Extracellular matrix stiffening promotes ovarian cancer progression by altering exosome secretion and contents

Extracellular matrix stiffening promotes ovarian cancer progression by altering exosome secretion and contents | 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 Article Extracellular matrix stiffening promotes ovarian cancer progression by altering exosome secretion and contents Yang Yu, Ye Xu, Lu Xu, Songyan Li, Hongyan Tian, Xinhan Zhao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6700847/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Extracellular matrix (ECM) stiffening plays a pivotal role in modulating the tumour microenvironment (TME) and thus promotes oncogenic transformation and cancer progression. Recent studies have underscored the correlations between ECM stiffening and the rates of tumour progression, invasive behaviour, and overall prognosis in patients with ovarian cancer. However, the specific mechanisms by which ECM stiffening influences the migratory and invasive behaviour of ovarian cancer cells remain elusive. In this study, we demonstrated that ECM stiffening in ovarian tumours not only promotes exosome secretion from tumour cells but also alters the protein composition and levels within these exosomes, thus impacting cell migration and invasion. Specifically, as the ECM stiffens, the proliferation of ovarian cancer cells increases significantly, accompanied by a marked increase in exosome secretion. Notably, ECM stiffening can change the levels of internal proteins such as p-mTOR, p-p70s6k, p-Akt (S473), p-4EBP1 (S65), and p-S6K (T389) and thus activate the Akt‒mTOR signalling pathway in ovarian cancer cells. Similarly, ECM stiffening also alters the levels of Jagged 1/2, Sox9, Hes1, and c-Myc and thus activates the Notch signalling pathway. Collectively, these findings demonstrate that ECM stiffening can significantly promote the proliferation, migration, and invasion of ovarian cancer cells, likely through the activation of both the Akt–mTOR and Notch signalling pathways. Biological sciences/Cancer Biological sciences/Cell biology ovarian cancer extracellular matrix exosome Akt–mTOR signalling pathway Notch signalling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction The remodelling and stiffening of the extracellular matrix (ECM) are critical during the development of solid tumours, facilitating and supporting an optimal environment for tumour growth 1 . ECM stiffening has thus been regarded as an important indicator for tumour diagnosis and prognosis 2 . Research indicates that ECM stiffening not only promotes cell proliferation 3 but is also intricately linked to epithelial‒mesenchymal transformation 4 , tumour metastasis 5 and drug resistance 6 . Moreover, the increased malignancy of tumours can be partly attributable to ECM stiffening 2 , with the enhanced mechanical signal transduction induced by ECM stiffening playing an important role. Although these studies focused primarily on the direct effects of ECM stiffening on tumour cells, the distal effects of ECM stiffening, such as how it can influence the influence of exosomes, remain to be investigated. During their progression, ovarian tumours undergo angiogenesis and inflammation, leading to tissue hardening to different extents. These findings underscore the dynamic interactions among ECM stiffening, cellular activities, and other microenvironmental factors. Emerging research reveals that cell-derived vesicles (exosomes), ranging from 30–150 nm in diameter, are prevalent in extracellular fluids and act as regulators of intercellular communication, thus impacting the extracellular microenvironment 7 – 10 . Tumour-derived exosomes contribute to various aspects of tumour progression, including metastasis, drug resistance, angiogenesis, and immune regulation 11 – 13 . Therefore, understanding the effects of ECM stiffening on the properties of exosomes is crucial. In this study, we showed that ECM stiffening promotes the secretion of exosomes from ovarian cancer cells and activates the AKT–mTOR and Notch signalling pathways within these exosomes, highlighting the important role of ECM stiffening in promoting the growth, invasion and metastasis of ovarian cancer cells. 2 Methods 2.1 Cell culture and reagents The human ovarian cancer cell lines A2780 and OVCAR-3 were cultured in DMEM (Gibco, Beijing, China) supplemented with 10% (v/v) foetal bovine serum (FBS) (Gibco, Beijing, China). Antibodies against mTOR, p-mTOR, P70s6k, p-P70s6k, Akt, p-Akt, S6, p-S6, 4EBP1, p-4EBP1, Jagged 1, Jagged 2, Sox9, HES1, c-Myc, Hrs, Alix, CD63, and TSG101 were used. 2.2 Preparation of polyacrylamide gel and cell transplantation Polyacrylamide (PA) gels of varying stiffnesses were prepared on coverslips and 60 mm or 150 mm tissue culture dishes following previously described methods. Coverslips were treated with 0.1 M NaOH, followed by 0.5% 3-APTMS (Sigma, CA, USA) and 0.5% glutaraldehyde solution, and then air-dried. Acrylamide and BIS acrylamide were dissolved in PBS in certain proportions (determined by hardness) to achieve the desired stiffness, with polymerization initiated by adding 10% APS at a ratio of 1/100 (v/v) (MkBio, Shanghai, China) and TEMED at a ratio of 3/1000 (v/v) (Invitrogen, CA, USA). The mixture was applied to a coverslip and carefully covered with another coverslip treated with Rain-X (Illinois Tool Works, Chicago, USA) to make a ‘sandwich’. After solidification, the ‘sandwich’ was washed with 0.05% Sulfo-SANPAH in PBS, exposed to ultraviolet light for 5 minutes (min), and incubated with 0.1 mg/ml collagen I (in PBS) for 2 hours (h) at room temperature. After the excess collagen was removed, the gels were ready and stored in PBS at 4°C for subsequent use. 2.3 Isolation and purification of exosomes Cells cultured on PA gels were washed three times with serum-free DMEM and incubated for 48 h in the same medium. Then, the culture medium was collected, and the exosomes were isolated and purified using the 'Differential Centrifugation Method' 14 , 15 as follows: the medium was centrifuged first at 3,000 × g for 20 min to remove dead cells and debris; the resulting supernatant was then centrifuged at 10,000 × g for 40 min to remove the larger vesicles; and the resulting supernatant was then centrifuged at 120,000 × g for 2 h at 4°C. The pellet containing the exosomes was resuspended in PBS for further analysis. 