Anti-tumor Effect of Glycopeptides with Different Morphologies

Abstract

Tumor progression is intricately associated with dysregulated extracellular matrix (ECM) remodeling. Supramolecular fibers composed of biomolecules exhibit structural and functional characteristics reminiscent of the native ECM. Leveraging this similarity, the construction of such fibers within the pericellular space may reconstruct the dysregulated ECM and act as a promising anticancer strategy, by remodeling the interactions between tumor cells and their microenvironment. In this study, we systematically evaluated a series of self-assembled glycopeptide fibers for their ability to inhibit tumor cell proliferation and angiogenesis. A truncated glycopeptide fiber demonstrated significantly enhanced efficacy in modulating the tumor microenvironment compared to other variants. Preliminary mechanistic investigations were also conducted, which revealed that the anti-tumor effect may be attributed to the physical interaction between fibers and tumor cells, thereby providing valuable insights into the mode of action. These findings not only highlight the therapeutic potential of glycopeptide-based fibrils but also pave the way for innovative approaches in cancer treatment.

Cancer has long been a significant challenge for humanity due to its complex pathogenesis and high mortality rates. Advances in modern medicine have given rise to various methods to treat cancer, such as surgical resection, chemotherapy, and tumor immunotherapy. − These treatments are widely used in clinical practice and have significantly improved the survival rate of cancer patients, but they still have some limitations that hinder their effectiveness, including high recurrence rates after surgical resection, significant toxic side effects, and relatively poor response to immunotherapy. − To address these issues, scientists have been actively exploring innovative treatment strategies in recent years. In materials science, researchers aim to design novel materials with high biocompatibility, the potential for continuous drug administration, and broad-spectrum anticancer capabilities for cancer treatment. −

The progression of tumors is greatly related to the remodeling of their extracellular matrix (ECM), which is primarily composed of fibrous structures formed by proteins and carbohydrates. ECM remodeling is widely recognized as a key process that facilitates tumor growth. It influences the behavior of immune cells by suppressing anti-tumor immune responses, thereby contributing to tumor progression. , For instance, collagen fibers often deposit around tumor cells, increasing the stiffness of the peritumoral matrix and forming a physical barrier that hinders immune surveillance by T cells. , Recently, inspired by ECM-derived fibrous architectures, it has been demonstrated that the assembly and formation of fibrous structures around tumor cells can enhance anti-tumor efficacy by leveraging the physical and chemical properties of the fibers. These fibrous assemblies have also exhibited broad-spectrum potential in inhibiting tumor migration. However, the impact of the morphological characteristics of these fibrous assemblies on tumor cell behavior remains largely unexplored. Accordingly, the rational design and precise construction of ECM-mimetic fibrous structures, coupled with in-depth mechanistic investigations, may represent a novel and effective approach for cancer therapy.

In this work, motivated by the demand for biomaterials with high therapeutic efficacy and low toxicity in cancer therapy, we hypothesize that fibrous structures with specific chemical functionalities and mesoscale packing features hold great potential in cancer treatment. Given that sialic acid is highly expressed on the outer glycocalyx layer of cells, , its incorporation into fibrous structures may enhance biocompatibility and targeting capability. Therefore, we propose a strategy that combines sialic acid modification with ECM-inspired fibrous architectures to construct highly biocompatible biomaterials and investigate their anti-tumor efficacy.

Here, we designed and synthesized a series of fibrous structures based on glycopeptide molecules with subtle structural variations. The morphologies of these self-assembled glycopeptide fibers were precisely controlled by tuning their molecular packing. The fiber surfaces were densely decorated with sialic acid to mimic the glycocalyx of cells. Sialic acid is known to be overexpressed on cancer cells and to contribute to the formation of an immunosuppressive microenvironment. Thus, this design is expected not only to mimic the fibrous architecture of the ECM, but also to present a glycocalyx-like surface resembling that of cancer cells, potentially competing with sialic acid-mediated immunosuppressive interactions. We demonstrated that the fibrous structures with comparable morphologies exhibit varying anti-tumor efficacies while maintaining high biocompatibility. Remarkably, even in the absence of chemotherapy or immunotherapy, the fibrous structures showed significant anti-tumor effects both in vitro and in vivo.

