Ovarian cancer derived extracellular vesicles promote the metastasis and angiogenesis by mediating M2 macrophages polarization

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However, the underlying molecular mechanism of these complicated crosstalk are still unclear. Methods Firstly, we explored the effect of tumor-associated macrophages (TAM) on the survival prognosis among patients with ovarian cancer. Then we isolated the extracellular vesicles derived from ovarian cancer cells (OV-EVs) through ultra-centrifugation, and then analyzed the effect of OV-EVs on regulating macrophages polarization in ovarian tumor and in whole peripheral blood. Meanwhile, we explored the roles of the OV-EVs induced macrophages in tumor progression through in vitro assay and in vivo assays. Results OV-EVs could be encapsulated by the macrophages and could induce macrophages into M2 phenotype. Meanwhile, the OV-EVs induced-M2 macrophage could promote the angiogenesis as well as the cancer metastasis in vitro and in vivo. In addition, OV-EVs induced macrophage could stimulate the angiogenesis in vivo through increasing the expression level of VEGF and the expression level of VEGFR in tumor. Conclusions The present study demonstrated that OV-EVs could induce the macrophages into M2 polarization and promote the metastasis of ovarian cancer. The study provides a new insight to understand the mechanism in ovarian cancer progression. Extracellular vesicles Ovarian cancer Macrophages polarization Metastasis Angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction It was widely acknowledged that ovarian cancer is becoming one of the most common malignant cancers in female reproductive system[ 1 ]. Due to the difficulty in diagnosis at early stage, millions of ovarian cancer patients are undergoing the cancer metastasis due to lacking timely treatment. Despite ongoing efforts of screening programs for ovarian cancer, only a small part of women could be diagnosed before the cancer spreads beyond the ovaries[ 2 ], and others might undergo the metastasizes throughout the peritoneal cavity, to the omentum[ 3 ], and even to the parenchyma of the liver[ 4 ] or lung. Thus, better understanding of the mechanism of metastasis of ovarian cancer would provide new insights in ovarian cancer progression and further treatments. Increasing evidence indicate that the process of tumor microenvironment remodeling is related to the cancer progression. In the tumor microenvironment, the interplay between diverse types of cells could influence the physiological process and pathological process of cancer cells. As well acknowledged, immune cells play vital roles in cancer metastasis as well as progression. Macrophages are reported as important components in the tumor microenvironment, which are associated with the cancer progression[ 5 ], drug resistance, immune escape[ 6 ], and so on[ 7 ]. Normally, macrophages were divided into M1 population and M2 population[ 8 ], for describing the two major and opposing activities of macrophages. M1 macrophages could inhibit the cell proliferation and cause tissue damage[ 9 ], while M2 macrophages could promote cell proliferation and tissue repairment[ 10 ]. It was reported that polarized M2 macrophages could induce premetastatic niche formation and cancer metastasis[ 11 ], which involved in the EMT process[ 12 ]. What’s more, some studies reported that VEGF production by lactate-polarized macrophages was increased, resulting in a positive feedback loop that further stimulated angiogenesis[ 13 , 14 ]. Extracellular vesicles (EVs) are particles derived from diverse types of cells[ 15 ], containing diverse cargos[ 16 , 17 ]. EVs could play roles in the cell communications and complicated interplay during the biological processes[ 18 ] and the pathological processes[ 19 ]. It was widely reported that extracellular vesicles could exert in cell proliferation[ 20 ], wound healing, drug resistance and so on. Also, extracellular vesicles mediated the progression of cancers[ 21 , 22 ], including chemoresistance, metastasis, and immune evasion. However, how the ovarian cancer derived extracellular vesicles interplay with macrophages in regulating the cancer progression, and the underlying molecular mechanisms of diverse types of crosstalk are still unclear. In the present study, we aimed to explore the effect of the ovarian cancer derived extracellular vesicles (OV-EVs) on the macrophage polarization and the cancer metastasis process. What’s more, we tried to explore the mechanism of extracellular vesicles in remodeling the tumor microenvironment. 2. Methods and Materials 2.1 Cells and cell culture The ovarian cancer cell line SKOV3 cells, THP-1 cells and HUVECs (Human Umbilical Vein Endothelial Cells) were purchased from the Chinese Academy of Sciences. We cultured the ovarian cancer cells SKOV3 in DMEM (added with 10% fetal bovine serum and 1% penicillin/streptomycin). 2.2 Isolation and identification of extracellular vesicles SKOV3 cells were cultured to the confluence reach to 70%, and then were cultured with medium (including extracellular vesicles-free FBS) for 48 hours. The supernatant of SKOV3 cells was collected for further extracellular vesicles isolation. We centrifuged the collected supernatant at the speed of 300g for 20minutes and then at 10000g for 60 minutes. Then we conducted the centrifugation at 120,000g for 2hours at 4°C, to isolate the extracellular vesicles. The extracellular vesicles pellet was washed by PBS to purify the isolated extracellular vesicles. 2.3 Isolation and identification of extracellular vesicles We characterized the shape of collected extracellular vesicles through the TEM (transmission electron microscopy) (FEI Tecnai G2 Spirit Twin, Philips, NL). Meanwhile, we also characterized the dimeters and the population of collected extracellular vesicles through the NanoSight NS300 (Malvern, Amesbury, GB). In addition, we tested the expression of the extracellular vesicles markers in the collected extracellular vesicles through western blot. 2.4 Cell viability assay The viability of cells was detected via the CCK8 assays. Firstly, we seeded 5*103 HUVECs (Human Umbilical Vein Endothelial Cells) per well into the 96 well plate and incubated for 12 hours. Then we treated the HUVEC cells with blank medium, conditioned medium from SKOV3 cells, or PBS (as control), respectively. After the incubation for 48 hours, 10ul/well CCK-8 kit was used for evaluating the cell viability, which was measured by the OD450 value using Varioskan LUX microplate reader (Thermo Fisher Scientific). 2.5 Tube-formation assay The angiogenesis ability was analyzed by tube-formation assay. Firstly, we prepare the 24 well plate (30 µl Matrigel (#356234, BD Biosciences, Oxford, UK) per well). Then we collected the HUVEC cells and seeded (10 5 cells per well in 24 well plate) into the prepared well (covered with Matrigel), cultured for 3 hours. The tube formation was captured by the microscopy (Leica DMi1), images of tube morphology were obtained at ×100 magnification and numbers of meshes were counted quantified by the ImageJ software (NIH Image, Bethesda, MD). 2.6 Inducing polarization assay We cultured the THP1 cells induced with PMA (phorbol 12-myristate-13-acetate) (100ng/ml) for 24h, for inducing the THP1 cells into M0 phenotype. And we treated the M0 macrophage cells with the culture supernatant or extracellular vesicles. Then we detected the proportion of CD163 + cells via flow cytometry to quantify the percentage of macrophages polarization. All the experiments were repeated for three times. 2.7 Immunofluorescence assay The uptake process of extracellular vesicles was analyzed by the immunofluorescence assay. HUVECs cells were seeded on autoclaved slides, which was laid in the 24-well plate. Then the cells were treated as below: the supernatant from OV-EVs treated macrophages, supernatant from blank medium treated macrophages and PBS for 72 hours. Then we fixed the cells with 4% paraformaldehyde, and then we added the FITC-VEGFR antibody to stain the VEGFR for 30 minutes. Images were captured by confocal laser scanning microscopy (Leica Microsystems, Wetzlar, GER) at different laser channels. And the images were merged, all the images were evaluated and quantified via the ImageJ software (NIH Image, Bethesda, MD) by independent pathologists from our hospital. 