Suppressive effect of dipyridamole on platelet-mediated epithelial-mesenchymal transition in breast cancer

Suppressive effect of dipyridamole on platelet-mediated epithelial-mesenchymal transition in breast cancer | 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 Suppressive effect of dipyridamole on platelet-mediated epithelial-mesenchymal transition in breast cancer Jie Xue, Hongyan Liu, Qige Xu, Yawen Guo, Haiyan Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8637051/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Epithelial-to-mesenchymal transition (EMT) is a key process implicated in cancer metastasis. Platelets are recognized as important initiators of EMT, with interactions between platelets and tumor cells contributing not only to the formation of tumor-associated thrombi but also the promotion of cancer metastasis. Dipyridamole, an antiplatelet agent commonly used in the treatment of cardiovascular and cerebrovascular diseases has recently been shown to enhance the efficacy of certain antitumor drugs. However, the standalone antitumor effects of dipyridamole, either in vitro or in vivo, have not yet been well-documented. This study aimed to investigate the impact of dipyridamole on platelet-mediated EMT in breast cancer through in vivo and in vitro models. The findings may uncover a novel antitumor mechanism of dipyridamole and suggest a potential new therapeutic approach for breast cancer. Biological sciences/Cancer Biological sciences/Cell biology Health sciences/Oncology Dipyridamole Platelet EMT Antitumor activity Breast Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Background Breast cancer is one of the leading causes of cancer-related mortality among women in China [ 1 ]. The average age of breast cancer diagnosis in Chinese women ranges from 45 to 55 years old, which is significantly younger than that in Western population [ 2 ]. Despite advances in diagnosis and treatment, the incidence and mortality rates of breast cancer continue to rise globally, including in developed and developing countries [ 3 ]. Metastasis is the primary cause of breast cancer-related death, and advanced metastatic breast cancer remains largely incurable [ 4 ]. Therefore, inhibiting breast cancer progression is crucial to reducing disease-related mortality. EMT is an important process that enhances cell motility, invasiveness, and the ability of cancer cells to disseminate within the tumor microenvironment [ 5 , 6 ]. Platelets, which play a central role in hemostasis, also contain various growth factors and cytokines, such as transforming growth factor beta (TGF-β) [ 7 ], platelet‑derived growth factor (PDGF) [ 8 – 10 ], and lysophosphatidic acid (LPA) [ 11 , 12 ], that serve as primary mediators of EMT in the tumor circulatory environment [ 13 – 15 ] (Fig. 1 ). These factors can trigger EMT through activation of signaling pathways, such as the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway [ 16 , 17 ]. Platelets interact with tumor cells after they enter the vascular system [ 18 ], and the cross-linking between platelets and tumor cells promotes the survival of tumor cells in circulation [ 19 , 20 ]. Dipyridamole is a vasodilator commonly used in combination with aspirin as an antithrombotic agent for secondary stroke prevention [ 21 ]. Its antiplatelet effects are primarily mediated through the inhibition of phosphodiesterase, which leads to elevated intracellular levels of cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP); inhibition of adenosine uptake by erythrocytes, resulting in increased extracellular adenosine levels; and suppression of thromboxane A2 (TXA2) [ 22 – 24 ]. Additionally, dipyridamole has been shown to impair cancer cell migration and metastasis by inhibiting the ERK1/2-MAPK signaling pathway [ 25 ]. However, the detailed mechanisms underlying its potential antitumor effects remain unclear. In this study, we demonstrate that platelets not only induce EMT in breast cancer cells but also promote their proliferation and migration. Notably, dipyridamole was found to counteract these effects by inhibiting platelet-induced EMT, as well as suppressing platelet-enhanced proliferation and migration of breast cancer cells. These findings suggest a novel antitumor mechanism of dipyridamole and support its potential as a therapeutic strategy for breast cancer. 2. Materials and methods 2.1 Cell culture Murine 4T1 and human MCF-7 breast cancer cell lines were used in this study. The cells were obtained from the Central Laboratory of the Affiliated Hospital of Qingdao University. All cell lines were cultured in RPMI-1640 complete medium supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. The cells were maintained in a humidified environment at 37℃ and 5% CO 2 . 2.2 Drug preparation Dipyridamole (100 mg) was dissolved in 660.55 µl of dimethylsulfoxide (DMSO) to prepare a 300 mM stock solution. The solution was aliquoted into 10 µl tubes and stored at -20℃. 2.3 Platelet preparation Platelets were isolated via secondary centrifugation. Whole blood anticoagulated with 3.2% sodium citrate was collected from healthy adult humans and rats. The blood was centrifuged at 1200 rpm for 15 min at room temperature. The light yellow upper layer and intermediate layer were transferred into new centrifuge tubes and centrifuged again at 900 rpm for 15 min. The resulting platelet-rich plasma was concentrated at 100 µl/tube and stored at -80℃. 2.4 Proliferation assay Cell proliferation activity was assessed using the Cell Counting Kit-8 (CCK-8) assay. MCF-7 and 4T1 cells (1×10 4 /well) were seeded into 96-well plates and treated with various concentrations of platelets (0.1%–5%) or dipyridamole (0, 6.25, 12.5, 25, 50, 100, and 200 µM) for 24 h. Subsequently, 10 µl of CCK-8 reagent was added to each well, followed by incubation for 2 h. Absorbance was measured at 450 nm. All experiments were performed in triplicate. 2.5 Cell migration assay MCF-7 (12×10 4 /well) and 4T1 (16×10 4 /well) cells were seeded into 6-well plates. After cells adhered, the medium was removed and cells were washed with sterile phosphate-buffered saline (PBS). A 20 µl sterile pipette tip was used to create a scratch perpendicular to the well’s surface. Treatment groups were applied according to the experimental design. Photographs were taken at 12 and 24 h under an inverted microscope. 2.6 Enzyme-linked immunosorbent assay MCF-7 and 4T1 cells were seeded into 6-well plates at densities of 12×10 4 and 16×10 4 /well, respectively. After 48 h of treatment, the culture supernatant was collected into sterile EP tubes and centrifuged at 1000 g for 20 min to remove residual platelets. According to the ELISA kit protocol, 100 µL of either the standard solution or sample was added to antigen-coated 96-well plates. Each condition was tested in triplicate. Plates were incubated at 37℃ for 90 min, followed by the addition of 100 µL biotinylated antibody working solution and incubated for 1 h at 37℃. Plates were then washed three times with 350 µL washing buffer. Subsequently, 100 µL of enzyme conjugate working solution was added, followed by a 30-minute incubation and five washes. Finally, 90 µL of substrate solution was added and incubated in the dark at 37℃ for 20 min. When a color gradient was visible in the standard wells, 50 µL of stop solution was added. Optical density was measured at 450 nm. 2.7 Fluorescence quantitative PCR MCF-7 (12×10 4 /well) and 4T1 (16×10 4 /well) cells were seeded into 6-well plates and treated for 48 h. After discarding the culture medium and washing with PBS, 1 ml of Trizol was added to each well to lyse the cells. Following the addition of 200 µL chloroform, the samples were vigorously mixed, allowed to stand for 5 min at room temperature, and centrifuged at 12,000 g at 4℃ for 15 min. The aqueous phase (500 µL) was collected add mixed with an equal volume of isopropanol. After standing for 10 min at room temperature, samples were centrifuged at 12,000 g at 4℃ for 10 min. The supernatant was discarded, and 1 ml of 75% ethanol was added to wash the RNA pellet. After centrifugation, the pellet was air-dried and dissolved in 20 µL DEPC-treated water. Complementary DNA (cDNA) was synthesized using reverse transcription. Gene expression was analyzed using real-time PCR. 2.8 Western blot experiment MCF-7 (12×10 4 /well) and 4T1 (16×10 4 /well) cells were seeded into 6-well plates and treated for 48 h. Cells were harvested, washed with PBS and lysed. The lysate was collected and centrifuged at 1000 g for five min. The cell pellet was stored at -80℃ for future analysis. Protein expression was assessed via SDS-PAGE, followed by membrane transfer, blocking, primary and secondary antibody incubation, and visualization using chemiluminescence. 