Tumor-derived exosomal miR-205 promotes ovarian cancer cell progression through M2 macrophage polarization via PI3K/Akt/mTOR pathway

doi:10.21203/rs.3.rs-5313475/v1

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Research Article

Tumor-derived exosomal miR-205 promotes ovarian cancer cell progression through M2 macrophage polarization via PI3K/Akt/mTOR pathway

https://doi.org/10.21203/rs.3.rs-5313475/v1

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Background

Tumour-associated macrophages (TAMs) are the most abundant immune cells in the tumour environment and are considered to be similar to M2 macrophages, which facilitate cancer progression. Exosomes, as important mediators of the cross-talk between tumor cells and tumour-associated macrophages, can facilitate the development and metastasis of ovarian cancer by mediates M2 macrophage polarization. However, the exact mechanisms underlying the communication between ovarian cancer (OC) cells and tumour-associated macrophages during ovarian cancer (OC) progression remain unclear.

Results

Herein, we demonstrated that high expression of miR-205 was associated with M2 macrophage infiltration which affected the prognosis of OC patients. Importantly, tumor-derived miR-205 could be transported from OC cells to macrophages via exosomes and promote cancer cell invasion and metastasis via inducing M2-like macrophages polarisation. Animal experiments further confirmed that exosomal-miR-205-induced M2 macrophages accelerate OC progression in vivo. Mechanistically, miR-205 downregulates PTEN, activating the PI3K/AKT/mTOR signaling pathway, which is critical for M2 polarization.

Conclusions

These results reveal that exosomal miR-205 plays a pivotal role in macrophage polarization within the OC microenvironment, highlighting its potential as a therapeutic target for OC treatment. This study not only enhances our understanding of the interactions between tumor and immune cells but also opens new avenues for targeted therapies against exosomal miR-205 in ovarian cancer.

Exosomes

miR-205

Ovarian cancer

Macrophage polarization

PI3K/AKT/mTOR pathway

Ovarian cancer (OC) is the most lethal malignant tumour of the gynaecological reproductive system [1, 2], characterized by complex interactions within its tumor microenvironment (TME) [3–6]. A key player in the TME is tumor-associated macrophages (TAMs), which can polarize into two distinct phenotypes: M1 and M2 [7, 8]. M1 macrophages are known for their pro-inflammatory properties and are linked to anti-tumor responses, whereas M2 macrophages tend to have immune-suppressive functions and are thought to aid in tumor progression and metastasis [9, 10]. The polarization of TAMs is influenced by various factors, including cytokines and microRNAs (miRNAs), which play a significant role in regulating gene expression and cellular processes within the TME [11–13].

Recent research has increasingly focused on how cell-cell communication can occur through exosomes, which are small extracellular vesicles released by different cell types [14–16]. Exosomes are extracellular vesicles released from various cell types and contain numerous molecules from the parental cells, including miRNAs, proteins and nucleic acids [17–19]. The transfer of miRNAs by exosomes is especially interesting, since exosomal miRNAs released from cancer cells could stimulate macrophages to switch their phenotype and role in promoting cancer progression and metastasis [20–22]. Recent studies have highlighted the pivotal role of miR-205 in cancer biology, particularly in the context of OC. miR-205 is implicated in the regulation of multiple cellular functions, including proliferation, apoptosis, and migration of cancer cells[23, 24]. Here, we demonstrated that miR-205 could modulate TAM polarization, promoting the M2 phenotype and thereby enhancing the tumor-promoting effects of macrophages. This interaction underscores the potential of miR-205 as a target for therapeutic intervention aimed at reprogramming the immune landscape in ovarian cancer.Recent research has increasingly focused on how cell-cell communication can occur through exosomes, which are small extracellular vesicles released by different cell types.

Despite the accumulating evidence regarding the role of TAMs and miRNAs in OC, there are still significant gaps in our understanding of the specific molecular mechanisms that drive TAM polarization and the exact contributions of miR-205. While previous studies have established links between TAM polarization and tumor progression, the direct effects of miR-205 on the signaling pathways involved in these processes remain inadequately explored. Investigating the pathways activated by miR-205 in TAMs could provide crucial insights into the molecular factors that drive ovarian tumor progression and aid in the development of innovative therapeutic strategies.

