Extracellular Vesicles Derived from Ovarian Cancer Cells Promote Tumor Progression through M2 Macrophage Polarization and Enhanced Angiogenesis

Extracellular Vesicles Derived from Ovarian Cancer Cells Promote Tumor Progression through M2 Macrophage Polarization and Enhanced Angiogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Extracellular Vesicles Derived from Ovarian Cancer Cells Promote Tumor Progression through M2 Macrophage Polarization and Enhanced Angiogenesis Yi Zhang, Tiantian Dai, Dandan Chu, Wei Zhang, Xujie Wang, Jinhua Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6834403/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2026 Read the published version in BMC Cancer → Version 1 posted 15 You are reading this latest preprint version Abstract Background : Ovarian cancer, among the most lethal gynecologic malignancies globally, features a tumor microenvironment crucial to disease progression. Extracellular vesicles (EVs) function as key intercellular communication mediators, though their role in ovarian cancer advancement via macrophage regulation remains inadequately characterized. Methods : EVs isolated from ovarian cancer cell lines (SKOV3, HO8910, ID8) underwent characterization through transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Macrophage polarization was evaluated following co-culture with cancer cells or their derived EVs. HUVEC angiogenic activity was assessed through tube formation, proliferation, and VEGFR expression analyses. In vivo studies examined tumor growth, macrophage infiltration, and angiogenesis in nude mice bearing SKOV3 tumors treated with cancer-derived EVs. Results : Ovarian cancer patients demonstrated significantly elevated M2 macrophage proportions in peripheral blood and tumor tissues compared to controls (p<0.05), with increased CD31 expression correlating with poor prognosis. In vitro, cancer cells and their derived EVs induced significant M2 polarization (p<0.0001) and enhanced HUVEC tube formation through VEGFR upregulation. The mouse model confirmed that cancer-derived EVs significantly promoted tumor growth (p<0.0001), M2 macrophage infiltration, and CD31 expression. Conclusions : This study demonstrates that ovarian cancer-derived EVs enhance tumor progression by inducing M2 macrophage polarization and stimulating angiogenesis, elucidating a novel tumor-microenvironment interaction mechanism and suggesting EV-targeted therapeutic approaches for ovarian cancer. Ovarian cancer Extracellular vesicles Tumor-associated macrophages Angiogenesis Tumor microenvironment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ovarian cancer is one of the most lethal gynecological malignancies [1] . Despite advances in surgical techniques and chemotherapeutic regimens, the 5-year survival rate for patients with advanced ovarian cancer remains below 30% [2] . The poor prognosis is largely attributed to late diagnosis, high recurrence rates, and the development of chemoresistance [3] . Understanding the molecular mechanisms underlying ovarian cancer progression is critical for developing more effective therapeutic strategies. The tumor microenvironment (TME) plays a pivotal role in cancer development, progression, and therapeutic response [4] . As a complex ecosystem, the TME consists of cancer cells, stromal cells, immune cells, blood vessels, and extracellular matrix components that interact in a dynamic manner [5] . Among the immune cells present in the TME, tumor-associated macrophages (TAMs) have emerged as key regulators of tumor progression [6] . Macrophages exhibit remarkable plasticity and can be polarized into distinct functional phenotypes: classically activated (M1) macrophages with anti-tumor properties and alternatively activated (M2) macrophages that generally promote tumor growth [7,8] . In ovarian cancer, increased infiltration of M2 TAMs has been associated with advanced disease stage, enhanced tumor angiogenesis, and poor patient outcomes [9-11] . However, the mechanisms by which ovarian cancer cells influence macrophage polarization remain incompletely understood. Recent evidence suggests that cancer cells can modulate the phenotype and function of surrounding cells through the secretion of extracellular vesicles (EVs) [12] . EVs are membrane-enclosed structures released by cells into the extracellular space, ranging from 30 to 1000 nm in diameter [13] . These vesicles contain bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, and microRNAs), and can transfer their cargo to recipient cells, thereby altering their phenotype and function [14,15] . Cancer-derived EVs have been implicated in various aspects of tumor biology, including immune modulation, angiogenesis, metastasis, and drug resistance [16,17] . Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is essential for tumor growth, progression, and metastasis [18-19] . CD31 (PECAM-1), a transmembrane glycoprotein primarily expressed on endothelial cells, is widely used as a marker for angiogenesis evaluation [20-21] . Previous studies have shown that high CD31 expression correlates with poor prognosis in various cancers, including ovarian cancer [22] . The vascular endothelial growth factor receptor (VEGFR) signaling pathway plays a central role in tumor angiogenesis and has been targeted for cancer therapy [23] . Emerging evidence suggests that EVs can modulate angiogenesis by transferring angiogenic factors or regulatory molecules to endothelial cells [24-26] . While studies have demonstrated that cancer-derived EVs can influence various aspects of tumor biology, the specific role of ovarian cancer-derived EVs in modulating macrophage polarization and subsequently affecting tumor angiogenesis and progression remains to be fully elucidated. Understanding these mechanisms could provide insights into novel therapeutic targets for ovarian cancer treatment. In this study, we investigated the effects of ovarian cancer-derived EVs on macrophage polarization and subsequent impacts on angiogenesis both in vitro and in vivo. We demonstrated that ovarian cancer patients exhibit increased proportions of M2 macrophages in peripheral blood and tumor tissues, along with elevated CD31 expression that correlates with poor prognosis. We found that ovarian cancer cells and their derived EVs promote M2 polarization of macrophages, which in turn enhances endothelial cell tube formation through upregulation of VEGFR expression. Furthermore, in a mouse model, we showed that ovarian cancer-derived EVs promote tumor growth, increase M2 macrophage infiltration, and enhance tumor angiogenesis. These findings reveal a novel mechanism by which ovarian cancer cells interact with the TME and suggest that targeting cancer-derived EVs could be a promising therapeutic strategy for ovarian cancer. Materials and Methods Cell Lines and Culture Ovarian cancer cell lines (SKOV3 and HO8910), murine ovarian cancer cell line (ID8), human monocytic cell line (THP-1), and human umbilical vein endothelial cells (HUVECs) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. SKOV3, HO8910, and ID8 cells were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin-streptomycin. THP-1 cells were maintained in RPMI-1640 medium containing 10 mM HEPES, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, 10% FBS, and 100 U/mL penicillin-streptomycin. HUVECs were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin. All cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. Patient Samples Peripheral blood and tissue samples were collected from ovarian cancer patients and non-ovarian cancer controls at the Department of Gynecology, Shanghai Changning Maternity and Infant Health Hospital. Fresh tissues were transported to the laboratory at 4°C and processed within 2 hours of collection. Fresh tissue samples were divided into two parts: one for flow cytometry analysis and one for immunohistochemistry. Isolation and Analysis of Peripheral Blood Mononuclear Cells (PBMCs) PBMCs were isolated using human peripheral blood lymphocyte separation medium (Beyotime) according to the manufacturer's instructions. Briefly, fresh blood was diluted 1:1 with PBS, carefully layered over the separation medium (ratio of separation medium to diluted blood = 1:2), and centrifuged at 800 g for 20 minutes at room temperature. The PBMC layer was collected, washed twice with PBS, and resuspended for flow cytometry analysis. For macrophage phenotype analysis, PBMCs were stained with FITC Mouse Anti-Human CD68 and APC Mouse Anti-Human CD206 (BD Pharmingen) according to manufacturer recommendations. Samples were incubated for 40 minutes at room temperature in the dark, washed, and analyzed using a flow cytometer with excitation at 488 nm (FITC) and 651 nm (APC). Data were analyzed using FlowJo software. Tumor Tissue Processing and Analysis For flow cytometry analysis, fresh tumor tissues were minced and digested with collagenase I (1 mg/mL, Beyotime) for 40 minutes at 37°C with shaking. The digestion was terminated by adding DMEM containing 10% FBS. The cell suspension was filtered through a 40 μm cell strainer, centrifuged at 500 g for 5 minutes at 4°C, and treated with red blood cell lysis buffer (Beyotime). After centrifugation, cells were stained with 647 Mouse Anti-Human CD68 and FITC Mouse Anti-Human CD206 (BD Pharmingen) for human samples, or Alexa Fluor 647 Rat Anti-Mouse F4/80 and Ms CD206 Alexa 488 (BD Pharmingen) for mouse samples. For immunohistochemistry, tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 μm thickness. Sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate buffer. Endogenous peroxidase activity was blocked, and sections were incubated with primary antibodies overnight at 4°C, followed by detection using the DAB method and hematoxylin counterstaining. Isolation and Culture of Bone Marrow-Derived Macrophages (BMDMs) BMDMs were isolated from 6-8 week-old female C57BL/6 mice. Femurs and tibias were collected under sterile conditions and bone marrow was flushed out with cold PBS. After red blood cell lysis, cells were resuspended in IMDM medium containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 ng/mL M-CSF, and cultured for 7 days, replacing medium every 3 days. BMDMs were identified by flow cytometry analysis of F4/80 and CD206 expression. Co-culture Experiments For co-culture experiments, Transwell inserts with 0.4 μm pore size were used. THP-1 cells (1×10⁵ cells/mL) were seeded in the lower chamber and treated with 150 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce differentiation into macrophages. The medium was then replaced, and SKOV3 or HO8910 cells (1×10⁵ cells/mL) were seeded in the upper chamber (400 μL). For mouse cell co-culture, BMDMs were seeded in the lower chamber and ID8 cells in the upper chamber. Control groups received an equal volume of PBS. After 48 hours of co-culture at 37°C with 5% CO₂, cells in the lower chamber were collected for flow cytometry analysis. Isolation and Characterization of Extracellular Vesicles (EVs) EVs were isolated from cell culture supernatants by differential ultracentrifugation. Cells were cultured to 60-70% confluence in T225 flasks, washed with PBS, and cultured in serum-free medium for 48 hours. Collected supernatants were centrifuged at 300 g for 20 minutes to remove cell debris. The resulting supernatant was ultracentrifuged at 10,000 g for 60 minutes, followed by ultracentrifugation at 125,000 g for 120 minutes using a Beckman Optima XPN-100 ultracentrifuge. The pellet was washed with PBS and ultracentrifuged again at 125,000 g for 120 minutes. The final EV pellet was resuspended in PBS and stored at -80°C. EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting for EV markers. For TEM, EV suspensions were placed on copper grids, stained with 2% uranyl acetate, and observed using a transmission electron microscope. For NTA, EVs were diluted in PBS and analyzed using a NanoSight NS300 instrument to determine particle size distribution and concentration. For western blotting, EV protein was extracted and analyzed for the expression of CD63 (ab134045, Abcam), TSG101 (ab133586, Abcam), and Alix (ab275377, Abcam). EV Uptake Assay EVs were labeled with PKH26 red fluorescent dye (Merck) according to the manufacturer's instructions. THP-1 cells were differentiated with PMA on glass coverslips for 24 hours, then incubated with PKH26-labeled EVs for 12 hours. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, stained with FITC-Actin Tracker (Beyotime) to visualize cell cytoskeleton, and mounted with DAPI-containing mounting medium. The uptake of labeled EVs by macrophages was observed using a confocal laser scanning microscope. EV Treatment of Macrophages THP-1 cells (1×10⁵ cells/mL) were seeded in 12-well plates and differentiated with 150 nM PMA for 24 hours. The medium was replaced, and cells were treated with SKOV3-EVs, HO8910-EVs, THP-1-EVs (all at a concentration of 10¹¹ particles/mL), or an equal volume of PBS. After 48 hours of incubation, cells were collected, stained for CD68 and CD206, and analyzed by flow cytometry. For BMDMs, similar procedures were followed using ID8-EVs, BMDM-EVs, or PBS as treatments, and cells were stained for F4/80 and CD206 before flow cytometry analysis. Tube Formation Assay Matrigel (Beyotime) was thawed overnight at 4°C, added to 96-well plates (40 μL/well), and incubated at 37°C for 30 minutes to solidify. HUVECs were co-cultured with macrophages pre-treated with SKOV3-EVs, HO8910-EVs, THP-1-EVs, or PBS in a Transwell system for 24 hours. HUVECs were then harvested, resuspended to 5×10⁴ cells/mL, and seeded on Matrigel (50 μL/well). After 4 hours of incubation, tube formation was observed under an inverted microscope, and images were captured from five random fields. Tube formation was quantified by measuring the number of branch points and total tube length using ImageJ software. Cell Proliferation Assay The proliferation of HUVECs co-cultured with EV-treated macrophages was assessed using the Cell Counting Kit-8 (CCK-8, MCE) according to the manufacturer's instructions. HUVECs were co-cultured with macrophages pre-treated with different EVs using a Transwell system. After co-culture, CCK-8 solution (10% v/v) was added to the lower chamber and incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Immunofluorescence Analysis HUVECs were seeded on glass coverslips in 24-well plates and co-cultured with macrophages pre-treated with different EVs for 12 hours. Cells were fixed with 4% paraformaldehyde, permeabilized, and incubated with anti-VEGFR2 antibody (ab315283, Abcam). After washing, cells were mounted with DAPI-containing mounting medium and observed under a confocal microscope. Western Blot Analysis Proteins were extracted from cells using RIPA lysis buffer (Pulaton) containing PMSF (Beyotime). Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 2 hours at room temperature and incubated with primary antibodies against VEGFR2 (ab315283, Abcam) and GAPDH (ab9485, Abcam) at 1:2000 dilution overnight at 4°C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies at 1:10000 dilution for 1 hour. Protein bands were visualized using ECL reagent and the Amersham-Imager 600 system. Animal Experiments Four-week-old female BALB/c nude mice were purchased from Jiesijie Laboratory Animal Co., Ltd. (Shanghai, China) and maintained in specific pathogen-free conditions. After acclimation for 2 weeks, mice were randomly divided into three groups (n=5 per group): PBS group, THP-1-EV group, and SKOV3-EV group. SKOV3 cells (1×10⁶) were injected subcutaneously into the right flank of each mouse. From day 1, all mice received daily intravenous injections of GW4869 (an inhibitor of EV secretion) to suppress the release of EVs from the tumor cells. From day 3, mice were intravenously injected with 100 μL of PBS, THP-1-EVs (1×10¹⁰ particles), or SKOV3-EVs (1×10¹⁰ particles) every 3 days for a total of 6 injections. Tumor dimensions were measured every 3 days, and tumor volume was calculated using the formula: V = 0.5 × (d²×D), where d and D represent the shortest and longest diameters, respectively. Mice were euthanized on day 21, and tumors were harvested, weighed, and processed for further analyses. For flow cytometry analysis of tumor-infiltrating macrophages, part of each tumor was digested to obtain single-cell suspensions, stained with Alexa Fluor 647 Rat Anti-Mouse F4/80 and Ms CD206 Alexa 488, and analyzed by flow cytometry. For immunohistochemical analysis, the other part of each tumor was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were stained for CD31, Ki67, and TUNEL to assess angiogenesis, proliferation, and apoptosis, respectively. Statistical Analysis Data were analyzed using SPSS Statistics 19 and GraphPad Prism 9.2.0. Results are presented as mean ± standard deviation (SD). Differences between two groups were analyzed by Student's t-test, and differences among multiple groups were analyzed by one-way ANOVA. p < 0.05 was considered statistically significant. Results Elevated M2 Macrophage Infiltration and CD31 Expression in Ovarian Cancer To investigate the potential role of tumor-associated macrophages in ovarian cancer, we first examined the proportion of M2-polarized macrophages in peripheral blood and tumor tissues from ovarian cancer patients and non-ovarian cancer controls. Flow cytometry analysis revealed that the proportion of CD163+/CD68+ M2 macrophages was significantly higher in peripheral blood of ovarian cancer patients compared to non-ovarian cancer controls ( p <0.05, Fig. 1A). Similarly, the proportion of CD206+/CD68+ M2 macrophages was also significantly elevated in the peripheral blood of ovarian cancer patients ( p <0.05, Fig. 1B). When examining tissue samples, we observed an even more pronounced difference. The proportion of CD163+/CD68+ M2 macrophages was markedly higher in ovarian cancer tissues compared to non-cancerous ovarian tissues ( p <0.01, Fig. 1C). Likewise, the CD206+/CD68+ M2 macrophage ratio was significantly elevated in ovarian cancer tissues ( p <0.01, Fig. 1D). Since angiogenesis plays a critical role in cancer progression, we also assessed the expression of CD31 (also known as PECAM-1), a marker of endothelial cells, in tissue samples. Immunohistochemical analysis demonstrated that CD31 expression was significantly higher in ovarian cancer tissues compared to non-cancerous ovarian tissues ( p <0.001, Fig. 1E). This finding was further confirmed by analysis of The Cancer Genome Atlas (TCGA) database, which showed elevated CD31 expression in ovarian cancer samples (Fig. 1F). Moreover, Kaplan-Meier survival analysis revealed that high CD31 expression correlated with poor overall survival in ovarian cancer patients (Fig. 1F), suggesting that increased angiogenesis is associated with worse clinical outcomes. Ovarian Cancer Cells Promote M2 Macrophage Polarization To determine whether ovarian cancer cells could directly influence macrophage polarization, we established co-culture systems using Transwell chambers. THP-1 monocytes were differentiated into macrophages and co-cultured with ovarian cancer cell lines SKOV3 or HO8910, with PBS or THP-1 cells alone serving as controls. Flow cytometry analysis showed that co-culture with SKOV3 cells significantly increased the proportion of CD206+ (M2) macrophages (14.6%) compared to THP-1 cells cultured alone (5.79%) or PBS controls (5.91%) ( p <0.001, Fig. 2A). Similarly, co-culture with HO8910 cells led to a significant increase in CD206+ macrophages (13.1%) compared to THP-1 cells alone (7.01%) or PBS controls (6.05%) ( p <0.0001, Fig. 2B). To validate these findings in a murine system, we first isolated and characterized bone marrow-derived macrophages (BMDMs) from mice. Flow cytometry analysis confirmed the successful isolation of BMDMs, with 62.2% of cells expressing the macrophage marker F4/80, while only 0.88% expressed the M2 marker CD206 at baseline (Fig. 2C). When BMDMs were co-cultured with the murine ovarian cancer cell line ID8, we observed a significant increase in the proportion of F4/80+CD206+ (M2) macrophages (16.6%) compared to BMDMs cultured alone (6.88%) or PBS controls (6.28%) ( p <0.0001, Fig. 2D). These results collectively demonstrate that ovarian cancer cells, regardless of their origin, can directly promote M2 polarization of macrophages. Characterization of Ovarian Cancer-Derived Extracellular Vesicles and Their Uptake by Macrophages Extracellular vesicles (EVs) serve as important mediators of intercellular communication in the tumor microenvironment. We isolated EVs from the culture supernatants of ovarian cancer cell lines SKOV3 and HO8910 by ultracentrifugation and characterized them using multiple approaches. Transmission electron microscopy (TEM) revealed that both SKOV3-EVs and HO8910-EVs exhibited typical cup-shaped morphology with diameters ranging from 30 to 1000 nm (Fig. 3A). Nanoparticle tracking analysis (NTA) confirmed a relatively uniform size distribution of the EVs, with most particles measuring approximately 200 nm in diameter (Fig. 3B), consistent with the TEM observations. Western blot analysis demonstrated that SKOV3-EVs and HO8910-EVs expressed characteristic EV markers, including CD63, Tsg101, and Alix (Fig. 3C), further confirming their identity as extracellular vesicles. To determine whether ovarian cancer-derived EVs could be internalized by macrophages, we labeled SKOV3-EVs with PKH26 (red fluorescence) and incubated them with THP-1 macrophages labeled with FITC-Actin Tracker (green fluorescence). Confocal microscopy revealed that PKH26-labeled SKOV3-EVs were efficiently taken up by THP-1 macrophages, as evidenced by the presence of red fluorescent signals within the cytoplasm of the green-labeled macrophages (Fig. 3D). This observation confirmed that ovarian cancer-derived EVs could be internalized by macrophages, providing a potential mechanism for cancer cell-macrophage communication. Ovarian Cancer-Derived Extracellular Vesicles Induce M2 Macrophage Polarization Having established that ovarian cancer-derived EVs could be internalized by macrophages, we next investigated whether these EVs could directly influence macrophage polarization. THP-1 macrophages were treated with SKOV3-EVs, HO8910-EVs, or THP-1-EVs (as a control), and macrophage polarization was assessed by flow cytometry. Treatment with SKOV3-EVs significantly increased the proportion of CD68+CD206+ (M2) macrophages compared to treatment with THP-1-EVs or PBS ( p <0.0001, Fig. 4A). Similarly, HO8910-EVs treatment led to a significant increase in CD68+CD206+ macrophages compared to THP-1-EVs or PBS treatment ( p <0.0001, Fig. 4B). To validate these findings in a murine system, BMDMs were treated with ID8-EVs, BMDM-EVs, or PBS. Flow cytometry analysis showed that ID8-EVs treatment significantly increased the proportion of F4/80+CD206+ (M2) macrophages compared to BMDM-EVs or PBS treatment ( p <0.0001, Fig. 4C). These results demonstrate that ovarian cancer-derived EVs directly promote M2 polarization of macrophages, similar to the effect observed with intact ovarian cancer cells. Ovarian Cancer EV-Treated Macrophages Enhance Angiogenesis Through VEGFR Upregulation Given the elevated CD31 expression observed in ovarian cancer tissues and the known pro-angiogenic properties of M2 macrophages, we investigated whether macrophages polarized by