2.8 Western Blot Extracellular vesicles were lysed in RIPA buffer. The protein concentration was determined by BCA method. Total of 20 µg protein was loaded on 10%-12% SDS-PAGE. The protein was transferred to PVDF membrane and blocked in 5% BSA for 12 h at 4°C. After washing with 1 × TBST, the membrane was incubated with following antibodies, CD63 (ab134045, 1:1000), Tsg101 (ab13e586, 1:1000) and Syntenin (ab133267, 1:1000) overnight at 4°C. After washing with 1 × TBST for three times, the membrane was incubated with HRP conjugated secondary antibodies. The membranes were visualized with enhanced chemiluminescence. 2.9 Immunohistochemistry The mice tumor tissue sections were assessed by two experienced pathologists without discrepancy in our hospital. The working dilution of anti-VEGFR antibodies (ab11939; Abcam) was 1:100. Slides were washed and incubated with the biotinylated secondary antibody (polyclonal goat anti-rabbit; Histostain-Plus IHC kit; Mingrui Biotech, Shanghai, China) for 45 min at 37°C and washed with PBS. To ensure the uniformity, all tissue sections were processed synchronically. 2.10 ELISA assay The ELISA assay was conducted to analyze the expression level of VEGF. We treated the cells with diverse medium and collected the cell supernatant for Elisa assay. Briefly, the detection of the serum levels of VEGF was conducted by the ELISA Kits (Yanhui, Shanghai, China). (***p < 0.001). 2.11 Flow cytometry Single cell suspensions were blocked with mouse FcR blocking reagent (Miltenyi Biotec) for 10 min at 4°C prior to surface staining. The following anti-mouse antibodies were used: FITC-CD11b, APC-CD163 from Biolegend. All flow cytometry data was acquired on FACS Calibur (BD, San Jose, USA) and analyzed by FlowJo V10.8 (TreeStar, Ashland, USA). 2.12 Animal assay The animal experiments were conducted in accordance with the criteria for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Obstetrics and Gynecologic hospital of Fudan University (Ethics No. SYXK2020-0032). As for the in vivo experiment, six weeks old female athymic nude mice (purchased from Jiesijie Laboratory Animal Co., Ltd., Shanghai) were divided randomly into three different sub-groups as below. Group #1 is OV-EVs group, group #2 is M0-EVs group, and group #3 is PBS group. Briefly, as depicted in Fig. 7 , SKOV3-Luciferase stable transfected cells were seeded via intraperitoneal injection at the concentration of 1.5 × 10 6 /mL per mouse at Day 0. Then intraperitoneally injections of the ovarian cancer derived extracellular vesicles (OV-EVs) (E 11 /mice per time), M0 derived extracellular vesicles (M0-EVs) (E 11 /mice per time), and PBS were conducted into group #1, group #2, and group #3 at Day 3, 6, 9, 12, 15, 18. We analyzed the metastasis ability of SKOV3-Luciferase stable transfected cells through the vivo bioluminescence imaging system. 2.13 Data analysis We conducted the survival prognosis evaluation in ovarian cancer, which based on the TCGA database ( https://tcga-data.nci.nih.gov ) and the GEPIA2 website ( http://gepia2.cancer-pku.cn/ ). We selected the “Expression Analysis”, followed by the “Survival Analysis”. We used the database by using CD115 search in “OV”, and plot as well as generate the Fig. 1 . 2.14 Statistical analysis All the experiments were repeated in triplicate, and experimental results were expressed as the means ± standard deviation (S.D). The statistical analysis was conducted via the SPSS 19.0 software and GraphPad Prism 9.3 software. 3. Results 3.1 The relationship of M2 phenotype and ovarian cancer The TCGA database were used to analyze the relationship of M2 phenotype and ovarian cancer. As shown in Fig. 1 A, we observed a higher expression of CD115 (a marker of M2 macrophages) in ovarian cancer cells. As shown in Fig. 1 B, we observed the better overall survival rate in the lower CD115 group, when compared to the higher expression of CD115 group among the ovarian cancer patients. 3.2 The effect of ovarian cancer cells in inducing macrophages polarization To explore the effect of ovarian cancer on the macrophage polarization, we evaluated the proportion of CD163+ (M2 macrophages marker) cells through the flow cytometry. As depicted in Fig. 2 A and B, the macrophages expressed high level of CD163 when treated with ovarian cancer cells derived conditioned medium. These results indicate that ovarian cancer cells derived conditioned medium could play a role in inducing the macrophages polarization into M2 phenotype. As depicted in Fig. 2 C and D, the macrophages expressed high level of CD163 when treated with ovarian cancer cells derived extracellular vesicles (OV-EVs). These results indicate that ovarian cancer cells derived extracellular vesicles could play a role in inducing the macrophages polarization into M2 phenotype. 3.3 Isolation and characterization of extracellular vesicles We isolated the extracellular vesicles from the ovarian cancer cells conditioned medium by ultracentrifuge. And we characterized the extracellular vesicles through diverse methods including TEM, NTA and Western blot. The shape of OV-EVs was measured by transmission electron microscope (Fig. 3 A). Meanwhile, the size distribution and size characterization of the OV-EVs were analyzed by nanoparticle tracking analysis (NTA) (Fig. 3 B). Furthermore, the markers of extracellular vesicles were detected through Western blot (Fig. 3 C, Figure S1 ). Thus, these above results indicate that the collected particles from the conditioned medium are extracellular vesicles. 3.4 The effect of ovarian cancer extracellular vesicles (OV-EVs) treated macrophages on inducing the angiogenesis ability To evaluate the influence of ovarian cancer derived extracellular vesicles (OV-EVs) treated macrophages on the angiogenesis ability and the proliferation ability. We conducted the cell viability assay and the tube formation assay via the HUVEC cells. As shown in Fig. 4 A and Fig. 4 B, OV-EVs induced macrophages could increase tube-formation ability, when compared with the M0-EVs induced macrophage and PBS. However, OV-EVs induced macrophages could not promote the HUVECs proliferation significantly when compared with the M0-EVs induced macrophage group and PBS group (Figure S2 A). What’s more, the OV-EVs could not promote the HUVECs proliferation either (Figure S2 B). These results indicate that ovarian cancer derived extracellular vesicles (OV-EVs) could promote the macrophage mediated angiogenesis in the cancer progression. 3.5 OV-EVs promote macrophages secretes VEGF in inducing angiogenesis To explore the mechanism of the increased the angiogenesis ability induced by OV-EVs treated macrophages, we detected the VEGFR expression on the diverse EVs treated HUVECs. As depicted in Fig. 5 (Figure S3 ), OV-EVs treated macrophages could induce the expression of VEGFR on the HUVECs. Furthermore, we detected the VEGF expression of the OV-EVs treated macrophages, M0-EVs treated macrophages and PBS treated macrophages, by the ELISA assay. As depicted in Fig. 6 A, the results revealed that VEGF expression increased significantly when treated with OV-EVs induced macrophages, when compared with the M0-EVs treated macrophages group and the PBS treated macrophages group. What’s more, as depicted in Fig. 6 B, OV-EVs induced macrophages could stimulate the expression of VEGFR (VEGF-Receptor) in HUVECs cells, while the blank medium treated macrophages and the PBS treated macrophages could not induce the VEGFR expression in HUVECs cells. As depicted in Fig. 6 C and 6 D, OV-EVs induced macrophages could stimulate the expression of VEGFR (VEGF-Receptor) in HUVECs cells, while the M0-EVs treated macrophages and the PBS treated macrophages could not induce the VEGFR expression in HUVECs cells. Above all, these results indicate that OV-EVs induced macrophages could play roles in inducing the angiogenesis process. 