https://orcid.org/0000-0003-3315-4797
Figures
Abstract
Gastric cancer is among the most common cancers and represents a major public health problem worldwide. New therapeutic strategies and drugs are needed. Anti-angiogenic agents targeting the Vascular Endothelial Growth Factor (VEGF) are used in combination therapy in the clinic, although their efficacy remains modest. We believe that these large anti-VEGF antibodies could be advantageously replaced by smaller peptides with better tissue penetration. In this study, we evaluate the efficacy of a previously described dimer peptide ligand of VEGF, D6, in inhibiting the proliferation of gastric cancer cells and the growth of the corresponding murine xenograft. The activity of the D6 peptide in these assays was comparable to that of bevacizumab, the positive control antibody, although the peptide required repeated injections at higher molar concentrations. These promising results justify the continued optimization of the peptide dimer, currently under investigation in our laboratory.
Citation: Ye X, Hu H, He Y, Ye F, Jin J, Olivier E, et al. (2026) A dimer peptide ligand of vascular endothelial growth factor slows the progression of human gastric tumors in mouse xenografts. PLoS One 21(8): e0344142. https://doi.org/10.1371/journal.pone.0344142
Editor: Alireza Shoari, Mayo Clinic Cancer Center, UNITED STATES OF AMERICA
Received: March 16, 2026; Accepted: July 15, 2026; Published: August 3, 2026
Copyright: © 2026 Ye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Gastric cancer is among the most common cancers, with approximately one million new cases diagnosed worldwide each year. Despite recent advances in treatment, this disease still causes more than 650,000 deaths, and the number of cases in people under 50 is increasing. The incidence of gastric cancer is higher in Asian, South American and Eastern European countries than that in the rest of the world [1]. Surgery offers a high chance of survival for operable gastric cancers. In patients with advanced, recurrent or metastatic cancers, systemic chemotherapy is the first line treatment, with possible additional treatments, such as biological agents targeting HER2 and the tight-junction CLDNP18.2 [1].
Vascular Endothelial Growth Factor (VEGF) is the most potent pro-angiogenic cytokine, and inhibition of its signaling pathway is one of the most effective strategies for blocking angiogenesis, a key process in the progression and metastasis of many solid tumors, including gastric adenocarcinoma [2–4]. In gastric cancer, the standard second-line treatment includes, in addition to paclitaxel, the anti-VEGF receptor-2 (VEGFR-2) monoclonal antibody ramucirumab, to block angiogenesis. Combinations with immune checkpoint inhibitors are also the subject of numerous studies [1,5]. Several tyrosine kinases (TK) inhibitors, including sorafenib, apatinib and regorafenib, targeting the VEGF receptors and other TK receptors with varying degrees of selectivity, have been tested in the clinic as combination therapy, resulting in some cases in beneficial effects, but with significant side-effects [2]. For example, apatinib, which selectively targets VEGFR-2, was approved for the treatment of advanced gastric cancer in China in 2014 [6]. Recently, the multikinase inhibitor regorafenib has shown improved survival compared to placebo in third line treatment [7]. The anti-VEGF antibody bevacizumab has been extensively evaluated in preclinical models and in the clinic in several phase II/III studies for the treatment of gastric cancer [2]. Phase II studies have shown that bevacizumab increases the effectiveness of chemotherapy for advanced gastric cancer, although two phase III trials failed to demonstrate any overall survival benefit. The authors note that a benefit may have been observed in non-Asian patients compared to Asian patients [3]. Following the results of a phase II/III resectable esophagogastric adenocarcinoma, bevacizumab was not recommended in combination with perioperative chemotherapy, due to the lack of improvement in survival and certain adverse effects [2]. These results could be explained by the density of the tumor stroma preventing sufficient perfusion of bevacizumab to the tumor, or by the activation of other angiogenic factors or angiogenic signaling pathways [8]. However, a recent retrospective study demonstrated that, in patients with locally advanced gastric cancer, the addition of neoadjuvant bevacizumab to chemotherapy resulted in longer survival, with tolerable adverse events. The authors of the study concluded that its application required further verification [9]. Overall, targeting the VEGF pathway to treat gastric cancer is a validated strategy, but it has led to variable results. While monotherapy has proven highly effective in preclinical animal models, combination therapy is required in clinical practice, and results depend on the target (VEGF, extracellular domain of VEGFR or TK domain), drug selectivity and affinity, cancer stage (first, second, or third line), patient history and combination drug. Therefore, it is necessary to develop new molecules targeting the VEGF pathway, exhibiting improved activity and different pharmacokinetic properties. In particular, we believe that tissue penetration properties should be improved. Indeed, the tumor microenvironment is complex, and the uncontrolled proliferation of blood vessels can prevent large biologics, such as antibodies, from reaching the tumor [10].