Based on our previous work, three amphiphilic glycopeptide molecules were synthesized by coupling different numbers of hydrophobic phenylalanine (F) with hydrophilic sialic acid (SL), termed 2F-SL, 3F-SL, and 4F-SL (Figure a, b and c). By dialysis in aqueous solution, these molecules self-assembled into three distinct architectures. Specifically, as we previously reported, 2F-SL formed nanoribbons through the association between multiple fiber strands (Figure d, g and S13a), 3F-SL assembled into ultra-long left-handed double-helical nanofibers (Figure e, h and S13b), whereas 4F-SL generated right-handed twisted nanoribbons (Figure f, j). Furthermore, to better compare the morphological effect, we selectively truncated ultra-long fibers of 3F-SL by ultrasonic treatment to obtain shorter fibers, namely 3F-SL Short. The transmission electron microscopy (TEM) observations suggested that the truncated fibers exhibited an average length of approximately 1 μm (Figure i), whereas 3F-SL lengths reached hundreds of microns, thereby confirming the successful fragmentation of the ultra-long left-handed double-helical fibers.

1.

Chemical structures and assemblies of glycopeptide molecules. (a–c) Chemical structures of 2F-SL, 3F-SL, and 4F-SL. (d–f) Proposed assembly process and mechanism of 2F-SL, 3F-SL and 4F-SL. (g) TEM image of multiple fiber strands formed by 2F-SL. (h) TEM image of double-helical nanofibers formed by 3F-SL. (i) TEM image of 3F-SL nanofibers after sonication treatment. (j) TEM image of right-handed twisted nanoribbons formed by 4F-SL.

By incubating different concentrations of these fibers with B16–F10 tumor cells, CCK-8 assay was employed to evaluate the effects of different morphologies on the proliferation of tumor cells (Figure a), in which PBS and free SL were set as control groups. The time-dependent results suggested that, compared with the PBS and SL groups, all of these fibers exhibited an inhibitory effect on cell proliferation after 48 h of treatment. Among them, the 3F-SL Short had a significantly enhanced inhibitory effect (Figure b, c). Furthermore, Calcein-AM/propidium iodide (Calcein AM/PI) staining was employed to investigate the viability of tumor cells. It revealed that, 2F-SL induced a slight increase in cell death, while 3F-SL and 4F-SL caused a moderate increase in cell death. Compared with other fibers, 3F-SL Short led to a significantly higher proportion of dead cells (Figure d). To verify the differences between these fibers, TEM analysis was performed on B16–F10 cells after treatment with the respective fibers. Compared with other groups, treatment with 3F-SL Short led to severe tumor cell membrane rupture and a notable increase in cellular lysosomes, which ultimately contributed to tumor cell death (Figure e). Additionally, based on our previous research suggesting that glycopeptide fibers can inhibit tumor cell migration, free SL and four types of glycopeptide fibers were co-incubated with B16–F10 cells, and their effects on B16–F10 cell migration were assessed using a transwell assay. The results demonstrated that both 3F-SL and 3F-SL Short inhibited B16–F10 cell migration, with the 3F-SL Short exhibiting the most significant inhibitory effect (Figure S14). These results collectively indicate that 3F-SL Short exhibits the most potent anti-tumor activity, which we hypothesize may be attributed to its unique fiber morphology, as supported by the similar effects of 3F-SL. Yet, the mechanisms by which distinct morphologies modulate therapeutic efficacy, particularly the superior anti-tumor activity observed in the 3F-SL Short group, remain to be fully elucidated and warrant systematic investigation in future mechanistic studies.

2.

Effects of different glycopeptide assemblies on the proliferation and viability of B16–F10 cells. (a) Schematic representation of in vitro experiments in B16–F10 cells. (b) B16–F10 cells proliferation of SL, 2F-SL, 3F-SL, 3F-SL Short and 4F-SL were measured with concentrations of 50 μM at 24, 48, and 72 h. (c) Growth curves of B16–F10 cell proliferation treated with different glycopeptides at 50 μM. (d) B16–F10 cell viability after 72 h co-incubation with SL, 2F-SL, 3F-SL, 3F-SL Short, and 4F-SL, assessed by Calcein-AM/PI staining. (e) TEM images of B16–F10 cell treated with PBS, 3F-SL and 3F-SL Short at 72 h (Yellow arrows indicate ruptured cell membranes; white arrows indicate increased lysosomes.). Bar graphs show the mean ± SD (n = 3, t-test or one-way ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001).