To address these gaps in knowledge, this study adopts a comprehensive approach that includes the use of human tissue samples, in vitro cell culture, and advanced molecular techniques such as RNA interference and exosome characterization. The goal is to clarify how tumor-derived exosomal miR-205 affects TAM polarization and the subsequent behaviors of tumor cells, including proliferation and invasion, within the OC microenvironment. Gaining a deeper understanding of the interaction between miR-205 and TAMs could reveal new therapeutic targets and strategies aimed at reprogramming the immune response in ovarian cancer patients.

In conclusion, exploring the relationship between miR-205 and TAM polarization offers a promising path for enhancing our understanding of OC biology and improving therapeutic outcomes. By examining the specific roles of tumor-derived exosomal miR-205 in shaping the immune landscape, this research seeks to contribute to the development of targeted therapies that could increase the effectiveness of current treatment options and ultimately improve patient prognosis in ovarian cancer.

2.1. Human tissue collection

A total of 74 cases of formalin-fixed paraffin-embedded samples, including 34 OC tissue specimens, 20 normal ovarian tissue specimens and 20 metastatic tissues, were collected from OC patients who underwent curative-intent surgery without prior radiotherapy and chemotherapy between 2014 and 2018 at the Department of Pathology, Xiangya Hospital of Central South University (CSU). All specimen diagnoses were confirmed by a qualified pathologist after surgery. The study was approved by the Institutional Ethics Committee of Xiangya Hospital of CSU and was conducted following the ethical guidelines of the Declaration of Helsinki. Each participant signed an informed consent form.

2.2. Cell culture

The epithelial OC cell lines HO-8910 and the monocytic cell line THP-1 were obtained from the Chinese Academy of Sciences Cell Bank and had been purchased from China Center for Type Culture Collection (CCTCC) and Cell Resource Center, IBMS, CAMS/PUMC (CRC/PUMC). HO-8910 cells stably overexpressing miR-205 and negative control cells were established by the Xiaoying Wu research group of the pathology laboratory of CSU, and the transfection efficiency was verified in a previous study [23]. All cell lines were cultured in DMEM or RPMI 1640 medium (Thermo Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (FBS) (Gibco, Gaithersburg, MD, USA). THP-1 cells (1×10⁶) were incubated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Solarbio, Beijing, China) for 24–48 h in vitro to induce their differentiation into macrophages. For the exosome treatment experiments, 1 µg/ml exosomes was added to the culture medium of recipient cells (2 × 10⁵).

2.3. Immunohistochemistry (IHC) and in situ hybridization (ISH)

A total of 74 paraffin-embedded OC tissues were assessed by IHC and ISH staining. The macrophage marker CD68 and the M2-type macrophage marker CD163 expression were measured with anti-CD68 (1:200, Abcam, Cambridge, UK) and anti-CD163 (1:200, Abcam) antibodies. ISH was performed on OC tissue samples using an LNA microRNA ISH miR-205 optimization kit (Exiqon; Woburn, MA, USA). The stained slides were scanned and photographed using digital pathological scanning equipment. The final score obtained from the Image-Pro plus (IPP, version 5.0, Media Cybernetics, Silver Spring, MD) analysis was used to identify the relative level of CD68, CD163 and miR-205 expression. The positivity rate was calculated as the integral optical density (IOD)/area sum. The experimental steps of IHC and ISH were the same as in our previous study [24].

2.4. Exosome isolation and characterization

The cell lines were cultured in normal medium until 60–70% confluence, thereafter, the medium was replaced with fresh medium supplemented with 10% exosome-depleted FBS. Then, the cell culture medium was harvested after 48 h and centrifuged (Beckman Coulter, Brea, CA, USA) at 2,000×g for 30 min at 4°C to remove residual cells, debris and remaining macropolymers. The supernatant was passed through a 0.22-µm filter (Millipore, Danvers, MA, USA). After centrifugation at 3,000×g for 30 min at 4°C in a dialysis tube (Millipore), the supernatant was incubated with ExoQuick-TC™ exosome precipitation solution (System Biosciences, Palo Alto, CA, USA) for 6 h to overnight at 4°C. Subsequently, the mixture was centrifuged at 1,500×g for 30 min at 4°C to harvest the yellow exosome pellets. The exosome pellets were resuspended in 100 µl phosphate-buffered saline (PBS) for further assays. Western blotting analysis was used to characterize the extracellular vesicle-associated protein markers Hsp70 (Abcam) and CD63 (Abcam) and the exosome-specific marker TSG101 (Abcam). For transmission electron microscopy (TEM), the exosomes were fixed with 1% glutaraldehyde, dropped in 300-mesh carbon/formvar-coated grids, stained with 2% uranyl acetate, dried and imaged by transmission electron microscopy (TEM) (FEI, Hillsboro, OR, USA), as previously reported [26]. The amount of exosomes was measured using the bicinchoninic acid (BCA) protein assay kit (Novagen, Merck Group, Madison, USA).