2.9 Tumor model Five-week-old female BALB/c mice (18–20 g) were housed in the Central Laboratory of the Affiliated Hospital of Qingdao University. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC). 4T1 cells (2×10⁶ in 100 µL PBS) in the logarithmic growth phase were injected into the right axillary region of each mice under isoflurane anesthesia. Mice were then treated with either vehicle (DMSO-PEG-Tween 80-normal saline) or dipyridamole (30 mg/kg/day) for 14 consecutive days, starting one week post-injection. 2.10 Hematoxylin and Eosin staining Mouse tissue samples were fixed with paraformaldehyde immediately after collection. Samples were processed into paraffin or frozen sections and stained with hematoxylin and eosin (HE). The stained slides were dehydrated, sealed, and examined microscopically. 2.11 Immunohistochemistry Tissue samples were fixed in paraformaldehyde and embedded in paraffin. After antigen retrieval and blocking of endogenous peroxidase activity, slides were incubated with primary and secondary antibodies. DAB substrate was used for color development, followed by nuclear countershading, dehydration, sealing, and microscopic evaluation. 2.12 Statistical analysis Data are presented as mean ± standard deviation. Comparisons between groups were made using the independent samples t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was used. A p-value < 0.05 was considered statistically significant. 3. Results 3.1 Dipyridamole impairs cell proliferation and reduces platelet-derived TGF-β1 release in vitro To explore the effects of dipyridamole on cell proliferation, we conducted in vitro assays using murine 4T1 and human MCF-7 breast cancer cell lines. Cells in the logarithmic growth phase were seeded into 96-well plates and treated with appropriate concentrations of platelets and dipyridamole according to experimental groups. Cell viability was assessed by CCK-8 assay to determine optimal platelet and drug concentrations. We observed that increasing platelet concentrations initially promoted breast cancer cell proliferation, with a peak at 0.7%, platelet concentration for MCF-7 and 4T1 cells. Therefore, 0.7% was selected as the working platelet concentration (Fig. 2 a, 2 b). Conversely, dipyridamole exhibited a concentration-dependent inhibitory effect on cell proliferation. Although low concentrations showed minimal cytotoxicity, a significant inhibitory effect was observed at 50 µM, which was subsequently used for further experiments (Fig. 2 c). After 48 h of treatment with 0.7% platelets, the concentration of TGF-β1 in the culture supernatant of MCF-7 and 4T1 cells was significantly higher compared to the control group. However, co-treatment with 0.7% platelets and 50 µM dipyridamole led to a marked reduction in TGF-β1 levels compared to the platelet-only group (Fig. 2 d). 3.2 Dipyridamole inhibits cell migration in vitro Cell migration was evaluated using a wound-healing assay. Log-phase MCF-7 and 4T1 cells were seeded into 6-well plates and mechanically scratched to create a wound. After applying the respective treatments, wound closure was monitored and imaged at 0, 12, and 24 h under an inverted microscope. As shown in Fig. 3 a and 3 b, 0.7% platelet treatment promoted scratch closure in MCF-7 cells after 24 h, while minimal effect was observed at 12 h, possibly owing to a delayed response. In contrast, co-treatment with dipyridamole significantly reduced the scratch-healing area at 12 and 24 h compared to the platelet-only group (Fig. 3 c, 3 d). Similar results were observed in 4T1 cells, where platelet treatment enhanced migration, and the addition of dipyridamole inhibited this effect. 3.3 Dipyridamole inhibits platelet-mediated EMT in vitro To assess EMT marker expression, MCF-7 and 4T1 cells were treated for 48 hours and analyzed via quantitative real-time PCR. Treatment with 0.7% platelets upregulated N- cadherin (N-cad) mRNA and downregulated E-cadherin (E-cad) mRNA compared to the control group (Fig. 4 a, 4 b). Western blot analysis further confirmed these findings: platelet-treated cells showed increased N-cad and decreased E-cad protein expression, while co-treatment with dipyridamole resulted in reduced N-cad and elevated E-cad levels (Fig. 4 c- 4 f). 3.4 Dipyridamole suppresses tumor growth and metastasis in vivo A murine breast cancer model was established to evaluate the in vivo effects of dipyridamole. Tumor volume measurements revealed that 0.7% platelet treatment significantly increased tumor size and growth rate compared to the control group. However, co-treatment with dipyridamole markedly reduced tumor growth (Fig. 5 a). Histopathological analysis via HE staining showed that tumors in all groups were nodular, with increased mitotic activity and evidence of local invasion. In the platelet treatment group, tumor cells were more likely to infiltrate surrounding striated muscle tissue. Importantly, lung metastasis were observed in the platelet group but not in the control or dipyridamole co-treatment groups, suggesting that dipyridamole can mitigate platelet-induced tumor cell migration. No liver metastases were detected in any group (Fig. 5 b). 3.5 Dipyridamole inhibits platelet-mediated EMT in vivo Immunohistochemical staining of tumor tissues showed that platelet treatment group upregulated N-cad and downregulated E-cad protein expression compared to controls. In contrast, co-treatment with dipyridamole reversed this trend, with N-cad downregulation and E-cad upregulation, indicating suppression of platelet-mediated EMT by dipyridamole in vivo (Fig. 5 c, 5 d). 4. Discussion Metastasis is a common clinical feature and the leading cause of mortality in many cancer patients [ 26 , 27 ]. Research indicates that EMT is a key mechanism underlying tumor metastasis [ 28 ]. TGF-β, a potent inducer of EMT released upon platelet activation, has been extensively studied in this context [ 29 , 30 ]. TGF-β and bone morphogenetic proteins activate signaling pathways such as ERK, JNK, and p38 MAPK, and induce PI3K/Akt/mTOR signaling, thereby participating in EMT regulation [ 31 ]. TGF-β can also induce ShcA recruitment to phosphorylated TbRI or TbRII, initiating the ERK signaling [ 32 , 33 ]. Additionally, TGF-β receptors and receptor tyrosine kinases activate non-SMAD pathways that facilitate EMT progression through non-transcriptional effects [ 34 ]. EMT is characterized by upregulation of mesenchymal markers (e.g., vimentin, N- cad, and fibronectin) [ 35 ], and downregulation epithelial adhesion proteins such as E- cad [ 36 ], leading to cellular dissociation and cytoskeletal reorganization. Additionally, these changes enhance the migratory and invasive capacity of tumor cells [ 37 ]. N-cad, typically expressed in nerve tissues, is upregulated in various cancers and associated with poor prognosis [ 38 – 40 ]. As a mesenchymal marker of EMT, N-cad contributes to tumor progression by facilitating cell detachment and antagonizing the tumor-suppressive effects of E-cad [ 41 , 42 , 43 ]. N-cad also enhances MMP-9 expression and activates Wnt/β-catenin and FGFR/MEK/ERK pathways, promoting metastasis [ 44 , 45 ]. Moreover, N-cad stabilizes FGFR at the cell surface, sustaining FGF signaling and activating PI3K/AKT and ERK pathways [ 44 , 46 , 47 ]. In contrast, the expression of E-cad is inversely correlated with cancer progression [ 48 ]. The abnormal expression of E- cadherin is related to the invasion and progression of various cancers (including liver, breast, colorectal, and lung cancers) [ 49 – 51 ]. Activated platelets combine with tumor cells to form a primary metastatic state, which promotes the changes of activated platelets and