2.4 Nanoparticle tracking analysis (NTA) of exosomes A total of 1 × 10 6 cells in DMEM were transplanted onto PA gels of varying stiffnesses. After 12 h, the cells were washed three times with serum-free DMEM and incubated with the same medium (i.e., serum-free DMEM) for 6 h. The culture medium was then collected and centrifuged at 3,000 × g for 20 min to remove dead cells and debris. The supernatant was further centrifuged at 10,000 × g for 40 min, and the pellets were collected, followed by NTA. 2.5 Characterization of exosomes by electron microscopy The purified exosomes in PBS were placed on nickel nets coated with Formvar carbon and immobilized with 2.5% glutaraldehyde. After being stained with 2% uranyl acetate, the samples were air-dried and examined under a JEM-1011 transmission electron microscope (TEM). 2.6 Western blot analysis After growing on PA gels for 24 h in complete medium, the cells were washed three times with serum-free DMEM and cultured in the same medium for 48 h. After being washed in cold PBS, the cells were centrifuged at 4°C for 5 min, and both the supernatant and the pellet were collected. The culture medium was used to collect the exosomes. The pellets and exosomes were then lysed in RIPA buffer at 4°C for 45 min to collect total proteins and proteins from exosomes, respectively. The protein concentrations were determined using a Bio-Rad assay kit (Bio-Rad, Hercules, CA). Protein samples were separated through 15% SDS‒PAGE and transferred to PVDF membranes (Roche, Bael, Switzerland), which were incubated in blocking buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.6], 0.1% Tween 20, and 5% nonfat dry milk) for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4°C and with secondary antibodies for 1.5 h at room temperature. The protein bands were visualized using Pierce™ ECL Western blotting Substrate (Thermo Scientific, MA, USA). 2.7 Statistical analysis All the data were obtained from experiments that were repeated at least 3 times and are expressed as the means ± SEMs. Statistical analyses were performed using SPSS 18.0, with multiple comparisons assessed by one-way ANOVA followed by Dunnett's test. Differences were considered significant at P < 0.05. 3 Results 3.1 ECM stiffening promotes the proliferation of ovarian cancer cells The behaviour of ovarian cancer cells cultured on PA gels of varying stiffnesses was assessed via optical microscopy. Compared with cells grown on 500 Pa gels, ovarian cancer cells grown on 10k Pa gels exhibited more extensive spreading and were more numerous (Fig. 1 ). These findings confirm that increased ECM stiffness is more conducive to the growth of ovarian cancer cells. 3.2 ECM stiffening promotes increased exosome secretion in ovarian cancer cells To assess the effect of ECM stiffness on exosome secretion, we fabricated two types of PA gels with distinct stiffness levels—500 Pascal (Pa, soft) and 10k Pascal (Pa, hard)—to simulate varying degrees of ECM stiffness. Exosomes were subsequently isolated from the supernatants of the cells that were grown on these PA gels and subjected to nanoparticle tracking analysis (NTA). We were able to detect exosomes in the medium as early as 4 h (Fig. 2 a). Notably, A2780 and OVCAR-3 cells cultured on 10k Pa gels exhibited a marked increase in exosome secretion compared with those cultured on 500 Pa gels (Fig. 2 a and b). These exosomes were further purified, and their size and morphology were characterized via TEM (Fig. 2 c). Furthermore, Western blot analysis revealed that, despite no change in total cellular protein levels, significant increases were noted in the levels of the exosomal marker proteins Jagged-1/2, Hrs, Alix, and CD63 in exosomes secreted by cells growing on 10k Pa gels (Fig. 2 d). These results confirmed that we had successfully isolated and purified exosomes from A2780 and OVCAR-3 cells. 3.3 ECM stiffening activates the Akt‒mTOR signalling pathway in ovarian cancer Given the significant increases in both the proliferation of ovarian cancer cells (Fig. 1 ) and exosome secretion (Fig. 2 ) under ECM stiffening conditions, we investigated the underlying mechanisms involved. The Akt–mTOR signalling pathway, which is crucial for cell growth, was the primary focus of our analysis. We determined the expression of proteins associated with the Akt‒mTOR signalling pathway in the exosomes. Our results revealed that the levels of phosphorylated mTOR (p-mTOR), p-p70s6k, p-Akt (S473), p-4EBP1 (S65), and p-S6K (T389) in exosomes from cells cultured on 10k Pa gels were significantly greater than those from cells cultured on 500 Pa gels (Fig. 3 ). These findings suggest that ECM stiffening can activate the Akt‒mTOR signalling pathway in ovarian cancer cells, thus contributing to enhanced cellular functions related to tumour cell growth. 