In this context, our objective is to study new small VEGF ligands capable of blocking its angiogenic activity, with tissue penetration properties different from those of large biologics such as antibodies. To our knowledge, there is no known small molecules capable of directly targeting VEGF and preventing its binding to VEGFRs. Peptides offer a compromise between their size (generally 1–5 kDa) and their ability to target such protein-protein interactions. Recent examples of successful peptide-based cancer therapeutics demonstrate their ability to target tumors and induce biological activity, as well as their versatility as molecular scaffolds for multivalent constructs. Tivdak (tisotumab vedotin) is an antibody-drug conjugate whose payload is the antineoplastic peptide monomethyl auristatin E, linked to the antibody by a cleavable peptide linker. It was approved by the FDA in 2021 and in the EU in 2025 for the treatment of recurrent or metastatic cervical cancer [11]. Pepaxti (melphalan flufenamide) is a fluorinated peptide prodrug of melphalan, approved by the EMA in 2022 for the treatment of multiple myeloma [12]. Lutathera ([177Lu]Lu-DOTA-TATE, containing the somatostatin receptor antagonist octreotate peptide) is the first “Peptide Receptor Radionuclide Therapy”, approved by the FDA in 2018 for adults with gastroenteropancreatic neuroendocrine tumors and in 2024 as first drug specifically for pediatric patients [13]. Lumisight (pegulicianine) is a multivalent molecule composed of a far-red fluorescent cyanine dye, a fluorescence quenching agent, a polyethylene glycol (PEG) side chain, and a tumor microenvironment-sensitive peptide linker. It was approved in 2024 as an optical imaging agent for the detection of cancerous tissues [14]. In our ongoing study to optimize VEGF-binding cyclic peptides previously identified by the phage-display technique [15], we shortened their size [16] and improved their affinity by introducing an additional cyclization [17]. From these bicyclic monomeric peptides, we synthesized dimeric peptides linked by a PEG linker capable of targeting the two symmetrical binding sites of the VEGF, which is a homodimeric protein. These peptide dimers bind to VEGF with high affinity, thereby preventing VEGFR activation and blocking VEGF-induced HUVEC proliferation and migration [18]. Herein, in order to determine their potential antitumor activity in gastric cancer, the most promising peptide dimer was evaluated on the human gastric cancer cell line SGC-7901 and on a xenografted murine model.
Materials and methods
Peptide synthesis
The amino acid coupling, cyclization and dimerization procedures followed the previously published synthesis, with some modifications [18]. The scale was increased to two batches of 0.2 mmol of resin, with Rink Amide MBHA resin (substitution 0.78 mmol/g or 0.52 mmol/g) instead of NovaSyn TGR resin (substitution 0.25 mmol/g). The coupling reactions were carried out on an Activo-P14 semi-automatic peptide synthesizer (Activotec) using Nα-Fmoc amino acids (5 equiv relative to the resin) activated with DIC (5 equiv) and OxymaPure (5 equiv) in dimethylformamide (DMF). HPLC analysis was performed using an Uptisphere C18 column (5 µm, 4.6 × 250 mm, Interchim) at a flow rate of 1 mL/min. The purifications were performed by semi-preparative HPLC on a Grace Alltima C18 column (5 µm, 10 × 250 mm, Vydac) with a gradient program from 20 to 100% B over 40 min and a flow rate of 2 mL/min. The solvents used were water containing 0.1% trifluoroacetic acid (TFA) (solvent A) and a 70% aqueous solution of acetonitrile containing 0.09% TFA (solvent B). The products were detected by UV at 220 and 254 nm.