thus induces EMT [ 52 , 53 ]. Dipyridamole, a phosphodiesterase 5 (PDE5) inhibitor [ 54 ], elevates cAMP and cGMP in endothelial and smooth muscle cells respectively [ 22 , 23 , 55 ]. Elevated cAMP/cGMP levels are associated with the inhibition of tumor growth [ 56 ], while many tumors downregulate these cyclic nucleotides through PDE overexpression [ 57 ]. Therefore, dipyridamole can increase the levels of cAMP and cGMP in cells by inhibiting PDEs [ 23 , 54 , 55 ]. However, cAMP and cGMP signaling pathways have been proved to be related to cancer progression [ 56 ]. EMT is a key step in tumor progression [ 28 , 58 ], and platelets are the primary promoter of EMT [ 59 , 60 ]. Dipyridamole can inhibit platelet aggregation by inhibiting PDE. Thus, we speculate that dipyridamole can affect platelet-induced EMT of tumor cells by affecting platelet aggregation. Our in vitro experiments showed that platelet treatment enhanced the proliferative activity of MCF-7 and 4T1 breast cancer cells in a concentration-dependent manner, peaking at 0.7% platelet concentration. Higher dipyridamole concentrations inhibited cell proliferation, with 50 µM demonstrating significant anti-proliferative effects. After platelets are co-cultured with breast cancer cells, platelets can promote the expression of TGF-β in breast cancer cells, and TGF-β is an effective EMT inducer. However, after platelet and dipyridamole co-cultured breast cancer cells, the expression of TGF-β decreased compared with platelet treatment group, indicating that dipyridamole can inhibit the expression of TGF-β in breast cancer cells induced by platelets. When platelets are co-cultured with breast cancer cells, the expression of N-cad mRNA and N-cad protein will also increase. N-cad is an interstitial marker of EMT, which is closely related to tumor progression and metastasis [ 41 , 42 ], while the expression of E-cad mRNA and E-cad protein decreases, and the expression of E-cad is usually related to cancer progression [ 48 ]. However, after platelet and dipyridamole co-cultured breast cancer cells, compared with platelet-treated group, the expression levels of N-cad mRNA and N-cad protein decreased, while the expression levels of E-cad mRNA and E-cad protein increased. The progress of EMT was mainly manifested by the up-regulation of interstitial markers, such as the up-regulation of N-cad protein [ 35 ] and the down-regulation of cell adhesion protein E-cad [ 36 ]. However, after platelet and dipyridamole co-cultured breast cancer cells, compared with platelet treatment group, the expression of these two proteins was opposite to the EMT process, which showed that dipyridamole had a certain inhibitory effect on platelet-induced EMT of breast cancer cells. The experimental results in vivo showed that compared with the control group, the mouse model of breast cancer treated with 0.7% platelet treatment group had larger tumor volume and faster growth rate. However, compared with the 0.7% platelet treatment group, the mouse model of breast cancer treated with 0.7% platelet treatment combined with 50 µM dipyridamole has a smaller tumor volume and slower growth rate. It shows that platelets can promote the growth of tumor, and promote the development of tumor to a certain extent, and dipyridamole can inhibit the growth of tumor promoted by platelets to a certain extent, which can improve the progress of tumor to some extent. After HE staining, it was found that the tumor in situ infiltrated into the surrounding normal tissues in different degrees in the control group, platelet treatment group and platelet combined with dipyridamole treatment group. After HE staining, it was found that no lung metastasis was found in the control group and dipyridamole combined with platelet group, but lung metastasis was found in platelet group, indicating that platelet can promote the migration of tumor cells to surrounding tissues, while dipyridamole can inhibit the migration of tumor cells to surrounding tissues promoted by platelet. Immunohistochemical staining showed that compared with the control group, the expression of N-cad protein was up-regulated and the expression of E-cad protein was down-regulated. Compared with the platelet treatment group, N-cad expression was downregulated in group co-treated with dipyridamole and E-cad expression was up-regulated. N-cad and E-cad proteins are important markers of epithelial mesenchymal transition, which are primarily manifested in the increase of N-cad and decrease of E-cad expression, and both of them are related to cancer progression. In vivo experiments show that platelets can promote EMT of breast cancer cells, while dipyridamole can inhibit EMT of breast cancer cells promoted by platelets. Overall, our study demonstrates that platelets promote EMT and enhance the proliferation and migration of breast cancer cells, processes central to metastasis [ 28 ]. EMT plays an important role in the invasion and spread of tumor cells. Epithelial tumor cells undergo a series of cellular changes and molecular events to gain interstitial motility and migration ability [ 6 , 61 ]. EMT process includes three steps, namely, loss of polarity, weakening of cell adhesion ability and acquisition of migration ability [ 62 – 64 ]. Dipyridamole was shown to inhibit these processes, thereby attenuating tumor progression. Despite the potential bleeding risk associated with dipyridamole, it has also been shown to enhance the cytotoxicity of several anticancer drugs in vitro. Therefore, combining dipyridamole with standard anticancer therapies may represent a promising strategy for future cancer treatment. 5. Conclusion This study investigated the effect of dipyridamole on platelet-induced EMT in breast cancer cells. Our results demonstrate that platelets promote breast cancer progression by enhancing proliferation, migration, and EMT. In vitro and in vivo experiments confirmed that dipyridamole suppresses these platelet-mediated effects, inducing reduced proliferation, inhibited EMT, and limited tumor migration. These findings suggest that dipyridamole may serve as an effective adjuvant to inhibit tumor progression driven by platelet interactions. Although further investigation is necessary to fully elucidate its mechanisms, this work provides a foundation for the future development of dipyridamole-based anticancer strategies. Abbreviations EMT, epithelial-to-mesenchymal transition; TGF-β, transforming growth factor beta; PDGF, platelet‑derived growth factor; LPA, lysophosphatidic acid; PMPs, Platelet‑derived microparticles; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; cGMP, cyclic guanine monophosphate; cAMP, cyclic adenosine monophosphate; TXA2, thromboxane A2. Declarations Ethical Approval and Consent to participate All animal experiments were conducted in accordance with the animal care guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Hospital of Qingdao University, and the assigned approval/accreditation number of the laboratory is AHQU-MAL20240619XJ. At the end of the experiment, all the mice were euthanized. They were anesthetized by inhaling 1.5%~2.5% isoflurane, and then they were killed by cervical dislocation. Consent for publication All authors consent to publication. Availability of supporting data The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. Competing interests The authors declare no competing interests. Funding This study was supported by the joint project on innovation development of the Shandong Provincial Natural Science Fund (Grant No. ZR2022LSW024) and the Clinical Medicine +X Project of Qingdao University (Grant No. 2020018). Authors' contributions XJ summarized and wrote this manuscript. LHY, XQG and GYW provided technical advice. WHY helped substantively revised it. All the authors have reviewed and agreed to submit this manuscript. Acknowledgements Not applicable. References Bray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 74 (3), 229–263 (2024). Fan, L. et al. Breast cancer in a transitional society over 18 years: trends and present status in Shanghai, China. Breast Cancer Res. Treat. 117 (2), 409–416 (2009). Fitzmaurice, C. et al. 