SL has been reported to inhibit endothelial cell growth, thus impeding the formation of tumor blood vessels. In the tumor microenvironment, tumor blood vessels play a critical role in supporting tumor growth and metastasis by supplying nutrients. Endothelial cells in these vessels typically form tubular structures to facilitate the supply of nutrients to tumors. To further investigate the effect of glycopeptide fibers on endothelial cell growth, we performed a CCK-8 assay (Figure a), revealing that 3F-SL Short exhibited the most effective inhibition of human umbilical vein endothelial cell (HUVEC) proliferation (Figures b, c, S15 and S16). Subsequently, HUVECs were employed to simulate in vitro tube formation, and they were able to form tubular structures within 18 h. The results showed that the free SL group and the 2F-SL group did not inhibit angiogenesis, while the 4F-SL group showed a slight inhibitory effect. In contrast, both 3F-SL and 3F-SL Short exhibited significant inhibition of tube formation (Figure d, e). This suggested that the left-handed double-helical nanofibers of 3F-SL and 3F-SL Short were more effective at suppressing HUVEC tube formation, indicating their potential for anti-tumor angiogenesis. To validate the anti-angiogenic effects of different glycopeptide fibers in vivo, we utilized commercial Matrigel® to simulate the tumor environment in mice. It was demonstrated that all these fibers exhibited the ability to inhibit tumor angiogenesis. Notably, complete inhibition of tumor angiogenesis was observed in both the 3F-SL and 3F-SL Short groups, whereas residual tumor blood vessels were observed in the 2F-SL and 4F-SL groups (Figure S17). These results highlighted that the fibrous morphology of sialylated glycopeptides was more conducive in suppressing tumor angiogenesis compared to free SL.

3.

Effects of different glycopeptide assemblies on HUVEC proliferation and tube formation. (a) Schematic illustration of in vitro experiments using B16–F10 cells. (b) Proliferation of HUVECs treated with SL, 2F-SL, 3F-SL, 3F-SL Short, and 4F-SL at a concentration of 50 μM, measured at 24, 48, and 72 h. (c) Growth curves of HUVEC proliferation after treatment with different glycopeptides at 50 μM. (d) Tube formation by HUVECs treated with SL, 2F-SL, 3F-SL, 3F-SL Short, and 4F-SL. (e) Quantification of tubular structures formed by HUVECs. Gene expression levels of (f) FAK, (g) RhoA, (h) ROCK1 and (i) ROCK2 measured by qPCR analysis. (j) Tumor volume was monitored daily during the treatment period. Bar graphs show the mean ± SD (n = 3, t-test or one-way ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001).

During the initiation and progression of tumors, tumor cells remodel the ECM, generating mechanical signals that regulate cellular behavior. In addition, physical parameters of externally applied materials, such as their morphology and stiffness, also influence the mechanical responses of cells. Focal adhesion kinase (FAK) is a signaling protein involved in the formation of focal adhesion (FA), and its downstream RhoA/ROCK signaling pathway has been shown to contribute to the activation of non-myosin II motor proteins and the formation of stress fibers. To systematically investigate the structural dependency of glycopeptide fibers in modulating mechanotransduction in HUVECs, we performed quantitative PCR (qPCR) analysis of FAK, RhoA, and ROCK, which are key mechanosensitive biomarkers. Quantitative analysis revealed that 3F-SL fibers induced significant upregulation of focal adhesion kinase (FAK) and Rho-associated protein kinase (ROCK) mRNA levels, with this regulatory effect demonstrating greater potency compared to the truncated 3F-SL Short fibers (Figure f, h, and i). These findings imply that the three-dimensional entanglement architecture intrinsic to 3F-SL fibers facilitates enhanced cellular adhesion through matrix engagement while amplifying mechanical signal transduction cascades. Notably, differential regulatory patterns were observed in RhoA expression profiles. While 3F-SL fibers exhibited no statistically significant alteration in RhoA mRNA levels relative to PBS control, other fiber glycopeptide architectures markedly suppressed RhoA expression (Figure g). This distinct regulatory pattern suggests that RhoA modulation may operate through non-canonical signaling mechanisms and this hypothesis warrants further validation through pathway-specific inhibition studies. These findings suggest that glycopeptide fibers could transmit mechanical signals to regulate cellular behavior, thereby enabling the realization of their intrinsic biological functions.