3.6 OV-EVs promote ovarian cancer metastasis by promoting the expression of VEGFR in vascular endothelial cell induced M2-macrophage in vivo As depicted in Fig. 7 , we conducted the animal assay. We used the SKOV3-Luciferase stable transfected cells to analyze the influence of OV-EVs induced macrophages on the metastasis ability of ovarian cancer. We divided animals into three groups (N = 3 mice per group) as below: OV-EVs group, M0-EVs group, as well as PBS group. We detected the cancer metastasis of ovarian cancer cells by measuring the luminescence of SKOV3-Luciferase cells through in vivo imaging system. As shown in Fig. 8 A and Fig. 8 B, OV-EVs could promote the metastasis of ovarian cancer. As shown in Fig. 8 C and Fig. 8 D, OV-EVs could induce the expression of VEGFR of the excised tumor tissues by the immunohistochemistry assay, the expression level of VEGFR in OV-EVs group are higher than other two groups. And the induced M2 macrophage could promote the VEGFR expression in tumor, which mediate the angiogenesis in the cancer progression. Furthermore, as depicted in Fig. 9 A, OV-EVs could induce more F4/80 + CD163 + macrophages infiltration in tumor. And as depicted in Fig. 9 B, OV-EVs could induce F4/80 + CD163 + macrophages in the whole peripheral blood. These results indicate that ovarian cancer derived extracellular vesicles (OV-EVs) could induce the macrophage into M2 phenotype, and infiltrate into the tumor. 4. Discussion Ovarian cancer is one of the intractable malignancies in female reproductive system[ 23 , 24 ]. Recently, various evidence witnessed the development of the understanding ovarian cancer progression[ 25 , 26 ]. However, it is still a huge challenge to treat the metastatic ovarian cancer. Macrophages are reported that mediate the tumor progression through diverse communications and crosstalk[ 27 , 28 ], which has been acknowledged as potential therapeutic targets[ 7 ]. In the tumor microenvironment, macrophages could secret diverse cytokines, which could contribute to maintain the inflammatory environment and promote cancer cell growth. Also, many studies have reported that tumor associated macrophages are associated to the cancer progression, including cancer proliferation[ 29 ], cancer metastasis[ 30 ], cancer drug resistance[ 31 ] and so on. Extracellular vesicles are known as membrane-bound vesicles containing different molecules that are involved in the crosstalk between diverse cells. Clinical data and experimental evidence showed that cancer cells derived extracellular vesicles could play vital roles in cancer progression. To explore the detailed mechanisms underlying the metastasis and angiogenesis, we first detected the effect of ovarian cancer on the macrophage polarization and found that ovarian cancer could induce the macrophages polarization. Then, we isolated the extracellular vesicles from ovarian cancer cells condition medium and finished the characterization by transmission electron microscope and NTA, which are widely acknowledged measurement. When treated the macrophages with extracellular vesicles derived from ovarian cancer cells, the results indicated the percentage of M2 phenotype increased. These above results are in line with the results from other groups. Then we treated the HUVEC cells with the conditioned medium of induced M2 macrophages, we found that induced M2 medium could promote the ability of tube formation. In addition, the ovarian cancer cells derived extracellular vesicles could induce the macrophage to secret the VEGF, which might explain the mechanism of increased tube-formation ability in many other studies. In animal experiment, we also found that the ovarian cancer cells derived extracellular vesicles could induce the M2 polarization in the whole peripheral blood, and the M2 macrophages infiltration in tumors. These processes could promote the metastasis of ovarian cancer, which is in line with the in vitro results. There are also limitations in the present study. Firstly, numerous ovarian cancer tissues are needed for further analysis in the further study for verifying the relationship between the macrophage infiltration and the ovarian cancer progression. Secondly, the mice were injected with SKOV3 cells, and then were injected with the OV-EVs for the same cells, which could produce OV-EVs to affect the microenvironment. What’s more, further experiments to exclude the effect from the seeded cells are needed. Thirdly, it should be explored that if there are any other types of cells in the environment to exert the similar functions to cancer progression. Above all, the present study exhibited the roles of OV-EVs in promoting macrophage polarization and angiogenesis. In addition, the study shows insights to explore the effect of cancer derived extracellular vesicles on the macrophages and the endothelia cells in the tumor micro-environment. 5. Conclusions We demonstrated that ovarian cancer derived extracellular vesicles (named “OV-EVs”) could contribute to the progression of ovarian cancer through skewing M2 macrophages polarization. Additionally, we explored that OV-EVs induced macrophages could enhance the angiogenesis ability of HUVECs. Furthermore, as the mechanism aspect, we found that OV-EVs induced macrophages could enhance the angiogenesis ability via increasing the high expression level of VEGF. Above all, our study elucidated a novel molecular mechanism between ovarian cancer, macrophages as well as the vascular endothelial cells in the tumor microenvironment. It was well acknowledged that extracellular vesicles could function as messengers between ovarian cancer and macrophages. These results provided new insights in understanding the development (including the metastasis and microenvironment remolding) of ovarian cancer as well as the theoretical basis for the further strategies against ovarian cancer. What’s more, the crosstalk between tumor cells and macrophages mediated extracellular vesicles might be a novel targeting strategy for the treatment of ovarian cancer. Declarations Data Availability Statement In this manuscript, data that support the findings of this study are available on request from the corresponding author. Inquiries should be communicated with the corresponding author, and we will consider all sufficiently specified and reasonable requests. Funding Statement The study was funded by the Scientific Research Project of Changning District Science and Technology Commission (CNKW2022Y32). Conflicts of Interest All the authors declare that there are no conflicts of interest. References Armstrong DK, Alvarez RD, Bakkum-Gamez JN, Barroilhet L, Behbakht K, Berchuck A, Chen LM, Cristea M, DeRosa M, Eisenhauer EL, et al. Ovarian Cancer, Version 2.2020, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2021;19:191–226. 10.6004/jnccn.2021.0007 . 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Additional Declarations No competing interests reported. Supplementary Files FigureS1.pdf Figure S1: The image of full membrane of Western blot for extracellular vesicles: the expression of CD63, Tsg101 and Syntenin in extracellular vesicles derived from SKOV3 ovarian cancer cells. FigureS2.pdf Figure S2: A. The proliferation of HUVECs treated by different macrophages. B. The proliferation of HUVECs treated by different EVs. Figure shows data of one out of three independent experiments done in triplicate with equivalent results. FigureS3.pdf Figure S3: The image of full membrane of Western blot for HUVECs: the expression of VEGFR in OV-EVs induced macrophages treated HUVECs, M0-EVs induced macrophages HUVECs. Cite Share Download PDF Status: Published Journal Publication published 24 Aug, 2024 Read the published version in Journal of Ovarian Research → Version 1 posted Editorial decision: Revision requested 28 May, 2024 Reviews received at journal 26 May, 2024 Reviewers agreed at journal 29 Apr, 2024 Reviews received at journal 25 Mar, 2024 Reviewers agreed at journal 21 Feb, 2024 Reviewers invited by journal 09 Feb, 2024 Editor assigned by journal 07 Feb, 2024 Submission checks completed at journal 05 Feb, 2024 First submitted to journal 05 Feb, 2024 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-3930707","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271148641,"identity":"57d84897-4b47-44ac-ba49-700b4a42b73f","order_by":0,"name":"Xue Tang","email":"","orcid":"","institution":"Maternal and Child Healthcare Hospital of Changning District Shanghai