2.5. Exosome labelling and tracking

To monitor exosome trafficking, purified exosomes isolated from the culture medium were collected and labelled with PKH67 red fluorescent membrane linker dye (Sigma-Aldrich, Merck KGaA) according to the manufacturer's instructions. Next, the PKH26-labelled exosomes were washed and centrifuged at 100,000×g for 20 min at 4°C to collect the exosomes, which were resuspended and added to unstained macrophages for exosome uptake studies. After treatment with PKH26-labelled exosomes for 4 h at 37°C, the cells were detected by fluorescence microscopy.

2.6. RNA interference, vector transfection and qRT-PCR

The miR-205 mimic (10 nM), inhibitor (20 nM), negative control (10 nM) and PTEN expression plasmid (2 µg, GeneCopoeia) were transfected into cells using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. The cells were collected for further assays at 24 or 48 h after transfection. The transfection efficiencies were verified by fluorescence microscopy and RT-PCR analysis. Total RNA from cells and exosomes was extracted using TRIzol reagent (Invitrogen), and complementary DNA was synthesized with a reverse transcription system (GeneCopoeia, Guangzhou, China) according to the manufacturer's instructions. Reverse transcription and qRT-PCR were performed as previously described [26]. The relative expression levels were evaluated using the ΔΔCt method. The 2^−ΔΔCt method was applied to analyse the results. The primers for miR-205 mimic, miR-205 inhibitor, miR-205 nc, CD163, CD68, IL10, CCL8, IL-1β, TNF-α and PTEN (GeneCopoeia) are available in the Supplementary Table 1.

2.7. Plasmids, transient transfections and luciferase assays

An miR-205 promoter (420 bp) was amplified from the mouse genomic DNA template and inserted into a pGL3 vector (Promega Corporation, Madison, WI, USA). Reporter vectors carrying the miR-205 target site were constructed by synthesizing a 3’-untranslated region (UTR) fragment containing the predicted target sites (miRWalk) for PTEN cDNA, and subsequently inserting the PTEN cDNA fragment into the multiple cloning site of a pMIR-REPORT™ luciferase miRNA expression reporter vector (Ambion Life Technologies, Carlsbad, CA, USA). For the luciferase reporter assay, HO-8910 cells were seeded in 24-well plates and transfected with the indicated plasmids. Cells were harvested 36 h after transfection. Luciferase activity was measured using the Dual-Luciferase® Reporter Assay System (Promega Corporation).

2.8. Western blot analysis

Proteins extracted from cells or exosomes were lysed in RIPA lysis buffer supplemented with protease inhibitor and quantified using a BCA protein assay kit (Novagen, Merck Group, Madison, USA). Approximately 30 µg of protein lysates (from each sample) were separated on 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% blocking buffer overnight at 4°C with primary antibodies, followed by incubation with a secondary antibody at room temperature. The primary antibodies included anti-CD63, anti-TSG101, anti-Hsp70, anti-E-cadherin, anti-vimentin, anti-PTEN, anti-mTOR, anti-p-mTOR, anti-AKT, anti-p-AKT, anti-4EBP1, anti-p-4EBP1 (Cell Signaling Technology, USA) and anti-α-tubulin (Proteintech, Chicago, USA). The chemiluminescence signal was detected using an enhanced chemiluminescence mixture (Sigma-Aldrich), and images were captured using a gel imaging system (Bio-Rad Laboratories, CA, USA).