3.4 ECM stiffening activates the Notch signalling pathway in ovarian cancer Notch proteins, an evolutionarily conserved family of type I transmembrane receptor proteins, play critical roles in tumorigenesis. Jagged 1, a key ligand of the Notch protein, activates this pathway by binding with Notch 1. Additionally, Hes1, an important effector downstream of the Notch pathway, is instrumental in maintaining the undifferentiated state of various precursor cells (Fig. 4 ). These findings indicate that the activated Notch signalling pathway in exosomes secreted by ovarian cancer cells might promote the development of ovarian cancer. Indeed, Western blotting analysis revealed significant upregulation of Jagged 1/2, Sox9, Hes1 and c-Myc, which are downstream effectors of Notch signal transduction, in exosomes from ovarian cancer cells grown on 10k Pa gels, indicating that ECM stiffening activates the Notch signalling pathway and thus promotes tumour development and progression. 4 Discussion The tumour microenvironment (TME), which encompasses elements such as blood vessels, fibroblasts, immune and inflammatory cells, signalling molecules, pH, and the ECM, plays a critical role in cancer progression. Crosstalk occurs between these components, leading to significant changes within the microenvironment. For example, studies have demonstrated that ECM stiffening can influence angiogenesis and inflammation 16 , 17 , although the underlying mechanisms remain unclear. In recent years, exosomes have been recognized as novel signal transducers within the TME. Previous studies have indicated that tumour-derived exosomes play multiple pathological roles in tumour progression, metastasis, and immune modulation 18 , 19 . However, the material basis and effects of ECM stiffening on exosomal functions are not fully understood. Our findings suggest that ECM stiffness regulates exosome secretion, suggesting that ECM stiffening could increase cancer risk and tumour progression by promoting exosome secretion and activating key proteins in the MAPK and PI3K pathways, thus modifying the TME. The Akt–mTOR signalling pathway is crucial for the survival of tumour cells. Key proteins in this pathway are overexpressed in various cancers, including breast, ovarian, and pancreatic cancers. The phosphorylation of mTORCs can activate ribosomal kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) 20 . The evidence suggests that various proto-oncogenic signalling pathways converge on mTOR, which is key for protein translation 21 . Abnormal activation of the mTOR signalling pathway can induce tumour growth, metastasis and angiogenesis 22 – 24 . Our study revealed that key proteins in the Akt‒mTOR signalling pathway in exosomes secreted by ovarian cancer cells cultured on hardened ECM were markedly activated, indicating that ECM stiffening not only increases the 'abundance' of exosomes but also enhances the 'content' of their functional proteins. In addition, we observed that ECM stiffening also activated the Notch signalling pathway in ovarian cancer. Cells cultured on stiffer gels presented increased expression levels of Notch ligands, such as Jagged 1/2. These findings suggest that ECM stiffening may promote ovarian cancer progression via Notch signalling, with increased Jagged 1/2 expression levels enhancing Notch activation in ECM stiffening-induced exosomes. This process indicates that the promotion of Notch signal transduction by ECM stiffening extends from ECM stiffening-affected cells to peripheral tissues and the surrounding microenvironment. A recent study reported that Jagged1 can induce angiogenesis through Notch signalling 25 . Given the critical role of angiogenesis in the progression of ovarian cancer, we hypothesize that Jagged 1/2 enrichment in ECM stiffening-induced exosomes promotes the progression of ovarian cancer. In conclusion, our study indicates that ECM stiffening not only increases the proliferation of ovarian cancer cells but also significantly increases the quantity of exosomes. Concurrently, we also observed that both the AKT–mTOR and Notch signalling pathways were activated in these exosomes, underscoring the significant impact of ECM stiffening on exosomes, which in turn influences the signalling and growth of ovarian cancer cells. Declarations Data availability All data generated or analyzed during this study are included in this published article. Acknowledgements This study was supported by the National Nature and Science Foundation of Jilin Province (No. YDZJ202201ZYTS165 and No. 20210101237JC). Author contributions Yang Yu: Study conception and design, acquisition of data, drafting of the article, analysis and interpretation of the data, and critical revision of the article. Ye Xu: Drafting of the article and analysis, acquisition of data, and interpretation of the data. Lu Xu: Drafting of the article and analysis and interpretation of the data. Songyan Li: Drafting of the article and analysis and interpretation of the data. Hongyan Tian: Drafting of the article and analysis and interpretation of the data. Xinhan Zhao: Study conception and design, analysis and interpretation of data, and drafting of the article. Additional information Competing interests The authors declare no competing interests. References Di Martino, J. S. et al. A tumor-derived type III collagen-rich ECM niche regulates tumor cell dormancy. Nat. Cancer 3, 90-107 (2022). Piersma, B., Hayward, M. K. & Weaver, V. M. Fibrosis and cancer: a strained relationship. Biochim. Biophys. Acta Rev. Cancer 1873, 188356 (2020). Wu, B. et al. Stiff matrix induces exosome secretion to promote tumour growth. Nat. Cell Biol. 25, 415-424 (2023). Patwardhan, S., Mahadik, P., Shetty, O. & Sen, S. ECM stiffness-tuned exosomes drive breast cancer motility through thrombospondin-1. Biomaterials 279, 121185 (2021). Nicolas-Boluda, A. et al. 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Liu, J. et al. The biology, function, and applications of exosomes in cancer. Acta Pharm. Sin. B 11, 2783-2797 (2021). Jafari, A., Babajani, A., Abdollahpour-Alitappeh, M., Ahmadi, N. & Rezaei-Tavirani, M. Exosomes and cancer: from molecular mechanisms to clinical applications. Med. Oncol. 