China","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Tang","suffix":""},{"id":271148642,"identity":"df9d67d3-2e35-4955-8473-a7034c4d90d7","order_by":1,"name":"Chengbin Ma","email":"","orcid":"","institution":"Maternal and Child Healthcare Hospital of Changning District Shanghai China","correspondingAuthor":false,"prefix":"","firstName":"Chengbin","middleName":"","lastName":"Ma","suffix":""},{"id":271148643,"identity":"44e73424-4d37-4d32-b0d5-f25ff2beca72","order_by":2,"name":"Qiongwei Wu","email":"","orcid":"","institution":"Maternal and Child Healthcare Hospital of Changning District Shanghai China","correspondingAuthor":false,"prefix":"","firstName":"Qiongwei","middleName":"","lastName":"Wu","suffix":""},{"id":271148644,"identity":"d4b38719-5f72-4712-889d-96f9f32c73b9","order_by":3,"name":"Meng Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYBAC9oYEBgkGBhsefvYGIrXwHABrSZOR7DlAmpbDNgY3HIjVwp6deJs35zwPww0Gxg8fc4jRwvN2szXvtts8jLMbmCVnbiNCi71E7jZpkBZmmQNszLzEaOGBaDnHwyaRQJqWAzw8xGsB+sVy7rZkHgmeg83E+YWHPXfjjbfb7Oztjzcf/PCRGC0gwMQDphgbiFQPUvuDeLWjYBSMglEwEgEAA+EzddeYoa8AAAAASUVORK5CYII=","orcid":"","institution":"Maternal and Child Healthcare Hospital of Changning District Shanghai China","correspondingAuthor":true,"prefix":"","firstName":"Meng","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-02-05 10:59:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3930707/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3930707/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13048-024-01497-y","type":"published","date":"2024-08-24T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50822867,"identity":"4e52b30a-48c3-4424-9223-c732ddac9370","added_by":"auto","created_at":"2024-02-07 22:05:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43306,"visible":true,"origin":"","legend":"\u003cp\u003eThe relationship of M2 phenotype expression and ovarian cancer.\u003c/p\u003e\n\u003cp\u003eA. The expression of CD115 in ovarian cancer tissues and normal tissues based on the TCGA dataset. B. the overall survival analysis (right) of CD115 in ovarian cancer based on the TCGA dataset. The 95% CI was added as dotted line in the figure.\u003c/p\u003e","description":"","filename":"Figure155.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/22f4d7bc0bb88c91e8be9ae5.png"},{"id":50822499,"identity":"86ac478e-8a1c-4bd4-a88d-81617f9be8eb","added_by":"auto","created_at":"2024-02-07 21:57:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":348538,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of ovarian cancer upon the polarization of macrophages.\u003c/p\u003e\n\u003cp\u003eA. The expression level of CD163 (M2 macrophage markers) in THP-1 cells after treated with PBS (Left), blank medium (Middle), and ovarian cancer conditioned medium (Right), respectively. B. Quantitative image of the CD163 expression of macrophages. C. The expression level of CD163 (M2 macrophage markers) in THP-1 cells after treated with PBS (Left), M0-EVs (Middle), and OV-EVs (Right), respectively. D. Quantitative image of the CD163 expression of macrophage cells. Figure shows data of one out of three independent experiments done in triplicate with equivalent results.\u003c/p\u003e","description":"","filename":"Figure249.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/413816db44ee315827c2df53.png"},{"id":50822501,"identity":"3ae360ad-2eb6-420c-9a82-d68f4dc257cc","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1324020,"visible":true,"origin":"","legend":"\u003cp\u003eThe characterization of extracellular vesicles.\u003c/p\u003e\n\u003cp\u003eA. The characterization result from transmission electron microscope of extracellular vesicles derived from SKOV3 ovarian cancer cells. B. The characterization result from NTA of extracellular vesicles derived from SKOV3 ovarian cancer cells. C. The characterization result from Western blot of extracellular vesicles: the expression of CD63, Tsg101 and Syntenin in extracellular vesicles derived from SKOV3 ovarian cancer cells.\u003c/p\u003e","description":"","filename":"Figure329.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/68d33b9a9e7bedfc244b4a16.png"},{"id":50822506,"identity":"858482a4-cbaf-4194-bb55-84ea219442c1","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2398826,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of ovarian cancer derived extracellular vesicles upon the angiogenesis ability.\u003c/p\u003e\n\u003cp\u003eA. Representative images of the tube formation ability of HUVECs treated with PBS (Left), M0-EVs treated macrophages (Middle), and OV-EVs treated macrophages (Right). B. Quantitative image of the formation ability of HUVECs. Figure shows data of one out of three independent experiments done in triplicate with equivalent results.\u003c/p\u003e","description":"","filename":"Figure426.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/746379dc1d46c37c566010ca.png"},{"id":50822504,"identity":"a379cbec-acfc-46a9-a645-51e1c4489ce4","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":107774,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of VEGFR in the surface of HUVECs.\u003c/p\u003e\n\u003cp\u003eThe expression level of VEGF from OV-EVs induced macrophages treated HUVECs, M0-EVs induced macrophages HUVECs.\u003c/p\u003e","description":"","filename":"Figure518.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/9d23fae4fdf260a40499a679.png"},{"id":50822510,"identity":"965e86b5-0efc-41cb-9d93-ef8b0255618b","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2113444,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression level of VEGF in macrophages and VEGFR in HUVECs after treated with OV-EVs.\u003c/p\u003e\n\u003cp\u003eA. The expression level of VEGF from OV-EVs induced macrophages, M0-EVs induced macrophages and PBS treated macrophages. B. The expression of VEGFR on the cell surface of HUVECs after treated with OV-EVs induced macrophages, M0-EVs induced macrophages, and PBS. C. Quantitative image of the VEGFR expression in HUVEC cells; D. The expression level of VEGFR in HUVECs after treated by OV-EVs treated macrophage, M0-EVs treated macrophage and PBS. Figure shows data of one out of three independent experiments done in triplicate with equivalent results.\u003c/p\u003e","description":"","filename":"Figure613.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/6221688ae1a96f64b5223bde.png"},{"id":50822509,"identity":"0264267e-6ca9-4ec6-831c-3a8cfa74411d","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":351005,"visible":true,"origin":"","legend":"\u003cp\u003eThe flow chart of the animal experiment.\u003c/p\u003e\n\u003cp\u003eA. Mice were grouped and seeded the cancer cells at Day 0, and were treated at Day 3, 6, 9, 12, 15, 18, followed the sacrificed. B. In the animal assay, SKOV3-Luciferase stable cells were seeded in nude mice. Mice were treated with OV-EVs in OV-EVs group, treated with M0-EVs in M0-EVs group, and PBS in PBS group. The metastasis level, the expression in tumor slides and the single cells derived from tumor were detected via bioluminescence imaging system, IHC and flow cytometry.\u003c/p\u003e","description":"","filename":"Figure77.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/d8edafaf12c2cb2c4301646f.png"},{"id":50822868,"identity":"73501d37-ff47-44b3-a611-fd548944ca91","added_by":"auto","created_at":"2024-02-07 22:05:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3641723,"visible":true,"origin":"","legend":"\u003cp\u003eThe influence of ovarian cancer derived extracellular vesicles induced macrophages on metastasis and angiogenesis in vivo.\u003c/p\u003e\n\u003cp\u003eA. Representative images of bioluminescent results were visualized to evaluate the metastasis of ovarian cancer after treated by OV-EVs induced macrophages, M0-EVs induced macrophages, and PBS in mice model. B. Representative images of the IHC staining for detecting the VEGFR expression in tumors (N=3). C. Representative images of IHC upon tumors treated by OV-EVs, M0-EVs and PBS in mice model; D. Quantitative image of the VEGFR expression in tumor slides (N=3).\u003c/p\u003e","description":"","filename":"Figure84.