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N-cadherin/FGFR promotes metastasis through epithelial-to-mesenchymal transition and stem/progenitor cell-like properties. Oncogene 33 (26), 3411–3421 (2014). Hollestelle, A. et al. Loss of E-cadherin is not a necessity for epithelial to mesenchymal transition in human breast cancer. Breast Cancer Res. Treat. 138 (1), 47–57 (2013). Kowalski, P. J., Rubin, M. A. & Kleer, C. G. E-cadherin expression in primary carcinomas of the breast and its distant metastases. Breast Cancer Res. 5 (6), R217–R222 (2003). Umbas, R. et al. Relation between aberrant alpha-catenin expression and loss of E-cadherin function in prostate cancer. Int. J. Cancer . 74 (4), 374–377 (1997). Christou, N. et al. E-cadherin: A potential biomarker of colorectal cancer prognosis. Oncol. Lett. 13 (6), 4571–4576 (2017). Xu, X. R., Yousef, G. M. & Ni, H. Cancer and platelet crosstalk: opportunities and challenges for aspirin and other antiplatelet agents. Blood 131 (16), 1777–1789 (2018). Ward, M. P., Mohamed, L. E. K. L. A. N., Kelly, B. M. & Bates, T. Platelets, immune cells and the coagulation cascade; friend or foe of the circulating tumour cell? Mol. Cancer . 20 (1), 59 (2021). Beavo, J. A. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol. Rev. 75 (4), 725–748 (1995). Kim, H. H. & Liao, J. K. Translational therapeutics of dipyridamole. Arterioscler. Thromb. Vasc Biol. 28 (3), s39–42 (2008). Maurice, D. H. et al. Advances in targeting cyclic nucleotide phosphodiesterases. Nat. Rev. Drug Discov . 13 (4), 290–314 (2014). Levy, I., Horvath, A., Azevedo, M., de Alexandre, R. B. & Stratakis, C. A. Phosphodiesterase function and endocrine cells: links to human disease and roles in tumor development and treatment. Curr. Opin. Pharmacol. 11 (6), 689–697 (2011). Lu, W. & Kang, Y. Epithelial-Mesenchymal Plasticity in Cancer Progression and Metastasis. Dev. Cell. 49 (3), 361–374 (2019). Xu, X. R. et al. Platelets are versatile cells: New discoveries in hemostasis, thrombosis, immune responses, tumor metastasis and beyond. Crit. Rev. Clin. Lab. Sci. 53 (6), 409–430 (2016). Lefrançais, E. et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature 544 (7648), 105–109 (2017). Bakir, B., Chiarella, A. M., Pitarresi, J. R. & Rustgi, A. K. EMT, MET, Plasticity, and Tumor Metastasis. Trends Cell. Biol. 30 (10), 764–776 (2020). Hay, E. D. An overview of epithelio-mesenchymal transformation. Acta Anat. (Basel) . 154 (1), 8–20 (1995). Nieto, M. A. Epithelial plasticity: a common theme in embryonic and cancer cells. Science 342 (6159), 1234850 (2013). Thiery, J. P., Acloque, H., Huang, R. Y. & Nieto, M. A. Epithelial-mesenchymal transitions in development and disease. Cell 139 (5), 871–890 (2009). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 26 Mar, 2026 Reviewers agreed at journal 10 Mar, 2026 Reviewers invited by journal 09 Mar, 2026 Editor assigned by journal 30 Jan, 2026 Editor invited by journal 22 Jan, 2026 Submission checks completed at journal 21 Jan, 2026 First submitted to journal 21 Jan, 2026 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8637051","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603998201,"identity":"e174c6c9-2415-4ea1-acab-7c5e5979f147","order_by":0,"name":"Jie Xue","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Xue","suffix":""},{"id":603998202,"identity":"23d9ee99-2b91-4364-9198-d9fcd80a41e7","order_by":1,"name":"Hongyan Liu","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Hongyan","middleName":"","lastName":"Liu","suffix":""},{"id":603998203,"identity":"59ff4661-4125-46aa-ab7e-54a4d5b514a6","order_by":2,"name":"Qige Xu","email":"","orcid":"","institution":"The Central Hospital of Qingdao Jiaozhou","correspondingAuthor":false,"prefix":"","firstName":"Qige","middleName":"","lastName":"Xu","suffix":""},{"id":603998204,"identity":"1b731aa5-20b2-410b-9dd9-03f95c6a8a6d","order_by":3,"name":"Yawen Guo","email":"","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Yawen","middleName":"","lastName":"Guo","suffix":""},{"id":603998205,"identity":"5c5c556a-dc9a-49da-8ebe-862feb307bce","order_by":4,"name":"Haiyan Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYDACCSBmbGCWY2NvP0CaFmM+njMJpGlJnCfhYECcDoPbPQbMhTus09skGBIYflRsI0LLnTMGzDPPpOe2STceYOw5c5uwFrMbOQbMvG2Hc9tkDiQwM7aRoCWdTSLBgDQtCcRrsb+RVsA8sy3dsA0YyAeJ8ovkjOQNzIVt1vLy7e0HH/yoIEILAwOH+W8Y8wAx6oGA/QEzkSpHwSgYBaNgpAIA+lQ6UkOFv4EAAAAASUVORK5CYII=","orcid":"","institution":"The Affiliated Hospital of Qingdao University","correspondingAuthor":true,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-01-19 08:44:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8637051/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8637051/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104554735,"identity":"0595d145-aa16-4451-9849-ab2d98721546","added_by":"auto","created_at":"2026-03-13 08:58:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1061541,"visible":true,"origin":"","legend":"\u003cp\u003eAfter platelet activation, alpha granule can release platelet-related factors, such as PDGF, TGF-β and LPA. Platelet-related factors combine with corresponding receptors to promote EMT transcription program through different signal pathways.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8637051/v1/391485dde668946aab3650b8.png"},{"id":104554728,"identity":"cae7d8e5-a66f-4e7f-9306-7839417433cb","added_by":"auto","created_at":"2026-03-13 08:57:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":446498,"visible":true,"origin":"","legend":"\u003cp\u003eDipyridamole impairs cell proliferation and release of platelet-related factors TGF-β1 in vitro. Control: control group, PLT: 0.7% platelet treatment group, PLT+DIP: 0.7% platelet combined with 50μM dipyridamole treatment group. (a) Effects of Platelets with Different Concentrations on Proliferation of Breast Cancer MCF-7 Cells. *: Compared with the control group, P \u0026lt; 0.05, * *: Compared with the control group, P \u0026lt; 0.01, the difference was statistically significant. (b) Effects of Platelets with Different Concentrations on Proliferation of Breast Cancer 4T1 Cells. *: Compared with the control group, P \u0026lt; 0.05, * *: Compared with the control group, P \u0026lt; 0.01, the difference was statistically significant. (c) Effects of Dipyridamole with Different Concentrations on Proliferation of Breast Cancer Cells. #: The control group, * *: Compared with the control group, P \u0026lt; 0.01，the difference was statistically significant. (d) Effect of dipyridamole combined with platelets on TGF-β1 content in breast cancer cell culture supernatant. #: Compared with the platelet treatment group, P \u0026lt; 0.01, * *: Compared with the control group, P \u0026lt; 0.01, the difference was statistically significant.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8637051/v1/1944f4bda3ae17f82820ef4c.png"},{"id":104554729,"identity":"5878496b-a88f-46f3-9f73-bb06fcdefa71","added_by":"auto","created_at":"2026-03-13 08:57:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1191644,"visible":true,"origin":"","legend":"\u003cp\u003eDipyridamole impairs in vitro cell migration. Control: control group, PLT: 0.7% platelet treatment group, PLT+DIP: 0.7% platelet combined with 50μM dipyridamole treatment group. (a) Effect of Dipyridamole combined with platelets on migration of breast cancer MCF-7 Cells. (b) Effect of Dipyridamole combined with platelets on migration of breast cancer 4T1 Cells. (c) Effect of dipyridamole combined with platelets on the scar healing area of breast cancer MCF-7 cells. #: Compared with the platelet treatment group, P \u0026lt; 0.01, * *: Compared with the control group, P \u0026lt; 0.01, the difference was statistically significant. (d) Effect of dipyridamole combined with platelets on the scar healing area of breast cancer 4T1 cells. #: Compared with the platelet treatment group, P \u0026lt; 0.01, * *: Compared with the control group, P \u0026lt; 0.01, the difference was statistically significant.