad2/3 into the nucleus is a hallmark of TGF-β pathway activation. The western blotting of cytoplasmic and nuclear proteins revealed increased nuclear translocation of Smad2 and Smad3 in meningioma cells cocultured with M2-MDEs (Figure 4(f)). In short, our study suggests that the activation of the TGF-β pathway may be a key biological event for M2-MDEs' promotion of tumor progression.
3.6. Inhibition of TGF-β Pathway Reverses the Tumor-Promoting Biological Effects Mediated by M2-MDEs. The above results raised the question of whether the TGF-β signaling pathway was responsible for the tumor-promoting effects of M2-MDEs. Thus, LY2109761, a classical selective TGF-β-receptor type-I inhibitor, was used to block TGF-β signaling. Western blotting demonstrated that LY2109761 not only effectively reduced the phosphorylation of Smad2/ 3 but also prevented their nuclear translocation, indicating that the upregulation of the TGF-β signaling in tumor cells   . In short, our study demonstrates that M2-MDEs play a promoting role in meningioma tumorigenesis, which can be partially reversed by blocking TGF-β signaling.

Discussion
Patients with malignant meningiomas tend to have poor prognoses and high mortality due to the uncontrollable proliferative activity and invasive ability of tumor cells. Despite improvements in the treatment of meningiomas over the past decade, the role of the TME in meningioma development remains unclear. Previous studies have suggested that a variety of stromal cells, including TAMs, interact with tumor cells in the TME, which contributes to tumor progression [14,15,17]. However, the molecular mechanisms underlying the integral role of infiltrating TAMs in the development of malignant meningiomas have not been fully studied. In recent years, the immunological landscape in meningiomas has been reported in the literature, and tumor-associated macrophages are the dominant immune    respectively. β-Tubulin and lamin B1 serve as house-keeping genes for cytoplasmic and nuclear proteins, respectively. * p < 0:05, * * p < 0:01, * * * p < 0:001, vs. the control and M0-MDE group. 12 Journal of Immunology Research  14 Journal of Immunology Research population in meningioma tissues, suggesting their criticality in tumor progression. In our study, the IHC analysis revealed that TAMs were enriched in meningioma tissue relative to meningeal and brain tissue. Infiltrating TAMs in meningiomas exhibit M2-biased polarization, which has been reported to assist tumor progression. Based on these results, we speculate that unique cell-to-cell communication in the TME might induce a transition in the functional state of the TAMs. It has been reported that the paracrine secretion of various malignant cells, including lung cancer and colorectal cancer cells, can induce the differentiation of TAMs to an anti-inflammatory phenotype. Our results from noncontact coculture experiments show that the paracrine signaling of meningioma cells induces the M2 polarization of TAMs. Accumulating evidence supports a unique role of exosomes in intercellular communication in the TME as widely distributed transport mediators in the stroma [32,33]. For example, Yang and colleagues proposed that exosomes from M2 macrophages promoted angiogenesis and tumor growth in pancreatic cancer in an E2F2-dependent manner [12]. The WHO classification of meningiomas is based on the number of proliferating cells. The most prominent features of grade III meningiomas are abnormally increased cell viability and mitotic rates, which pose challenges for the treatment of malignant meningiomas. In this study, we found that M2-MDEs could significantly promote the cell viability and inhibit the apoptosis of meningioma cells, which may explain the abnormal cell proliferation in malignant meningioma. Targeting infiltrating macrophages and M2-MDEs in tumor tissues may provide ideas for clinical therapeutic strategies to curb the proliferation of malignant meningiomas. Furthermore, increasing evidence suggests that M2 TAM-derived exosomes promote malignant tumor invasion and metastasis. Lan et al. demonstrated that M2-MDEs promoted colon cancer cell invasiveness and metastasis by delivering miR-21-5p and miR-155-5p [34]. High-grade meningiomas often exhibit creeping growth due to their enhanced invasive capacity, which leads to higher surgical risk and poorer prognosis [35,36]. Interestingly, our study showed that coculture with M2-MDEs conferred enhanced migratory and invasive abilities to meningioma cells. Furthermore, M2-MDEs facilitated the upregulation of vimentin, N-cadherin, slug, and snail in meningioma, suggesting that M2-MDEs contribute to the mesenchymal transition of meningioma cells. Multiple signaling pathways have been shown to be involved in M2-MDE-mediated biological effects. Here, we performed RNA-seq to analyze the differential transcrip-tomes of tumor cells cocultured with M2-MDEs and M0-MDEs individually. We identified the TGF-β signaling pathway as a potential mechanism responsible for the regulation of tumor development and invasion by M2-MDEs. Previous studies have also shown that the TGF-β pathway is involved in the regulation of tumor EMT and mobility. Similar to previous studies, our study shows that M2-MDEs can effectively induce the phosphorylation of Smad2/3 and promote their nuclear accumulation relative to M0-MDEs. Correspondingly, blocking the TGF-β pathway with selective smallmolecule inhibitors significantly reversed the increase in

18
Journal of Immunology Research meningioma cell viability and invasive capacity induced by M2-MDEs. Therefore, targeting the M2-MDE-mediated activation of the TGF-β pathway in tumor cells may be a potential therapeutic strategy for meningiomas. Subsequently, we further explored the effects of M2-MDEs on tumorigenesis and tumor development by constructing 3D tumorspheres and xenograft tumor models. Compared with that of 2D culture, the growth environment of 3D tumorspheres allows meningioma cells to exhibit characteristics closer to those in the natural conditions in vivo [37]. Multiple studies suggest that M2-macrophage infiltration contributes to tumor development. However, depending on the cell type and environmental characteristics, the TGF-β signaling pathway plays a binary role in tumorigenesis. In our study, coculture with M2-MDEs effectively promoted tumor growth and cell viability in 3D tumorspheres and xenografts. Consistent with 2D cultures, these biological effects were reversed by treatment with LY2109761.
It is worth noting that our work also has certain limitations. First, our study did not focus on the source of infiltrating macrophages in the meningioma microenvironment, which may contribute to understanding the mechanisms of meningioma tumorigenesis. Second, the transformation of the functional state of infiltrating macrophages in meningioma tissue and its molecular mechanisms remains to be further explored. Based on the results of this study, characterizing the contents of infiltrating macrophage-derived exosomes to explore the molecular mechanisms of their protumor biological effects in meningiomas will be a further research strategy.
In short, our study confirmed that M2-MDEs promote the proliferation, migration, and invasion of meningioma cells by activating the TGF-β signaling pathway. Therefore, targeting TAM-derived exosomes is expected to be a promising treatment modality for meningiomas. Based on the underlying intercellular communication network in the TME, this article provides a novel idea for drug treatment strategies for meningioma.

Data Availability
All data supporting this study is available from the corresponding author on reasonable request.

Ethical Approval
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the West China Hospital of Sichuan University (protocol code 2020990).

Consent
Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.  Table S1: list of primary antibodies.