38, 45 (2021). Mukherjee, S. et al. Unlocking exosome-based theragnostic signatures: deciphering secrets of ovarian cancer metastasis. ACS Omega 8, 36614-36627 (2023). Zhang, Q., Jeppesen, D. K., Higginbotham, J. N., Franklin, J. L. & Coffey, R. J. Comprehensive isolation of extracellular vesicles and nanoparticles. Nat. Protoc. 18, 1462-1487 (2023). Crescitelli, R., Lässer, C. & Lötvall, J. Isolation and characterization of extracellular vesicle subpopulations from tissues. Nat. Protoc. 16, 1548-1580 (2021). Yuan, T. et al. Bioprinted, spatially defined breast tumor microenvironment models of intratumoral heterogeneity and drug resistance. Trends Biotechnol. 42, 1523-1550 (2024). 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Mishra, R., Patel, H., Alanazi, S., Kilroy, M. K. & Garrett, J. T. PI3K inhibitors in cancer: clinical implications and adverse effects. Int. J. Mol. Sci. 22, 3464 (2021). Liang, J. H. et al. Dopamine signaling from ganglion cells directs layer-specific angiogenesis in the retina. Curr. Biol. 33, 3821-3834.e5 (2023). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.jpg SupplementaryFigure2.jpg SupplementaryFigure3.jpg Cite Share Download PDF Status: Posted Version 1 posted 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-6700847","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":482510884,"identity":"13fbf99d-81f2-495c-ae99-873ac28e2c62","order_by":0,"name":"Yang Yu","email":"","orcid":"","institution":"Department of Medical Oncology,The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yu","suffix":""},{"id":482510885,"identity":"3f03da9d-95ea-4066-ac86-9bae30bdd558","order_by":1,"name":"Ye Xu","email":"","orcid":"","institution":"The School of Basic Medical Sciences, Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Xu","suffix":""},{"id":482510886,"identity":"786798f2-d769-457b-be3e-233311958400","order_by":2,"name":"Lu Xu","email":"","orcid":"","institution":"The School of Basic Medical Sciences, Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Xu","suffix":""},{"id":482510887,"identity":"168d35ea-d7e0-4ab0-b135-c5624533b6af","order_by":3,"name":"Songyan Li","email":"","orcid":"","institution":"The School of Basic Medical Sciences, Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Songyan","middleName":"","lastName":"Li","suffix":""},{"id":482510888,"identity":"d733ec54-c543-49ec-a597-cc93a5f9800a","order_by":4,"name":"Hongyan Tian","email":"","orcid":"","institution":"The School of Basic Medical Sciences, Jilin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Tian","suffix":""},{"id":482510889,"identity":"64fec13d-d6bc-4350-8cb3-5fc685201dcd","order_by":5,"name":"Xinhan Zhao","email":"data:image/png;base64,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","orcid":"","institution":"Department of Medical Oncology,The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Xinhan","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2025-05-19 16:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6700847/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6700847/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86395535,"identity":"624922fe-dd01-48cc-8050-e052b5879e54","added_by":"auto","created_at":"2025-07-10 07:49:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":101617,"visible":true,"origin":"","legend":"\u003cp\u003eECM stiffening promotes the growth of A2780 and OVCAR-3 cells.\u003c/p\u003e\n\u003cp\u003eA2780 and OVCAR-3 cells were cultured in DMEM supplemented with 10% (v/v) FBS. After reaching the logarithmic growth phase, the cells were transplanted on the surface of PA gels with stiffnesses of 500 and 10k Pa.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6700847/v1/d155abcd83424b68ea511928.png"},{"id":86395537,"identity":"1394974a-ee91-4f26-a0de-42c692cf9c55","added_by":"auto","created_at":"2025-07-10 07:49:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":220908,"visible":true,"origin":"","legend":"\u003cp\u003eECM stiffening promotes exosome secretion by A2780 and OVCAR-3 cells.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea, b\u003c/strong\u003e) Nanoparticle tracking analysis (NTA) of exosomes revealed that the fold change in exosome number was greater for cells grown on 10 kPa PA gels than for those grown on 500 Pa PA gels. The data are presented as the means ± SEMs of three independent experiments; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 vs. 500 Pa. (\u003cstrong\u003ec\u003c/strong\u003e) The exosomes were imaged via TEM. (\u003cstrong\u003ed\u003c/strong\u003e) Protein samples prepared from exosomes and cells were subjected to Western blot analysis using antibodies against Jagged1/2, Hrs, Alix, and CD63. GAPDH was used as the loading control, original blots/gels are presented in Supplementary Figure 1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6700847/v1/da2619fd125a15135be4149f.png"},{"id":86396433,"identity":"85e44635-7f2d-4576-b728-1354376cf128","added_by":"auto","created_at":"2025-07-10 07:57:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":173718,"visible":true,"origin":"","legend":"\u003cp\u003eECM stiffening alters the levels of key proteins in the Akt–mTOR signalling pathway.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Protein samples prepared from exosomes under 500 and 10k Pa conditions were prepared and subjected to Western blot analysis using antibodies against mTOR, p-mTOR, p70s6k, p-p70s6k, Akt, p-Akt, S6, p-S6,4EBP1, and p-4EBP1. GAPDH was used as the loading control, original blots/gels are presented in Supplementary Figure 2.\u003c/p\u003e\n\u003cp\u003e. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of the expression levels of mTOR, p-mTOR, p70s6k, p-p70s6k, Akt, p-Akt, S6, p-S6,4EBP1, and p-4EBP1 in exosomes from OVCAR-3 cells, as shown in (\u003cstrong\u003ea\u003c/strong\u003e). The data are presented as the means ± SEMs of three independent experiments; *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 vs. 500 Pa. (\u003cstrong\u003ec\u003c/strong\u003e) Quantification of the expression levels of mTOR, p-mTOR, p70s6k, p-p70s6k, Akt, p-Akt, S6, p-S6,4EBP1, and p-4EBP1 in exosomes from A2780 cells, as shown in (\u003cstrong\u003ea\u003c/strong\u003e). The data are presented as the means ± SEMs of three independent experiments; *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 vs. 500 Pa.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6700847/v1/1caac5c5e93109834c861f0c.png"},{"id":86395561,"identity":"abe65051-3718-4f15-91f4-e71f1727f4a2","added_by":"auto","created_at":"2025-07-10 07:49:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":149803,"visible":true,"origin":"","legend":"\u003cp\u003eECM stiffening increases the levels of key proteins in the Notch signalling pathway.\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Protein samples from exosomes from OVCAR-3 cells were prepared and subjected to Western blot analysis using antibodies against Jagged-1, Sox-9, Hes1, and c-Myc. GAPDH was used as the loading control, original blots/gels are presented in Supplementary Figure 3. (\u003cstrong\u003eb\u003c/strong\u003e) Quantification of Jagged-1, Sox-9, Hes1, and c-Myc expression levels in exosomes from OVCAR-3 cells, as shown in (\u003cstrong\u003ea\u003c/strong\u003e). The data are presented as the means ± SEMs of three independent experiments; *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 vs. 500 Pa. (\u003cstrong\u003ec\u003c/strong\u003e) Protein samples from exosomes from A-2780 cells were prepared and subjected to Western blot analysis using antibodies against Jagged-2, Sox-9, Hes1, and c-Myc. GAPDH was used as the loading control, original blots/gels are presented in Supplementary Figure 3. (\u003cstrong\u003ed\u003c/strong\u003e) Quantification of Jagged-2, Sox-9, Hes1, and c-Myc expression levels in exosomes from A2780 cells, as shown in (\u003cstrong\u003ec\u003c/strong\u003e). The data are presented as the means ± SEMs of three independent experiments; *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05 vs. 500 Pa.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6700847/v1/8f98fcfb6bca56f2e3c4fa5b.png"},{"id":90162586,"identity":"7c4dbd21-cebd-48dd-b182-49052611a518","added_by":"auto","created_at":"2025-08-29 09:32:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1278279,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6700847/v1/45592317-be6d-4076-a596-5836103339fa.pdf"},{"id":86396936,"identity":"9b82bb84-64e4-4fe0-b9f7-8db84e53406c","added_by":"auto","created_at":"2025-07-10 08:05:51","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":3934963,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6700847/v1/f995701a9956bd43c739f299.jpg"},{"id":86395544,"identity":"48883ef9-efc6-4615-a80a-eea85b7bed16","added_by":"auto","created_at":"2025-07-10 07:49:51","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2459778,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6700847/v1/c5a3c08e8fd4d9cbcdc3b9fe.jpg"},{"id":86396431,"identity":"c32af7c3-eb66-496b-8f41-1e716cae1f6f","added_by":"auto","created_at":"2025-07-10 07:57:51","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1148786,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6700847/v1/e3aa6be3ee40b6920a52824f.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extracellular matrix stiffening promotes ovarian cancer progression by altering exosome secretion and contents","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe remodelling and stiffening of the extracellular matrix (ECM) are critical during the development of solid tumours, facilitating and supporting an optimal environment for tumour growth\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. ECM stiffening has thus been regarded as an important indicator for tumour diagnosis and prognosis\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Research indicates that ECM stiffening not only promotes cell proliferation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e but is also intricately linked to epithelial‒mesenchymal transformation\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, tumour metastasis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e and drug resistance\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Moreover, the increased malignancy of tumours can be partly attributable to ECM stiffening\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, with the enhanced mechanical signal transduction induced by ECM stiffening playing an important role. Although these studies focused primarily on the direct effects of ECM stiffening on tumour cells, the distal effects of ECM stiffening, such as how it can influence the influence of exosomes, remain to be investigated.\u003c/p\u003e\u003cp\u003eDuring their progression, ovarian tumours undergo angiogenesis and inflammation, leading to tissue hardening to different extents. These findings underscore the dynamic interactions among ECM stiffening, cellular activities, and other microenvironmental factors. Emerging research reveals that cell-derived vesicles (exosomes), ranging from 30\u0026ndash;150 nm in diameter, are prevalent in extracellular fluids and act as regulators of intercellular communication, thus impacting the extracellular microenvironment\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Tumour-derived exosomes contribute to various aspects of tumour progression, including metastasis, drug resistance, angiogenesis, and immune regulation\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Therefore, understanding the effects of ECM stiffening on the properties of exosomes is crucial. In this study, we showed that ECM stiffening promotes the secretion of exosomes from ovarian cancer cells and activates the AKT\u0026ndash;mTOR and Notch signalling pathways within these exosomes, highlighting the important role of ECM stiffening in promoting the growth, invasion and metastasis of ovarian cancer cells.