GDF15 at Cancer-Relevant Concentrations Promotes Angiogenesis

GDF15 at Cancer-Relevant Concentrations Promotes Angiogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article GDF15 at Cancer-Relevant Concentrations Promotes Angiogenesis Max Hüllwegen, Reiner Andag, Fatemeh Hosami, Nathalia Soares da Cruz, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9276203/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Growth Differentiation Factor 15 (GDF15) is a key molecule of cellular stress responses and an established biomarker in cancer, where elevated serum concentrations correlate with cachexia and poor prognosis. Although the metabolic actions of circulating GDF15 are primarily attributed to its interactions with the GDNF family receptor alpha (GFRAL) expressed in the hindbrain, GDF15 also exerts pleiotropic effects in multiple other tissues, indicating the presence of GFRAL-independent signaling pathways. Earlier studies reported pro-angiogenic effects of GDF15 on endothelial cells, but they predominantly employed higher concentrations of recombinant protein than common in health or disease. Here, we examined how GDF15 levels commonly observed in the serum of cancer patients influence the behavior of primary human endothelial cells. At these concentrations, GDF15 enhanced endothelial cell-cycle progression, proliferation, migration, tube formation and aerobic glycolysis. Notably, pharmacological blockade of GFRAL did not diminish these responses, supporting the existence of alternative receptor mechanisms mediating GDF15 activity in the vasculature. The results suggest that GDF15 at cancer-associated concentrations has a protective effect on endothelial cells. GDF15 Angiogenesis Endothelium Cachexia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background GDF15 is a critical mediator of the physiological stress response. Although many cell types can release GDF15, circulating plasma GDF15 concentration are typically low (0.5–1.2 ng/mL (1,2)). Excessive physical exercise, pregnancy, cancer, infections, heart failure, chronic kidney disease, and other pathologies can markedly elevate GDF15 levels in the bloodstream (2–10 ng/ml) (3). The full range of GDF15 functions remains incompletely understood. GDF15 released during cellular stress may exert protective effects, for example by mitigating obesity, insulin resistance, and fatty liver disease (4–7) and by reducing inflammation. However, chronically elevated GDF15, as seen in severe chronic diseases such as advanced cancer, heart failure or chronic kidney disease, may drive maladaptive processes (7). In cancer patients, GDF15 serum levels are frequently chronically elevated (around 2–10 ng/mL(2)). GDF15 levels correlate with advanced tumor stage, metastasis, and cachexia, (8–10) a wasting disorder characterized by progressive loss of skeletal muscle and adipose tissue (11). GDF15 suppresses appetite and food intake via GFRAL receptors (12) expressed exclusively in the hindbrain (13). Notably, GDF15-blocking antibodies have shown promising results in animal models and in early-phase clinical trials in patients with various advanced cancers, leading to significant weight gain (14,15) and potential restoration of CD8 + T-cell infiltration, which may help to overcome immunotherapy resistance (16). Endothelial cells, which form the inner lining of all blood vessels, play a major role in tumor progression (17), metastasis (18) and cachexia development (19,20). These cells are in direct contact with circulating GDF15 and may respond to it. Previous work suggests that GDF15 can exert protective effects on endothelial cells under stress, inhibiting apoptosis and inflammation, and promoting angiogenesis by the PI3K/AKT (Phosphoinositide 3-kinase/protein kinase B) and ERK1/2 (extracellular-signal regulated kinases) signaling pathways(21–23). Because endothelial cells lack GFRAL receptors (13), these effects are thought to be mediated by alternative signaling mechanisms. However, prior studies used doses of recombinant GDF15 protein (14,24), which are up to 10-fold higher than levels typically observed in serum of patients with advanced cancer. Therefore, the aim of this study is to investigate how GDF15 in cancer-associated concentrations (approximately 5–10 ng/ml) affect cultured primary human endothelial cells. Materials and Methods Cell culture Primary human umbilical venous endothelial cells (HUVECs) were isolated from umbilical cords (Ethics approval no. 28/1/23, Göttingen, see ‘Declarations’) and used until passage 4. HUVECs were cultured in complete Endopan 3 Medium (P04-0010K, PAN-Biotech) with 3% FCS (fetal calf serum) at humidified 37°C and 5% CO 2 . Medium was changed every 24 hours. HEK293 (Human Embryonic Kidney) cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) with 10% FCS. HEK293 cells were transfected with a plasmid encoding GDF15 (HG10936-UT; Sino Biological) or empty control and conditioned medium was harvested 16 hours after transfection. Recombinant proteins and antibodies Human recombinant GDF15 was purchased from R&D systems, (cat. no. 957-GD) and dissolved in 4 mmol HCL. Anti-GDF15 (NBP3-28880; Novus Biologicals) and anti-GFRAL (BPS-101351; BPS bioscience) were used in 5-fold and 10-fold molar excess compared to GDF15. Cell number and cell proliferation HUVECs (200000 cells/well) were seeded in 6-well plates and allowed to attach for 24 h. Cell culture medium (including additives) was renewed every 24 h. Experimental conditions (n = 3 per group) included: a control group, 10 ng/mL rhGDF15 (recombinant human GDF15), 10 ng/mL rhGDF15 + anti-GDF15 (NBP3 28880; Novus Biologicals) at a 5-fold molar excess, 10 ng/mL rhGDF15 + anti-GFRAL (BPS 101351; BPS bioscience) at a 10-fold molar excess, 10 ng/mL HEK293-derived GDF15 (plasmid: HG10936-UT; Sino Biological), and 10 ng/mL HEK293-derived GDF15 + anti-GDF15 at a 5-fold molar excess. Image preprocessing ( supp. Figure 1 ) included stacking, 8-bit conversion, and background correction (Subtract Background: 50px rolling-ball radius). Frequency-domain denoising using FFT Bandpass Filter (large structures ≥ 40px, small structures ≤3px). Segmentation used Auto Threshold (Otsu) followed by watershed separation of touching objects. Particles were quantified (Analyze Particles: 50–500px², circularity 0.20–1.00) from three randomly selected fields per well. Per-well means (technical replicates) were normalized to baseline (0 h = 100%) of each biological replicate. Supp. Figure 1 Wound closure assay HUVECs were grown to 90% confluence, wounded with a single linear scratch using a 100 µL pipette tip and washed twice with Phosphate-buffered saline (PBS). Scratch edges were inspected for continuity and uniformity. Serum-reduced media (1% FCS) was added to suppress proliferation, and cells were treated with 10 ng/mL rhGDF15 (n = 3). Phase-contrast images were captured at 0, 8, 16, 24, 48, and 72 h using fixed plate coordinates. Wound width was quantified using the ImageJ Wound Healing Size Tool (25) (variance window radius 20, saturated pixels 0.001%, threshold 100, global scale and diagonal wound correction enabled). A rectangular ROI encompassing the wound was defined per image, and outputs were exported to CSV. Wound area was normalized to the matched control at each timepoint. Cell cycle and Flow cytometry analysis HUVECs were allowed to attach for 24 h, followed by 24 h treatment with 10 ng/mL rhGDF15 (n = 4). Cells were harvested by trypsinization, counted, and normalized to the lowest cell number. After fixation in 70% ethanol for 2 h at 4°C, cells were stained with a DAPI (4′,6-Diamidin-2-phenylindol)/Triton X-100 solution, prepared by adding 10 µL of a 1 mg/mL DAPI stock (D9542; Sigma-Aldrich) to 10 mL of 0.1% (v/v) Triton X-100 (T9284; Sigma-Aldrich) in PBS. Cells were subsequently analyzed by flow cytometry (BD FACS Canto II). Data analysis was performed with FlowJo v10.10.0 (Ashland, OR: Becton, Dickinson and Company; 2023). Cell populations were gated to exclude debris and aggregates. The automatic cell cycle algorithm determined G1 and G2/M phases using an asynchronous control culture as reference. Data was normalized to the untreated control within each biological replicate. Tube Formation Assay Tube formation assays were performed in µ-Slide 15 well 3D chambers to assess GDF15 effects on endothelial network assembly. Matrigel (10 µL/well) was thawed on ice and polymerized at 37°C for 30 min. HUVECs were seeded at a low density 24 h prior, then harvested and resuspended at 4 × 10⁵ cells/mL. Cell suspension (25 µL; 1.0 × 10⁴ cells) and treatment medium (25 µL) were added per well for final rhGDF15 concentrations of 0 and 10 ng/mL. Phase-contrast images (4x magnification) were acquired at 4, 8 and 24 h under constant settings. Images analyzed using WimTube AI Tool (Onimagin Technologies) to quantify angiogenic parameters, especially total number of loops. Nine biological replicates across two runs were planned; two wells with poor adhesion were excluded a priori (final n = 7). Mitochondrial Functional Assay in HUVECs Mitochondrial baseline function and stress responses were assessed by measuring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using the Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies) in combination with the Seahorse XF Cell Mito Stress Test Kit (103015-100, Agilent Technologies). Human umbilical vein endothelial cells (HUVECs) were seeded at 10,000 cells/well in complete cell culture medium and allowed to adhere overnight. The following morning, the medium was replaced and stimulation with GDF15 (10 ng/mL) was initiated. 4 mM HCl was used as the vehicle control (vol/vol). The medium was replaced after 24 h and fresh GDF15 (10 ng/mL) or vehicle control was added, followed by an additional 24 h incubation prior to measurement. On the day of the experiment, cells were washed with PBS and transferred to Seahorse XF DMEM Medium (103575-100, Agilent Technologies) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. Cells were incubated for 1 h at 37°C in a non-CO₂ incubator prior to the assay. The assay protocol consisted of five baseline measurement cycles (Mix 3 min, Wait 2 min, Measure 3 min). After sequential injections of oligomycin, FCCP (Carbonylcyanid-p-trifluoromethoxyphenylhydrazon), and rotenone/antimycin A five measurement cycles were performed for each compound using the same Mix:Wait:Measure timing (3:2:3). Final working concentrations were: Oligomycin: 1.5 µM, FCCP: 2 µM, Rotenone/antimycin A: 0.5 µM Data were normalized to cell content using crystal violet staining performed immediately after completion of the Seahorse assay. Crystal Violet Staining Following completion of the Seahorse assay, cells were fixed with 200 µL/well of 10% formalin for 15 min at room temperature (RT). Cells were then washed three times with distilled H₂O (400 µL/well, 2 min per wash). Cells were stained with 0.02% crystal violet solution in 10% ethanol for 30 min at RT, followed by three washes with distilled H₂O (400 µL/well, 2 min per wash). Statistical analyses Results are presented as mean ± SD. Statistical significance was assessed by ANOVA followed by pairwise comparisons within each timepoint, controlling the False Discovery Rate (FDR) using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q = 0.05). Q < 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism (Version 10.3.1, GraphPad Software, Boston, Massachusetts USA). In the tube formation assay, potential outliers have been defined using robust regression and outlier removal method (ROUT), Q = 1%. Images were preprocessed in Fiji (v2.16.0/1.54p; ImageJ distribution,(26)). Results GDF15 promotes endothelial cell proliferation and cell cycle progression GDF15 can exert protective effects and promote proliferation of different cell types (27–29). Previous reports suggested that GDF15 increases endothelial cell proliferation (24), but at concentrations about 10-fold higher than those observed in the blood of cancer patients. We treated primary human vein umbilical endothelial cells (HUVECs) with 10 ng/ml recombinant GDF15 to assess cell number kinetics. At cancer-associated levels, rhGDF15 robustly increased endothelial cell proliferation, with higher cell numbers observed at 24, 48, and 72 hours (Fig. 1 A). Controlling for potential contaminating factors, previous rhGDF15 preparations have been reported to contain TGF-β (30). To ensure the observed effects were specifically due to GDF15, we treated HUVECs with rhGDF15 in the presence or absence of a monoclonal GDF15-blocking antibody. Addition of the GDF15 antibody fully prevented the pro-proliferative effects of GDF15 on endothelial cells (Fig. 1 A). To investigate how GDF15 acts on cell cycle, HUVECs were treated with rhGDF15 for 24 hours and analyzed by flow cytometry. GDF15 reduced the G1 fraction by 12.73% and increased the G2/M fraction by 17.54% relative to control (Fig. 2 ), indicating accelerated cell cycle progression and enhanced proliferative capacity. We also tested whether cell secreted GDF15 could enhance endothelial cell proliferation. HUVECs were exposed to conditioned medium from HEK293 cells transfected with a full-length GDF15 expression plasmid. Compared with conditioned medium from non-transfected HEK293 cells, GDF15-containing conditioned medium (10 ng/ml HEK293-derived GDF15) promoted HUVEC proliferation (Fig. 1 B) We aimed to determine whether the GDF15-mediated effect on endothelial cell proliferation is mediated by GFRAL, the only well-established receptor for GDF15. It is widely assumed that GFRAL expression is restricted to neurons in the hindbrain (12) and that GDF15 potentially acts through other poorly understood mechanisms on other cell types (12,31). Analysis of published single cell RNA sequencing data sets (13,32) support the hypothesis that GFRAL is not expressed by endothelial cells. Nevertheless, we treated HUVEC with rhGDF15 in the presence or absence of GFRAL-blocking antibodies (administered at 10-fold molecular excess over GDF15). GFRAL blockade did not abolish the pro-proliferative effects of GDF15 (Fig. 1 C), indicating that GFRAL-independent signaling pathways exist in endothelial cells. GDF15 enhances endothelial cell migration Endothelial regeneration requires cell proliferation and migration. To determine GDF15's effects on cell migration, we performed a gap (wound) closure assay. Cell proliferation was inhibited by use of low serum condition (1% FCS). Under these conditions, rhGDF15 significantly accelerated gap closure (Fig. 3 ), indicating that GDF15 promotes endothelial cell migration. GDF15 promotes endothelial tube formation To evaluate effects on angiogenic network formation, we performed a Matrigel tube formation assay. Treatment with 10 ng/ml rhGDF15 significantly enhanced tube formation compared to control (Fig. 4 ). GDF15 enhances glycolysis and mitochondrial activity During angiogenesis, endothelial cells require readily available energy for migration and proliferation. Therefore, glycolysis is the preferred metabolic pathway, even under normoxic conditions (33,34). To further understand the mechanism underlying the observed pro-proliferative effects, we performed a Seahorse assay to asses cellular metabolism. The extracellular acidification rate (ECAR), a surrogate of glycolytic proton efflux, was increased in the GDF15–stimulated cells (Fig. 5 A). In addition, the oxygen consumption rate (OCR) was also elevated in rhGDF15 treated cells under basal conditions, as well as during maximal respiration, while non-mitochondrial oxygen consumption was similar between groups (Fig. 5 B). Discussion GDF15 is a stress-responsive cytokine overexpressed in