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8637051/v1/f962979074df501134616d27.png"},{"id":104554725,"identity":"73d47e66-2869-4156-a78a-5f90413178fb","added_by":"auto","created_at":"2026-03-13 08:57:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":564029,"visible":true,"origin":"","legend":"\u003cp\u003eDipyridamole inhibits in vitro platelet-mediated EMT. Control: control group, PLT: 0.7% platelet treatment group, PLT+DIP: 0.7% platelet combined with 50μM dipyridamole treatment group. (a) Effect of dipyridamole combined with platelets on N-cad gene expression in breast cancer cells. (b) Effect of dipyridamole combined with platelets on E-cad gene expression in breast cancer cells. (c) Effects of dipyridamole combined with platelets on the expression of N-cad and E-cad proteins in breast cancer MCF-7 cells. (d) Effect of dipyridamole combined with platelets on N-cad expression in breast cancer cells. (e) Effect of dipyridamole combined with platelets on E-cad expression in breast cancer cells. (f) Effects of dipyridamole combined with platelets on the expression of N-cad and E-cad proteins in breast cancer 4T1 cells. #: Compared with the platelet treatment group, P \u0026lt; 0.01, * *: Compared with the control group, P \u0026lt; 0.01, the difference was statistically significant.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8637051/v1/7fa330f08746a47d2cbf9a48.png"},{"id":104554744,"identity":"2f17b28e-e0c3-462a-af56-d4c342bcdf9b","added_by":"auto","created_at":"2026-03-13 08:58:02","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1089163,"visible":true,"origin":"","legend":"\u003cp\u003eDipyridamole impairs the growth and migration of primary tumor in vivo and inhibits platelet-mediated EMT in vivo. Control: control group, PLT: 0.7%platelet treatment group, PLT+DIP: 0.7% platelet combined with 50μM dipyridamole treatment group. (a) Effect of dipyridamole combined with platelet on tumor size of breast cancer mouse model. (b) Effect of dipyridamole combined with platelets on the relative expression of N-cad and E-cad proteins in mouse model of breast cancer. #: Compared with the platelet treatment group, P \u0026lt; 0.01, * *: Compared with the control group, P \u0026lt; 0.01, the difference was statistically significant. (c) HE staining in mouse model of breast cancer. (d) Immunohistochemical staining of mouse model of breast cancer.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8637051/v1/c0d1d8b3263a907acb8d90db.jpeg"},{"id":104554810,"identity":"e036ffa8-a5e4-4ebe-9d71-d06a2b9e921d","added_by":"auto","created_at":"2026-03-13 08:58:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5089094,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8637051/v1/4f16b7ba-9fa0-4c07-b7e0-1ff3d27e4921.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Suppressive effect of dipyridamole on platelet-mediated epithelial-mesenchymal transition in breast cancer","fulltext":[{"header":"1. Background","content":"\u003cp\u003eBreast cancer is one of the leading causes of cancer-related mortality among women in China [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The average age of breast cancer diagnosis in Chinese women ranges from 45 to 55 years old, which is significantly younger than that in Western population [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite advances in diagnosis and treatment, the incidence and mortality rates of breast cancer continue to rise globally, including in developed and developing countries [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Metastasis is the primary cause of breast cancer-related death, and advanced metastatic breast cancer remains largely incurable [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, inhibiting breast cancer progression is crucial to reducing disease-related mortality.\u003c/p\u003e \u003cp\u003eEMT is an important process that enhances cell motility, invasiveness, and the ability of cancer cells to disseminate within the tumor microenvironment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Platelets, which play a central role in hemostasis, also contain various growth factors and cytokines, such as transforming growth factor beta (TGF-β) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], platelet‑derived growth factor (PDGF) [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and lysophosphatidic acid (LPA) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], that serve as primary mediators of EMT in the tumor circulatory environment [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These factors can trigger EMT through activation of signaling pathways, such as the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Platelets interact with tumor cells after they enter the vascular system [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and the cross-linking between platelets and tumor cells promotes the survival of tumor cells in circulation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDipyridamole is a vasodilator commonly used in combination with aspirin as an antithrombotic agent for secondary stroke prevention [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Its antiplatelet effects are primarily mediated through the inhibition of phosphodiesterase, which leads to elevated intracellular levels of cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP); inhibition of adenosine uptake by erythrocytes, resulting in increased extracellular adenosine levels; and suppression of thromboxane A2 (TXA2) [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, dipyridamole has been shown to impair cancer cell migration and metastasis by inhibiting the ERK1/2-MAPK signaling pathway [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the detailed mechanisms underlying its potential antitumor effects remain unclear.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that platelets not only induce EMT in breast cancer cells but also promote their proliferation and migration. Notably, dipyridamole was found to counteract these effects by inhibiting platelet-induced EMT, as well as suppressing platelet-enhanced proliferation and migration of breast cancer cells. These findings suggest a novel antitumor mechanism of dipyridamole and support its potential as a therapeutic strategy for breast cancer.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell culture\u003c/h2\u003e \u003cp\u003eMurine 4T1 and human MCF-7 breast cancer cell lines were used in this study. The cells were obtained from the Central Laboratory of the Affiliated Hospital of Qingdao University. All cell lines were cultured in RPMI-1640 complete medium supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. The cells were maintained in a humidified environment at 37℃ and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Drug preparation\u003c/h2\u003e \u003cp\u003eDipyridamole (100 mg) was dissolved in 660.55 \u0026micro;l of dimethylsulfoxide (DMSO) to prepare a 300 mM stock solution. The solution was aliquoted into 10 \u0026micro;l tubes and stored at -20℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Platelet preparation\u003c/h2\u003e \u003cp\u003ePlatelets were isolated via secondary centrifugation. Whole blood anticoagulated with 3.2% sodium citrate was collected from healthy adult humans and rats. The blood was centrifuged at 1200 rpm for 15 min at room temperature. The light yellow upper layer and intermediate layer were transferred into new centrifuge tubes and centrifuged again at 900 rpm for 15 min. The resulting platelet-rich plasma was concentrated at 100 \u0026micro;l/tube and stored at -80℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Proliferation assay\u003c/h2\u003e \u003cp\u003eCell proliferation activity was assessed using the Cell Counting Kit-8 (CCK-8) assay. MCF-7 and 4T1 cells (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) were seeded into 96-well plates and treated with various concentrations of platelets (0.1%\u0026ndash;5%) or dipyridamole (0, 6.25, 12.5, 25, 50, 100, and 200 \u0026micro;M) for 24 h. Subsequently, 10 \u0026micro;l of CCK-8 reagent was added to each well, followed by incubation for 2 h. Absorbance was measured at 450 nm. All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell migration assay\u003c/h2\u003e \u003cp\u003eMCF-7 (12\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) and 4T1 (16\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) cells were seeded into 6-well plates. After cells adhered, the medium was