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/9df752d3ca4298d74e7cf237.png"},{"id":50822511,"identity":"690bb566-549b-4f37-856a-aa2b803509fe","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":799887,"visible":true,"origin":"","legend":"\u003cp\u003eThe percentage of induced M2 macrophages in tumor and in whole peripheral blood in mice.\u003c/p\u003e\n\u003cp\u003eA. The percentage of induced M2 macrophages in tumor single cell suspension; B; Quantitative image of the M2 macrophages in tumor single cell suspension; C: The percentage of induced M2 macrophages in whole peripheral blood; D: Quantitative image of the M2 macrophages in whole peripheral blood. Figure shows data of one out of three independent experiments done in triplicate with equivalent results.\u003c/p\u003e","description":"","filename":"Figure91.png","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/dd8240dca36c77882394fc1c.png"},{"id":63300226,"identity":"7c4217b3-f225-488b-8473-716d61adf293","added_by":"auto","created_at":"2024-08-26 16:12:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13558956,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/f5fd6f26-2f0f-46ad-83eb-ee50b9e3e78a.pdf"},{"id":50822502,"identity":"a0b46b94-2374-40f1-9f0c-7cabbd29e6e9","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":64839,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1: The image of full membrane of Western blot for extracellular vesicles: the expression of CD63, Tsg101 and Syntenin in extracellular vesicles derived from SKOV3 ovarian cancer cells.\u003c/p\u003e","description":"","filename":"FigureS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/b16421863b63568bab45a849.pdf"},{"id":50822505,"identity":"6643f6a4-e1aa-47e8-9d48-c5b74f424c2e","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":200154,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S2: A. The proliferation of HUVECs treated by different macrophages. B. The proliferation of HUVECs treated by different EVs. Figure shows data of one out of three independent experiments done in triplicate with equivalent results.\u003c/p\u003e","description":"","filename":"FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/d21e6640ecfa3bdd29b14b3f.pdf"},{"id":50822507,"identity":"713aae13-6a45-4c34-9f61-76877d04e4e9","added_by":"auto","created_at":"2024-02-07 21:57:17","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":197028,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S3: The image of full membrane of Western blot for HUVECs: the expression of VEGFR in OV-EVs induced macrophages treated HUVECs, M0-EVs induced macrophages HUVECs.\u003c/p\u003e","description":"","filename":"FigureS3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3930707/v1/80565d8ffc8d6be71fd22268.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ovarian cancer derived extracellular vesicles promote the metastasis and angiogenesis by mediating M2 macrophages polarization","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIt was widely acknowledged that ovarian cancer is becoming one of the most common malignant cancers in female reproductive system[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Due to the difficulty in diagnosis at early stage, millions of ovarian cancer patients are undergoing the cancer metastasis due to lacking timely treatment. Despite ongoing efforts of screening programs for ovarian cancer, only a small part of women could be diagnosed before the cancer spreads beyond the ovaries[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and others might undergo the metastasizes throughout the peritoneal cavity, to the omentum[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and even to the parenchyma of the liver[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] or lung. Thus, better understanding of the mechanism of metastasis of ovarian cancer would provide new insights in ovarian cancer progression and further treatments.\u003c/p\u003e \u003cp\u003eIncreasing evidence indicate that the process of tumor microenvironment remodeling is related to the cancer progression. In the tumor microenvironment, the interplay between diverse types of cells could influence the physiological process and pathological process of cancer cells. As well acknowledged, immune cells play vital roles in cancer metastasis as well as progression. Macrophages are reported as important components in the tumor microenvironment, which are associated with the cancer progression[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], drug resistance, immune escape[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and so on[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Normally, macrophages were divided into M1 population and M2 population[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], for describing the two major and opposing activities of macrophages. M1 macrophages could inhibit the cell proliferation and cause tissue damage[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], while M2 macrophages could promote cell proliferation and tissue repairment[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. It was reported that polarized M2 macrophages could induce premetastatic niche formation and cancer metastasis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], which involved in the EMT process[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. What\u0026rsquo;s more, some studies reported that VEGF production by lactate-polarized macrophages was increased, resulting in a positive feedback loop that further stimulated angiogenesis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) are particles derived from diverse types of cells[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], containing diverse cargos[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. EVs could play roles in the cell communications and complicated interplay during the biological processes[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and the pathological processes[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It was widely reported that extracellular vesicles could exert in cell proliferation[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], wound healing, drug resistance and so on. Also, extracellular vesicles mediated the progression of cancers[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], including chemoresistance, metastasis, and immune evasion. However, how the ovarian cancer derived extracellular vesicles interplay with macrophages in regulating the cancer progression, and the underlying molecular mechanisms of diverse types of crosstalk are still unclear.\u003c/p\u003e \u003cp\u003eIn the present study, we aimed to explore the effect of the ovarian cancer derived extracellular vesicles (OV-EVs) on the macrophage polarization and the cancer metastasis process. What\u0026rsquo;s more, we tried to explore the mechanism of extracellular vesicles in remodeling the tumor microenvironment.\u003c/p\u003e"},{"header":"2. Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cells and cell culture\u003c/h2\u003e \u003cp\u003eThe ovarian cancer cell line SKOV3 cells, THP-1 cells and HUVECs (Human Umbilical Vein Endothelial Cells) were purchased from the Chinese Academy of Sciences. We cultured the ovarian cancer cells SKOV3 in DMEM (added with 10% fetal bovine serum and 1% penicillin/streptomycin).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation and identification of extracellular vesicles\u003c/h2\u003e \u003cp\u003eSKOV3 cells were cultured to the confluence reach to 70%, and then were cultured with medium (including extracellular vesicles-free FBS) for 48 hours. The supernatant of SKOV3 cells was collected for further extracellular vesicles isolation. We centrifuged the collected supernatant at the speed of 300g for 20minutes and then at 10000g for 60 minutes. Then we conducted the centrifugation at 120,000g for 2hours at 4\u0026deg;C, to isolate the extracellular vesicles.