\u003c/p\u003e"},{"header":"2 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Cell culture and reagents\u003c/h2\u003e\u003cp\u003eThe human ovarian cancer cell lines A2780 and OVCAR-3 were cultured in DMEM (Gibco, Beijing, China) supplemented with 10% (v/v) foetal bovine serum (FBS) (Gibco, Beijing, China). Antibodies against mTOR, p-mTOR, P70s6k, p-P70s6k, Akt, p-Akt, S6, p-S6, 4EBP1, p-4EBP1, Jagged 1, Jagged 2, Sox9, HES1, c-Myc, Hrs, Alix, CD63, and TSG101 were used.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of polyacrylamide gel and cell transplantation\u003c/h2\u003e\u003cp\u003ePolyacrylamide (PA) gels of varying stiffnesses were prepared on coverslips and 60 mm or 150 mm tissue culture dishes following previously described methods. Coverslips were treated with 0.1 M NaOH, followed by 0.5% 3-APTMS (Sigma, CA, USA) and 0.5% glutaraldehyde solution, and then air-dried. Acrylamide and BIS acrylamide were dissolved in PBS in certain proportions (determined by hardness) to achieve the desired stiffness, with polymerization initiated by adding 10% APS at a ratio of 1/100 (v/v) (MkBio, Shanghai, China) and TEMED at a ratio of 3/1000 (v/v) (Invitrogen, CA, USA). The mixture was applied to a coverslip and carefully covered with another coverslip treated with Rain-X (Illinois Tool Works, Chicago, USA) to make a \u0026lsquo;sandwich\u0026rsquo;. After solidification, the \u0026lsquo;sandwich\u0026rsquo; was washed with 0.05% Sulfo-SANPAH in PBS, exposed to ultraviolet light for 5 minutes (min), and incubated with 0.1 mg/ml collagen I (in PBS) for 2 hours (h) at room temperature. After the excess collagen was removed, the gels were ready and stored in PBS at 4\u0026deg;C for subsequent use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Isolation and purification of exosomes\u003c/h2\u003e\u003cp\u003eCells cultured on PA gels were washed three times with serum-free DMEM and incubated for 48 h in the same medium. Then, the culture medium was collected, and the exosomes were isolated and purified using the 'Differential Centrifugation Method'\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e as follows: the medium was centrifuged first at 3,000 \u0026times; g for 20 min to remove dead cells and debris; the resulting supernatant was then centrifuged at 10,000 \u0026times; g for 40 min to remove the larger vesicles; and the resulting supernatant was then centrifuged at 120,000 \u0026times; g for 2 h at 4\u0026deg;C. The pellet containing the exosomes was resuspended in PBS for further analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Nanoparticle tracking analysis (NTA) of exosomes\u003c/h2\u003e\u003cp\u003eA total of 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells in DMEM were transplanted onto PA gels of varying stiffnesses. After 12 h, the cells were washed three times with serum-free DMEM and incubated with the same medium (i.e., serum-free DMEM) for 6 h. The culture medium was then collected and centrifuged at 3,000 \u0026times; g for 20 min to remove dead cells and debris. The supernatant was further centrifuged at 10,000 \u0026times; g for 40 min, and the pellets were collected, followed by NTA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Characterization of exosomes by electron microscopy\u003c/h2\u003e\u003cp\u003eThe purified exosomes in PBS were placed on nickel nets coated with Formvar carbon and immobilized with 2.5% glutaraldehyde. After being stained with 2% uranyl acetate, the samples were air-dried and examined under a JEM-1011 transmission electron microscope (TEM).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Western blot analysis\u003c/h2\u003e\u003cp\u003eAfter growing on PA gels for 24 h in complete medium, the cells were washed three times with serum-free DMEM and cultured in the same medium for 48 h. After being washed in cold PBS, the cells were centrifuged at 4\u0026deg;C for 5 min, and both the supernatant and the pellet were collected. The culture medium was used to collect the exosomes. The pellets and exosomes were then lysed in RIPA buffer at 4\u0026deg;C for 45 min to collect total proteins and proteins from exosomes, respectively. The protein concentrations were determined using a Bio-Rad assay kit (Bio-Rad, Hercules, CA). Protein samples were separated through 15% SDS‒PAGE and transferred to PVDF membranes (Roche, Bael, Switzerland), which were incubated in blocking buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.6], 0.1% Tween 20, and 5% nonfat dry milk) for 2 h at room temperature, followed by incubation with primary antibodies overnight at 4\u0026deg;C and with secondary antibodies for 1.5 h at room temperature. The protein bands were visualized using Pierce\u0026trade; ECL Western blotting Substrate (Thermo Scientific, MA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll the data were obtained from experiments that were repeated at least 3 times and are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs. Statistical analyses were performed using SPSS 18.0, with multiple comparisons assessed by one-way ANOVA followed by Dunnett's test. Differences were considered significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.1 ECM stiffening promotes the proliferation of ovarian cancer