Briefly, after the synthesis of the first 5 amino acids, the orthogonal protecting groups phenyl isopropyl (Pip) and methyl trityl (Mtt) were removed by treating the peptide resin with 3% TFA in dichloromethane, followed by neutralization with 5% DIEA in DMF. Cyclization was achieved on resin in DMF using 4 equiv of PyBOP and 10 equiv of DIEA relative to the resin, at room temperature for 14 h. Once cyclization was complete, the peptide was elongated to its full length by standard SPPS and acetylated. The peptide was cleaved from the resin with TFA/H2O/2,2’-ethylenedioxy diethane thiol (EDDT)/triisopropylsilane (TIPS) (94:2.5:2.5:1.0) for 3 h. The intramolecular disulfide bond was formed by dissolving the crude peptide in dimethyl sulfoxide and adding drops of aqueous ammonia to maintain a basic pH. The reaction was monitored by HPLC. After completion of the cyclization, the crude bicyclic peptide monomer M1 was purified by semi-preparative HPLC and lyophilized to obtain a white solid. Two batches of monomer peptide were synthesized. In the first batch, 0.2 mmol Rink Amide MBHA resin (0.78 mmol/g) gave 300 mg crude peptide, resulting in 27 mg of pure M1 (7% yield). The second batch of 0.2 mmol Rink Amide MBHA resin at 0.52 mmol/g gave 437 mg crude peptide, resulting again in 27 mg of pure M1 (7% yield), for a total of 54 mg. The D6 dimer was synthesized by reacting the peptide monomer M1, DIEA (10 equiv relative to peptide), and NHS-PEG25-NHS (0.5 equiv relative to peptide) in DMF. The reaction was monitored by HPLC, and the dimer was purified by semi-preparative HPLC, yielding 36 mg of D6 (51% yield, purity > 98% by HPLC). The identity of D6 was confirmed by HRMS with ESI.
VEGF-induced human gastric cancer SGC-7901 cell proliferation assay
The assay was performed as previously described [19]. Briefly, SGC-7901 human gastric cancer cells (2 × 103 cells/well) were seeded onto a 96-well plate in RPMI (Roswell Park Memorial Institute) 1640 medium (Gibco, Life Technologies) containing 2% FBS (Gibco, Life Technologies), and incubated overnight at 37°C, with 5% CO2. The medium was then removed and a new serum-free medium was added, in the presence or absence of 50 ng/mL VEGF-A (R&D Systems) and with different concentrations of D6 (0.4, 2, 10 and 50 μM), or bevacizumab (6.5 μM, 1 mg/mL, Roche), or control group (serum-free medium). The plate was incubated for an additional 48 h (6 wells/concentration/group). Cell proliferation was quantified by Cell Counting Kit-8 (CCK-8, Sigma) assay according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (AMR-100, ALLSHENG, Hangzhou). The experiments were repeated three times.
Cell viability assay on HCE-T
Human corneal epithelial (HCE-T) cells (RIKEN biobank, Tsukuba, Japan) [20] were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 0.5% penicillin-streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2 and handled under sterile conditions in a laminar flow cabinet. Culture medium was renewed regularly, and cells were passaged upon reaching approximately 80–90% confluence. For subculture, cells were detached using trypsin, resuspended in complete medium, and seeded into 96-well plates at a density of 90,000 cells/mL prior to experimental treatments.
The peptide D6 was dissolved in sterile water to obtain a stock solution of 5 mM. A working solution was prepared by diluting the stock solution 1:100 resulting in a final concentration of 50 µM in culture medium supplemented with 2.5% fetal bovine serum (FBS), 1% L-glutamine, and 0.5% penicillin-streptomycin. Cells were exposed to the peptide-containing medium for 24 h under standard culture conditions (37°C, 5% CO2). Control cells received an equivalent volume of sterile water corresponding to the final vehicle concentration (1%, v/v).