removed and cells were washed with sterile phosphate-buffered saline (PBS). A 20 \u0026micro;l sterile pipette tip was used to create a scratch perpendicular to the well\u0026rsquo;s surface. Treatment groups were applied according to the experimental design. Photographs were taken at 12 and 24 h under an inverted microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Enzyme-linked immunosorbent assay\u003c/h2\u003e \u003cp\u003eMCF-7 and 4T1 cells were seeded into 6-well plates at densities of 12\u0026times;10\u003csup\u003e4\u003c/sup\u003e and 16\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well, respectively. After 48 h of treatment, the culture supernatant was collected into sterile EP tubes and centrifuged at 1000 g for 20 min to remove residual platelets. According to the ELISA kit protocol, 100 \u0026micro;L of either the standard solution or sample was added to antigen-coated 96-well plates. Each condition was tested in triplicate. Plates were incubated at 37℃ for 90 min, followed by the addition of 100 \u0026micro;L biotinylated antibody working solution and incubated for 1 h at 37℃. Plates were then washed three times with 350 \u0026micro;L washing buffer. Subsequently, 100 \u0026micro;L of enzyme conjugate working solution was added, followed by a 30-minute incubation and five washes. Finally, 90 \u0026micro;L of substrate solution was added and incubated in the dark at 37℃ for 20 min. When a color gradient was visible in the standard wells, 50 \u0026micro;L of stop solution was added. Optical density was measured at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Fluorescence quantitative PCR\u003c/h2\u003e \u003cp\u003eMCF-7 (12\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) and 4T1 (16\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) cells were seeded into 6-well plates and treated for 48 h. After discarding the culture medium and washing with PBS, 1 ml of Trizol was added to each well to lyse the cells. Following the addition of 200 \u0026micro;L chloroform, the samples were vigorously mixed, allowed to stand for 5 min at room temperature, and centrifuged at 12,000 g at 4℃ for 15 min. The aqueous phase (500 \u0026micro;L) was collected add mixed with an equal volume of isopropanol. After standing for 10 min at room temperature, samples were centrifuged at 12,000 g at 4℃ for 10 min. The supernatant was discarded, and 1 ml of 75% ethanol was added to wash the RNA pellet. After centrifugation, the pellet was air-dried and dissolved in 20 \u0026micro;L DEPC-treated water. Complementary DNA (cDNA) was synthesized using reverse transcription. Gene expression was analyzed using real-time PCR.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot experiment\u003c/h2\u003e \u003cp\u003eMCF-7 (12\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) and 4T1 (16\u0026times;10\u003csup\u003e4\u003c/sup\u003e/well) cells were seeded into 6-well plates and treated for 48 h. Cells were harvested, washed with PBS and lysed. The lysate was collected and centrifuged at 1000 g for five min. The cell pellet was stored at -80℃ for future analysis. Protein expression was assessed via SDS-PAGE, followed by membrane transfer, blocking, primary and secondary antibody incubation, and visualization using chemiluminescence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Tumor model\u003c/h2\u003e \u003cp\u003eFive-week-old female BALB/c mice (18\u0026ndash;20 g) were housed in the Central Laboratory of the Affiliated Hospital of Qingdao University. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC). 4T1 cells (2\u0026times;10⁶ in 100 \u0026micro;L PBS) in the logarithmic growth phase were injected into the right axillary region of each mice under isoflurane anesthesia. Mice were then treated with either vehicle (DMSO-PEG-Tween 80-normal saline) or dipyridamole (30 mg/kg/day) for 14 consecutive days, starting one week post-injection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Hematoxylin and Eosin staining\u003c/h2\u003e \u003cp\u003eMouse tissue samples were fixed with paraformaldehyde immediately after collection. Samples were processed into paraffin or frozen sections and stained with hematoxylin and eosin (HE). The stained slides were dehydrated, sealed, and examined microscopically.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eTissue samples were fixed in paraformaldehyde and embedded in paraffin. After antigen retrieval and blocking of endogenous peroxidase activity, slides were incubated with primary and secondary antibodies. DAB substrate was used for color development, followed by nuclear countershading, dehydration, sealing, and microscopic evaluation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons between groups were made using the independent samples t-test. For comparisons among multiple groups, one-way analysis of variance (ANOVA) was used. A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Dipyridamole impairs cell proliferation and reduces platelet-derived TGF-β1 release in vitro\u003c/h2\u003e \u003cp\u003eTo explore the effects of dipyridamole on cell proliferation, we conducted in vitro assays using murine 4T1 and human MCF-7 breast cancer cell lines. Cells in the logarithmic growth phase were seeded into 96-well plates and treated with appropriate concentrations of platelets and dipyridamole according to experimental groups. Cell viability was assessed by CCK-8 assay to determine optimal platelet and drug concentrations. We observed that increasing platelet concentrations initially promoted breast cancer cell proliferation, with a peak at 0.7%, platelet concentration for MCF-7 and 4T1 cells. Therefore, 0.7% was selected as the working platelet concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Conversely, dipyridamole exhibited a concentration-dependent inhibitory effect on cell proliferation. Although low concentrations showed minimal cytotoxicity, a significant inhibitory effect was observed at 50 \u0026micro;M, which was subsequently used for further experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). After 48 h of treatment with 0.7% platelets, the concentration of TGF-β1 in the culture supernatant of MCF-7 and 4T1 cells was significantly higher compared to the control group. However, co-treatment with 0.7% platelets and 50 \u0026micro;M dipyridamole led to a marked reduction in TGF-β1 levels compared to the platelet-only group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Dipyridamole inhibits cell migration in vitro\u003c/h2\u003e \u003cp\u003eCell migration was evaluated using a wound-healing assay. Log-phase MCF-7 and 4T1 cells were seeded into 6-well plates and mechanically scratched to create a wound. After applying the respective treatments, wound closure was monitored and imaged at 0, 12, and 24 h under an inverted microscope. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, 0.7% platelet treatment promoted scratch closure in MCF-7 cells after 24 h, while minimal effect was observed at 12 h, possibly owing to a delayed response. In contrast, co-treatment with dipyridamole significantly reduced the scratch-healing area at 12 and 24 h compared to the platelet-only group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). Similar results were observed in 4T1 cells, where platelet treatment enhanced migration, and the addition of dipyridamole inhibited this effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Dipyridamole inhibits platelet-mediated EMT in vitro\u003c/h2\u003e \u003cp\u003eTo assess EMT marker expression, MCF-7 and 4T1 cells were treated for 48 hours and analyzed via quantitative real-time PCR. Treatment with 0.7% platelets upregulated N- cadherin (N-cad) mRNA and downregulated E-cadherin (E-cad) mRNA compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Western blot analysis further confirmed these findings: platelet-treated cells showed increased N-cad and decreased E-cad protein expression, while co-treatment with dipyridamole resulted in reduced N-cad and elevated E-cad levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Dipyridamole suppresses tumor growth and metastasis in vivo\u003c/h2\u003e \u003cp\u003eA murine breast cancer model was established to evaluate the in