2.9. Co-culture, cell migration and invasion assays

Before starting the assays, THP-1 cells (1×10⁴) were seeded on the bottom chamber of 8-mm Transwell inserts (Corning, Sigma-Aldrich) with PMA (100 ng/ml) for 48 h. Then, the THP-1 cells were differentiated into M0 macrophages, and the supernatant was replaced with normal medium containing 10% FBS for 24 h to eliminate the effect of PMA. Tumour cells (5×10⁵) were seeded on the upper side of the co-culture system before the co-culture assay. After treatment with miR-205-Exos or miR-205 mimic/NC/inhibitor (RiboBio, Guangzhou, China), M0 macrophages were co-cultured with HO-8910 cells. Cell migration and invasion assays were performed as previously described [23].

2.10. Animal experiments

All animal experiments were performed in accordance with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and according to the protocols approved by the Animal Care and Use Committee at CSU. Female BALB/c nude mice (4–5 weeks old, 18 ~ 20 g) were housed in a specific pathogen-free environment in the Animal Laboratory Unit of CSU and were randomly divided into two groups (n = 6/group). HO-8910 cells (1.5×10⁶ cells) mixed with conditioned macrophages pretreated with NC-Exos or miR-205-Exos were injected intraperitoneally (i.p.) into different groups. The procedure of living animal bioluminescence imaging was described previously [24].

2.11. Statistical analysis

All experiments were performed in triplicate. Data are presented as the mean ± standard error of the mean (SEM). Differences between treated and control groups were analysed using Student’s t test or one-way ANOVA. Survival curves were estimated using the Kaplan-Meier method, and the log-rank test was used to calculate differences between the curves [25]. A P-value less than 0.05 was considered statistically significant. Statistical analyses were performed by GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA, USA).

3.1. The expression levels of miR-205 correlate with TAMs infiltration in OC patients

Macrophages are among the most abundant immune-related cells recruited to the tumour site and play significant roles in TME. M2 macrophages, which are believed to be the main phenotype of TAMs, promote the progression and metastasis of tumours. In this study, an anti-CD163 antibody was first used to identify M2 macrophages by IHC. This result revealed a significantly increased expression of CD163 in primary tumors that was even higher in metastasic tumors (Fig. 1A and B). In addition, the upregulation of CD163 is associated with poor prognosis in OC patients (Fig. 1C and D). Moreover, we found that CD163 expression levels were higher in OC specimens with high miR-205 expression than in those with low miR-205 expression (Fig. 1E). Moreover, Spearman correlation analysis suggested that there was a corresponding upregulation of miR-205 when CD163 was increased (Fig. 1F). These results suggest that miR-205 expression is associated with TAM infiltration. Interestingly, we found that high miR-205 expression OC tissues appeared to involve more miR-205-positive immune cells infiltration (Fig. 1G). Based on their morphology, we reasoned that the majority of miR-205-positive immune cells observed in OC might be TAMs. To further test this hypothesis, ISH and IHC were performed to detect miR-205, the macrophage marker CD68 and the M2-type macrophage marker CD163 in the same two independent sets of OC specimens. The following staining patterns were observed: miR-205 staining on both tumour cells and CD68 + and CD163 + cells in most of the OC tissue samples (31/54, Fig. 1H). Therefore, according to these results, we hypothesized that miR-205 in OC cells may play an important role in macrophage polarization.

3.2. Exosomes from OC cells are able to polarize macrophages towards M2 type by transporting miR-205

The transfer of miRNAs by exosomes is especially interesting, since exosomal miRNAs released from cancer cells could stimulate macrophages to switch their phenotype and role in promoting cancer progression and metastasis. Here, we investigated whether exosomal miR-205 derived from OC cells can be transported to macrophages then reprogram the phenotype of TAMs. To reduce the effect of other factors of exosomes, a model system was used to alter exosomal miR-205 expression. The OC cell line HO-8910 with stable overexpression of miR-205 and the negative control cell line were established. Next, exosomes with relatively high miR-205 content (miR-205-Exos) and relatively low miR-205 content (NC-Exos) were isolated and confirmed expression of miR-205 by RT-PCR (Fig. 2A). As shown in Fig. 2B, the typical round particles ranged in diameter from 30 to 100 nm and were characterized by teacup-like shapes. Western blot analysis confirmed the presence of the exosomal proteins CD63, TSG101 and Hsp70 (Fig. 2C). After labelling with the fluorescent dye PKH67, miR-205-Exos from OC cells were incubated with macrophages. As shown in Fig. 2D, the PKH67-labelled exosomes could be internalized by macrophages. After treatment with exosomes, miR-205 expression was significantly increased in macrophages treated with miR-205-Exos compared with NC-Exos or control (PBS) (Fig. 2E).