ovarian cancer-derived EVs could promote angiogenesis. Human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophages previously treated with SKOV3-EVs, HO8910-EVs, THP-1-EVs, or PBS, and their tube formation capacity was assessed. HUVECs co-cultured with macrophages pre-treated with SKOV3-EVs or HO8910-EVs demonstrated significantly enhanced tube formation compared to those co-cultured with macrophages pre-treated with THP-1-EVs or PBS ( p <0.0001, Fig. 5A-B). This result suggests that ovarian cancer EV-treated macrophages promote angiogenesis. Interestingly, when we assessed the proliferation of HUVECs using the CCK-8 assay, we found no significant differences among the various treatment groups (Fig. 5C). This indicates that the enhanced tube formation was not due to increased endothelial cell proliferation but likely resulted from other pro-angiogenic mechanisms. To explore the underlying mechanism, we examined the expression of vascular endothelial growth factor receptor (VEGFR) in HUVECs co-cultured with differently treated macrophages. Immunofluorescence analysis revealed that HUVECs co-cultured with macrophages pre-treated with SKOV3-EVs or HO8910-EVs showed markedly increased VEGFR expression compared to those co-cultured with macrophages pre-treated with THP-1-EVs or PBS (Fig. 5D). This observation was further confirmed by Western blot analysis, which showed significantly higher VEGFR protein levels in HUVECs co-cultured with macrophages pre-treated with ovarian cancer-derived EVs (Fig. 5E). Ovarian Cancer-Derived Extracellular Vesicles Promote Tumor Progression In Vivo To validate our in vitro findings, we established a xenograft model by subcutaneously injecting SKOV3 cells into nude mice. To eliminate the confounding effects of endogenous tumor-derived EVs, all mice were treated daily with GW4869, an inhibitor of EV secretion. Mice were then intravenously injected with SKOV3-EVs, THP-1-EVs, or PBS every three days. Mice treated with SKOV3-EVs developed significantly larger tumors compared to those treated with THP-1-EVs or PBS, as evidenced by gross examination (Fig. 6A), tumor weight measurements ( p <0.0001, Fig. 6B), and tumor volume growth curves ( p <0.0001, Fig. 6C). There were no significant differences in body weight among the three groups, suggesting that the treatments were well-tolerated (Fig. 6D). Immunohistochemical analysis of tumor sections revealed that tumors from SKOV3-EV-treated mice exhibited significantly higher expression of Ki67, a marker of cell proliferation, compared to tumors from THP-1-EV or PBS-treated mice ( p <0.0001, Fig. 6E). Conversely, TUNEL staining showed decreased apoptosis in tumors from SKOV3-EV-treated mice compared to the control groups ( p <0.0001, Fig. 6F). These findings indicate that ovarian cancer-derived EVs promote tumor growth by enhancing cell proliferation and suppressing apoptosis. To assess the effect of ovarian cancer-derived EVs on macrophage polarization in vivo, we analyzed the proportion of M2 macrophages in tumor tissues by flow cytometry. Tumors from mice treated with SKOV3-EVs contained a significantly higher proportion of CD206+/F4/80+ (M2) macrophages compared to tumors from mice treated with THP-1-EVs or PBS ( p <0.0001, Fig. 6G). This finding confirms that ovarian cancer-derived EVs promote M2 macrophage polarization in vivo. Discussion In this study, we investigated the role of extracellular vesicles (EVs) derived from ovarian cancer cells in regulating tumor-associated macrophage polarization and tumor progression. Our findings demonstrate that ovarian cancer-derived EVs promote M2 macrophage polarization, which subsequently enhances angiogenesis through VEGFR upregulation, ultimately contributing to tumor growth. These results provide novel insights into the mechanisms by which ovarian cancer cells interact with the tumor microenvironment and suggest potential therapeutic strategies targeting EV-mediated communication. The tumor microenvironment plays a crucial role in cancer progression, with tumor-associated macrophages (TAMs) being key components that influence tumor growth, invasion, and response to therapy [27-28] . Consistent with previous studies [29-30] , we observed a significant increase in M2-polarized macrophages in both peripheral blood and tumor tissues of ovarian cancer patients compared to non-cancer controls. This M2 predominance supports the notion that ovarian cancer creates an immunosuppressive microenvironment that favors tumor progression. The correlation between increased M2 macrophage infiltration and elevated CD31 expression in ovarian cancer tissues suggests a link between M2 polarization and enhanced angiogenesis, which is further supported by our finding that high CD31 expression is associated with poor overall survival in ovarian cancer patients. Our co-culture experiments demonstrated that ovarian cancer cells directly promote M2 polarization of macrophages, suggesting a paracrine effect. This finding aligns with previous reports showing that cancer cells can release soluble factors that influence macrophage function [31-32] . However, the precise mediators of this intercellular communication remained unclear. Given the emerging role of EVs in cell-to-cell communication, we hypothesized that ovarian cancer-derived EVs might be involved in this process. We successfully isolated and characterized EVs from ovarian cancer cell lines, confirming their identity through multiple approaches including TEM, NTA, and Western blot analysis for established EV markers. The demonstration that these EVs could be internalized by macrophages provided a potential mechanism for the transfer of bioactive molecules from cancer cells to macrophages. Indeed, when macrophages were treated with ovarian cancer-derived EVs, they exhibited increased M2 polarization compared to those treated with control EVs or PBS. This result was consistent across both human and murine systems, highlighting the conserved nature of this mechanism. The M2 polarization induced by ovarian cancer-derived EVs has functional consequences, as evidenced by the enhanced tube formation capacity of HUVECs co-cultured with EV-treated macrophages. Interestingly, this effect was not due to increased endothelial cell proliferation but was associated with upregulation of VEGFR expression in endothelial cells. This finding suggests that ovarian cancer EV-polarized M2 macrophages promote angiogenesis through modulation of VEGF signaling, consistent with the known pro-angiogenic properties of M2 macrophages [33-34] . The in vivo experiments provided compelling evidence for the relevance of our findings in a physiological context. By using the EV secretion inhibitor GW4869 to block endogenous tumor-derived EVs, we were able to specifically assess the effects of exogenously administered EVs. The observation that SKOV3-EV treatment led to increased tumor growth, enhanced cell proliferation, reduced apoptosis, increased M2 macrophage infiltration, and elevated CD31 expression supports a model in which ovarian cancer-derived EVs promote tumor progression through multiple mechanisms, including modulation of the tumor microenvironment. Our findings contribute to the growing body of evidence implicating EVs in cancer progression and immune modulation. Our study comprehensively demonstrate this effect in ovarian cancer and to link it directly to enhanced angiogenesis and tumor growth. This mechanism may explain, at least in part, the high levels of M2 macrophages and CD31 expression observed in ovarian cancer tissues. The content of ovarian cancer-derived EVs that mediates their effect on macrophage polarization remains to be elucidated. EVs contain various bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, and microRNAs) [35] , any of which could potentially influence macrophage function. Previous studies have shown that miRNAs contained in cancer-derived EVs can modulate gene expression in recipient cells [36] . Future research should focus on identifying the specific EV cargo responsible for inducing M2 polarization and determining how these molecules alter macrophage phenotype and function. From a clinical perspective, our results suggest that targeting EV-mediated communication between ovarian cancer cells and macrophages could be a promising therapeutic strategy. Inhibiting EV production or release, blocking EV uptake by macrophages, or neutralizing specific EV components could potentially reduce M2 polarization and angiogenesis, thereby limiting tumor growth. Additionally, the presence of M2 macrophages and specific EV markers in peripheral blood could serve as potential biomarkers for ovarian cancer diagnosis, prognosis, or treatment response. Several limitations of our study should be acknowledged. First, although we demonstrated the effects of ovarian cancer-derived EVs on macrophage polarization and angiogenesis, we did not identify the specific molecular mediators of these effects. Second, while our in vivo model provided valuable insights, it may not fully recapitulate the complexity of the human tumor microenvironment. Third, we focused on two specific ovarian cancer cell lines, and the extent to which our findings can be generalized to other ovarian cancer subtypes remains to be determined. In conclusion, our study demonstrates that ovarian cancer-derived EVs promote M2 macrophage polarization, which in turn enhances angiogenesis through VEGFR upregulation, ultimately contributing to tumor progression. These findings provide novel insights into the mechanisms by which ovarian cancer cells interact with the tumor microenvironment and suggest potential therapeutic strategies targeting EV-mediated communication. Abbreviations BMDMs：bone marrow-derived macrophages EVs：Extracellular vesicles FBS：fetal bovine serum HUVECs：Human umbilical vein endothelial cells NTA：Nanoparticle tracking analysis PBMCs：Peripheral Blood Mononuclear Cells TAMs：tumor-associated macrophages TEM：Transmission electron microscopy TME：The tumor microenvironment VEGFR：vascular endothelial growth factor receptor Declarations Data availability The ovarian cancer dataset used in this study was obtained from The Cancer Genome Atlas (TCGA) public database. All data are freely available through the TCGA data portal (https://portal.gdc.cancer.gov/) or GDC Data Transfer Tool. The analyses presented in this paper are based on these publicly available data and do not require additional permission restrictions. Acknowledgements None Funding There is no funding to report. Author information Yi Zhang and Tiantian Dai have contributed equally to this article and should be considered co-first authors. Contributions Yi Zhang and Tiantian Dai contributed equally to this work and should be considered co-first authors. Yi Zhang performed the animal experiments. Tiantian Dai collected clinical data and analyzed the data. Dandan Chu and Wei Zhang prepared all figures. Yi Zhang and Tiantian Dai wrote this manuscript. Xujie Wang and Jinhua Zhou conceived, designed, and supervised the project and contributed equally to this work. Corresponding authors Correspondence to Xujie Wang or Jinhua Zhou. Xujie Wang, [email protected] Jinhua Zhou, [email protected] Conflict of interest The authors declare no competing interests. Ethics approval All patients authorized the use of their specimens by written informed consent. The protocols used in our study were approved by the Ethics Committee of Changning Maternity and Infant Health Hospital(CNFBLLKT-2023-01）. The procedures for the care and use of animals were approved by the Ethics Committee of The First Affiliated Hospital of Soochow University, and all applicable institutional and governmental regulations concerning the ethical use of animals were followed. References Konstantinopoulos PA, Matulonis UA. Clinical and translational advances in ovarian cancer therapy. Nat Cancer. 