Deciphering the cell surface glyco-code: a promising perspective on unveiling the vulnerability of cancer stem cells

Structural characteristics of glycans in cancer stem cells (CSCs)

Cellular glycosylation represents a complex molecular language, wherein the human glycose “alphabet” encompasses 10 monosaccharides that can be interlinked in a myriad of permutations. Among the prevalent membrane protein glycosylation modalities, N- and O-glycosylation play pivotal roles. N-glycosylation involves the attachment of a glycan chain to the Amide nitrogen group of an asparagine residue within the polypeptide sequence (Asn-X-Ser/Thr, with X being any amino acid other than proline) with three main types identified (high mannose, heterozygous, and complex N-glycans). A high mannose N-glycan has terminal branches consisting only of mannose. Complex and heterozygous glycans may contain residues of galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), N-acetylneuraminic acid (NeuAc), and N-glycylneuraminic acid (NeuGc). A heterozygous glycan contains an unsubstituted terminal mannose residue¹. O-glycosylation refers to the attachment of GalNAc to the hydroxyl group of serine (Ser), threonine (Thr), or tyrosine (Tyr) residues within polypeptides. This initial modification is subsequently elaborated by glycosyltransferases within the medial and trans-Golgi compartments, leading to the formation of diverse O-glycan structures, ranging from core structure 1 to core structure 8¹. The term “glyco-code” refers to the specific patterns and structures of glycans that encode biological information, thereby modulating cellular communication, immune responses, and disease progression. The glyco-code is deciphered by glycan-binding proteins, which recognize and bind to specific glycan structures, thereby mediating various cellular processes². Understanding the glyco-code is crucial for developing targeted therapies for diseases, including cancer and autoimmune disorders.

Metabolism has a crucial role in regulating the structure and function of glycoproteins through complex modifications to glycans. In the context of cancer, tumor cells undergo metabolic reprogramming, resulting in significant modifications to glycan structures on glycoproteins via the up- or down-regulation of specific glycosyltransferases and glycosidases. Such aberrant glycosylation patterns can contribute to tumor progression, immune evasion, and metastasis³. The characteristic glycans are thus promising candidates for clinical diagnostic markers and therapeutic targets. For example, the MUC1 glycoprotein is often overexpressed in tumors and associates with CSC characteristics, positioning the MUC1 glycoprotein as a potential target for vaccine development. Similarly, dinutuximab, which targets the disialoganglioside, GD2, is used clinically to treat neuroblastoma⁴.

Unique glycan structures distinguish CSCs from non-stem cancer cells. For example, liver cancer CSCs exhibit significantly increased fucosylation, particularly α-1,2 fucosylation, along with elevated expression of the fucosylation-related gene, FUT1, which is associated with increased resistance to drugs⁵. Breast CSC-like cells display high levels of α-2,3 sialylated core type 2 O-linked glycans⁶. Furthermore, CSCs in liver cancer and glioma possess high levels of mannose-type N-glycans on the surface due to low expression of mannosidase⁷. Notably, the expression of the ganglioside, GD2, is markedly increased in CSCs and downregulation of the GD2 level causes a phenotypic change from a CSC phenotype to a non-CSC phenotype⁸. These glycosylation changes not only serve as important biochemical markers but also offer insights into the mechanisms underlying the pronounced resistance of CSCs to conventional therapies. In practical clinical applications, research efforts could focus on developing biomarkers associated with these glycosylation features to identify and monitor CSC dynamics at an early stage. These methods would enable more precise treatment regimens, improve treatment efficiency, and reduce the likelihood of cancer recurrence.