To further investigate whether exosomal miR-205 alters differentiation and function of macrophages, we assessed the immune-phenotype and cytokine profifile of macrophages that were differentiated from macrophage-like cells presented with exosomes. Human THP-1 monocytes were differentiated into macrophage-like cells by an incubation in the presence of phorbol 12-myristate 13-acetate (PMA), which is characterized by the adherent morphology and the expression of recognized macrophage marker CD68 (Fig. 2F-G). we assessed the possible role of exosomal miR-205 on macrophages polarization. The expression levels of M2 cytokines (CD163, IL10 and CCL8) in macrophages were significantly increased when the macrophage-like cells were incubated with miR-205-Exos compared with NC-Exos or control Fig. 2H, I and J), while the expression of M1 cytokines (IL-1β and TNF-α) decreased significantly (Fig. 2K and L). Therefore, the above results demonstrate that exosomal miR-205 can activate macrophage differentiation into the M2 phenotype.

3.3. Tumor-derived exosomal miR-205 promotes OC cell migration and invasion via altering the polarisation of M2-like macrophage in vitro

TAMs are widely known for their crucial roles in various aspects of cancer progression, including tumour cell invasion, motility, angiogenesis and premetastatic site formation. Next, a co-culture system was established to investigate the effects of M2 macrophages induced by cancer-derived exosomal miR-205 in the migration, invasion and EMT of OC cells (Fig. 3A). As shown in Fig. 3B and C, miR-205-Exos-treated macrophages significantly increased the migration and invasion abilities of OC cells compared to NC-Exos-treated macrophages. After transfected miR-205 mimic or inhibitor, miR-205 was up- or down-regulated in OC cells (Fig. 3B), respectively. could enhance or attenuate the cell migration and invasion (Fig. 3D and E). Next, we sought to evaluate whether exosomal miR-205-induced macrophages affect EMT in OC cells. Compared to co-culture with NC-Exos-treated or NC-transfected cells, co-culture with miR-205-Exos-treated or miR-205 mimic-transfected macrophages significantly decreased E-cadherin expression levels and increased vimentin and MMP7 expression levels, and these effects were reversed by the miR-205 inhibitor (Fig. 3F and G). Taken together, these results suggest that exosomal miR-205-induced M2 macrophages could enhance the migration, invasion and EMT of OC cells.

3.4. Exosomal miR-205-induced M2 macrophages promote the development of OC in vivo

Next, we further evaluated the effects of exosomal miR-205-induced M2 phenotype polarization on cancer progression. As shown in Fig. 4A, macrophages were pretreated with NC-Exos or miR-205-Exos for 48 h. Then, luciferase-labelled HO-8910 cells mixed with the pretreated macrophages were injected into the abdominal cavities of nude mice. Subsequently, the mice were intraperitoneally injected with the same number of macrophages pretreated with NC-Exos or miR-205-Exos every three days. The IVIS system was used to detect the luciferase activity levels, which reflected the tumour volume in the peritoneal cavity. In comparison with those in the NC-Exos group, the luciferase activity levels of the miR-205-Exos group increased significantly, especially at three and four weeks after the first injection (Fig. 4B and C). In addition, we found that the number of peritoneal implants in the miR-205-Exos group was greater than that in the NC-Exos group (Fig. 4D). Compared with mice in the NC-Exos group, mice in the miR-205-Exos group showed shorter survival times (Fig. 4E). IHC and HE staining showed that the expression levels of CD163 (an M2-type macrophage marker) were significantly increased in the miR-205-Exos group compared with the NC-Exos group (Fig. 4F and G), suggesting that there were more M2 macrophages infiltrating around the cancer tissues in the miR-205-Exos group than in the NC-Exos group. These results indicate that exosomal miR-205 induces M2-type polarization of macrophages in the TME and promotes cancer progression in vivo.