vivo effects of dipyridamole. Tumor volume measurements revealed that 0.7% platelet treatment significantly increased tumor size and growth rate compared to the control group. However, co-treatment with dipyridamole markedly reduced tumor growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Histopathological analysis via HE staining showed that tumors in all groups were nodular, with increased mitotic activity and evidence of local invasion. In the platelet treatment group, tumor cells were more likely to infiltrate surrounding striated muscle tissue. Importantly, lung metastasis were observed in the platelet group but not in the control or dipyridamole co-treatment groups, suggesting that dipyridamole can mitigate platelet-induced tumor cell migration. No liver metastases were detected in any group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Dipyridamole inhibits platelet-mediated EMT in vivo\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining of tumor tissues showed that platelet treatment group upregulated N-cad and downregulated E-cad protein expression compared to controls. In contrast, co-treatment with dipyridamole reversed this trend, with N-cad downregulation and E-cad upregulation, indicating suppression of platelet-mediated EMT by dipyridamole in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMetastasis is a common clinical feature and the leading cause of mortality in many cancer patients [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Research indicates that EMT is a key mechanism underlying tumor metastasis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. TGF-β, a potent inducer of EMT released upon platelet activation, has been extensively studied in this context [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. TGF-β and bone morphogenetic proteins activate signaling pathways such as ERK, JNK, and p38 MAPK, and induce PI3K/Akt/mTOR signaling, thereby participating in EMT regulation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. TGF-β can also induce ShcA recruitment to phosphorylated TbRI or TbRII, initiating the ERK signaling [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, TGF-β receptors and receptor tyrosine kinases activate non-SMAD pathways that facilitate EMT progression through non-transcriptional effects [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. EMT is characterized by upregulation of mesenchymal markers (e.g., vimentin, N- cad, and fibronectin) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and downregulation epithelial adhesion proteins such as E- cad [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], leading to cellular dissociation and cytoskeletal reorganization. Additionally, these changes enhance the migratory and invasive capacity of tumor cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. N-cad, typically expressed in nerve tissues, is upregulated in various cancers and associated with poor prognosis [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. As a mesenchymal marker of EMT, N-cad contributes to tumor progression by facilitating cell detachment and antagonizing the tumor-suppressive effects of E-cad [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. N-cad also enhances MMP-9 expression and activates Wnt/β-catenin and FGFR/MEK/ERK pathways, promoting metastasis [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Moreover, N-cad stabilizes FGFR at the cell surface, sustaining FGF signaling and activating PI3K/AKT and ERK pathways [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In contrast, the expression of E-cad is inversely correlated with cancer progression [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The abnormal expression of E- cadherin is related to the invasion and progression of various cancers (including liver, breast, colorectal, and lung cancers) [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Activated platelets combine with tumor cells to form a primary metastatic state, which promotes the changes of activated platelets and thus induces EMT [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDipyridamole, a phosphodiesterase 5 (PDE5) inhibitor [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], elevates cAMP and cGMP in endothelial and smooth muscle cells respectively [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Elevated cAMP/cGMP levels are associated with the inhibition of tumor growth [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], while many tumors downregulate these cyclic nucleotides through PDE overexpression [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Therefore, dipyridamole can increase the levels of cAMP and cGMP in cells by inhibiting PDEs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, cAMP and cGMP signaling pathways have been proved to be related to cancer progression [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. EMT is a key step in tumor progression [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], and platelets are the primary promoter of EMT [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Dipyridamole can inhibit platelet aggregation by inhibiting PDE. Thus, we speculate that dipyridamole can affect platelet-induced EMT of tumor cells by affecting platelet aggregation.\u003c/p\u003e \u003cp\u003eOur in vitro experiments showed that platelet treatment enhanced the proliferative activity of MCF-7 and 4T1 breast cancer cells in a concentration-dependent manner, peaking at 0.7% platelet concentration. Higher dipyridamole concentrations inhibited cell proliferation, with 50 \u0026micro;M demonstrating significant anti-proliferative effects. After platelets are co-cultured with breast cancer cells, platelets can promote the expression of TGF-β in breast cancer cells, and TGF-β is an effective EMT inducer. However, after platelet and dipyridamole co-cultured breast cancer cells, the expression of TGF-β decreased compared with platelet treatment group, indicating that dipyridamole can inhibit the expression of TGF-β in breast cancer cells induced by platelets. When platelets are co-cultured with breast cancer cells, the expression of N-cad mRNA and N-cad protein will also increase. N-cad is an interstitial marker of EMT, which is closely related to tumor progression and metastasis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], while the expression of E-cad mRNA and E-cad protein decreases, and the expression of E-cad is usually related to cancer progression [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. However, after platelet and dipyridamole co-cultured breast cancer cells, compared with platelet-treated group, the expression levels of N-cad mRNA and N-cad protein decreased, while the expression levels of E-cad mRNA and E-cad protein increased. The progress of EMT was mainly manifested by the up-regulation of interstitial markers, such as the up-regulation of N-cad protein [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and the down-regulation of cell adhesion protein E-cad [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, after platelet and dipyridamole co-cultured breast cancer cells, compared with platelet treatment group, the expression of these two proteins was opposite to the EMT process, which showed that dipyridamole had a certain inhibitory effect on platelet-induced EMT of breast cancer cells.