2023, 4(9): 1239-57. Havasi A, Cainap SS, Havasi AT, et al. Ovarian Cancer-Insights into Platinum Resistance and Overcoming It. Medicina (Kaunas) . 2023, 59(3). Wong-Brown MW, van der Westhuizen A, Bowden N A. Targeting DNA Repair in Ovarian Cancer Treatment Resistance. Clin Oncol (R Coll Radiol). 2020, 32(8): 518-26. Xiao Y, Yu D. Tumor microenvironment as a therapeutic target in cancer. Pharmacol Ther . 2021;221:107753. Vitale I, Manic G, Coussens LM, Kroemer G, Galluzzi L. Macrophages and Metabolism in the Tumor Microenvironment. Cell Metab . 2019;30(1):36-50. Jin MZ, Jin WL. The updated landscape of tumor microenvironment and drug repurposing. Signal Transduct Target Ther . 2020;5(1):166. Genin M, Clement F, Fattaccioli A, Raes M, Michiels C. M1 and M2 macrophages derived from THP-1 cells differentially modulate the response of cancer cells to etoposide. BMC Cancer, 2015, 15: 577. Wu L, Cheng D, Yang X, et al. M2-TAMs promote immunoresistance in lung adenocarcinoma by enhancing METTL3-mediated m6A methylation. Ann Transl Med. 2022, 10(24): 1380. Bui I, Bonavida B. Polarization of M2 Tumor-Associated Macrophages (TAMs) in Cancer Immunotherapy. Crit Rev Oncog. 2024, 29(4): 75-95. Wang Y, Zhang J, Shi H, et al. M2 Tumor-Associated Macrophages-Derived Exosomal MALAT1 Promotes Glycolysis and Gastric Cancer Progression. Adv Sci (Weinh). 2024, 11(24): e2309298. Fu LQ, Du WL, Cai MH, Yao JY, Zhao YY, Mou XZ. The roles of tumor-associated macrophages in tumor angiogenesis and metastasis. Cell Immunol. 2020, 353: 104119. Urabe F, Kosaka N, Ito K, Kimura T, Egawa S, Ochiya T. Extracellular vesicles as biomarkers and therapeutic targets for cancer. Am J Physiol Cell Physiol . 2020;318(1):C29-C39. Gupta D, Zickler AM, El Andaloussi S. Dosing extracellular vesicles. Adv Drug Deliv Rev . 2021;178:113961. Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: novel frontiers in regenerative medicine. Stem Cell Res Ther . 2018;9(1):63. Marar C, Starich B, Wirtz D. Extracellular vesicles in immunomodulation and tumor progression. Nat Immunol . 2021;22(5):560-570. Hill AF. Extracellular Vesicles and Neurodegenerative Diseases. J Neurosci . 2019;39(47):9269-9273. Namee NM, O'Driscoll L. Extracellular vesicles and anti-cancer drug resistance. Biochim Biophys Acta Rev Cancer . 2018;1870(2):123-136. Griffioen AW, Dudley AC. The rising impact of angiogenesis research. Angiogenesis . 2022;25(4):435-437. La Mendola D, Trincavelli ML, Martini C. Angiogenesis in Disease. 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Extracellular Vesicles Maintain Blood-Brain Barrier Integrity by the Suppression of Caveolin-1/CD147/VEGFR2/MMP Pathway After Ischemic Stroke. Int J Nanomedicine . 2024;19:1451-1467 Zhang P, Lim SB, Jiang K, Chew TW, Low BC, Lim CT. Distinct mRNAs in Cancer Extracellular Vesicles Activate Angiogenesis and Alter Transcriptome of Vascular Endothelial Cells. Cancers (Basel) . 2021;13(9):2009. Wang L, Zhang L, Zhang Z, Wu P, Zhang Y, Chen X. Advances in targeting tumor microenvironment for immunotherapy. Front Immunol . 2024;15:1472772. Kumari S, Advani D, Sharma S, Ambasta RK, Kumar P. Combinatorial therapy in tumor microenvironment: Where do we stand?. Biochim Biophys Acta Rev Cancer . 2021;1876(2):188585. Pan Y, Yu Y, Wang X, Zhang T. Tumor-Associated Macrophages in Tumor Immunity [published correction appears in Front Immunol. 2021 Dec 10;12:775758. doi: 10.3389/fimmu.2021.775758.]. Front Immunol . 2020;11:583084. Wang S, Liu G, Li Y, Pan Y. Metabolic Reprogramming Induces Macrophage Polarization in the Tumor Microenvironment. Front Immunol . 2022;13:840029. Li M, Yang Y, Xiong L, Jiang P, Wang J, Li C. Metabolism, metabolites, and macrophages in cancer. J Hematol Oncol . 2023;16(1):80. Published 2023 Jul 25. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell . 2010;141(1):39-51. Zhao Y, Guo S, Deng J, et al. VEGF/VEGFR-Targeted Therapy and Immunotherapy in Non-small Cell Lung Cancer: Targeting the Tumor Microenvironment. Int J Biol Sci . 2022;18(9):3845-3858. Wang H, Yung MMH, Ngan HYS, Chan KKL, Chan DW. The Impact of the Tumor Microenvironment on Macrophage Polarization in Cancer Metastatic Progression. Int J Mol Sci . 2021;22(12):6560. Minciacchi VR, Freeman MR, Di Vizio D. Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes. Semin Cell Dev Biol . 2015;40:41-51. Parthasarathy G, Hirsova P, Kostallari E, Sidhu GS, Ibrahim SH, Malhi H. Extracellular Vesicles in Hepatobiliary Health and Disease. Compr Physiol . 2023;13(3):4631-4658. Additional Declarations No competing interests reported. Supplementary Files uncroppedblotsofWesternblot.docx Cite Share Download PDF Status: Published Journal Publication published 24 Apr, 2026 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 28 Jul, 2025 Reviews received at journal 22 Jul, 2025 Reviews received at journal 22 Jul, 2025 Reviewers agreed at journal 16 Jul, 2025 Reviewers agreed at journal 14 Jul, 2025 Reviewers agreed at journal 13 Jul, 2025 Reviews received at journal 12 Jul, 2025 Reviewers agreed at journal 12 Jul, 2025 Reviews received at journal 10 Jul, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers invited by journal 09 Jul, 2025 Editor invited by journal 08 Jul, 2025 Editor assigned by journal 22 Jun, 2025 Submission checks completed at journal 22 Jun, 2025 First submitted to journal 06 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6834403","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":484729979,"identity":"241f3f71-ba26-43a7-80a7-adfc04fea23d","order_by":0,"name":"Yi Zhang","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhang","suffix":""},{"id":484729980,"identity":"6975be52-3569-4688-a8c7-eadcdc6785d1","order_by":1,"name":"Tiantian Dai","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Tiantian","middleName":"","lastName":"Dai","suffix":""},{"id":484729981,"identity":"e6d4c7aa-643a-45da-9140-0792f97d8da6","order_by":2,"name":"Dandan Chu","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Dandan","middleName":"","lastName":"Chu","suffix":""},{"id":484729982,"identity":"717a048e-2d23-4e96-90d2-80d79fd2cb58","order_by":3,"name":"Wei Zhang","email":"","orcid":"","institution":"East China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Zhang","suffix":""},{"id":484729983,"identity":"e24a546c-718d-4f1b-8ce2-7fe2af9b6062","order_by":4,"name":"Xujie Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIie3PvarCMBTA8RMCmcLtegpSXyHi4uarJAjdBMcKghGlHVRcu/kKjo4RoVPcHSuC+12uDhc/dsXUzSG/+fw55wB43hdiweJ8weQWkWWpSpkM3MkPGoEtS5tUy6MobeFOIpAC+ylVmVan8DChFQ4DI8WeMTLX2zhRmkGQTeX7hGqjcs5pOErjvVrXAO1u5diy0VtEZI0xfySWgcCuK+mQ8b8QvF0Efz2V0ipJTAGlRDKDGKol+LgFjREkhw5KW3DnL/XFjEF4NcNRDur3kgyiIJu/T57wz8Y9z/O8l+7RJUmCvp10vAAAAABJRU5ErkJggg==","orcid":"","institution":"East China Normal University","correspondingAuthor":true,"prefix":"","firstName":"Xujie","middleName":"","lastName":"Wang","suffix":""},{"id":484729984,"identity":"109f7fc7-fad0-487e-af5d-a91fece51124","order_by":5,"name":"Jinhua Zhou","email":"","orcid":"","institution":"The First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jinhua","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-06-06 07:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6834403/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6834403/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12885-026-16059-2","type":"published","date":"2026-04-24T15:59:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86670708,"identity":"06526c99-6354-4542-a4fe-d24b646e3f4e","added_by":"auto","created_at":"2025-07-14 11:30:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":453813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased M2 macrophage infiltration and CD31 expression in ovarian cancer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Flow cytometry analysis of CD163+/CD68+ macrophages in peripheral blood from ovarian cancer patients and non-cancer controls. *\u003c/strong\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.05. (B) Flow cytometry analysis of CD206+/CD68+ macrophages in peripheral blood. *\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.05. (C) Flow cytometry analysis of CD163+/CD68+ macrophages in ovarian tissue. **\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.01. (D) Flow cytometry analysis of CD206+/CD68+ macrophages in ovarian tissue. **\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.01. (E) Representative immunohistochemical images of CD31 expression (left panel) and quantification (right panel). Scale bar, 100 μm. ***\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.001. (F) CD31 expression in ovarian cancer compared to normal tissue from TCGA database (left) and its correlation with patient survival (right).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6834403/v1/40de9635dedbde022d0083f6.png"},{"id":86672046,"identity":"06209333-e436-439e-8bcd-d417e017afb7","added_by":"auto","created_at":"2025-07-14 11:38:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261493,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOvarian cancer cells induce M2 polarization of macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Flow cytometry of CD68+CD206+ macrophages after co-culture with PBS, THP-1 cells, or SKOV3 cells (left) and quantification (right). ***\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.001. (B) Flow cytometry of CD68+CD206+ macrophages after co-culture with PBS, THP-1 cells, or HO8910 cells (left) and quantification (right). ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (C) Flow cytometry of F4/80 expression (left) and CD206 expression (right) in primary BMDMs. (D) Flow cytometry of F4/80+CD206+ BMDMs after co-culture with PBS, BMDMs, or ID8 cells (left) and quantification (right). ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6834403/v1/88e7ef558d93eff2364bbba9.png"},{"id":86670714,"identity":"f721486f-dee7-49e2-94bc-16920612691d","added_by":"auto","created_at":"2025-07-14 11:30:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":280822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of ovarian cancer-derived extracellular vesicles and their uptake by macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) TEM images of SKOV3-EVs (left) and HO8910-EVs (right). Scale bar, 200 nm. (B) Size distribution of SKOV3-EVs (left) and HO8910-EVs (right) by NTA. (C) Western blot analysis of EV markers in SKOV3-EVs (left) and HO8910-EVs (right). (D) Confocal microscopy showing uptake of PKH26-labeled SKOV3-EVs (red) by THP-1 macrophages labeled with FITC-Actin Tracker (green); nuclei stained with DAPI (blue). Scale bar, 10 μm.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6834403/v1/7c5a28c52d6f53dd6c2f5e1e.png"},{"id":86670707,"identity":"fa299245-2072-4fd0-b746-b5b878f66c53","added_by":"auto","created_at":"2025-07-14 11:30:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOvarian cancer-derived extracellular vesicles promote M2 polarization of macrophages.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Flow cytometry of CD68+CD206+ macrophages after treatment with PBS, THP-1-EVs, or SKOV3-EVs (left) and quantification (right). ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (B) Flow cytometry of CD68+CD206+ macrophages after treatment with PBS, THP-1-EVs, or HO8910-EVs (left) and quantification (right). ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (C) Flow cytometry of F4/80+CD206+ BMDMs after treatment with PBS, BMDM-EVs, or ID8-EVs (left) and quantification (right). ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6834403/v1/1fa40829c7d9c31e489c6ba0.png"},{"id":86672048,"identity":"c08e51b1-2da5-471b-aea5-bbdeae7ea866","added_by":"auto","created_at":"2025-07-14 11:38:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":589025,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOvarian cancer EV-treated macrophages enhance angiogenesis through VEGFR upregulation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Tube formation by HUVECs co-cultured with macrophages pre-treated with PBS, THP-1-EVs, SKOV3-EVs, or HO8910-EVs. Scale bar, 200 μm. (B) Quantification of branch points in tube formation assay. ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (C) HUVEC proliferation measured by CCK-8 assay. ns, not significant. (D) Immunofluorescence of VEGFR (green) in HUVECs co-cultured with differently pre-treated macrophages; nuclei stained with DAPI (blue). Scale bar, 50 μm. (E) Western blot of VEGFR expression in HUVECs co-cultured with differently pre-treated macrophages.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6834403/v1/cf91558277c114d22160a176.png"},{"id":86670717,"identity":"a80d5197-427d-4e7d-8b5e-da6e555edd20","added_by":"auto","created_at":"2025-07-14 11:30:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":519739,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOvarian cancer-derived extracellular vesicles promote tumor progression in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) Representative images of xenograft tumors from mice treated with PBS, THP-1-EVs, or SKOV3-EVs. (B) Tumor weight analysis. ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (C) Tumor growth curves. ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (D) Body weight monitoring. ns, not significant. (E) Ki67 immunohistochemistry (left) and quantification (right). Scale bar, 100 μm. ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (F) TUNEL staining (left) and quantification (right). Scale bar, 100 μm. ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (G) Flow cytometry of CD206+/F4/80+ macrophages in tumor tissues (left) and quantification (right). ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001. (H) CD31 immunohistochemistry in tumor sections (left) and quantification (right). Scale bar, 100 μm. ****\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026lt;0.0001.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6834403/v1/a62b3b2a06899fd0a32dd6ee.png"},{"id":107927962,"identity":"b95713d2-cbcc-405c-8fd8-61e59b4a086b","added_by":"auto","created_at":"2026-04-27 16:06:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2571317,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6834403/v1/0b618dbe-466a-4fc3-9e20-ed07a0da6d13.pdf"},{"id":86673485,"identity":"47c31e9c-9d74-44cf-812d-3501b9190160","added_by":"auto","created_at":"2025-07-14 11:46:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":208851,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedblotsofWesternblot.docx","url":"https://assets-eu.researchsquare.com/files/rs-6834403/v1/e72ecb78c5346c634175e4ba.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extracellular Vesicles Derived from Ovarian Cancer Cells Promote Tumor Progression through M2 Macrophage Polarization and Enhanced Angiogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOvarian cancer is one of the most lethal gynecological malignancies\u003csup\u003e[1]\u003c/sup\u003e. Despite advances in surgical techniques and chemotherapeutic regimens, the 5-year survival rate for patients with advanced ovarian cancer remains below 30%\u003csup\u003e[2]\u003c/sup\u003e. The poor prognosis is largely attributed to late diagnosis, high recurrence rates, and the development of chemoresistance\u003csup\u003e[3]\u003c/sup\u003e. Understanding the molecular mechanisms underlying ovarian cancer progression is critical for developing more effective therapeutic strategies.\u003c/p\u003e\n\u003cp\u003eThe tumor microenvironment (TME) plays a pivotal role in cancer development, progression, and therapeutic response \u003csup\u003e[4]\u003c/sup\u003e. As a complex ecosystem, the TME consists of cancer cells, stromal cells, immune cells, blood vessels, and extracellular matrix components that interact in a dynamic manner\u003csup\u003e\u0026nbsp;[5]\u003c/sup\u003e. Among the immune cells present in the TME, tumor-associated macrophages (TAMs) have emerged as key regulators of tumor progression\u003csup\u003e[6]\u003c/sup\u003e. Macrophages exhibit remarkable plasticity and can be polarized into distinct functional phenotypes: classically activated (M1) macrophages with anti-tumor properties and alternatively activated (M2) macrophages that generally promote tumor growth\u003csup\u003e[7,8]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn ovarian cancer, increased infiltration of M2 TAMs has been associated with advanced disease stage, enhanced tumor angiogenesis, and poor patient outcomes \u003csup\u003e[9-11]\u003c/sup\u003e. However, the mechanisms by which ovarian cancer cells influence macrophage polarization remain incompletely understood. Recent evidence suggests that cancer cells can modulate the phenotype and function of surrounding cells through the secretion of extracellular vesicles (EVs)\u003csup\u003e[12]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eEVs are membrane-enclosed structures released by cells into the extracellular space, ranging from 30 to 1000 nm in diameter\u003csup\u003e[13]\u003c/sup\u003e. These vesicles contain bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, and microRNAs), and can transfer their cargo to recipient cells, thereby altering their phenotype and function\u003csup\u003e[14,15]\u003c/sup\u003e. Cancer-derived EVs have been implicated in various aspects of tumor biology, including immune modulation, angiogenesis, metastasis, and drug resistance\u003csup\u003e[16,17]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAngiogenesis, the formation of new blood vessels from pre-existing vasculature, is essential for tumor growth, progression, and metastasis\u003csup\u003e[18-19]\u003c/sup\u003e. CD31 (PECAM-1), a transmembrane glycoprotein primarily expressed on endothelial cells, is widely used as a marker for angiogenesis evaluation\u003csup\u003e[20-21]\u003c/sup\u003e. Previous studies have shown that high CD31 expression correlates with poor prognosis in various cancers, including ovarian cancer\u003csup\u003e[22]\u003c/sup\u003e. The vascular endothelial growth factor receptor (VEGFR) signaling pathway plays a central role in tumor angiogenesis and has been targeted for cancer therapy\u003csup\u003e[23]\u003c/sup\u003e. Emerging evidence suggests that EVs can modulate angiogenesis by transferring angiogenic factors or regulatory molecules to endothelial cells\u003csup\u003e[24-26]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eWhile studies have demonstrated that cancer-derived EVs can influence various aspects of tumor biology, the specific role of ovarian cancer-derived EVs in modulating macrophage polarization and subsequently affecting tumor angiogenesis and progression remains to be fully elucidated. Understanding these mechanisms could provide insights into novel therapeutic targets for ovarian cancer treatment.\u003c/p\u003e\n\u003cp\u003eIn this study, we investigated the effects of ovarian cancer-derived EVs on macrophage polarization and subsequent impacts on angiogenesis both in vitro and in vivo. We demonstrated that ovarian cancer patients exhibit increased proportions of M2 macrophages in peripheral blood and tumor tissues, along with elevated CD31 expression that correlates with poor prognosis. We found that ovarian cancer cells and their derived EVs promote M2 polarization of macrophages, which in turn enhances endothelial cell tube formation through upregulation of VEGFR expression. Furthermore, in a mouse model, we showed that ovarian cancer-derived EVs promote tumor growth, increase M2 macrophage infiltration, and enhance tumor angiogenesis. These findings reveal a novel mechanism by which ovarian cancer cells interact with the TME and suggest that targeting cancer-derived EVs could be a promising therapeutic strategy for ovarian cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell Lines and Culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOvarian cancer cell lines (SKOV3 and HO8910), murine ovarian cancer cell line (ID8), human monocytic cell line (THP-1), and human umbilical vein endothelial cells (HUVECs) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences. SKOV3, HO8910, and ID8 cells were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 U/mL penicillin-streptomycin. THP-1 cells were maintained in RPMI-1640 medium containing 10 mM HEPES, 1 mM sodium pyruvate, 50 \u0026mu;M \u0026beta;-mercaptoethanol, 10% FBS, and 100 U/mL penicillin-streptomycin. HUVECs were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin-streptomycin. All cells were maintained at 37\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient Samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeripheral blood and tissue samples were collected from ovarian cancer patients and non-ovarian cancer controls at the Department of Gynecology, Shanghai Changning Maternity and Infant Health Hospital. Fresh tissues were transported to the laboratory at 4\u0026deg;C and processed within 2 hours of collection. Fresh tissue samples were divided into two parts: one for flow cytometry analysis and one for immunohistochemistry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Analysis of Peripheral Blood Mononuclear Cells (PBMCs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePBMCs were isolated using human peripheral blood lymphocyte separation medium (Beyotime) according to the manufacturer\u0026apos;s instructions. Briefly, fresh blood was diluted 1:1 with PBS, carefully layered over the separation medium (ratio of separation medium to diluted blood = 1:2), and centrifuged at 800 g for 20 minutes at room temperature. The PBMC layer was collected, washed twice with PBS, and resuspended for flow cytometry analysis.\u003c/p\u003e\n\u003cp\u003eFor macrophage phenotype analysis, PBMCs were stained with FITC Mouse Anti-Human CD68 and APC Mouse Anti-Human CD206 (BD Pharmingen) according to manufacturer recommendations. Samples were incubated for 40 minutes at room temperature in the dark, washed, and analyzed using a flow cytometer with excitation at 488 nm (FITC) and 651 nm (APC). Data were analyzed using FlowJo software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumor Tissue Processing and Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor flow cytometry analysis, fresh tumor tissues were minced and digested with collagenase I (1 mg/mL, Beyotime) for 40 minutes at 37\u0026deg;C with shaking. The digestion was terminated by adding DMEM containing 10% FBS. The cell suspension was filtered through a 40 \u0026mu;m cell strainer, centrifuged at 500 g for 5 minutes at 4\u0026deg;C, and treated with red blood cell lysis buffer (Beyotime). After centrifugation, cells were stained with 647 Mouse Anti-Human CD68 and FITC Mouse Anti-Human CD206 (BD Pharmingen) for human samples, or Alexa Fluor 647 Rat Anti-Mouse F4/80 and Ms CD206 Alexa 488 (BD Pharmingen) for mouse samples.