3.5. Exosomal miR-205 polarized the M2 macrophage via activating PI3K/AKT/mTOR axis

To deeply explore the mechanism by which exosomal miR-205 modulates macrophage polarization, TargetScan and miRBase were used to determine possible miR-205 target genes. The candidate target gene PTEN was chosen for its role in the regulation of macrophage polarization, and luciferase reporter assays were performed to confirm the direct targeting inhibitory effect of miR-205 on PTEN (Fig. 5A). Consistent with the luciferase reporter analysis, miR-205 mimics or miR-205-Exos significantly decreased PTEN mRNA and protein levels in macrophages, while miR-205 inhibitor revealed the opposite effects (Fig. 5B and C). As shown in Fig. 5D and E, the expression level of PTEN suppressed by miR-205 mimics or miR-205-Exos in macrophage was rescued by treatment with PTEN plasmid. Furthemore, the role of PTEN in miR-205-induced macrophage polarization and cytokine production was investigated using PTEN plasmid, miR-205 mimics and miR-205-Exos. Notably, overexpression of miR-205 markedly upregulated the expression levels of M2-type markers (CD163 and IL10).

PTEN is a negative regulator of the PI3K pathway and has been shown to reprogram tumor-infifiltrating macrophage. We next examined whether exosomal miR-205 regulates macrophage polarization through the PTEN/PI3K axis. Notably, treatment with the miR-205 mimics, miR-205-Exos resulted in significantly decreased expression of PTEN and increased expression levels of p-AKT, p-mTOR and p-4EBP-1 in macrophages, and these effects were reversed by PTEN expression (Fig. 6A and B). We then evaluated the effect of PI3K/AKT/mTOR signalling pathway-specific blockade on M2 macrophage polarization induced by exosomal miR-205. Western blot analysis confirmed that the PI3K inhibitor GDC-0941, AKT inhibitor MK-2206 and mTOR inhibitor rapamycin effectively blocked the activation of the PI3K/AKT/mTOR pathway induced by miR-205 upregulation (Fig. 6C). As shown in Fig. 6D-K, the effects of miR-205 overexpression on M2 macrophage polarization were significantly abolished by GDC-0941, MK-2206 and rapamycin. These data indicate that exosomal miR-205 activates the PI3K/AKT/mTOR axis by downregulating PTEN expression in macrophages, thus inducing the polarization of macrophages toward M2 phenotypes.

With the increased popularity of clinical immunotherapy in recent years, the regulation of the immune microenvironment in OC has gained increasing attention [27, 28]. The tumor microenvironment, particularly the presence and polarization of TAMs, plays a critical role in the progression and metastasis of ovarian cancer by influencing tumor growth and modulating immune responses [29–31]. Many studies report that the communication between cancer cells and macrophages could be mediated by exosomes, and exosomal miRNAs released from cancer cells could definitely reprogram macrophages to establish a kind of tumour immunosuppressive microenvironment that may promote the invasion and metastasis of tumour cells [32, 33].

Our study focuses on elucidating the molecular mechanisms through which miR-205 mediates the polarization of TAMs towards the M2 phenotype within the ovarian cancer microenvironment. Prior research has suggested that miRNAs significantly regulate the interactions between tumor cells and immune cells, yet the specific role of miR-205 in ovarian cancer remains poorly defined. Here, we present key findings demonstrating that exosomal miR-205 derived from OC cells can induce M2 polarization of macrophages, thereby promoting tumor progression in vitro and in vivo. The activation of the PI3K/AKT/mTOR signaling pathway in this context further underscores the potential of targeting miR-205 as a therapeutic strategy in ovarian cancer management.