diverse conditions including cancer. While signaling through GFRAL in the hindbrain regulates appetite, nausea, and insulin sensitivity (14,35), compelling evidences indicate that GDF15 also acts on cell types that do not express GFRAL, including endothelial cells. Endothelial cells occupy a strategic location to sense circulation metabolic messengers, such as GDF15 (2), and integrate systemic and local signals. There is substantial evidence that endothelial cells respond to cancer-associated signals throughout the body (18,36), as well as to metabolic disturbances (37). Although high-dose GDF15 (on the order of 100 ng/ml) has been reported to promote angiogenesis in vitro (24), such concentrations are typically observed only in pregnancy, whereas most cancer patients exhibit serum GDF15 in the 2–10 ng/ml range (2,7,38). These recent observations motivated our fucus on cancer-associated GDF15 levels and their effects on primary human endothelial cells. Our results show that cancer-associated concentrations of GDF15 promote endothelial proliferation, cell cycle progression, migration, and tube formation. Using GDF15-blocking antibodies and conditioned medium, the presence of potential contaminations of recombinant GDF15 protein preparations could be ruled out. Even under physiological conditions, endothelial cells derive most of their energy from glycolysis (up to ~ 85%) (39). Endothelial cells use glycolysis for proliferation, migration and cell cycle progression; increased glycolysis supports DNA synthesis and cell division/proliferation (33,34,40). A Seahorse assay enabled us to better assess the metabolic situation of the HUVECs. The increased glycolytic activity of the cells shown here under 10 ng/mL GDF15 indicates a metabolic reprogramming of the cells, which could strongly support the overall pro-angiogenic effect of GDF15. In the context of cancer, malignant tumors are a major source of GDF15 (10,41) and elevated serum GDF15 levels are associated with higher epithelial-to-mesenchymal transition (EMT) of cancer cells and metastasis rates (42–44). The protective effect of GDF15 on the endothelium may facilitate generation of a vascular network within tumors even under metabolic stress. (42,43,45). Thus, GDF15 appears to act as a dual regulator, contributing to both pro-vascular and pro-tumorigenic phenotypes. Conclusions Our findings further contribute to the concept of diverse roles of GDF15 in the context of cancer. While GDF15 reprograms systemic metabolism by suppressing appetite via its receptor GFRAL expressed in the hindbrain, it also influences other cell types like the endothelium through poorly characterized receptors. As such, novel therapies using GDF15 inhibitors might not only target energy intake of patients with cancer but also other cellular functions such as tumor angiogenesis, a hallmark of cancer progression. This needs to be addressed in future studies. Study Limitations The employed cellular models cannot fully mimic the tumor microenvironment's complexity. We also evaluated relatively short-term responses; long-term exposure to GDF15 and its effects on endothelial physiology and pathology remain to be explored in animal models. Further research is needed to identify endothelial receptor and downstream signaling mediators that link GDF15 to proliferation and angiogenesis. Abbreviations (rh)GDF15 (recombinant human) Growth Differentiation Factor-15 GFRAL GDNF family receptor alpha ERK Extracellular-signal regulated kinases AKT Protein kinase B HUVEC Primary human umbilical venous endothelial cells FCS Fetal calf serum DMEM Dulbecco’s Modified Eagle’s Medium HEK293 Human embryotic kidney cell line 293 PBS Phosphate-buffered saline DAPI 4′,6-Diamidin-2-phenylindol ECAR Extracellular acidification rate OCR Oxygen consumption rate FCCP Carbonylcyanid-p-trifluoromethoxyphenylhydrazon RT Room temperature FDR False Discovery Rate EMT Epithelial-to-mesenchymal transition Declarations Ethics approval and consent to participate: All procedures involving human-derived material were performed in accordance with the Declaration of Helsinki and approved by the ethics board of University Medical Center Göttingen, approval number 28/1/23. HUVECs were isolated from human umbilical cord tissue. Written informed consent was obtained from all donors prior to tissue collection. Consent for publication: Not applicable The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The authors declare that they have no competing interests This work was supported by Deutsche Forschungsgemeinschaft (DFG) under grant number 394046768-SFB1366 'Vascular control of organ function' (project C04) awarded to M.H. and A.F. Authors' contributions: MH: Data acquisition and curation, formal analysis, visualization, and writing - original draft, review & editing . RA, FH, SK: supervision and investigation. NSdC: Data acquisition, supervision and investigation. EL: Conduction and analysis of the Seahorse Assay and writing - review & editing. MFH: Provided the HUVEC cells. JG, PS, JH: supervision. SSH: supervision, writing - review & editing. AF: conceptualization, supervision, funding acquisition, and writing - review & editing. 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Endothelial cell in tumor angiogenesis: Origins, mechanisms, and therapeutic implication. Genes & Diseases. 2025 Nov;12(6):101611. doi:10.1016/j.gendis.2025.101611 Tran V, De Silva TM, Sobey CG, Lim K, Drummond GR, Vinh A, et al. The Vascular Consequences of Metabolic Syndrome: Rodent Models, Endothelial Dysfunction, and Current Therapies. Front Pharmacol. 2020 Mar 4;11:148. doi:10.3389/fphar.2020.00148 Lerner L, Hayes TG, Tao N, Krieger B, Feng B, Wu Z, et al. Plasma growth differentiation factor 15 is associated with weight loss and mortality in cancer patients. J Cachexia Sarcopenia Muscle. 2015 Dec;6(4):317–24. doi:10.1002/jcsm.12033 PubMed PMID: 26672741; PubMed Central PMCID: PMC4670740. Xu S, Liao J, Liu B, Zhang C, Xu X. Aerobic glycolysis of vascular endothelial cells: a novel perspective in cancer therapy. Mol Biol Rep. 2024 Dec;51(1):717. doi:10.1007/s11033-024-09588-1 Xu Y, An X, Guo X, Habtetsion TG, Wang Y, Xu X, et al. Endothelial PFKFB3 Plays a Critical Role in Angiogenesis. ATVB. 2014 Jun;34(6):1231–9. doi:10.1161/ATVBAHA.113.303041 Chen SJ, Karan D, Johansson SL, Lin FF, Zeckser J, Singh AP, et al. Prostate-derived factor as a paracrine and autocrine factor for the proliferation of androgen receptor-positive human prostate cancer cells. Prostate. 2007 Apr 1;67(5):557–71. doi:10.1002/pros.20551 PubMed PMID: 17221842. Li C, Wang J, Kong J, Tang J, Wu Y, Xu E, et al. GDF15 promotes EMT and metastasis in colorectal cancer. Oncotarget. 2015 Oct 22;7(1):860–72. PubMed PMID: 26497212; PubMed Central PMCID: PMC4808038. Wang X, Yang Z, Tian H, Li Y, Li M, Zhao W, et al. Circulating MIC-1/GDF15 is a complementary screening biomarker with CEA and correlates with liver metastasis and poor survival in colorectal cancer. Oncotarget. 2017 Feb 11;8(15):24892–901. doi:10.18632/oncotarget.15279 PubMed PMID: 28206963; PubMed Central PMCID: PMC5421897. Allgayer H, Mahapatra S, Mishra B, Swain B, Saha S, Khanra S, et al. Epithelial-to-mesenchymal transition (EMT) and cancer metastasis: the status quo of methods and experimental models 2025. Mol Cancer. 2025 Jun 7;24(1):167. doi:10.1186/s12943-025-02338-2 Zhang Y, Wang X, Zhang M, Zhang Z, Jiang L, Li L. GDF15 promotes epithelial-to-mesenchymal transition in colorectal. Artificial Cells, Nanomedicine, and Biotechnology. 2018 Nov 5;46(sup2):652–8. doi:10.1080/21691401.2018.1466146 PubMed PMID: 29771147. Additional Declarations No competing interests reported. Supplementary Files supp.Fig.1.tif Supp. Figure 1: Image editing workflow for cell number analysis; 1: original picture; 2: converted image to 8-bit; 3: background subtraction; 4: Bandpass filter; 5: Otsu threshold; 6: watershed; 7: after ImageJ “analyze particles” plugin. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 14 May, 2026 Reviewers invited by journal 28 Apr, 2026 Editor assigned by journal 02 Apr, 2026 Submission checks completed at journal 01 Apr, 2026 First submitted to journal 01 Apr, 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-9276203","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":622469188,"identity":"186e0025-51ea-4eea-a130-624b28f22d13","order_by":0,"name":"Max Hüllwegen","email":"","orcid":"","institution":"Universitätsmedizin Göttingen","correspondingAuthor":false,"prefix":"","firstName":"Max","middleName":"","lastName":"Hüllwegen","suffix":""},{"id":622469189,"identity":"06358eb6-2c0b-40ea-b705-94aa2555857b","order_by":1,"name":"Reiner Andag","email":"","orcid":"","institution":"Universitätsmedizin Göttingen","correspondingAuthor":false,"prefix":"","firstName":"Reiner","middleName":"","lastName":"Andag","suffix":""},{"id":622469190,"identity":"2d2b7d3d-971d-4368-bc2e-7b9d185688a0","order_by":2,"name":"Fatemeh Hosami","email":"","orcid":"","institution":"Medical Faculty Mannheim, Heidelberg University","correspondingAuthor":false,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Hosami","suffix":""},{"id":622469191,"identity":"4f52b871-588a-4df3-900f-5fd0e6c513e7","order_by":3,"name":"Nathalia Soares da Cruz","email":"","orcid":"","institution":"Medical Faculty Mannheim, Heidelberg University","correspondingAuthor":false,"prefix":"","firstName":"Nathalia","middleName":"Soares da","lastName":"Cruz","suffix":""},{"id":622469192,"identity":"988997ba-c987-4845-b434-4bdef724298c","order_by":4,"name":"Ekaterina Legchenko","email":"","orcid":"","institution":"Medical Faculty Mannheim, Heidelberg University","correspondingAuthor":false,"prefix":"","firstName":"Ekaterina","middleName":"","lastName":"Legchenko","suffix":""},{"id":622469193,"identity":"ec08a0b9-eab7-4984-bab1-1d335d2520e4","order_by":5,"name":"Mir Fuad Hasanov","email":"","orcid":"","institution":"Universitätsmedizin Göttingen","correspondingAuthor":false,"prefix":"","firstName":"Mir","middleName":"Fuad","lastName":"Hasanov","suffix":""},{"id":622469194,"identity":"1de39647-023f-4016-989a-876c901da0a7","order_by":6,"name":"Stefan Küffer","email":"","orcid":"","institution":"Universitätsmedizin Göttingen","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Küffer","suffix":""},{"id":622469195,"identity":"10be904e-d3c8-40a5-b340-2da1332cb9c7","order_by":7,"name":"Julia Gallwas","email":"","orcid":"","institution":"Universitätsmedizin Göttingen","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Gallwas","suffix":""},{"id":622469196,"identity":"f71bb085-7d26-4763-8c56-96a294953ab4","order_by":8,"name":"Philipp Ströbel","email":"","orcid":"","institution":"Universitätsmedizin Göttingen","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Ströbel","suffix":""},{"id":622469197,"identity":"ef4eb7f0-9f7f-4462-83b0-d412ecfdca3c","order_by":9,"name":"Jörg Heineke","email":"","orcid":"","institution":"Medical Faculty Mannheim, Heidelberg University","correspondingAuthor":false,"prefix":"","firstName":"Jörg","middleName":"","lastName":"Heineke","suffix":""},{"id":622469198,"identity":"8ff189b0-c988-46f5-858f-6e1c7259140c","order_by":10,"name":"Sana Safatul Hasan","email":"","orcid":"","institution":"Medical Faculty Mannheim, Heidelberg University","correspondingAuthor":false,"prefix":"","firstName":"Sana","middleName":"Safatul","lastName":"Hasan","suffix":""},{"id":622469199,"identity":"36d4c67a-bdda-4843-a622-beb3f8a203b5","order_by":11,"name":"Andreas Fischer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYHACAzDJ3szAwAyk5cA8HmK08ByGaDEmQcsBiJbEBkJa+Gc3b/vwoaaOgYed/eHngop76RuunU5geFOBW4vEnWPFM2ccO8zAw8xjLD3jTHHuhtu5GxjnnMFjzY0cY2YetgMM9kCSmbctAawFyMCtQx6k5c8/oMOY2Z8x8/5LSDcAa/mHW4sBSAtjGzNQC4MZM29DQgJESwNuLYY30ooZe/sO84D9wnMswXAmUMvBOcdwa5G7kbyZ4ce3Ojke/uMPP/PUJMjz3c7d+OBNDR7vQwFqRBwgrGEUjIJRMApGAT4AAJnyTDwXR7O9AAAAAElFTkSuQmCC","orcid":"","institution":"Medical Faculty Mannheim, Heidelberg University","correspondingAuthor":true,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Fischer","suffix":""}],"badges":[],"createdAt":"2026-03-31 07:39:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9276203/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9276203/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106878177,"identity":"10bc6706-587b-4ed1-b89b-0c3ed51909d9","added_by":"auto","created_at":"2026-04-14 10:44:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7590525,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCell number:\u003c/em\u003erelative cell number compared to control group is shown on the X-axis. Cells were treated with 10 ng/mL rhGDF15 and a GDF15-blocking Antibody in 5-fold molar excess to 10ng/mL rhGDF15 (\u003cstrong\u003eA\u003c/strong\u003e), with 10 ng/mL HEK293-derived GDF15 and a GDF15-blocking Antibody in 5-fold molar excess to 10 ng/mL HEK293-derived GDF15 (\u003cstrong\u003eB\u003c/strong\u003e) and with a GFRAL-blocking Antibody in 10-fold molar excess to 10 ng/mL rhGDF15 (\u003cstrong\u003eC\u003c/strong\u003e) every 24 h for up to 72 h; on the Y-axis, three random images per condition from three biological replicates were averaged and normalized to 0 h (100% = replicate‑matched control), data depicted as mean with SD, adjusted p-values shown, Two-way RM ANOVA: main effect of treatment, p\u0026lt;0.0001 (\u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003eB\u003c/strong\u003e,\u003cstrong\u003eC\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"Fig.1cellcount.png","url":"https://assets-eu.researchsquare.com/files/rs-9276203/v1/e40044131ceb1d1e85494104.png"},{"id":106878179,"identity":"d3f114b7-dc39-4d8a-bbc1-9b61f890020d","added_by":"auto","created_at":"2026-04-14 10:44:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":361893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003elow Cytometry Cell Cyle Analysis:\u003c/em\u003e Phase shifts relative to control group. On the X-Axis, Phases reported as G1, S, and combined G2/M for DAPI DNA‑content analysis. HUVECS were treated for 24 h in cell culture medium with or without 10 ng/mL rhGDF15; n = 4 biological replicates from two independent runs with same conditions were normalized to the untreated control (100% = replicate‑matched control) at 24 h; gating and automated cell cycle analysis conducted in FlowJo; data depicted as mean with SD, adjusted p-values shown\u003c/p\u003e","description":"","filename":"Fig.2FACScellcylce.png","url":"https://assets-eu.researchsquare.com/files/rs-9276203/v1/e4261cb93d41d62215321631.png"},{"id":106960735,"identity":"35dc87cd-bfc7-4d1e-953f-d5d14259112f","added_by":"auto","created_at":"2026-04-15 09:22:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5029075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eScratch Assay\u003c/em\u003e: Scratch width [µm] is shown on the Y-axis. Cells were treated until gap closure for 72 h in total, with serum-reduced (1%) cell culture medium (with or without rhGDF15), media was changed every 24 h, n = 3 biological replicates; Standardized pictures with pre-programmed coordinates for each well were taken at 0, 8, 16, 24 and 48 h and analyzed in ImageJ with the Wound Healing Size Tool by Suarez-Arnedo et al., data depicted as mean with SD, adjusted p-values shown\u003c/p\u003e","description":"","filename":"Fig.3scratch.png","url":"https://assets-eu.researchsquare.com/files/rs-9276203/v1/007092d5ce17531d48835d42.png"},{"id":106878182,"identity":"58f661b0-d830-405f-90f6-5f9d1f211f76","added_by":"auto","created_at":"2026-04-14 10:44:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":681785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eTube formation Assay\u003c/em\u003e: Cells were seeded in µ-slides