\u003c/p\u003e \u003cp\u003eThe experimental results in vivo showed that compared with the control group, the mouse model of breast cancer treated with 0.7% platelet treatment group had larger tumor volume and faster growth rate. However, compared with the 0.7% platelet treatment group, the mouse model of breast cancer treated with 0.7% platelet treatment combined with 50 \u0026micro;M dipyridamole has a smaller tumor volume and slower growth rate. It shows that platelets can promote the growth of tumor, and promote the development of tumor to a certain extent, and dipyridamole can inhibit the growth of tumor promoted by platelets to a certain extent, which can improve the progress of tumor to some extent. After HE staining, it was found that the tumor in situ infiltrated into the surrounding normal tissues in different degrees in the control group, platelet treatment group and platelet combined with dipyridamole treatment group. After HE staining, it was found that no lung metastasis was found in the control group and dipyridamole combined with platelet group, but lung metastasis was found in platelet group, indicating that platelet can promote the migration of tumor cells to surrounding tissues, while dipyridamole can inhibit the migration of tumor cells to surrounding tissues promoted by platelet. Immunohistochemical staining showed that compared with the control group, the expression of N-cad protein was up-regulated and the expression of E-cad protein was down-regulated. Compared with the platelet treatment group, N-cad expression was downregulated in group co-treated with dipyridamole and E-cad expression was up-regulated. N-cad and E-cad proteins are important markers of epithelial mesenchymal transition, which are primarily manifested in the increase of N-cad and decrease of E-cad expression, and both of them are related to cancer progression. In vivo experiments show that platelets can promote EMT of breast cancer cells, while dipyridamole can inhibit EMT of breast cancer cells promoted by platelets.\u003c/p\u003e \u003cp\u003eOverall, our study demonstrates that platelets promote EMT and enhance the proliferation and migration of breast cancer cells, processes central to metastasis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. EMT plays an important role in the invasion and spread of tumor cells. Epithelial tumor cells undergo a series of cellular changes and molecular events to gain interstitial motility and migration ability [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. EMT process includes three steps, namely, loss of polarity, weakening of cell adhesion ability and acquisition of migration ability [\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Dipyridamole was shown to inhibit these processes, thereby attenuating tumor progression. Despite the potential bleeding risk associated with dipyridamole, it has also been shown to enhance the cytotoxicity of several anticancer drugs in vitro. Therefore, combining dipyridamole with standard anticancer therapies may represent a promising strategy for future cancer treatment.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study investigated the effect of dipyridamole on platelet-induced EMT in breast cancer cells. Our results demonstrate that platelets promote breast cancer progression by enhancing proliferation, migration, and EMT. In vitro and in vivo experiments confirmed that dipyridamole suppresses these platelet-mediated effects, inducing reduced proliferation, inhibited EMT, and limited tumor migration. These findings suggest that dipyridamole may serve as an effective adjuvant to inhibit tumor progression driven by platelet interactions. Although further investigation is necessary to fully elucidate its mechanisms, this work provides a foundation for the future development of dipyridamole-based anticancer strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eEMT, epithelial-to-mesenchymal transition;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta;,\u0026nbsp;transforming growth factor beta;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePDGF, platelet‑derived growth factor;\u003c/p\u003e\n\u003cp\u003eLPA, lysophosphatidic acid;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePMPs, Platelet‑derived microparticles;\u003c/p\u003e\n\u003cp\u003eERK, extracellular signal-regulated kinase;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMAPK, mitogen-activated protein kinase;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ecGMP, cyclic guanine monophosphate;\u003c/p\u003e\n\u003cp\u003ecAMP, cyclic adenosine monophosphate;\u003c/p\u003e\n\u003cp\u003eTXA2, thromboxane A2.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical Approval and Consent to participate\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the animal care guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Hospital of Qingdao University, and the assigned approval/accreditation number of the laboratory is AHQU-MAL20240619XJ. At the end of the experiment, all the mice were euthanized. They were anesthetized by inhaling 1.5%~2.5% isoflurane, and then they were killed by cervical dislocation.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eAll authors consent to publication.\u003c/p\u003e\n\u003cp\u003eAvailability of supporting data\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis study was supported by the joint project on innovation development of the Shandong Provincial Natural Science Fund (Grant No. ZR2022LSW024) and the Clinical Medicine +X Project of Qingdao University (Grant No. 2020018).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions\u003c/p\u003e\n\u003cp\u003eXJ summarized and wrote this manuscript. LHY, XQG and GYW provided technical advice. WHY helped substantively revised it. All the authors have reviewed and agreed to submit this manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J. Clin.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e (3), 229\u0026ndash;263 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan, L. et al. Breast cancer in a transitional society over 18 years: trends and present status in Shanghai, China. \u003cem\u003eBreast Cancer Res. Treat.\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e (2), 409\u0026ndash;416 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFitzmaurice, C. et al. 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Epithelial-mesenchymal transitions in development and disease. \u003cem\u003eCell\u003c/em\u003e \u003cb\u003e139\u003c/b\u003e (5), 871\u0026ndash;890 (2009).\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Dipyridamole, Platelet, EMT, Antitumor activity, Breast Cancer","lastPublishedDoi":"10.21203/rs.3.rs-8637051/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8637051/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEpithelial-to-mesenchymal transition (EMT) is a key process implicated in cancer metastasis. Platelets are recognized as important initiators of EMT, with interactions between platelets and tumor cells contributing not only to the formation of tumor-associated thrombi but also the promotion of cancer metastasis. Dipyridamole, an antiplatelet agent commonly used in the treatment of cardiovascular and cerebrovascular diseases has recently been shown to enhance the efficacy of certain antitumor drugs. However, the standalone antitumor effects of dipyridamole, either in vitro or in vivo, have not yet been well-documented. This study aimed to investigate the impact of dipyridamole on platelet-mediated EMT in breast cancer through in vivo and in vitro models. The findings may uncover a novel antitumor mechanism of dipyridamole and suggest a potential new therapeutic approach for breast cancer.\u003c/p\u003e","manuscriptTitle":"Suppressive effect of dipyridamole on platelet-mediated epithelial-mesenchymal transition in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-13 08:56:14","doi":"10.21203/rs.3.rs-8637051/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-26T13:29:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"182844603332340488330226970320673540229","date":"2026-03-10T10:02:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-10T02:41:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-30T17:45:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-22T11:32:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-22T02:04:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-22T01:56:23+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aa930f4a-24b6-4cd5-9206-864737615b9e","owner":[],"postedDate":"March 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64275429,"name":"Biological sciences/Cancer"},{"id":64275430,"name":"Biological sciences/Cell biology"},{"id":64275431,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-03-13T08:56:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-13 08:56:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8637051","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8637051","identity":"rs-8637051","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}