\u003c/p\u003e \u003cp\u003eThe extracellular vesicles pellet was washed by PBS to purify the isolated extracellular vesicles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Isolation and identification of extracellular vesicles\u003c/h2\u003e \u003cp\u003eWe characterized the shape of collected extracellular vesicles through the TEM (transmission electron microscopy) (FEI Tecnai G2 Spirit Twin, Philips, NL). Meanwhile, we also characterized the dimeters and the population of collected extracellular vesicles through the NanoSight NS300 (Malvern, Amesbury, GB). In addition, we tested the expression of the extracellular vesicles markers in the collected extracellular vesicles through western blot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell viability assay\u003c/h2\u003e \u003cp\u003eThe viability of cells was detected via the CCK8 assays. Firstly, we seeded 5*103 HUVECs (Human Umbilical Vein Endothelial Cells) per well into the 96 well plate and incubated for 12 hours. Then we treated the HUVEC cells with blank medium, conditioned medium from SKOV3 cells, or PBS (as control), respectively. After the incubation for 48 hours, 10ul/well CCK-8 kit was used for evaluating the cell viability, which was measured by the OD450 value using Varioskan LUX microplate reader (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Tube-formation assay\u003c/h2\u003e \u003cp\u003eThe angiogenesis ability was analyzed by tube-formation assay. Firstly, we prepare the 24 well plate (30 \u0026micro;l Matrigel (#356234, BD Biosciences, Oxford, UK) per well). Then we collected the HUVEC cells and seeded (10\u003csup\u003e5\u003c/sup\u003e cells per well in 24 well plate) into the prepared well (covered with Matrigel), cultured for 3 hours. The tube formation was captured by the microscopy (Leica DMi1), images of tube morphology were obtained at \u0026times;100 magnification and numbers of meshes were counted quantified by the ImageJ software (NIH Image, Bethesda, MD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Inducing polarization assay\u003c/h2\u003e \u003cp\u003eWe cultured the THP1 cells induced with PMA (phorbol 12-myristate-13-acetate) (100ng/ml) for 24h, for inducing the THP1 cells into M0 phenotype. And we treated the M0 macrophage cells with the culture supernatant or extracellular vesicles. Then we detected the proportion of CD163\u0026thinsp;+\u0026thinsp;cells via flow cytometry to quantify the percentage of macrophages polarization. All the experiments were repeated for three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Immunofluorescence assay\u003c/h2\u003e \u003cp\u003eThe uptake process of extracellular vesicles was analyzed by the immunofluorescence assay. HUVECs cells were seeded on autoclaved slides, which was laid in the 24-well plate. Then the cells were treated as below: the supernatant from OV-EVs treated macrophages, supernatant from blank medium treated macrophages and PBS for 72 hours. Then we fixed the cells with 4% paraformaldehyde, and then we added the FITC-VEGFR antibody to stain the VEGFR for 30 minutes. Images were captured by confocal laser scanning microscopy (Leica Microsystems, Wetzlar, GER) at different laser channels. And the images were merged, all the images were evaluated and quantified via the ImageJ software (NIH Image, Bethesda, MD) by independent pathologists from our hospital.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western Blot\u003c/h2\u003e \u003cp\u003eExtracellular vesicles were lysed in RIPA buffer. The protein concentration was determined by BCA method. Total of 20 \u0026micro;g protein was loaded on 10%-12% SDS-PAGE. The protein was transferred to PVDF membrane and blocked in 5% BSA for 12 h at 4\u0026deg;C. After washing with 1 \u0026times; TBST, the membrane was incubated with following antibodies, CD63 (ab134045, 1:1000), Tsg101 (ab13e586, 1:1000) and Syntenin (ab133267, 1:1000) overnight at 4\u0026deg;C. After washing with 1 \u0026times; TBST for three times, the membrane was incubated with HRP conjugated secondary antibodies. The membranes were visualized with enhanced chemiluminescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eThe mice tumor tissue sections were assessed by two experienced pathologists without discrepancy in our hospital. The working dilution of anti-VEGFR antibodies (ab11939; Abcam) was 1:100. Slides were washed and incubated with the biotinylated secondary antibody (polyclonal goat anti-rabbit; Histostain-Plus IHC kit; Mingrui Biotech, Shanghai, China) for 45 min at 37\u0026deg;C and washed with PBS. To ensure the uniformity, all tissue sections were processed synchronically.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 ELISA assay\u003c/h2\u003e \u003cp\u003eThe ELISA assay was conducted to analyze the expression level of VEGF. We treated the cells with diverse medium and collected the cell supernatant for Elisa assay. Briefly, the detection of the serum levels of VEGF was conducted by the ELISA Kits (Yanhui, Shanghai, China). (***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Flow cytometry\u003c/h2\u003e \u003cp\u003eSingle cell suspensions were blocked with mouse FcR blocking reagent (Miltenyi Biotec) for 10 min at 4\u0026deg;C prior to surface staining. The following anti-mouse antibodies were used: FITC-CD11b, APC-CD163 from Biolegend. All flow cytometry data was acquired on FACS Calibur (BD, San Jose, USA) and analyzed by FlowJo V10.8 (TreeStar, Ashland, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Animal assay\u003c/h2\u003e \u003cp\u003e The animal experiments were conducted in accordance with the criteria for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Obstetrics and Gynecologic hospital of Fudan University (Ethics No. SYXK2020-0032). As for the in vivo experiment, six weeks old female athymic nude mice (purchased from Jiesijie Laboratory Animal Co., Ltd., Shanghai) were divided randomly into three different sub-groups as below. Group #1 is OV-EVs group, group #2 is M0-EVs group, and group #3 is PBS group. Briefly, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e7\u003c/span\u003e, SKOV3-Luciferase stable transfected cells were seeded via intraperitoneal injection at the concentration of 1.5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e/mL per mouse at Day 0. Then intraperitoneally injections of the ovarian cancer derived extracellular vesicles (OV-EVs) (E\u003csup\u003e11\u003c/sup\u003e/mice per time), M0 derived extracellular vesicles (M0-EVs) (E\u003csup\u003e11\u003c/sup\u003e/mice per time), and PBS were conducted into group #1, group #2, and group #3 at Day 3, 6, 9, 12, 15, 18. We analyzed the metastasis ability of SKOV3-Luciferase stable transfected cells through the vivo bioluminescence imaging system.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Data analysis\u003c/h2\u003e \u003cp\u003eWe conducted the survival prognosis evaluation in ovarian cancer, which based on the TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tcga-data.nci.nih.gov\u003c/span\u003e\u003cspan address=\"https://tcga-data.nci.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and the GEPIA2 website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia2.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia2.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). We selected the \u0026ldquo;Expression Analysis\u0026rdquo;, followed by the \u0026ldquo;Survival Analysis\u0026rdquo;. We used the database by using CD115 search in \u0026ldquo;OV\u0026rdquo;, and plot as well as generate the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the experiments were repeated in triplicate, and experimental results were expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (S.D). The statistical analysis was conducted via the SPSS 19.0 software and GraphPad Prism 9.3 software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The relationship of M2 phenotype and ovarian cancer\u003c/h2\u003e \u003cp\u003eThe TCGA database were used to analyze the relationship of M2 phenotype and ovarian cancer. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, we observed a higher expression of CD115 (a marker of M2 macrophages) in ovarian cancer cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, we observed the better overall survival rate in the lower CD115 group, when compared to the higher expression of CD115 group among the ovarian cancer patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The effect of ovarian cancer cells in inducing macrophages polarization\u003c/h2\u003e \u003cp\u003eTo explore the effect of ovarian cancer on the macrophage polarization, we evaluated the proportion of CD163+ (M2 macrophages marker) cells through the flow cytometry. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and B, the macrophages expressed high level of CD163 when treated with ovarian cancer cells derived conditioned medium. These results indicate that ovarian cancer cells derived conditioned medium could play a role in inducing the macrophages polarization into M2 phenotype. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D, the macrophages expressed high level of CD163 when treated with ovarian cancer cells derived extracellular vesicles (OV-EVs). These results indicate that ovarian cancer cells derived extracellular vesicles could play a role in inducing the macrophages polarization into M2 phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Isolation and characterization of extracellular vesicles\u003c/h2\u003e \u003cp\u003eWe isolated the extracellular vesicles from the ovarian cancer cells conditioned medium by ultracentrifuge. And we characterized the extracellular vesicles through diverse methods including TEM, NTA and Western blot. The shape of OV-EVs was measured by transmission electron microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Meanwhile, the size distribution and size characterization of the OV-EVs were analyzed by nanoparticle tracking analysis (NTA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Furthermore, the markers of extracellular vesicles were detected through Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Thus, these above results indicate that the collected particles from the conditioned medium are extracellular vesicles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 The effect of ovarian cancer extracellular vesicles (OV-EVs) treated macrophages on inducing the angiogenesis ability\u003c/h2\u003e \u003cp\u003eTo evaluate the influence of ovarian cancer derived extracellular vesicles (OV-EVs) treated macrophages on the angiogenesis ability and the proliferation ability. We conducted the cell viability assay and the tube formation assay via the HUVEC cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, OV-EVs induced macrophages could increase tube-formation ability, when compared with the M0-EVs induced macrophage and PBS. However, OV-EVs induced macrophages could not promote the HUVECs proliferation significantly when compared with the M0-EVs induced macrophage group and PBS group (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). What\u0026rsquo;s more, the OV-EVs could not promote the HUVECs proliferation either (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). These results indicate that ovarian cancer derived extracellular vesicles (OV-EVs) could promote the macrophage mediated angiogenesis in the cancer progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 OV-EVs promote macrophages secretes VEGF in inducing angiogenesis\u003c/h2\u003e \u003cp\u003eTo explore the mechanism of the increased the angiogenesis ability induced by OV-EVs treated macrophages, we detected the VEGFR expression on the diverse EVs treated HUVECs. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e), OV-EVs treated macrophages could induce the expression of VEGFR on the HUVECs. Furthermore, we detected the VEGF expression of the OV-EVs treated macrophages, M0-EVs treated macrophages and PBS treated macrophages, by the ELISA assay. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the results revealed that VEGF expression increased significantly when treated with OV-EVs induced macrophages, when compared with the M0-EVs treated macrophages group and the PBS treated macrophages group. What\u0026rsquo;s more, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, OV-EVs induced macrophages could stimulate the expression of VEGFR (VEGF-Receptor) in HUVECs cells, while the blank medium treated macrophages and the PBS treated macrophages could not induce the VEGFR expression in HUVECs cells. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eC and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, OV-EVs induced macrophages could stimulate the expression of VEGFR (VEGF-Receptor) in HUVECs cells, while the M0-EVs treated macrophages and the PBS treated macrophages could not induce the VEGFR expression in HUVECs cells. Above all, these results indicate that OV-EVs induced macrophages could play roles in inducing the angiogenesis process.\u003c/p\u003e \u003ch2\u003e3.6 OV-EVs promote ovarian cancer metastasis by promoting the expression of VEGFR in vascular endothelial cell induced M2-macrophage in vivo\u003c/h2\u003e \u003cp\u003eAs depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e7\u003c/span\u003e, we conducted the animal assay. We used the SKOV3-Luciferase stable transfected cells to analyze the influence of OV-EVs induced macrophages on the metastasis ability of ovarian cancer. We divided animals into three groups (N\u0026thinsp;=\u0026thinsp;3 mice per group) as below: OV-EVs group, M0-EVs group, as well as PBS group. We detected the cancer metastasis of ovarian cancer cells by measuring the luminescence of SKOV3-Luciferase cells through in vivo imaging system. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, OV-EVs could promote the metastasis of ovarian cancer. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003eD, OV-EVs could induce the expression of VEGFR of the excised tumor tissues by the immunohistochemistry assay, the expression level of VEGFR in OV-EVs group are higher than other two groups. And the induced M2 macrophage could promote the VEGFR expression in tumor, which mediate the angiogenesis in the cancer progression. Furthermore, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eA, OV-EVs could induce more F4/80\u0026thinsp;+\u0026thinsp;CD163\u0026thinsp;+\u0026thinsp;macrophages infiltration in tumor. And as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e9\u003c/span\u003eB, OV-EVs could induce F4/80\u0026thinsp;+\u0026thinsp;CD163\u0026thinsp;+\u0026thinsp;macrophages in the whole peripheral blood. These results indicate that ovarian cancer derived extracellular vesicles (OV-EVs) could induce the macrophage into M2 phenotype, and infiltrate into the tumor.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOvarian cancer is one of the intractable malignancies in female reproductive system[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Recently, various evidence witnessed the development of the understanding ovarian cancer progression[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, it is still a huge challenge to treat the metastatic ovarian cancer. Macrophages are reported that mediate the tumor progression through diverse communications and crosstalk[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which has been acknowledged as potential therapeutic targets[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the tumor microenvironment, macrophages could secret diverse cytokines, which could contribute to maintain the inflammatory environment and promote cancer cell growth. Also, many studies have reported that tumor associated macrophages are associated to the cancer progression, including cancer proliferation[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], cancer metastasis[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], cancer drug resistance[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and so on. Extracellular vesicles are known as membrane-bound vesicles containing different molecules that are involved in the crosstalk between diverse cells. Clinical data and experimental evidence showed that cancer cells derived extracellular vesicles could play vital roles in cancer progression.