Cell viability was assessed using the Neutral Red Uptake Assay, which is based on the ability of viable cells to incorporate and retain the supravital dye Neutral Red within lysosomes, as previously described [21]. Briefly, a Neutral Red stock solution (0.4% w/v in distilled water) was prepared, diluted 1:80 in culture medium without serum, and centrifuged prior to use to remove potential dye aggregates. Following exposure to the test conditions, cells were incubated with the Neutral Red working solution for 3 h at 37°C in a humidified atmosphere containing 5% CO2. Cells were then washed with phosphate-buffered saline (PBS), and the incorporated dye was extracted using a water/ethanol/acetic acid (49.5/49.5/1, v/v/v) solution. After homogenization, fluorescence intensity was measured using a Spark microplate reader (Tecan, Männedorf, Switzerland) at an excitation wavelength of 540 nm and an emission wavelength of 600 nm. A 1% Triton X-100 solution was used as a positive control for cytotoxicity. Cell viability was expressed as a percentage of the untreated control, which was set at 100%.
In vivo antitumor study on a xenografted mouse model
The assay was performed as previously described [19]. The evaluation of antitumoral activity was carried out at the Laboratory of Experimental Animal Science, Hangzhou Normal University (Hangzhou, China), maintained under standardized environmental conditions, with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Zhejiang Chinese Medical University (written approval No. IACUC-20230410–02). All efforts were made throughout the experiments to minimize mice suffering and distress. They were weighed and their behavior was monitored every two days. They were housed in cages measuring 290 mm × 178 mm × 160 mm, with 6 mice per cage, and were provided with food and water ad libitum. Research staff underwent the required training in animal welfare, handling techniques, anesthesia, and humane endpoint recognition before participating in the experiment. The humane endpoint criteria were: tumor volume greater than 2000 mm3; tumor diameter greater than 2 cm in any direction; body weight loss greater than 20%; ulceration, necrosis, or infection of the tumor; severe lethargy, impaired mobility, or inability to access food/water; signs of pain or distress unrelieved by supporting care.
SGC-7901 human gastric cancer cells (1 × 106 cells/500 μL) from the Cell Bank of the Chinese Academy of Sciences (Shanghai) were subcutaneously injected into the right flank of 8-weeks-old BALB/c female mice (a total of 24 mice). When the tumor size reached 100–300 mm3, BALB/c mice were randomly divided into groups (n = 6). D6 peptide (5 mg/kg/day and 15 mg/kg/day) and PBS (control group) were administrated intravenously for 2 weeks, and bevacizumab (5 mg/kg) was administrated intravenously once, as a positive control. The tumor volume was measured every 2 days with a digital Vernier caliper, using the following formula: v = a2 × b × 0.52 (where a is the shortest diameter and b is the longest diameter of the tumor). No mortality was observed and no animal met humane endpoint criteria before the end of the study. After 14 days of treatment, the mice were euthanized within 24 hours using CO2 to induce analgesia and anesthesia, followed by cervical dislocation to ensure euthanasia, and the solid tumors were separated from the bodies, photographed and weighed (balance OHAUS Adventure). For this study, no informed consent was required as the experiment did not include human participants.
Statistical analysis
For Fig 2A and Fig 3, the data are expressed as the arithmetic mean ± SEM of at least three different experiments. The statistical significance of the results was assessed by a one-way analysis of variance (ANOVA). For Fig 2B, results were normalized to the untreated control group and expressed as percentage of the control (control = 100%) and presented as mean ± SD. The normality of data distribution was assessed using the Shapiro-Wilk test. Statistical differences between groups were evaluated by one-way ANOVA followed by Dunnett’s multiple comparisons test using the untreated control as the reference group.