\u003c/p\u003e\n\u003cp\u003eFor immunohistochemistry, tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 \u0026mu;m thickness. Sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate buffer. Endogenous peroxidase activity was blocked, and sections were incubated with primary antibodies overnight at 4\u0026deg;C, followed by detection using the DAB method and hematoxylin counterstaining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Culture of Bone Marrow-Derived Macrophages (BMDMs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMDMs were isolated from 6-8 week-old female C57BL/6 mice. Femurs and tibias were collected under sterile conditions and bone marrow was flushed out with cold PBS. After red blood cell lysis, cells were resuspended in IMDM medium containing 10% FBS, 100 U/mL penicillin, 100 \u0026mu;g/mL streptomycin, and 10 ng/mL M-CSF, and cultured for 7 days, replacing medium every 3 days. BMDMs were identified by flow cytometry analysis of F4/80 and CD206 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-culture Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor co-culture experiments, Transwell inserts with 0.4 \u0026mu;m pore size were used. THP-1 cells (1\u0026times;10⁵ cells/mL) were seeded in the lower chamber and treated with 150 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce differentiation into macrophages. The medium was then replaced, and SKOV3 or HO8910 cells (1\u0026times;10⁵ cells/mL) were seeded in the upper chamber (400 \u0026mu;L). For mouse cell co-culture, BMDMs were seeded in the lower chamber and ID8 cells in the upper chamber. Control groups received an equal volume of PBS. After 48 hours of co-culture at 37\u0026deg;C with 5% CO₂, cells in the lower chamber were collected for flow cytometry analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and Characterization of Extracellular Vesicles (EVs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEVs were isolated from cell culture supernatants by differential ultracentrifugation. Cells were cultured to 60-70% confluence in T225 flasks, washed with PBS, and cultured in serum-free medium for 48 hours. Collected supernatants were centrifuged at 300 g for 20 minutes to remove cell debris. The resulting supernatant was ultracentrifuged at 10,000 g for 60 minutes, followed by ultracentrifugation at 125,000 g for 120 minutes using a Beckman Optima XPN-100 ultracentrifuge. The pellet was washed with PBS and ultracentrifuged again at 125,000 g for 120 minutes. The final EV pellet was resuspended in PBS and stored at -80\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting for EV markers. For TEM, EV suspensions were placed on copper grids, stained with 2% uranyl acetate, and observed using a transmission electron microscope. For NTA, EVs were diluted in PBS and analyzed using a NanoSight NS300 instrument to determine particle size distribution and concentration. For western blotting, EV protein was extracted and analyzed for the expression of CD63 (ab134045, Abcam), TSG101 (ab133586, Abcam), and Alix (ab275377, Abcam).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEV Uptake Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEVs were labeled with PKH26 red fluorescent dye (Merck) according to the manufacturer\u0026apos;s instructions. THP-1 cells were differentiated with PMA on glass coverslips for 24 hours, then incubated with PKH26-labeled EVs for 12 hours. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, stained with FITC-Actin Tracker (Beyotime) to visualize cell cytoskeleton, and mounted with DAPI-containing mounting medium. The uptake of labeled EVs by macrophages was observed using a confocal laser scanning microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEV Treatment of Macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTHP-1 cells (1\u0026times;10⁵ cells/mL) were seeded in 12-well plates and differentiated with 150 nM PMA for 24 hours. The medium was replaced, and cells were treated with SKOV3-EVs, HO8910-EVs, THP-1-EVs (all at a concentration of 10\u0026sup1;\u0026sup1; particles/mL), or an equal volume of PBS. After 48 hours of incubation, cells were collected, stained for CD68 and CD206, and analyzed by flow cytometry.\u003c/p\u003e\n\u003cp\u003eFor BMDMs, similar procedures were followed using ID8-EVs, BMDM-EVs, or PBS as treatments, and cells were stained for F4/80 and CD206 before flow cytometry analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTube Formation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMatrigel (Beyotime) was thawed overnight at 4\u0026deg;C, added to 96-well plates (40 \u0026mu;L/well), and incubated at 37\u0026deg;C for 30 minutes to solidify. HUVECs were co-cultured with macrophages pre-treated with SKOV3-EVs, HO8910-EVs, THP-1-EVs, or PBS in a Transwell system for 24 hours. HUVECs were then harvested, resuspended to 5\u0026times;10⁴ cells/mL, and seeded on Matrigel (50 \u0026mu;L/well). After 4 hours of incubation, tube formation was observed under an inverted microscope, and images were captured from five random fields. Tube formation was quantified by measuring the number of branch points and total tube length using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Proliferation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proliferation of HUVECs co-cultured with EV-treated macrophages was assessed using the Cell Counting Kit-8 (CCK-8, MCE) according to the manufacturer\u0026apos;s instructions. HUVECs were co-cultured with macrophages pre-treated with different EVs using a Transwell system. After co-culture, CCK-8 solution (10% v/v) was added to the lower chamber and incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHUVECs were seeded on glass coverslips in 24-well plates and co-cultured with macrophages pre-treated with different EVs for 12 hours. Cells were fixed with 4% paraformaldehyde, permeabilized, and incubated with anti-VEGFR2 antibody (ab315283, Abcam). After washing, cells were mounted with DAPI-containing mounting medium and observed under a confocal microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from cells using RIPA lysis buffer (Pulaton) containing PMSF (Beyotime). Protein concentrations were determined using the BCA Protein Assay Kit (Beyotime). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk for 2 hours at room temperature and incubated with primary antibodies against VEGFR2 (ab315283, Abcam) and GAPDH (ab9485, Abcam) at 1:2000 dilution overnight at 4\u0026deg;C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies at 1:10000 dilution for 1 hour. Protein bands were visualized using ECL reagent and the Amersham-Imager 600 system.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour-week-old female BALB/c nude mice were purchased from Jiesijie Laboratory Animal Co., Ltd. (Shanghai, China) and maintained in specific pathogen-free conditions. After acclimation for 2 weeks, mice were randomly divided into three groups (n=5 per group): PBS group, THP-1-EV group, and SKOV3-EV group.\u003c/p\u003e\n\u003cp\u003eSKOV3 cells (1\u0026times;10⁶) were injected subcutaneously into the right flank of each mouse. From day 1, all mice received daily intravenous injections of GW4869 (an inhibitor of EV secretion) to suppress the release of EVs from the tumor cells. From day 3, mice were intravenously injected with 100 \u0026mu;L of PBS, THP-1-EVs (1\u0026times;10\u0026sup1;⁰ particles), or SKOV3-EVs (1\u0026times;10\u0026sup1;⁰ particles) every 3 days for a total of 6 injections. Tumor dimensions were measured every 3 days, and tumor volume was calculated using the formula: V = 0.5 \u0026times; (d\u0026sup2;\u0026times;D), where d and D represent the shortest and longest diameters, respectively. Mice were euthanized on day 21, and tumors were harvested, weighed, and processed for further analyses.\u003c/p\u003e\n\u003cp\u003eFor flow cytometry analysis of tumor-infiltrating macrophages, part of each tumor was digested to obtain single-cell suspensions, stained with Alexa Fluor 647 Rat Anti-Mouse F4/80 and Ms CD206 Alexa 488, and analyzed by flow cytometry. For immunohistochemical analysis, the other part of each tumor was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Sections were stained for CD31, Ki67, and TUNEL to assess angiogenesis, proliferation, and apoptosis, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using SPSS Statistics 19 and GraphPad Prism 9.2.0. Results are presented as mean \u0026plusmn; standard deviation (SD). Differences between two groups were analyzed by Student\u0026apos;s t-test, and differences among multiple groups were analyzed by one-way ANOVA. \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eElevated M2 Macrophage Infiltration and CD31 Expression in Ovarian Cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the potential role of tumor-associated macrophages in ovarian cancer, we first examined the proportion of M2-polarized macrophages in peripheral blood and tumor tissues from ovarian cancer patients and non-ovarian cancer controls. Flow cytometry analysis revealed that the proportion of CD163+/CD68+ M2 macrophages was significantly higher in peripheral blood of ovarian cancer patients compared to non-ovarian cancer controls (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, Fig. 1A). Similarly, the proportion of CD206+/CD68+ M2 macrophages was also significantly elevated in the peripheral blood of ovarian cancer patients (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, Fig. 1B).\u003c/p\u003e\n\u003cp\u003eWhen examining tissue samples, we observed an even more pronounced difference. The proportion of CD163+/CD68+ M2 macrophages was markedly higher in ovarian cancer tissues compared to non-cancerous ovarian tissues (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, Fig. 1C). Likewise, the CD206+/CD68+ M2 macrophage ratio was significantly elevated in ovarian cancer tissues (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, Fig. 1D).\u003c/p\u003e\n\u003cp\u003eSince angiogenesis plays a critical role in cancer progression, we also assessed the expression of CD31 (also known as PECAM-1), a marker of endothelial cells, in tissue samples. Immunohistochemical analysis demonstrated that CD31 expression was significantly higher in ovarian cancer tissues compared to non-cancerous ovarian tissues (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, Fig. 1E). This finding was further confirmed by analysis of The Cancer Genome Atlas (TCGA) database, which showed elevated CD31 expression in ovarian cancer samples (Fig. 1F). Moreover, Kaplan-Meier survival analysis revealed that high CD31 expression correlated with poor overall survival in ovarian cancer patients (Fig. 1F), suggesting that increased angiogenesis is associated with worse clinical outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOvarian Cancer Cells Promote M2 Macrophage Polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether ovarian cancer cells could directly influence macrophage polarization, we established co-culture systems using Transwell chambers. THP-1 monocytes were differentiated into macrophages and co-cultured with ovarian cancer cell lines SKOV3 or HO8910, with PBS or THP-1 cells alone serving as controls.\u003c/p\u003e\n\u003cp\u003eFlow cytometry analysis showed that co-culture with SKOV3 cells significantly increased the proportion of CD206+ (M2) macrophages (14.6%) compared to THP-1 cells cultured alone (5.79%) or PBS controls (5.91%) (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, Fig. 2A). Similarly, co-culture with HO8910 cells led to a significant increase in CD206+ macrophages (13.1%) compared to THP-1 cells alone (7.01%) or PBS controls (6.05%) (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 2B).\u003c/p\u003e\n\u003cp\u003eTo validate these findings in a murine system, we first isolated and characterized bone marrow-derived macrophages (BMDMs) from mice. Flow cytometry analysis confirmed the successful isolation of BMDMs, with 62.2% of cells expressing the macrophage marker F4/80, while only 0.88% expressed the M2 marker CD206 at baseline (Fig. 2C).