Contribution of glycans to CSC characteristics and the underlying mechanism

Glycosylation is crucial in modulating the properties of CSCs and influencing cancer progression (Figure 1). For example, N-glycosylation of EpCAM enhances self-renewal, epithelial-mesenchymal transition (EMT), and CSC stemness properties⁹. CD133, a widely recognized CSC marker, interacts with glycosyltransferase 8 domain 1 (GLT8D1) under hypoxic conditions to inhibit lysosomal degradation, thereby maintaining the glioma stem cell characteristics¹⁰. Specifically, the high mannose-type N-glycan of CD133 strengthens the CD133-DNMT1 interaction, which activates the transcription of p21 and p27, thereby maintaining stem cell stemness⁷. Additionally, sialylation has an effect on CSC characteristics. Upregulation of another important glycosyltransferase, ST6Gal-I, has been implicated in imparting CSC traits to cells¹¹. Moreover, in breast CSCs, α-2,3 sialylation of the core type 2 O-glycan chain of CD44 facilitates the interaction with hyaluronic acid, thereby enhancing CSC properties⁶. Finally, GALNT1, a key O-glycosylation enzyme, activates SHH signaling, which is crucial for self-renewal¹². Glycosylation has a critical role in influencing the stability and degradation pathways of key proteins, as well as maintaining CSC characteristics by regulating important signaling pathways. This insight paves the way for the development of effective cancer therapies and presents challenging yet promising research topics for the future.

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Figure 1

The unique glycan chain structures on the surface of CSCs have an important role in maintaining the characteristics of CSCs. (A) The high-mannose type N-glycan of CD133 is necessary for its interaction with DNMT1. The collaboration between CD133 and DNMT1 facilitates the repression of p21 and p27 through a promoter methylation mechanism, thereby enhancing the tumorigenesis of CSCs. (B) The O-linked, core 2 α-2,3 sialoglycan expressed on CD44 promotes its interaction with hyaluronic acid (HA), triggering the recruitment of Src family kinases, which are critical for phosphorylation of various signaling proteins. The downstream consequences of these phosphorylations lead to the activation of STAT3, a transcription factor that, upon dimerization and nuclear translocation, induces the expression of numerous genes, including VEGF, PD-1, and TGF-β. This sequence of events enables CSCs to secrete cytokines and growth factors that suppress immune cell functions, thus evading anti-tumor immune responses. (C) GALNT1 mediates O-linked glycosylation of SHH to promote the activation of sonic hedgehog (SHH) signaling. Upon binding of SHH to the Ptch receptor, a conformational change occurs that alleviates inhibitory control of Ptch over Smo. The release of this inhibition activates Smo, triggering a cascade of signaling events. In the presence of SHH, GLI proteins are converted into the active forms, which then translocate to the nucleus. Within the nucleus, these proteins bind to specific promoter sequences, activating target genes, such as Gli1 and CCND1, which are critical for the self-renewal and maintenance of CSCs (figure generated in Figdraw).

Significance of glycan-lectin interactions in the CSC microenvironment

The interactions between glycans and lectins within the cancer microenvironment significantly impact cancer biology. Lectins, which are glycan-binding proteins, recognize these carbohydrate structures and facilitate cell-cell and cell-matrix interactions¹³. In the context of CSCs, these interactions are vital for regulating tumor progression, metastasis, and immune evasion. For example, galactin-3, which is secreted by CSCs in various tumor tissues, promotes apoptosis of T cells by binding to the immune checkpoint molecule, PD-1¹⁴. Additionally, high levels of mannose-type N-glycans on the surface of hepatocellular carcinoma stem cells promote tumor formation and immune escape by interacting with mannose-receptors on lymphatic endothelial cells¹⁵. Finally, a TIM-3/galectin-9 autocrine stimulatory loop drives the self-renewal of human myeloid leukemia stem cells and leukemic progression¹⁶. Consequently, glycans can act as signaling molecules that initiate various biological processes within the CSC microenvironment. Understanding these molecular interactions provides vital insights into the mechanisms underlying cancer progression and may reveal potential targets for therapeutic intervention.

Application of glycans in sorting CSCs

Lectins have long been instrumental in characterizing cell surface glycan structures because of specific selectivity for branching, linkage, and terminal modification of glycans. Lectins are frequently used in the affinity purification of glycoproteins and glycopeptides¹³. Glycan capture technology, which utilizes lectins, represents a systematic method for identifying glycoproteins, glycosylation sites, and glycan profiles. Lectin microarrays are commonly used to directly analyze the surface glycosomes of living mammalian cells. These microarrays, which are composed of fluorescently labeled lectins, are incubated with living cells, allowing for the reporting of glycan structure data through fluorescence scanning¹³.