cells\u003c/h2\u003e\u003cp\u003eThe behaviour of ovarian cancer cells cultured on PA gels of varying stiffnesses was assessed via optical microscopy. Compared with cells grown on 500 Pa gels, ovarian cancer cells grown on 10k Pa gels exhibited more extensive spreading and were more numerous (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings confirm that increased ECM stiffness is more conducive to the growth of ovarian cancer cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.2 ECM stiffening promotes increased exosome secretion in ovarian cancer cells\u003c/h2\u003e\u003cp\u003eTo assess the effect of ECM stiffness on exosome secretion, we fabricated two types of PA gels with distinct stiffness levels\u0026mdash;500 Pascal (Pa, soft) and 10k Pascal (Pa, hard)\u0026mdash;to simulate varying degrees of ECM stiffness. Exosomes were subsequently isolated from the supernatants of the cells that were grown on these PA gels and subjected to nanoparticle tracking analysis (NTA). We were able to detect exosomes in the medium as early as 4 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Notably, A2780 and OVCAR-3 cells cultured on 10k Pa gels exhibited a marked increase in exosome secretion compared with those cultured on 500 Pa gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and b). These exosomes were further purified, and their size and morphology were characterized via TEM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Furthermore, Western blot analysis revealed that, despite no change in total cellular protein levels, significant increases were noted in the levels of the exosomal marker proteins Jagged-1/2, Hrs, Alix, and CD63 in exosomes secreted by cells growing on 10k Pa gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). These results confirmed that we had successfully isolated and purified exosomes from A2780 and OVCAR-3 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.3 ECM stiffening activates the Akt‒mTOR signalling pathway in ovarian cancer\u003c/h2\u003e\u003cp\u003eGiven the significant increases in both the proliferation of ovarian cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and exosome secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) under ECM stiffening conditions, we investigated the underlying mechanisms involved. The Akt\u0026ndash;mTOR signalling pathway, which is crucial for cell growth, was the primary focus of our analysis. We determined the expression of proteins associated with the Akt‒mTOR signalling pathway in the exosomes. Our results revealed that the levels of phosphorylated mTOR (p-mTOR), p-p70s6k, p-Akt (S473), p-4EBP1 (S65), and p-S6K (T389) in exosomes from cells cultured on 10k Pa gels were significantly greater than those from cells cultured on 500 Pa gels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These findings suggest that ECM stiffening can activate the Akt‒mTOR signalling pathway in ovarian cancer cells, thus contributing to enhanced cellular functions related to tumour cell growth.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.4 ECM stiffening activates the Notch signalling pathway in ovarian cancer\u003c/h2\u003e\u003cp\u003eNotch proteins, an evolutionarily conserved family of type I transmembrane receptor proteins, play critical roles in tumorigenesis. Jagged 1, a key ligand of the Notch protein, activates this pathway by binding with Notch 1. Additionally, Hes1, an important effector downstream of the Notch pathway, is instrumental in maintaining the undifferentiated state of various precursor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings indicate that the activated Notch signalling pathway in exosomes secreted by ovarian cancer cells might promote the development of ovarian cancer. Indeed, Western blotting analysis revealed significant upregulation of Jagged 1/2, Sox9, Hes1 and c-Myc, which are downstream effectors of Notch signal transduction, in exosomes from ovarian cancer cells grown on 10k Pa gels, indicating that ECM stiffening activates the Notch signalling pathway and thus promotes tumour development and progression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe tumour microenvironment (TME), which encompasses elements such as blood vessels, fibroblasts, immune and inflammatory cells, signalling molecules, pH, and the ECM, plays a critical role in cancer progression. Crosstalk occurs between these components, leading to significant changes within the microenvironment. For example, studies have demonstrated that ECM stiffening can influence angiogenesis and inflammation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, although the underlying mechanisms remain unclear.\u003c/p\u003e\u003cp\u003eIn recent years, exosomes have been recognized as novel signal transducers within the TME. Previous studies have indicated that tumour-derived exosomes play multiple pathological roles in tumour progression, metastasis, and immune modulation\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. However, the material basis and effects of ECM stiffening on exosomal functions are not fully understood. Our findings suggest that ECM stiffness regulates exosome secretion, suggesting that ECM stiffening could increase cancer risk and tumour progression by promoting exosome secretion and activating key proteins in the MAPK and PI3K pathways, thus modifying the TME.