A. Cellular assays of D6 and bevacizumab on a gastric cancer cell line. Human gastric cancer cells SGC-7901 were treated without (-) or with (+) VEGF, and supplemented with D6 (at the indicated concentrations), or bevacizumab, or serum-free medium as control groups. Relative cell proliferation (%) was analyzed with GraphPad Prism 8 software. The data are presented as mean ± SD, compared to the control group by one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, “ns” non-significant. B. Effect of D6 incubation at 50 µM on HCE-T cell viability. Cell viability was evaluated in HCE-T cells after 24 h exposure using the Neutral Red Uptake Assay. Results are expressed as percentage of the untreated cells (set at 100%) and presented as mean ± SD of three independent experiments. The control line represents the solvent effect (1% water). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test against the untreated control. p < 0.05 was considered statistically significant. ****p < 0.0001 versus untreated control.
(A) Quantification of tumor volume every 2 days. (B) Quantification of tumor weight after 14 days. (C) Images of tumors taken from mice after 14 days of treatment with D6 (5 mg/kg/day and 15 mg/kg/day), with bevacizumab (5 mg/kg, once) or with PBS (control). (D) Body weight of mice was quantified every 2 days. Data are presented as mean ± SD, compared to the control group by a one-way ANOVA statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Data were analyzed using GraphPad Prism (version 8; GraphPad Software, San Diego, CA, USA), and statistical significance was set at p < 0.05.
Results
The D6 dimer was chosen because it exhibited the highest affinity for VEGF, with Kd,20°C = 9 nM and Kd,37°C = 26 nM, and the strongest anti-angiogenic activity in HUVEC assays. The new synthesis was adapted from our previously published procedures to provide sufficient quantity to allow in vivo experiments at two doses on six mice [18]. Monomer M1 was synthesized from two batches of 0.4 mmol of Rink amide MBHA resin pooled together. The purified monomer was then homodimerized via its Lys 6 side chain with a PEG25 linker activated by N-hydroxy succinimide groups, to yield 36 mg of D6 (MW = 5128 g/mol), whose HPLC chromatogram (purity > 98%) and mass spectrometry spectra matched previously published data (Fig 1) [18].
The D6 dimer was assayed on the human gastric cancer cell line SGC-7901 (Fig 2A). In the groups supplemented with VEGF (50 ng/mL), D6 induced a dose-dependent inhibition of SGC-7901 cell proliferation at concentrations of 0.4, 2, 10 and 50 μM. At 50 μM, it exhibited antiproliferative activity similar to that of bevacizumab at 6.5 μM, used as positive control. In the groups without VEGF supplementation, no significant effects on cell proliferation were observed, indicating that peptide D6 did not induce cytotoxicity at concentrations up to 50 μM (Fig 2A). Safety was confirmed by cell viability assays on the non-cancer human corneal epithelial cell line (HCE-T). As shown in Fig 2B, the vehicle control (1% water) did not affect the viability of HCE-T cells compared to untreated cells. As expected, the positive control Triton X-100 markedly reduced cell viability to 62.1% of that of the untreated control. In contrast, treatment with D6 at 50 µM for 24 h did not alter cell viability, which remained at 101.4% compared to untreated cells.
D6 (5 mg/kg/day and 15 mg/kg/day) and bevacizumab (5 mg/kg, as a single injection, used as a positive control) were administrated by intravenous injection for 2 weeks on BALB/c nude mice bearing a subcutaneous xenograft of SGC-7901 cells. Tumor volume and body weight of mice were measured every 2 days. Peptide D6 displayed dose-dependent inhibition of tumor growth. Compared to the PBS group, administrated at a dose of 5 mg/kg/day, it reduced tumor volume by 54% and tumor weight by 56%, and administrated at a dose of 15 mg/kg/day, it reduced tumor volume by 68% and tumor weight by 69%. Bevacizumab reduced tumor volume by 75% and tumor weight by 78% with a single administration of 5 mg/kg (Fig 3A, 3B and 3C). During the 2 weeks of the experiment, the inhibitory effect of D6 administrated at 15 mg/kg/day on tumor volume was comparable to that of bevacizumab administrated at 5 mg/kg once (Fig 3A). No mortality was observed in mice during the 2 weeks of treatment, and monitoring of body weight showed no significant variation compared to the PBS control group, with a slow and steady increase in body weight, as expected (Fig 3D). Therefore, no obvious toxicity of D6 was observed up to 15 mg/kg/day.