on Matrigel to induce capillary-like tube formation in vitro and treated with or without 10 ng/mL rhGDF15. Standardized images were taken at 4 and 8 h and analyzed with the Wimasis AI tool for tube formation. The graph shows n = 7 biological replicates from two independent runs with same conditions. The Y-axis shows \u003cstrong\u003etotal number of loops formed\u003c/strong\u003e(closed regions bounded by tubes at the 4 h timepoint, \u003cstrong\u003eA\u003c/strong\u003e; data depicted as mean with SD, adjusted p-values shown\u003c/p\u003e","description":"","filename":"Fig.4TubeFormation.png","url":"https://assets-eu.researchsquare.com/files/rs-9276203/v1/7f1bd5293fb230a9cbb13204.png"},{"id":106878181,"identity":"3ca3a9a4-c218-4211-8d2c-c7b511d5af91","added_by":"auto","created_at":"2026-04-14 10:44:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":224708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMitochondrial Functional Assay\u003c/em\u003e: Normalized OCR (oxygen consumption rate, \u003cstrong\u003eA\u003c/strong\u003e) and ECAR (extracellular acidification rate, used as a proxy for glycolytic proton efflux, \u003cstrong\u003eB\u003c/strong\u003e) Data. Assay inhibitors and control detergents (Oligomycin, FCCP, Rot/AA) were added at the depicted time points on the X-Axis. Cells were pre-incubated for 48h with 10 ng/mL rhGDF15; data was normalized using a Crystal Violet Staining afterwards; data depicted as mean with SD, p-values shown\u003c/p\u003e","description":"","filename":"Fig.5normalizedOCRECARdata.png","url":"https://assets-eu.researchsquare.com/files/rs-9276203/v1/850bbcdf338b5853afc6e5f0.png"},{"id":106961713,"identity":"a2734475-5010-4c94-81a4-578ba0adc0b8","added_by":"auto","created_at":"2026-04-15 09:26:32","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":773626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupp. Figure 1:\u003c/strong\u003e Image editing workflow for cell number analysis; 1: original picture; 2: converted image to 8-bit; 3: background subtraction; 4: Bandpass filter; 5: Otsu threshold; 6: watershed; 7: after ImageJ “analyze particles” plugin.\u003c/p\u003e","description":"","filename":"supp.Fig.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-9276203/v1/421c9b84c737ff20554aa4e3.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"GDF15 at Cancer-Relevant Concentrations Promotes Angiogenesis","fulltext":[{"header":"Background","content":"\u003cp\u003eGDF15 is a critical mediator of the physiological stress response. Although many cell types can release GDF15, circulating plasma GDF15 concentration are typically low (0.5\u0026ndash;1.2 ng/mL (1,2)). Excessive physical exercise, pregnancy, cancer, infections, heart failure, chronic kidney disease, and other pathologies can markedly elevate GDF15 levels in the bloodstream (2\u0026ndash;10 ng/ml) (3). The full range of GDF15 functions remains incompletely understood. GDF15 released during cellular stress may exert protective effects, for example by mitigating obesity, insulin resistance, and fatty liver disease (4\u0026ndash;7) and by reducing inflammation. However, chronically elevated GDF15, as seen in severe chronic diseases such as advanced cancer, heart failure or chronic kidney disease, may drive maladaptive processes (7).\u003c/p\u003e \u003cp\u003eIn cancer patients, GDF15 serum levels are frequently chronically elevated (around 2\u0026ndash;10 ng/mL(2)). GDF15 levels correlate with advanced tumor stage, metastasis, and cachexia, (8\u0026ndash;10) a wasting disorder characterized by progressive loss of skeletal muscle and adipose tissue (11). GDF15 suppresses appetite and food intake via GFRAL receptors (12) expressed exclusively in the hindbrain (13). Notably, GDF15-blocking antibodies have shown promising results in animal models and in early-phase clinical trials in patients with various advanced cancers, leading to significant weight gain (14,15) and potential restoration of CD8\u003csup\u003e+\u003c/sup\u003e T-cell infiltration, which may help to overcome immunotherapy resistance (16).\u003c/p\u003e \u003cp\u003eEndothelial cells, which form the inner lining of all blood vessels, play a major role in tumor progression (17), metastasis (18) and cachexia development (19,20). These cells are in direct contact with circulating GDF15 and may respond to it. Previous work suggests that GDF15 can exert protective effects on endothelial cells under stress, inhibiting apoptosis and inflammation, and promoting angiogenesis by the PI3K/AKT (Phosphoinositide 3-kinase/protein kinase B) and ERK1/2 (extracellular-signal regulated kinases) signaling pathways(21\u0026ndash;23). Because endothelial cells lack GFRAL receptors (13), these effects are thought to be mediated by alternative signaling mechanisms. However, prior studies used doses of recombinant GDF15 protein (14,24), which are up to 10-fold higher than levels typically observed in serum of patients with advanced cancer.\u003c/p\u003e \u003cp\u003eTherefore, the aim of this study is to investigate how GDF15 in cancer-associated concentrations (approximately 5\u0026ndash;10 ng/ml) affect cultured primary human endothelial cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003ePrimary human umbilical venous endothelial cells (HUVECs) were isolated from umbilical cords (Ethics approval no. 28/1/23, G\u0026ouml;ttingen, see \u0026lsquo;Declarations\u0026rsquo;) and used until passage 4. HUVECs were cultured in complete Endopan 3 Medium (P04-0010K, PAN-Biotech) with 3% FCS (fetal calf serum) at humidified 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Medium was changed every 24 hours.\u003c/p\u003e \u003cp\u003eHEK293 (Human Embryonic Kidney) cells were cultured in DMEM (Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium) with 10% FCS. HEK293 cells were transfected with a plasmid encoding GDF15 (HG10936-UT; Sino Biological) or empty control and conditioned medium was harvested 16 hours after transfection.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRecombinant proteins and antibodies\u003c/h3\u003e\n\u003cp\u003eHuman recombinant GDF15 was purchased from R\u0026amp;D systems, (cat. no. 957-GD) and dissolved in 4 mmol HCL. Anti-GDF15 (NBP3-28880; Novus Biologicals) and anti-GFRAL (BPS-101351; BPS bioscience) were used in 5-fold and 10-fold molar excess compared to GDF15.\u003c/p\u003e\n\u003ch3\u003eCell number and cell proliferation\u003c/h3\u003e\n\u003cp\u003eHUVECs (200000 cells/well) were seeded in 6-well plates and allowed to attach for 24 h. Cell culture medium (including additives) was renewed every 24 h. Experimental conditions (n\u0026thinsp;=\u0026thinsp;3 per group) included: a control group, 10 ng/mL rhGDF15 (recombinant human GDF15), 10 ng/mL rhGDF15\u0026thinsp;+\u0026thinsp;anti-GDF15 (NBP3 28880; Novus Biologicals) at a 5-fold molar excess, 10 ng/mL rhGDF15\u0026thinsp;+\u0026thinsp;anti-GFRAL (BPS 101351; BPS bioscience) at a 10-fold molar excess, 10 ng/mL HEK293-derived GDF15 (plasmid: HG10936-UT; Sino Biological), and 10 ng/mL HEK293-derived GDF15\u0026thinsp;+\u0026thinsp;anti-GDF15 at a 5-fold molar excess.\u003c/p\u003e \u003cp\u003eImage preprocessing (\u003cb\u003esupp.\u003c/b\u003e Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e) included stacking, 8-bit conversion, and background correction (Subtract Background: 50px rolling-ball radius). Frequency-domain denoising using FFT Bandpass Filter (large structures \u0026ge;\u0026thinsp;40px, small structures \u0026le;3px). Segmentation used Auto Threshold (Otsu) followed by watershed separation of touching objects. Particles were quantified (Analyze Particles: 50\u0026ndash;500px\u0026sup2;, circularity 0.20\u0026ndash;1.00) from three randomly selected fields per well. Per-well means (technical replicates) were normalized to baseline (0 h\u0026thinsp;=\u0026thinsp;100%) of each biological replicate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSupp. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003ch3\u003eWound closure assay\u003c/h3\u003e\n\u003cp\u003eHUVECs were grown to 90% confluence, wounded with a single linear scratch using a 100 \u0026micro;L pipette tip and washed twice with Phosphate-buffered saline (PBS). Scratch edges were inspected for continuity and uniformity. Serum-reduced media (1% FCS) was added to suppress proliferation, and cells were treated with 10 ng/mL rhGDF15 (n\u0026thinsp;=\u0026thinsp;3). Phase-contrast images were captured at 0, 8, 16, 24, 48, and 72 h using fixed plate coordinates.\u003c/p\u003e \u003cp\u003eWound width was quantified using the ImageJ Wound Healing Size Tool (25) (variance window radius 20, saturated pixels 0.001%, threshold 100, global scale and diagonal wound correction enabled). A rectangular ROI encompassing the wound was defined per image, and outputs were exported to CSV. Wound area was normalized to the matched control at each timepoint.\u003c/p\u003e\n\u003ch3\u003eCell cycle and Flow cytometry analysis\u003c/h3\u003e\n\u003cp\u003eHUVECs were allowed to attach for 24 h, followed by 24 h treatment with 10 ng/mL rhGDF15 (n\u0026thinsp;=\u0026thinsp;4). Cells were harvested by trypsinization, counted, and normalized to the lowest cell number. After fixation in 70% ethanol for 2 h at 4\u0026deg;C, cells were stained with a DAPI (4\u0026prime;,6-Diamidin-2-phenylindol)/Triton X-100 solution, prepared by adding 10 \u0026micro;L of a 1 mg/mL DAPI stock (D9542; Sigma-Aldrich) to 10 mL of 0.1% (v/v) Triton X-100 (T9284; Sigma-Aldrich) in PBS. Cells were subsequently analyzed by flow cytometry (BD FACS Canto II). Data analysis was performed with FlowJo v10.10.0 (Ashland, OR: Becton, Dickinson and Company; 2023). Cell populations were gated to exclude debris and aggregates. The automatic cell cycle algorithm determined G1 and G2/M phases using an asynchronous control culture as reference. Data was normalized to the untreated control within each biological replicate.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTube Formation Assay\u003c/h2\u003e \u003cp\u003eTube formation assays were performed in \u0026micro;-Slide 15 well 3D chambers to assess GDF15 effects on endothelial network assembly. Matrigel (10 \u0026micro;L/well) was thawed on ice and polymerized at 37\u0026deg;C for 30 min. HUVECs were seeded at a low density 24 h prior, then harvested and resuspended at 4 \u0026times; 10⁵ cells/mL. Cell suspension (25 \u0026micro;L; 1.0 \u0026times; 10⁴ cells) and treatment medium (25 \u0026micro;L) were added per well for final rhGDF15 concentrations of 0 and 10 ng/mL. Phase-contrast images (4x magnification) were acquired at 4, 8 and 24 h under constant settings. Images analyzed using WimTube AI Tool (Onimagin Technologies) to quantify angiogenic parameters, especially total number of loops. Nine biological replicates across two runs were planned; two wells with poor adhesion were excluded a priori (final n\u0026thinsp;=\u0026thinsp;7).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMitochondrial Functional Assay in HUVECs\u003c/h3\u003e\n\u003cp\u003eMitochondrial baseline function and stress responses were assessed by measuring the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) using the Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies) in combination with the Seahorse XF Cell Mito Stress Test Kit (103015-100, Agilent Technologies).\u003c/p\u003e \u003cp\u003eHuman umbilical vein endothelial cells (HUVECs) were seeded at 10,000 cells/well in complete cell culture medium and allowed to adhere overnight. The following morning, the medium was replaced and stimulation with GDF15 (10 ng/mL) was initiated. 4 mM HCl was used as the vehicle control (vol/vol). The medium was replaced after 24 h and fresh GDF15 (10 ng/mL) or vehicle control was added, followed by an additional 24 h incubation prior to measurement. On the day of the experiment, cells were washed with PBS and transferred to Seahorse XF DMEM Medium (103575-100, Agilent Technologies) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. Cells were incubated for 1 h at 37\u0026deg;C in a non-CO₂ incubator prior to the assay.\u003c/p\u003e \u003cp\u003eThe assay protocol consisted of five baseline measurement cycles (Mix 3 min, Wait 2 min, Measure 3 min). After sequential injections of oligomycin, FCCP (Carbonylcyanid-p-trifluoromethoxyphenylhydrazon), and rotenone/antimycin A five measurement cycles were performed for each compound using the same Mix:Wait:Measure timing (3:2:3). Final working concentrations were: Oligomycin: 1.5 \u0026micro;M, FCCP: 2 \u0026micro;M, Rotenone/antimycin A: 0.5 \u0026micro;M\u003c/p\u003e \u003cp\u003eData were normalized to cell content using crystal violet staining performed immediately after completion of the Seahorse assay.\u003c/p\u003e\n\u003ch3\u003eCrystal Violet Staining\u003c/h3\u003e\n\u003cp\u003eFollowing completion of the Seahorse assay, cells were fixed with 200 \u0026micro;L/well of 10% formalin for 15 min at room temperature (RT). Cells were then washed three times with distilled H₂O (400 \u0026micro;L/well, 2 min per wash). Cells were stained with 0.02% crystal violet solution in 10% ethanol for 30 min at RT, followed by three washes with distilled H₂O (400 \u0026micro;L/well, 2 min per wash).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eResults are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical significance was assessed by ANOVA followed by pairwise comparisons within each timepoint, controlling the False Discovery Rate (FDR) using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q\u0026thinsp;=\u0026thinsp;0.05). Q\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism (Version 10.3.1, GraphPad Software, Boston, Massachusetts USA). In the tube formation assay, potential outliers have been defined using robust regression and outlier removal method (ROUT), Q\u0026thinsp;=\u0026thinsp;1%. Images were preprocessed in Fiji (v2.16.0/1.54p; ImageJ distribution,(26)).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGDF15 promotes endothelial cell proliferation and cell cycle progression\u003c/h2\u003e \u003cp\u003eGDF15 can exert protective effects and promote proliferation of different cell types (27\u0026ndash;29). Previous reports suggested that GDF15 increases endothelial cell proliferation (24), but at concentrations about 10-fold higher than those observed in the blood of cancer patients. We treated primary human vein umbilical endothelial cells (HUVECs) with 10 ng/ml recombinant GDF15 to assess cell number kinetics. At cancer-associated levels, rhGDF15 robustly increased endothelial cell proliferation, with higher cell numbers observed at 24, 48, and 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eControlling for potential contaminating factors, previous rhGDF15 preparations have been reported to contain TGF-β (30). To ensure the observed effects were specifically due to GDF15, we treated HUVECs with rhGDF15 in the presence or absence of a monoclonal GDF15-blocking antibody. Addition of the GDF15 antibody fully prevented the pro-proliferative effects of GDF15 on endothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eTo investigate how GDF15 acts on cell cycle, HUVECs were treated with rhGDF15 for 24 hours and analyzed by flow cytometry. GDF15 reduced the G1 fraction by 12.73% and increased the G2/M fraction by 17.54% relative to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating accelerated cell cycle progression and enhanced proliferative capacity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also tested whether cell secreted GDF15 could enhance endothelial cell proliferation. HUVECs were exposed to conditioned medium from HEK293 cells transfected with a