\u003c/p\u003e\u003cp\u003eThe Akt\u0026ndash;mTOR signalling pathway is crucial for the survival of tumour cells. Key proteins in this pathway are overexpressed in various cancers, including breast, ovarian, and pancreatic cancers. The phosphorylation of mTORCs can activate ribosomal kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1)\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. The evidence suggests that various proto-oncogenic signalling pathways converge on mTOR, which is key for protein translation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Abnormal activation of the mTOR signalling pathway can induce tumour growth, metastasis and angiogenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Our study revealed that key proteins in the Akt‒mTOR signalling pathway in exosomes secreted by ovarian cancer cells cultured on hardened ECM were markedly activated, indicating that ECM stiffening not only increases the 'abundance' of exosomes but also enhances the 'content' of their functional proteins.\u003c/p\u003e\u003cp\u003eIn addition, we observed that ECM stiffening also activated the Notch signalling pathway in ovarian cancer. Cells cultured on stiffer gels presented increased expression levels of Notch ligands, such as Jagged 1/2. These findings suggest that ECM stiffening may promote ovarian cancer progression via Notch signalling, with increased Jagged 1/2 expression levels enhancing Notch activation in ECM stiffening-induced exosomes. This process indicates that the promotion of Notch signal transduction by ECM stiffening extends from ECM stiffening-affected cells to peripheral tissues and the surrounding microenvironment. A recent study reported that Jagged1 can induce angiogenesis through Notch signalling\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Given the critical role of angiogenesis in the progression of ovarian cancer, we hypothesize that Jagged 1/2 enrichment in ECM stiffening-induced exosomes promotes the progression of ovarian cancer.\u003c/p\u003e\u003cp\u003eIn conclusion, our study indicates that ECM stiffening not only increases the proliferation of ovarian cancer cells but also significantly increases the quantity of exosomes. Concurrently, we also observed that both the AKT\u0026ndash;mTOR and Notch signalling pathways were activated in these exosomes, underscoring the significant impact of ECM stiffening on exosomes, which in turn influences the signalling and growth of ovarian cancer cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Nature and Science Foundation of Jilin Province (No. YDZJ202201ZYTS165 and No. 20210101237JC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYang Yu: Study conception and design, acquisition of data, drafting of the article, analysis and interpretation of the data, and critical revision of the article. Ye Xu: Drafting of the article and analysis, acquisition of data, and interpretation of the data. Lu Xu: Drafting of the article and analysis and interpretation of the data. Songyan Li: Drafting of the article and analysis and interpretation of the data. Hongyan Tian: Drafting of the article and analysis and interpretation of the data. Xinhan Zhao: Study conception and design, analysis and interpretation of data, and drafting of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDi Martino, J. S. et al. A tumor-derived type III collagen-rich ECM niche regulates tumor cell dormancy. \u003cem\u003eNat. 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Biol.\u003c/em\u003e \u003cstrong\u003e33,\u003c/strong\u003e 3821-3834.e5 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ovarian cancer, extracellular matrix, exosome, Akt–mTOR signalling pathway, Notch signalling pathway","lastPublishedDoi":"10.21203/rs.3.rs-6700847/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6700847/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExtracellular matrix (ECM) stiffening plays a pivotal role in modulating the tumour microenvironment (TME) and thus promotes oncogenic transformation and cancer progression. Recent studies have underscored the correlations between ECM stiffening and the rates of tumour progression, invasive behaviour, and overall prognosis in patients with ovarian cancer. However, the specific mechanisms by which ECM stiffening influences the migratory and invasive behaviour of ovarian cancer cells remain elusive. In this study, we demonstrated that ECM stiffening in ovarian tumours not only promotes exosome secretion from tumour cells but also alters the protein composition and levels within these exosomes, thus impacting cell migration and invasion. Specifically, as the ECM stiffens, the proliferation of ovarian cancer cells increases significantly, accompanied by a marked increase in exosome secretion. Notably, ECM stiffening can change the levels of internal proteins such as p-mTOR, p-p70s6k, p-Akt (S473), p-4EBP1 (S65), and p-S6K (T389) and thus activate the Akt‒mTOR signalling pathway in ovarian cancer cells. Similarly, ECM stiffening also alters the levels of Jagged 1/2, Sox9, Hes1, and c-Myc and thus activates the Notch signalling pathway. Collectively, these findings demonstrate that ECM stiffening can significantly promote the proliferation, migration, and invasion of ovarian cancer cells, likely through the activation of both the Akt\u0026ndash;mTOR and Notch signalling pathways.\u003c/p\u003e","manuscriptTitle":"Extracellular matrix stiffening promotes ovarian cancer progression by altering exosome secretion and contents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-10 07:49:46","doi":"10.21203/rs.3.rs-6700847/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dd3e114b-3fd4-4319-8c86-f226f6ae0ac8","owner":[],"postedDate":"July 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51232122,"name":"Biological sciences/Cancer"},{"id":51232123,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-08-29T09:24:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-10 07:49:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6700847","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6700847","identity":"rs-6700847","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}