Discussion
Angiogenesis is a key process in the growth of solid tumors. Folkman’s initial report suggested that “anti-angiogenesis” treatments should prevent the formation of new blood vessels within the solid tumor, resulting in several therapeutic benefits [22]. Since then, anti-angiogenic drugs targeting the main pro-angiogenic growth factor VEGF have been used successfully, often in combination with other drugs, to treat cancers, including gastric cancer [2]. There remains a need for new, more potent anti-angiogenic drugs with designed pharmacological properties. In preclinical studies aimed at developing such anti-VEGF drugs, activity assays with on HUVECs and cancers cells requiring this growth factor for their proliferation are standard practice.
We have previously reported a series of VEGF dimer peptide ligands, among which the D6 dimer showed the best inhibitory activity in HUVEC-based assays [18]. Therefore, we tested here its ability to inhibit VEGF-induced proliferation of human SGC-7901 gastric cancer cells and the growth of solid tumor xenografts of the same cell line in nude mice. This cell line was chosen because it is one of the most widely used gastric cell line in preclinical anti-tumor and anti-angiogenic assays, both for cell-based assays and xenografts in mice. It has high mRNA levels of VEGF and VEGFR-2, and requires VEGF for its proliferation [23].
We show here that the binding of D6 to VEGF was able to effectively suppress the proliferation of SGC-7901 cells in a dose-dependent manner, with inhibitory activity observed from 0.4 µM. The peptide was not toxic to SGC-7901 cells and to HCE-T up to concentrations of 50 µM, an important safety feature that has already been demonstrated on HUVEC [18]. In mouse xenografts, the D6 dimer peptide significantly inhibited tumor growth of SGC-7901 cells at a dose of 5 mg/kg/day, and its efficacy at 15 mg/kg/day was comparable to that of bevacizumab at 5 mg/kg. We performed an in vivo evaluation of the peptide at doses 5 mg/kg/day and 15 mg/kg/day, as peptides are generally considered to have low in vivo stability [24]. It is interesting to note that the peptide was administered by intravenous injection, demonstrating its ability to reach the tumor xenograft from the bloodstream. These results can be explained by its bicyclic nature and the presence of the PEG linker, which may improve its in vivo stability compared to unmodified linear peptides [25].
We have previously described cyclic peptide ligands of the VEGFRs, designed from a VEGF epitope, which have anti-angiogenic activity in vitro. Among these peptides, we tested B-cL1 under the same conditions as those used here, i.e., the SGC-7901 gastric cancer cell line and the corresponding mouse xenograft. The B-cL1 peptide inhibited tumor growth with similar efficacy to bevacizumab at a lower dose of 5 mg/kg/day, which is slightly better than D6 [19]. This result could indicate that targeting the receptor would be a more effective strategy than targeting circulating VEGF in the context of gastric cancer. Bevacizumab (150 kDa) was administered by intravenous injection every 14 days at a dose of 5 mg/kg, the dose usually used in clinical practice [8]. Comparing the active concentrations in mol/L of bevacizumab and D6 (5.1 kDa), it appears that a higher molar concentration of the peptide is required for a comparable therapeutic effect, in agreement with the differences in affinity values measured under the same assay conditions (for the binding of bevacizumab to VEGF, Kd,20°C = 0.66 nM) [18]. Therefore, new peptides with improved affinities are currently being developed within our research team.
Overall, the promising results obtained with D6 in mouse xenograft models warrant further pharmacokinetic and pharmacodynamic studies. The demonstration of the therapeutic potential of this anti-VEGF peptide dimer in gastric cancer opens the way to a wide range of VEGF-dependent pathologies, including not only different types of cancers, but also ocular pathologies such as age-related macular degeneration, diabetic retinopathy, and corneal neovascularization.
Acknowledgments
We thank the Laboratory of Experimental Animal Science, Hangzhou Normal University (Hangzhou, China) for animal assay. We thank Université Paris Cité, the CNRS, and INSERM for their support.
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