\u003c/p\u003e\n\u003cp\u003eWhen BMDMs were co-cultured with the murine ovarian cancer cell line ID8, we observed a significant increase in the proportion of F4/80+CD206+ (M2) macrophages (16.6%) compared to BMDMs cultured alone (6.88%) or PBS controls (6.28%) (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 2D). These results collectively demonstrate that ovarian cancer cells, regardless of their origin, can directly promote M2 polarization of macrophages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of Ovarian Cancer-Derived Extracellular Vesicles and Their Uptake by Macrophages\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExtracellular vesicles (EVs) serve as important mediators of intercellular communication in the tumor microenvironment. We isolated EVs from the culture supernatants of ovarian cancer cell lines SKOV3 and HO8910 by ultracentrifugation and characterized them using multiple approaches.\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy (TEM) revealed that both SKOV3-EVs and HO8910-EVs exhibited typical cup-shaped morphology with diameters ranging from 30 to 1000 nm (Fig. 3A). Nanoparticle tracking analysis (NTA) confirmed a relatively uniform size distribution of the EVs, with most particles measuring approximately 200 nm in diameter (Fig. 3B), consistent with the TEM observations.\u003c/p\u003e\n\u003cp\u003eWestern blot analysis demonstrated that SKOV3-EVs and HO8910-EVs expressed characteristic EV markers, including CD63, Tsg101, and Alix (Fig. 3C), further confirming their identity as extracellular vesicles.\u003c/p\u003e\n\u003cp\u003eTo determine whether ovarian cancer-derived EVs could be internalized by macrophages, we labeled SKOV3-EVs with PKH26 (red fluorescence) and incubated them with THP-1 macrophages labeled with FITC-Actin Tracker (green fluorescence). Confocal microscopy revealed that PKH26-labeled SKOV3-EVs were efficiently taken up by THP-1 macrophages, as evidenced by the presence of red fluorescent signals within the cytoplasm of the green-labeled macrophages (Fig. 3D). This observation confirmed that ovarian cancer-derived EVs could be internalized by macrophages, providing a potential mechanism for cancer cell-macrophage communication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOvarian Cancer-Derived Extracellular Vesicles Induce M2 Macrophage Polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaving established that ovarian cancer-derived EVs could be internalized by macrophages, we next investigated whether these EVs could directly influence macrophage polarization. THP-1 macrophages were treated with SKOV3-EVs, HO8910-EVs, or THP-1-EVs (as a control), and macrophage polarization was assessed by flow cytometry.\u003c/p\u003e\n\u003cp\u003eTreatment with SKOV3-EVs significantly increased the proportion of CD68+CD206+ (M2) macrophages compared to treatment with THP-1-EVs or PBS (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 4A). Similarly, HO8910-EVs treatment led to a significant increase in CD68+CD206+ macrophages compared to THP-1-EVs or PBS treatment (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 4B).\u003c/p\u003e\n\u003cp\u003eTo validate these findings in a murine system, BMDMs were treated with ID8-EVs, BMDM-EVs, or PBS. Flow cytometry analysis showed that ID8-EVs treatment significantly increased the proportion of F4/80+CD206+ (M2) macrophages compared to BMDM-EVs or PBS treatment (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 4C). These results demonstrate that ovarian cancer-derived EVs directly promote M2 polarization of macrophages, similar to the effect observed with intact ovarian cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOvarian Cancer EV-Treated Macrophages Enhance Angiogenesis Through VEGFR Upregulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the elevated CD31 expression observed in ovarian cancer tissues and the known pro-angiogenic properties of M2 macrophages, we investigated whether macrophages polarized by ovarian cancer-derived EVs could promote angiogenesis. Human umbilical vein endothelial cells (HUVECs) were co-cultured with macrophages previously treated with SKOV3-EVs, HO8910-EVs, THP-1-EVs, or PBS, and their tube formation capacity was assessed.\u003c/p\u003e\n\u003cp\u003eHUVECs co-cultured with macrophages pre-treated with SKOV3-EVs or HO8910-EVs demonstrated significantly enhanced tube formation compared to those co-cultured with macrophages pre-treated with THP-1-EVs or PBS (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 5A-B). This result suggests that ovarian cancer EV-treated macrophages promote angiogenesis.\u003c/p\u003e\n\u003cp\u003eInterestingly, when we assessed the proliferation of HUVECs using the CCK-8 assay, we found no significant differences among the various treatment groups (Fig. 5C). This indicates that the enhanced tube formation was not due to increased endothelial cell proliferation but likely resulted from other pro-angiogenic mechanisms.\u003c/p\u003e\n\u003cp\u003eTo explore the underlying mechanism, we examined the expression of vascular endothelial growth factor receptor (VEGFR) in HUVECs co-cultured with differently treated macrophages. Immunofluorescence analysis revealed that HUVECs co-cultured with macrophages pre-treated with SKOV3-EVs or HO8910-EVs showed markedly increased VEGFR expression compared to those co-cultured with macrophages pre-treated with THP-1-EVs or PBS (Fig. 5D). This observation was further confirmed by Western blot analysis, which showed significantly higher VEGFR protein levels in HUVECs co-cultured with macrophages pre-treated with ovarian cancer-derived EVs (Fig. 5E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOvarian Cancer-Derived Extracellular Vesicles Promote Tumor Progression In Vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate our in vitro findings, we established a xenograft model by subcutaneously injecting SKOV3 cells into nude mice. To eliminate the confounding effects of endogenous tumor-derived EVs, all mice were treated daily with GW4869, an inhibitor of EV secretion. Mice were then intravenously injected with SKOV3-EVs, THP-1-EVs, or PBS every three days.\u003c/p\u003e\n\u003cp\u003eMice treated with SKOV3-EVs developed significantly larger tumors compared to those treated with THP-1-EVs or PBS, as evidenced by gross examination (Fig. 6A), tumor weight measurements (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 6B), and tumor volume growth curves (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 6C). There were no significant differences in body weight among the three groups, suggesting that the treatments were well-tolerated (Fig. 6D).\u003c/p\u003e\n\u003cp\u003eImmunohistochemical analysis of tumor sections revealed that tumors from SKOV3-EV-treated mice exhibited significantly higher expression of Ki67, a marker of cell proliferation, compared to tumors from THP-1-EV or PBS-treated mice (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.0001, Fig. 6E). Conversely, TUNEL staining showed decreased apoptosis in tumors from SKOV3-EV-treated mice compared to the control groups (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 6F). These findings indicate that ovarian cancer-derived EVs promote tumor growth by enhancing cell proliferation and suppressing apoptosis.\u003c/p\u003e\n\u003cp\u003eTo assess the effect of ovarian cancer-derived EVs on macrophage polarization in vivo, we analyzed the proportion of M2 macrophages in tumor tissues by flow cytometry. Tumors from mice treated with SKOV3-EVs contained a significantly higher proportion of CD206+/F4/80+ (M2) macrophages compared to tumors from mice treated with THP-1-EVs or PBS (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001, Fig. 6G). This finding confirms that ovarian cancer-derived EVs promote M2 macrophage polarization in vivo.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the role of extracellular vesicles (EVs) derived from ovarian cancer cells in regulating tumor-associated macrophage polarization and tumor progression. Our findings demonstrate that ovarian cancer-derived EVs promote M2 macrophage polarization, which subsequently enhances angiogenesis through VEGFR upregulation, ultimately contributing to tumor growth. These results provide novel insights into the mechanisms by which ovarian cancer cells interact with the tumor microenvironment and suggest potential therapeutic strategies targeting EV-mediated communication.\u003c/p\u003e\n\u003cp\u003eThe tumor microenvironment plays a crucial role in cancer progression, with tumor-associated macrophages (TAMs) being key components that influence tumor growth, invasion, and response to therapy\u003csup\u003e\u0026nbsp;[27-28]\u003c/sup\u003e. Consistent with previous studies \u003csup\u003e[29-30]\u003c/sup\u003e, we observed a significant increase in M2-polarized macrophages in both peripheral blood and tumor tissues of ovarian cancer patients compared to non-cancer controls. This M2 predominance supports the notion that ovarian cancer creates an immunosuppressive microenvironment that favors tumor progression. The correlation between increased M2 macrophage infiltration and elevated CD31 expression in ovarian cancer tissues suggests a link between M2 polarization and enhanced angiogenesis, which is further supported by our finding that high CD31 expression is associated with poor overall survival in ovarian cancer patients.\u003c/p\u003e\n\u003cp\u003eOur co-culture experiments demonstrated that ovarian cancer cells directly promote M2 polarization of macrophages, suggesting a paracrine effect. This finding aligns with previous reports showing that cancer cells can release soluble factors that influence macrophage function\u003csup\u003e[31-32]\u003c/sup\u003e. However, the precise mediators of this intercellular communication remained unclear. Given the emerging role of EVs in cell-to-cell communication, we hypothesized that ovarian cancer-derived EVs might be involved in this process.\u003c/p\u003e\n\u003cp\u003eWe successfully isolated and characterized EVs from ovarian cancer cell lines, confirming their identity through multiple approaches including TEM, NTA, and Western blot analysis for established EV markers. The demonstration that these EVs could be internalized by macrophages provided a potential mechanism for the transfer of bioactive molecules from cancer cells to macrophages. Indeed, when macrophages were treated with ovarian cancer-derived EVs, they exhibited increased M2 polarization compared to those treated with control EVs or PBS. This result was consistent across both human and murine systems, highlighting the conserved nature of this mechanism.\u003c/p\u003e\n\u003cp\u003eThe M2 polarization induced by ovarian cancer-derived EVs has functional consequences, as evidenced by the enhanced tube formation capacity of HUVECs co-cultured with EV-treated macrophages. Interestingly, this effect was not due to increased endothelial cell proliferation but was associated with upregulation of VEGFR expression in endothelial cells. This finding suggests that ovarian cancer EV-polarized M2 macrophages promote angiogenesis through modulation of VEGF signaling, consistent with the known pro-angiogenic properties of M2 macrophages \u003csup\u003e[33-34]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe in vivo experiments provided compelling evidence for the relevance of our findings in a physiological context. By using the EV secretion inhibitor GW4869 to block endogenous tumor-derived EVs, we were able to specifically assess the effects of exogenously administered EVs. The observation that SKOV3-EV treatment led to increased tumor growth, enhanced cell proliferation, reduced apoptosis, increased M2 macrophage infiltration, and elevated CD31 expression supports a model in which ovarian cancer-derived EVs promote tumor progression through multiple mechanisms, including modulation of the tumor microenvironment.