To further investigate the anti-tumor efficacy of different glycopeptide fibers in vivo, their effects were evaluated using the B16–F10 melanoma model which was established in C57BL/6 mice. Notably, on the 8th day of treatment, the 3F-SL Short group began to exhibit significant inhibition of tumor growth. Moreover, as the treatment progressed, the tumor growth inhibition effect of the 3F-SL Short group became increasingly significant, exhibiting markedly greater efficacy than the other glycopeptide fiber groups. The effects of the other glycopeptide fibers were comparable, and all demonstrated a certain degree of tumor growth inhibition relative to the PBS control group (Figure j). The in vivo experiments further suggested that the morphology of glycopeptide assemblies was strongly correlated with their anti-tumor effect, and 3F-SL Short still showed the strongest anti-tumor effect.

In addition, to explore the combined effect on tumor immunity of glycopeptide fibers as a supporting material with other drugs, we selected 3F-SL due to its unique structural characteristics. Specifically, 3F-SL possesses a high aspect ratio and abundant surface carboxyl groups, which not only facilitate stable spatial entanglement but also provide abundant nucleation sites for mineralization. Therefore, the carboxyl groups on 3F-SL were directly mineralized with calcium carbonate (CaCO₃) on the fiber surface. TEM revealed that the surface of 3F-SL was uniformly coated with spherical CaCO₃ nanoparticles approximately 50 nm in diameter (Figure a). Atomic force microscopy (AFM) further confirmed that CaCO₃ was predominantly deposited on the fiber surface (Figure b). Upon magnification, CaCO₃ was observed to be effectively mineralized on the fiber, forming a uniform spherical morphology (CaCO₃/Fiber) (Figure c). Further investigation of the immunomodulatory effects of CaCO₃/Fiber combined with CD47 antibody (CaCO₃/Fiber + CD47) was performed in the tumor microenvironment. In the B16–F10 melanoma model of C57BL/6 mice, CaCO₃/Fiber + CD47 treatment downregulated the expression of CD206 in M2-type macrophages and upregulated the expression of CD80 in M1-type macrophages (Figure d, e), indicating the regulation of the tumor microenvironment. Further analysis of dendritic cells (DCs) in the tumor microenvironment showed that CaCO₃/Fiber + CD47 treatment promoted the high expression of CD80 and CD86 molecules, suggesting enhanced activation of DCs (Figure f). Immunohistochemical analysis revealed that both the CaCO₃/Fiber and CaCO₃/Fiber + CD47 treatment groups downregulated regulatory T cells (Foxp3⁺), with the combined treatment showing the most significant effect, indicating that CaCO₃/Fiber can enhance the regulatory effect of CD47 antibody on immunosuppressive T cells (Figure S18). Compared with the control group, histological examination of the major organs (heart, liver, spleen, lung, and kidney) of treated mice in the CaCO₃/Fiber and CaCO₃/Fiber + CD47 groups showed no abnormalities, indicating the safety of this treatment method (Figure S19). These results suggest that using 3F-SL as a supporting material for mineralized CaCO₃ (CaCO₃/Fiber) can regulate the tumor microenvironment without causing organ damage.

4.

3F-SL fiber after CaCO₃ mineralization combined with CD47 antibody anti-tumor effect. (a, b) TEM and AFM images of SL-3F fibers after CaCO₃ mineralization. (c) AFM magnified image of SL-3F fibers after CaCO₃ mineralization. (d) M2-like macrophage (CD206^hi) and (e) M1-like macrophage (CD80^hi) gating on F4/80⁺CD11b⁺CD45⁺ cells. (f) Representative flow cytometric analysis images and relative quantification of CD80⁺CD86⁺ dendritic cells gating on CD45⁺CD11c⁺ cells. Bar graphs show the mean ± SD (n = 3, t-test or one-way ANOVA, *p < 0.05; **p < 0.01; ***p < 0.001).

In this study, we synthesized a series of glycopeptide molecules and self-assembled glycopeptide fibers with distinct morphologies in aqueous solutions. Our findings indicate that all glycopeptide fibers, regardless of their morphology, exhibited notable anti-tumor activity. Among them, the truncated glycopeptide fibers demonstrated the most pronounced anti-tumor effects. We hypothesize that this enhanced efficacy is due to the stronger interactions with tumor cells. Moreover, the morphology of glycopeptide fibers plays a critical role in modulating their anti-tumor activity. This observation underscores the importance of further investigating how the assembly morphology and spatial arrangement of glycopeptides influence tumor cell behavior, tumor progression, and potential therapeutic strategies. Such insights may open new pathways for the development of novel cancer treatments.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/polymscitech.5c00093.

• Experimental reagents and equipment; synthesis route and ¹H NMR characterization of glycopeptide molecules; the experimental steps involved in this study; some experimental data mentioned in the text (PDF)

The authors declare no competing financial interest.