Lectins have been utilized to sort and enrich CSCs across various tumor types (Figure 2). For example, breast cancer CSCs can be effectively enriched in vitro and in vivo by using the lectin. SLBR-N, which binds to the α-2,3 sialic acid of the O-glycan chain. Notably, SLBR-N demonstrates superior efficacy in CSC enrichment compared to CD44 because the application circumvents the complications associated with CD44 splicing and the variations in glycan chain status⁶. Moreover, SSEA-1/CD15/Lewis X serves as an enrichment marker for tumor-initiating cells in human glioblastoma¹⁷. Furthermore, a novel method combining anti-CD133 antibodies with cyanovirin-N (CVN) lectins, which recognize Manα1,2-Man, has been developed to enhance CSC enrichment. It has also been confirmed that CD133⁺/Manα1,2-Man⁺ cells exhibit more pronounced tumor stem-like characteristics compared to CD133⁺/Manα1,2-Man⁻ cells¹⁸.

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Figure 2

Application of glycans in CSC sorting and targeted therapy. Lectins specifically recognize and bind the polysaccharide structures on the CSC surface, facilitating their classification and enrichment. (A) The lectin SLBR-N recognizing α-2,3 sialic acid of the O-glycine chain can promote efficient enrichment of breast cancer stem cells through purification. (B) The purification of CSC is enhanced by the combination of anti-CD133 antibody and CVN lectin that recognizes Manα1,2-Man. (C) Lewis X is a purified and enrichment marker for human GBM stem cells. Moreover, specific polysaccharide chain structure inhibition can significantly reduce the biology of CSCs. (D) Inhibition of fucosylation by 2-deoxy-D-galactose contributes to the eradication of CSCs. (E) Blocking the interaction between Siglec-10 and CD24 by anti-CD24 antibody restores the phagocytosis of cancer cells by macrophages. (F) Interfering with GD3S expression, a key enzyme that affects expression of the breast cancer stem cell marker, GD2, can reduce the CSC population and its associated properties (figure created with BioRender.com).

The identification and enrichment of CSCs are of particular importance in clinical research because these cells are considered the primary drivers of tumor recurrence and resistance. Consequently, the development of efficient CSC isolation technologies has emerged as a leading topic in contemporary cancer research. Utilization of lectins in the study of CSCs not only elucidates the distinct biological properties of CSCs but also opens new pathways for targeted cancer therapies.

Value of glycans in targeted CSC therapy

Emerging therapeutic strategies targeting CSCs are garnering increasing attention in cancer research. CSCs have become pivotal targets due to their critical role in maintaining tumor growth and recurrence⁶. Researchers have discovered that emerging therapies targeting CSCs focus on inhibiting unique glycan structures to curb tumor formation and metastasis (Figure 2). This approach not only complements traditional chemotherapy regimens but also offers innovative perspectives for improving cancer treatment outcomes. For example, silencing the glycosylase, GALNT1, could disrupt SHH signaling activated by O-glycosylation in bladder cancer CSCs, thereby inhibiting the capacity to retain stem-like properties. This reduction in CSC viability has demonstrated a level of tumor growth inhibition comparable to that achieved with the chemotherapy drug, cyclopamine¹⁹. Furthermore, inhibition of fucosylation by 2-deoxy-o-galactose increases the efficacy of sorafenib, while effectively eradicating CSCs⁵. Additionally, targeting sialic acid in CSCs can affect the interaction between sialic acid and its receptors, thereby programming the tumor microenvironment and assisting CSCs in evading immune detection. For example, cluster of differentiation 24 (CD24) has been identified as a marker for CSCs in several types of solid cancers. CD24-positive cancer cells escape immune recognition through interaction with Siglec-10. Blocking CD24 binding to Siglec-10 using monoclonal antibodies enhances phagocytosis of CD24⁺ cells, thereby improving the survival outcomes of tumor-bearing mice²⁰. Lastly, ganglioside GD2, a sphingolipid, is increasingly recognized as a marker for breast CSCs. GD3 synthetase (GD3S), the enzyme responsible for converting GD3-to-GD2, is highly expressed in breast CSCs. Targeting GD3S expression use of shRNA or employing pharmacologic inhibitors can significantly diminish the CSC population and disrupt the associated properties, contributing to potential therapeutic strategies against breast cancer⁸. Consequently, research focused on altering glycan structures and associated enzymes not only offers novel strategies for CSC treatment but also provides potential approaches for enhancing tumor immunotherapy.