\u003c/p\u003e\n\u003cp\u003eOur findings contribute to the growing body of evidence implicating EVs in cancer progression and immune modulation. Our study comprehensively demonstrate this effect in ovarian cancer and to link it directly to enhanced angiogenesis and tumor growth. This mechanism may explain, at least in part, the high levels of M2 macrophages and CD31 expression observed in ovarian cancer tissues.\u003c/p\u003e\n\u003cp\u003eThe content of ovarian cancer-derived EVs that mediates their effect on macrophage polarization remains to be elucidated. EVs contain various bioactive molecules, including proteins, lipids, and nucleic acids (DNA, mRNAs, and microRNAs)\u003csup\u003e[35]\u003c/sup\u003e, any of which could potentially influence macrophage function. Previous studies have shown that miRNAs contained in cancer-derived EVs can modulate gene expression in recipient cells\u003csup\u003e[36]\u003c/sup\u003e. Future research should focus on identifying the specific EV cargo responsible for inducing M2 polarization and determining how these molecules alter macrophage phenotype and function.\u003c/p\u003e\n\u003cp\u003eFrom a clinical perspective, our results suggest that targeting EV-mediated communication between ovarian cancer cells and macrophages could be a promising therapeutic strategy. Inhibiting EV production or release, blocking EV uptake by macrophages, or neutralizing specific EV components could potentially reduce M2 polarization and angiogenesis, thereby limiting tumor growth. Additionally, the presence of M2 macrophages and specific EV markers in peripheral blood could serve as potential biomarkers for ovarian cancer diagnosis, prognosis, or treatment response.\u003c/p\u003e\n\u003cp\u003eSeveral limitations of our study should be acknowledged. First, although we demonstrated the effects of ovarian cancer-derived EVs on macrophage polarization and angiogenesis, we did not identify the specific molecular mediators of these effects. Second, while our in vivo model provided valuable insights, it may not fully recapitulate the complexity of the human tumor microenvironment. Third, we focused on two specific ovarian cancer cell lines, and the extent to which our findings can be generalized to other ovarian cancer subtypes remains to be determined.\u003c/p\u003e\n\u003cp\u003eIn conclusion, our study demonstrates that ovarian cancer-derived EVs promote M2 macrophage polarization, which in turn enhances angiogenesis through VEGFR upregulation, ultimately contributing to tumor progression. These findings provide novel insights into the mechanisms by which ovarian cancer cells interact with the tumor microenvironment and suggest potential therapeutic strategies targeting EV-mediated communication.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBMDMs：bone marrow-derived macrophages\u003c/p\u003e\n\u003cp\u003eEVs：Extracellular vesicles\u003c/p\u003e\n\u003cp\u003eFBS：fetal bovine serum\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHUVECs：Human umbilical vein endothelial cells\u003c/p\u003e\n\u003cp\u003eNTA：Nanoparticle tracking analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePBMCs：Peripheral Blood Mononuclear Cells\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTAMs：tumor-associated macrophages\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTEM：Transmission electron microscopy\u003c/p\u003e\n\u003cp\u003eTME：The tumor microenvironment\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eVEGFR：vascular endothelial growth factor receptor\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ovarian cancer dataset used in this study was obtained from The Cancer Genome Atlas (TCGA) public database. All data are freely available through the TCGA data portal (https://portal.gdc.cancer.gov/) or GDC Data Transfer Tool. The analyses presented in this paper are based on these publicly available data and do not require additional permission restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere is no funding to report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYi Zhang and Tiantian Dai have contributed equally to this article\u0026nbsp;and\u0026nbsp;should be considered co-first authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYi Zhang and Tiantian Dai contributed equally to this work and should be considered co-first authors. Yi Zhang performed the animal experiments. Tiantian Dai collected clinical data and analyzed the data. Dandan Chu and Wei Zhang prepared all figures. Yi Zhang and Tiantian Dai wrote this manuscript.\u0026nbsp;Xujie Wang and Jinhua Zhou conceived, designed, and supervised the project\u0026nbsp;and contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Xujie Wang or Jinhua Zhou.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Xujie Wang, [email protected]\u003c/p\u003e\n\u003cp\u003eJinhua Zhou, [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients authorized the use of their specimens by written informed consent. The protocols used in our study were approved by the Ethics Committee of Changning Maternity and Infant Health Hospital(CNFBLLKT-2023-01）. The procedures for the care and use of animals were approved by the Ethics Committee of The First Affiliated Hospital of Soochow University, and all applicable institutional and governmental regulations concerning the ethical use of animals were followed.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKonstantinopoulos PA, Matulonis UA. Clinical and translational advances in ovarian cancer therapy. Nat Cancer. 2023, 4(9): 1239-57.\u003c/li\u003e\n \u003cli\u003eHavasi A, Cainap SS, Havasi AT, et al. Ovarian Cancer-Insights into Platinum Resistance and Overcoming It. \u003cem\u003eMedicina (Kaunas)\u003c/em\u003e. 2023, 59(3).\u003c/li\u003e\n \u003cli\u003eWong-Brown MW, van der Westhuizen A, Bowden N A. Targeting DNA Repair in Ovarian Cancer Treatment Resistance. 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Macrophage diversity enhances tumor progression and metastasis. \u003cem\u003eCell\u003c/em\u003e. 2010;141(1):39-51.\u003c/li\u003e\n \u003cli\u003eZhao Y, Guo S, Deng J, et al. VEGF/VEGFR-Targeted Therapy and Immunotherapy in Non-small Cell Lung Cancer: Targeting the Tumor Microenvironment. \u003cem\u003eInt J Biol Sci\u003c/em\u003e. 2022;18(9):3845-3858.\u003c/li\u003e\n \u003cli\u003eWang H, Yung MMH, Ngan HYS, Chan KKL, Chan DW. The Impact of the Tumor Microenvironment on Macrophage Polarization in Cancer Metastatic Progression. \u003cem\u003eInt J Mol Sci\u003c/em\u003e. 2021;22(12):6560.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMinciacchi VR, Freeman MR, Di Vizio D. Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes. \u003cem\u003eSemin Cell Dev Biol\u003c/em\u003e. 2015;40:41-51.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eParthasarathy G, Hirsova P, Kostallari E, Sidhu GS, Ibrahim SH, Malhi H. Extracellular Vesicles in Hepatobiliary Health and Disease. \u003cem\u003eCompr Physiol\u003c/em\u003e. 2023;13(3):4631-4658.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Ovarian cancer, Extracellular vesicles, Tumor-associated macrophages, Angiogenesis, Tumor microenvironment","lastPublishedDoi":"10.21203/rs.3.rs-6834403/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6834403/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Ovarian cancer, among the most lethal gynecologic malignancies globally, features a tumor microenvironment crucial to disease progression. Extracellular vesicles (EVs) function as key intercellular communication mediators, though their role in ovarian cancer advancement via macrophage regulation remains inadequately characterized.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: EVs isolated from ovarian cancer cell lines (SKOV3, HO8910, ID8) underwent characterization through transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Macrophage polarization was evaluated following co-culture with cancer cells or their derived EVs. HUVEC angiogenic activity was assessed through tube formation, proliferation, and VEGFR expression analyses. In vivo studies examined tumor growth, macrophage infiltration, and angiogenesis in nude mice bearing SKOV3 tumors treated with cancer-derived EVs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Ovarian cancer patients demonstrated significantly elevated M2 macrophage proportions in peripheral blood and tumor tissues compared to controls (p\u0026lt;0.05), with increased CD31 expression correlating with poor prognosis. In vitro, cancer cells and their derived EVs induced significant M2 polarization (p\u0026lt;0.0001) and enhanced HUVEC tube formation through VEGFR upregulation. The mouse model confirmed that cancer-derived EVs significantly promoted tumor growth (p\u0026lt;0.0001), M2 macrophage infiltration, and CD31 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: This study demonstrates that ovarian cancer-derived EVs enhance tumor progression by inducing M2 macrophage polarization and stimulating angiogenesis, elucidating a novel tumor-microenvironment interaction mechanism and suggesting EV-targeted therapeutic approaches for ovarian cancer.\u003c/p\u003e","manuscriptTitle":"Extracellular Vesicles Derived from Ovarian Cancer Cells Promote Tumor Progression through M2 Macrophage Polarization and Enhanced Angiogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 11:30:44","doi":"10.21203/rs.3.rs-6834403/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-28T14:39:10+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T19:11:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T17:44:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32105582804074238618505265533011438264","date":"2025-07-16T10:49:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"322310264379315402040272805696955557626","date":"2025-07-14T19:08:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"26410769438742355262128919397317478252","date":"2025-07-14T02:22:06+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-12T18:32:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334386118142961116352281281029194566476","date":"2025-07-12T16:29:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-11T02:03:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330390147642838477115806573286722897159","date":"2025-07-10T00:38:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-09T14:08:50+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-08T11:51:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-22T22:18:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-22T22:17:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-06-06T06:56:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7ebd0734-850f-4eae-a8de-32cb82bc9177","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:04:12+00:00","versionOfRecord":{"articleIdentity":"rs-6834403","link":"https://doi.org/10.1186/s12885-026-16059-2","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2026-04-24 15:59:16","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2025-07-14 11:30:44","video":"","vorDoi":"10.1186/s12885-026-16059-2","vorDoiUrl":"https://doi.org/10.1186/s12885-026-16059-2","workflowStages":[]},"version":"v1","identity":"rs-6834403","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6834403","identity":"rs-6834403","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}