\u003c/p\u003e \u003cp\u003eTo explore the detailed mechanisms underlying the metastasis and angiogenesis, we first detected the effect of ovarian cancer on the macrophage polarization and found that ovarian cancer could induce the macrophages polarization. Then, we isolated the extracellular vesicles from ovarian cancer cells condition medium and finished the characterization by transmission electron microscope and NTA, which are widely acknowledged measurement. When treated the macrophages with extracellular vesicles derived from ovarian cancer cells, the results indicated the percentage of M2 phenotype increased. These above results are in line with the results from other groups. Then we treated the HUVEC cells with the conditioned medium of induced M2 macrophages, we found that induced M2 medium could promote the ability of tube formation. In addition, the ovarian cancer cells derived extracellular vesicles could induce the macrophage to secret the VEGF, which might explain the mechanism of increased tube-formation ability in many other studies. In animal experiment, we also found that the ovarian cancer cells derived extracellular vesicles could induce the M2 polarization in the whole peripheral blood, and the M2 macrophages infiltration in tumors. These processes could promote the metastasis of ovarian cancer, which is in line with the in vitro results.\u003c/p\u003e \u003cp\u003eThere are also limitations in the present study. Firstly, numerous ovarian cancer tissues are needed for further analysis in the further study for verifying the relationship between the macrophage infiltration and the ovarian cancer progression. Secondly, the mice were injected with SKOV3 cells, and then were injected with the OV-EVs for the same cells, which could produce OV-EVs to affect the microenvironment. What\u0026rsquo;s more, further experiments to exclude the effect from the seeded cells are needed. Thirdly, it should be explored that if there are any other types of cells in the environment to exert the similar functions to cancer progression.\u003c/p\u003e \u003cp\u003eAbove all, the present study exhibited the roles of OV-EVs in promoting macrophage polarization and angiogenesis. In addition, the study shows insights to explore the effect of cancer derived extracellular vesicles on the macrophages and the endothelia cells in the tumor micro-environment.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWe demonstrated that ovarian cancer derived extracellular vesicles (named \u0026ldquo;OV-EVs\u0026rdquo;) could contribute to the progression of ovarian cancer through skewing M2 macrophages polarization. Additionally, we explored that OV-EVs induced macrophages could enhance the angiogenesis ability of HUVECs. Furthermore, as the mechanism aspect, we found that OV-EVs induced macrophages could enhance the angiogenesis ability via increasing the high expression level of VEGF. Above all, our study elucidated a novel molecular mechanism between ovarian cancer, macrophages as well as the vascular endothelial cells in the tumor microenvironment. It was well acknowledged that extracellular vesicles could function as messengers between ovarian cancer and macrophages. These results provided new insights in understanding the development (including the metastasis and microenvironment remolding) of ovarian cancer as well as the theoretical basis for the further strategies against ovarian cancer. What\u0026rsquo;s more, the crosstalk between tumor cells and macrophages mediated extracellular vesicles might be a novel targeting strategy for the treatment of ovarian cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this manuscript, data that support the findings of this study are available on request from the corresponding author. Inquiries should be communicated with the corresponding author, and we will consider all sufficiently specified and reasonable requests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was funded by the Scientific Research Project of Changning District Science and Technology Commission (CNKW2022Y32).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declare that there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArmstrong DK, Alvarez RD, Bakkum-Gamez JN, Barroilhet L, Behbakht K, Berchuck A, Chen LM, Cristea M, DeRosa M, Eisenhauer EL, et al. Ovarian Cancer, Version 2.2020, NCCN Clinical Practice Guidelines in Oncology. 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Cancer Cell. 2015;27:462\u0026ndash;72. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ccell.2015.02.015\u003c/span\u003e\u003cspan address=\"10.1016/j.ccell.2015.02.015\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\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":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Extracellular vesicles, Ovarian cancer, Macrophages polarization, Metastasis, Angiogenesis","lastPublishedDoi":"10.21203/rs.3.rs-3930707/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3930707/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eExtracellular vesicles involve in the interplay between the cancer cells and other cells (including tumor associated macrophages) surrounding the tumor microenvironment, to remodel the tumor microenvironment and subsequently regulate the tumor progression. However, the underlying molecular mechanism of these complicated crosstalk are still unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFirstly, we explored the effect of tumor-associated macrophages (TAM) on the survival prognosis among patients with ovarian cancer. Then we isolated the extracellular vesicles derived from ovarian cancer cells (OV-EVs) through ultra-centrifugation, and then analyzed the effect of OV-EVs on regulating macrophages polarization in ovarian tumor and in whole peripheral blood. Meanwhile, we explored the roles of the OV-EVs induced macrophages in tumor progression through in vitro assay and in vivo assays.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOV-EVs could be encapsulated by the macrophages and could induce macrophages into M2 phenotype. Meanwhile, the OV-EVs induced-M2 macrophage could promote the angiogenesis as well as the cancer metastasis in vitro and in vivo. In addition, OV-EVs induced macrophage could stimulate the angiogenesis in vivo through increasing the expression level of VEGF and the expression level of VEGFR in tumor.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe present study demonstrated that OV-EVs could induce the macrophages into M2 polarization and promote the metastasis of ovarian cancer. The study provides a new insight to understand the mechanism in ovarian cancer progression.\u003c/p\u003e","manuscriptTitle":"Ovarian cancer derived extracellular vesicles promote the metastasis and angiogenesis by mediating M2 macrophages polarization","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 21:57:12","doi":"10.21203/rs.3.rs-3930707/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-28T20:47:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-26T20:51:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63037821365943322407641535361249103530","date":"2024-04-29T17:04:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-25T14:05:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"53e132de-c571-4952-8167-11bcc27b7cc7","date":"2024-02-21T05:47:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-09T19:09:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-08T00:36:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-05T14:20:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Ovarian Research","date":"2024-02-05T10:55:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-ovarian-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jovr","sideBox":"Learn more about [Journal of Ovarian Research](http://ovarianresearch.biomedcentral.com)","snPcode":"13048","submissionUrl":"https://submission.nature.com/new-submission/13048/3","title":"Journal of Ovarian Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"370915be-f69c-48ed-8b9b-7bce87d115ff","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-26T16:02:24+00:00","versionOfRecord":{"articleIdentity":"rs-3930707","link":"https://doi.org/10.1186/s13048-024-01497-y","journal":{"identity":"journal-of-ovarian-research","isVorOnly":false,"title":"Journal of Ovarian Research"},"publishedOn":"2024-08-24 15:57:32","publishedOnDateReadable":"August 24th, 2024"},"versionCreatedAt":"2024-02-07 21:57:12","video":"","vorDoi":"10.1186/s13048-024-01497-y","vorDoiUrl":"https://doi.org/10.1186/s13048-024-01497-y","workflowStages":[]},"version":"v1","identity":"rs-3930707","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3930707","identity":"rs-3930707","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}