full-length GDF15 expression plasmid. Compared with conditioned medium from non-transfected HEK293 cells, GDF15-containing conditioned medium (10 ng/ml HEK293-derived GDF15) promoted HUVEC proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)\u003c/p\u003e \u003cp\u003eWe aimed to determine whether the GDF15-mediated effect on endothelial cell proliferation is mediated by GFRAL, the only well-established receptor for GDF15. It is widely assumed that GFRAL expression is restricted to neurons in the hindbrain (12) and that GDF15 potentially acts through other poorly understood mechanisms on other cell types (12,31). Analysis of published single cell RNA sequencing data sets (13,32) support the hypothesis that GFRAL is not expressed by endothelial cells. Nevertheless, we treated HUVEC with rhGDF15 in the presence or absence of GFRAL-blocking antibodies (administered at 10-fold molecular excess over GDF15). GFRAL blockade did not abolish the pro-proliferative effects of GDF15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating that GFRAL-independent signaling pathways exist in endothelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eGDF15 enhances endothelial cell migration\u003c/h2\u003e \u003cp\u003eEndothelial regeneration requires cell proliferation and migration. To determine GDF15's effects on cell migration, we performed a gap (wound) closure assay. Cell proliferation was inhibited by use of low serum condition (1% FCS). Under these conditions, rhGDF15 significantly accelerated gap closure (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e), indicating that GDF15 promotes endothelial cell migration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGDF15 promotes endothelial tube formation\u003c/h2\u003e \u003cp\u003eTo evaluate effects on angiogenic network formation, we performed a Matrigel tube formation assay. Treatment with 10 ng/ml rhGDF15 significantly enhanced tube formation compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGDF15 enhances glycolysis and mitochondrial activity\u003c/h2\u003e \u003cp\u003eDuring angiogenesis, endothelial cells require readily available energy for migration and proliferation. Therefore, glycolysis is the preferred metabolic pathway, even under normoxic conditions (33,34). To further understand the mechanism underlying the observed pro-proliferative effects, we performed a Seahorse assay to asses cellular metabolism.\u003c/p\u003e \u003cp\u003eThe extracellular acidification rate (ECAR), a surrogate of glycolytic proton efflux, was increased in the GDF15\u0026ndash;stimulated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In addition, the oxygen consumption rate (OCR) was also elevated in rhGDF15 treated cells under basal conditions, as well as during maximal respiration, while non-mitochondrial oxygen consumption was similar between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eGDF15 is a stress-responsive cytokine overexpressed in diverse conditions including cancer. While signaling through GFRAL in the hindbrain regulates appetite, nausea, and insulin sensitivity (14,35), compelling evidences indicate that GDF15 also acts on cell types that do not express GFRAL, including endothelial cells. Endothelial cells occupy a strategic location to sense circulation metabolic messengers, such as GDF15 (2), and integrate systemic and local signals. There is substantial evidence that endothelial cells respond to cancer-associated signals throughout the body (18,36), as well as to metabolic disturbances (37). Although high-dose GDF15 (on the order of 100 ng/ml) has been reported to promote angiogenesis in vitro (24), such concentrations are typically observed only in pregnancy, whereas most cancer patients exhibit serum GDF15 in the 2\u0026ndash;10 ng/ml range (2,7,38). These recent observations motivated our fucus on cancer-associated GDF15 levels and their effects on primary human endothelial cells.\u003c/p\u003e \u003cp\u003eOur results show that cancer-associated concentrations of GDF15 promote endothelial proliferation, cell cycle progression, migration, and tube formation. Using GDF15-blocking antibodies and conditioned medium, the presence of potential contaminations of recombinant GDF15 protein preparations could be ruled out.\u003c/p\u003e \u003cp\u003eEven under physiological conditions, endothelial cells derive most of their energy from glycolysis (up to ~\u0026thinsp;85%) (39). Endothelial cells use glycolysis for proliferation, migration and cell cycle progression; increased glycolysis supports DNA synthesis and cell division/proliferation (33,34,40). A Seahorse assay enabled us to better assess the metabolic situation of the HUVECs. The increased glycolytic activity of the cells shown here under 10 ng/mL GDF15 indicates a metabolic reprogramming of the cells, which could strongly support the overall pro-angiogenic effect of GDF15.\u003c/p\u003e \u003cp\u003eIn the context of cancer, malignant tumors are a major source of GDF15 (10,41) and elevated serum GDF15 levels are associated with higher epithelial-to-mesenchymal transition (EMT) of cancer cells and metastasis rates (42\u0026ndash;44). The protective effect of GDF15 on the endothelium may facilitate generation of a vascular network within tumors even under metabolic stress. (42,43,45). Thus, GDF15 appears to act as a dual regulator, contributing to both pro-vascular and pro-tumorigenic phenotypes.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings further contribute to the concept of diverse roles of GDF15 in the context of cancer. While GDF15 reprograms systemic metabolism by suppressing appetite via its receptor GFRAL expressed in the hindbrain, it also influences other cell types like the endothelium through poorly characterized receptors. As such, novel therapies using GDF15 inhibitors might not only target energy intake of patients with cancer but also other cellular functions such as tumor angiogenesis, a hallmark of cancer progression. This needs to be addressed in future studies.\u003c/p\u003e"},{"header":"Study Limitations","content":"The employed cellular models cannot fully mimic the tumor microenvironment's complexity. We also evaluated relatively short-term responses; long-term exposure to GDF15 and its effects on endothelial physiology and pathology remain to be explored in animal models. Further research is needed to identify endothelial receptor and downstream signaling mediators that link GDF15 to proliferation and angiogenesis. "},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e(rh)GDF15\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e(recombinant human) Growth Differentiation Factor-15\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGFRAL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGDNF family receptor alpha\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eERK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular-signal regulated kinases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAKT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein kinase B\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHUVEC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePrimary human umbilical venous endothelial cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFCS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFetal calf serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHEK293\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman embryotic kidney cell line 293\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDAPI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4\u0026prime;,6-Diamidin-2-phenylindol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eECAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular acidification rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxygen consumption rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFCCP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCarbonylcyanid-p-trifluoromethoxyphenylhydrazon\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoom temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFDR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFalse Discovery Rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEMT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEpithelial-to-mesenchymal transition\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cu\u003eEthics approval and consent to participate:\u003c/u\u003e All procedures involving human-derived material were performed in accordance with the Declaration of Helsinki and approved by the ethics board of University Medical Center G\u0026ouml;ttingen, approval number 28/1/23. HUVECs were isolated from human umbilical cord tissue. Written informed consent was obtained from all donors prior to tissue collection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003eThis work was supported by Deutsche Forschungsgemeinschaft (DFG) under grant number 394046768-SFB1366 \u0026apos;Vascular control of organ function\u0026apos; (project C04) awarded to M.H. and A.F.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: MH: Data acquisition and curation, formal analysis, visualization, and writing - original draft, review \u0026amp; editing . RA, FH, SK: supervision and investigation. NSdC: Data acquisition, supervision and investigation. EL: Conduction and analysis of the Seahorse Assay and writing - review \u0026amp; editing. MFH: Provided the HUVEC cells. JG, PS, JH: supervision. SSH: supervision, writing - review \u0026amp; editing. AF: conceptualization, supervision, funding acquisition, and writing - review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors gratefully acknowledge the Scientific Core Facility Cell Sorting at the University Medical Center G\u0026ouml;ttingen (Germany) for the support. Statistical analyses and Graphs were created with GraphPadPrism and edited with Inkscape.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWelsh P, Kimenai DM, Marioni RE, Hayward C, Campbell A, Porteous D, et al. Reference ranges for GDF-15, and risk factors associated with GDF-15, in a large general population cohort. Clinical Chemistry and Laboratory Medicine (CCLM). 2022 Oct 26;60(11):1820\u0026ndash;9. doi:10.1515/cclm-2022-0135\u003c/li\u003e\n\u003cli\u003eBreit SN, Tsai VW. Metabolic Messenger: growth differentiation factor 15. Nat Metab. 2025 Sep;7(9):1732\u0026ndash;44. doi:10.1038/s42255-025-01353-3 PubMed PMID: 40825850.\u003c/li\u003e\n\u003cli\u003eSigvardsen CM, Richter MM, Engelbeen S, Kleinert M, Richter EA. GDF15 is still a mystery hormone. Trends in Endocrinology \u0026amp; Metabolism. 2024 Oct;S1043276024002546. doi:10.1016/j.tem.2024.09.002 PubMed PMID: 39472228.\u003c/li\u003e\n\u003cli\u003eWang D, Jabile MJT, Lu J, Townsend LK, Valvano CM, Gautam J, et al. Fatty Acids Increase GDF15 and Reduce Food Intake Through a GFRAL Signaling Axis. Diabetes. 2024 Jan 1;73(1):51\u0026ndash;6. doi:10.2337/db23-0495 PubMed PMID: 37847913; PubMed Central PMCID: PMC10784653.\u003c/li\u003e\n\u003cli\u003eWang D, Day EA, Townsend LK, Djordjevic D, J\u0026oslash;rgensen SB, Steinberg GR. GDF15: emerging biology and therapeutic applications for obesity and cardiometabolic disease. Nat Rev Endocrinol. 2021 Oct;17(10):592\u0026ndash;607. doi:10.1038/s41574-021-00529-7 PubMed PMID: 34381196.\u003c/li\u003e\n\u003cli\u003eSj\u0026oslash;berg KA, Sigvardsen CM, Alvarado-Diaz A, Andersen NR, Larance M, Seeley RJ, et al. GDF15 increases insulin action in the liver and adipose tissue via a \u0026beta;-adrenergic receptor-mediated mechanism. Cell Metabolism. 2023 Aug;35(8):1327-1340.e5. doi:10.1016/j.cmet.2023.06.016 PubMed PMID: 37473755.\u003c/li\u003e\n\u003cli\u003eH\u0026uuml;llwegen M, Kleinert M, Von Haehling S, Fischer A. GDF15: from biomarker to target in cancer cachexia. Trends in Cancer. 2025 Nov;11(11):1093\u0026ndash;105. doi:10.1016/j.trecan.2025.06.007 PubMed PMID: 40640073.\u003c/li\u003e\n\u003cli\u003eSiddiqui JA, Pothuraju R, Khan P, Sharma G, Muniyan S, Seshacharyulu P, et al. Pathophysiological role of growth differentiation factor 15 (GDF15) in obesity, cancer, and cachexia. Cytokine \u0026amp; Growth Factor Reviews. 2022 Apr;64:71\u0026ndash;83. doi:10.1016/j.cytogfr.2021.11.002\u003c/li\u003e\n\u003cli\u003eLing T, Zhang J, Ding F, Ma L. Role of growth differentiation factor 15 in cancer cachexia (Review). Oncology Letters. 2023 Nov 1;26(5):1\u0026ndash;12. doi:10.3892/ol.2023.14049\u003c/li\u003e\n\u003cli\u003eAl-Sawaf O, Weiss J, Skrzypski M, Lam JM, Karasaki T, Zambrana F, et al. Body composition and lung cancer-associated cachexia in TRACERx. Nat Med. 2023 Apr;29(4):846\u0026ndash;58. doi:10.1038/s41591-023-02232-8\u003c/li\u003e\n\u003cli\u003eFearon K, Strasser F, Anker SD, Bosaeus I, Bruera E, Fainsinger RL, et al. Definition and classification of cancer cachexia: an international consensus. 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Artificial Cells, Nanomedicine, and Biotechnology. 2018 Nov 5;46(sup2):652\u0026ndash;8. doi:10.1080/21691401.2018.1466146 PubMed PMID: 29771147.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"GDF15, Angiogenesis, Endothelium, Cachexia","lastPublishedDoi":"10.21203/rs.3.rs-9276203/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9276203/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGrowth Differentiation Factor 15 (GDF15) is a key molecule of cellular stress responses and an established biomarker in cancer, where elevated serum concentrations correlate with cachexia and poor prognosis. Although the metabolic actions of circulating GDF15 are primarily attributed to its interactions with the GDNF family receptor alpha (GFRAL) expressed in the hindbrain, GDF15 also exerts pleiotropic effects in multiple other tissues, indicating the presence of GFRAL-independent signaling pathways. Earlier studies reported pro-angiogenic effects of GDF15 on endothelial cells, but they predominantly employed higher concentrations of recombinant protein than common in health or disease. Here, we examined how GDF15 levels commonly observed in the serum of cancer patients influence the behavior of primary human endothelial cells. At these concentrations, GDF15 enhanced endothelial cell-cycle progression, proliferation, migration, tube formation and aerobic glycolysis. Notably, pharmacological blockade of GFRAL did not diminish these responses, supporting the existence of alternative receptor mechanisms mediating GDF15 activity in the vasculature. The results suggest that GDF15 at cancer-associated concentrations has a protective effect on endothelial cells.\u003c/p\u003e","manuscriptTitle":"GDF15 at Cancer-Relevant Concentrations Promotes Angiogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 10:44:53","doi":"10.21203/rs.3.rs-9276203/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"89052997855406836540109593021537767347","date":"2026-05-14T13:42:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-28T11:31:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-02T05:34:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-01T13:30:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2026-04-01T11:38:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2bb9516e-81ec-4849-9266-3d3e3b6672ec","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"89052997855406836540109593021537767347","date":"2026-05-14T13:42:50+00:00","index":69,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T11:39:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 10:44:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9276203","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9276203","identity":"rs-9276203","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}