Exosomes are important components of the TME and act as an important messenger that mediates the cross-talk between tumour cells and immune cells [33, 34]. Numerous studies suggest that exosomal miRNAs can significantly influence the phenotypic transformation of macrophages through various targets[35, 36]. Moreover, the release of exosomal miRNAs from cancer cells promotes the progression of tumor cells [37]. The findings of this study highlight the critical role of the tumor-associated macrophages (TAMs) in ovarian cancer (OC) progression, particularly focusing on the modulation of miR-205 in the tumor microenvironment. Our results demonstrate that exosomes derived from OC cells significantly influence macrophage polarization towards an M2 phenotype, which is associated with enhanced tumor growth and metastasis. This polarization is mediated through the activation of the PTEN-PI3K/AKT/mTOR pathway, underscoring the importance of miR-205 as a potential therapeutic target in OC. Previous studies have established a link between TAMs and tumor advancement, particularly in how M2 macrophages contribute to immune evasion and promote a favorable microenvironment for tumor growth [38–41]. Moreover, the modulation of miR-205 in this context suggests that targeting this microRNA may provide a novel approach to reprogram the tumor microenvironment and enhance the efficacy of existing therapies.

Additionally, the results suggest that the activation of the PI3K/AKT/mTOR signaling pathway by miR-205 is critical for the M2 polarization of macrophages, which plays a significant role in OC progression. This aligns with findings from other studies indicating that the PI3K/AKT pathway is crucial for macrophage differentiation and function, particularly in cancer settings [42–46]. The dysregulation of this signaling pathway in TAMs may not only contribute to the immunosuppressive tumor microenvironment but also facilitate the aggressive behavior of OC cells. Furthermore, our study indicates that high levels of miR-205 correlate with increased levels of M2 markers in macrophages, providing insights into potential biomarkers for disease progression and therapeutic targets for intervention.

This study is not without its limitations. The relatively small sample size may hinder the generalizability of the findings, as it does not fully represent the heterogeneity observed in ovarian cancer patient populations. Furthermore, the lack of comprehensive clinical validation limits the ability to draw robust conclusions regarding the translational potential of miR-205 as a therapeutic target. Additionally, while the focus on macrophage polarization provides important insights, the functional differences among various macrophage subtypes were not extensively explored, which may obscure a complete understanding of their roles in the tumor microenvironment. Potential batch effects during data analysis could also introduce variability, affecting the reliability of the results. Addressing these limitations in future studies is crucial to enhancing the validity and applicability of the findings.

In conclusion, this research elucidates the molecular mechanisms by which miR-205 modulates macrophage polarization through extracellular vesicles within the ovarian cancer microenvironment. The findings underscore the critical role of miR-205 in tumor progression and highlight its potential as a therapeutic target. By providing new insights into the dynamic interplay between tumor cells and immune cells, this study lays the groundwork for future investigations aimed at developing innovative strategies for ovarian cancer treatment. Continued exploration of miR-205 and its regulatory pathways may ultimately contribute to improved therapeutic outcomes and a deeper understanding of cancer immunobiology.

CD163: macrophage mannose receptors 163; EMT: epithelial-mesenchymal transition; FBS: foetal bovine serum; ISH: in situ hybridization; IHC: immunohistochemistry; miRNAs: microRNAs; IL-6: interleukin-6; IL-1β: interleukin-1β; IL10: interleukin-10; IL8: interleukin-8; miRNAs: microRNAs; OC: ovarian cancer; PBS: phosphate-buffered saline; PMA: phorbol 12-myristate 13-acetate; TEM: transmission electron microscopy; TAMs: Tumour-associated macrophages; TME: tumour microenvironment; IFN-γ: interferon-γ; TNF-α: tumor necrosis factor-α.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Xiangya Hospital ( Central South University, Changsha, China) and conducted in adherence with the Declaration of Helsinki. The patients were informed about the sample collection and had signed informed consent forms.

Funding

This work was supported by grants from the National Natural Science Foundation of China (grant no. 81172469), the Open Sharing Fund for the Large-scale Instruments and Equipments of Central South University (grant no. CSUZC202039), the Scientific Research Launch Project for new employees of the Second Xiangya Hospital of Central South University and Hunan Provincial Natural Science Foundation of China (grant no. 2019JJ40394 and 2022JJ40687).

Author Contribution

LQH conceived, designed the study, prepared all the figures and wrote the manuscript. QC contributed to the study design and performed the experiments. XYW contributed to the study design and reviewed and edited the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

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Scheme 1: Schematic diagram of the role of tumor-derived exosomal miR-205 in M2 macrophage polarization during cancer progression.

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Tumor-derived exosomal miR-205 promotes ovarian cancer cell progression through M2 macrophage polarization via PI3K/Akt/mTOR pathway

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