Targeting glycans associated with CSCs holds significant potential but presents several challenges. One major issue is the presence of multiple identical glycan structures on membrane proteins, which can lead to off-target effects. To mitigate this effect, bispecific antibodies may be used to selectively target specific glycans on designated proteins. Additionally, tumor cell drug resistance can lead to alterations in the glycan profiles, enabling tumor cells to evade therapeutic interventions. Furthermore, variability in glycan expression among patients can result in differing effectiveness of glycan-targeted therapies, leading to heterogeneous responses within the patient population. Continued in-depth research and clinical applications of these discoveries are expected to improve overall cancer treatment outcomes, ultimately benefiting a broader patient population.

Future perspectives

The structure of glycan in CSCs is not static, rather the structure is modulated by various factors and displays dynamic characteristics during tumor progression. Future researches are expected to focus more on the temporal dynamics of glycan structures and the specific expression patterns at different stages of the CSC life cycle. For example, single-cell glycemic technology allows for detailed analysis of changes in glycan chain structures within individual CSCs across different developmental phases, revealing specific roles in tumor evolution. These studies will enhance our understanding of the specific mechanisms by which glycans influence tumor development and metastasis, thereby providing more accurate diagnostic and therapeutic targets.

The tumor microenvironment is crucial for the maintenance and function of CSCs, with glycans serving as core molecules in this context. Forthcoming research will increasingly focus on how glycans mediate interactions between CSCs and the microenvironment. Specifically, glycans are known to regulate interactions between CSCs and stromal, immune, and vascular endothelial cells, thereby influencing tumor growth and metastasis. Investigating the underlying mechanisms of these glycan-mediated cell-cell interactions will contribute to the advancement of novel anticancer therapies targeting inhibition of CSC function through intervention in these interactions.

The diversity and complexity of glycan chains make glycan chains ideal markers for identifying CSCs. Future research will focus on the creation of highly sensitive and specific glycan chain detection techniques to identify and to capture these cells. For example, mass spectrometry and glycan chip technology can rapidly and efficiently screen for glycan chain structure uniquely expressed by CSCs. Additionally, future research will combine bioinformatics and machine learning algorithms to identify potential glycan chain markers from large-scale glycemic data, thereby providing robust support for precision medicine of CSCs. The synthesis and modification of glycan chains depend on intracellular glucose metabolic pathways with reprogramming of these metabolic pathways being particularly evident in CSCs. Future research will further elucidate the relationship between metabolic reprogramming and glycan structures, as well as their roles in the maintenance of CSCs. For example, by studying the regulation of key signaling pathways, such as PI3K/Akt and mTOR, on glucose metabolism and glycan chain synthesis, the metabolic adaptation mechanism underlying glycosylation in CSCs can be uncovered. This understanding will facilitate the development of pharmacologic agents targeting glycan chain synthesis and modification, thereby inhibiting the growth and spread of CSCs.

Summary

This review emphasized the latest developments in the structural features of glycan chains found in CSCs, the influence of these glycan chains on the properties of CSCs, and their potential as therapeutic targets in treatment applications (Figure 3). The study of glycan structures in CSCs presents numerous challenges alongside significant opportunities and potential. Future research will prioritize several key areas, including the dynamic changes of glycan structures, their interactions with the microenvironment, the identification of more specific glycan markers, the regulation of glycan-related metabolic pathways, and their applications in cancer therapies. By leveraging advanced glycemic technologies, single-cell analysis, and bioinformatics methods, researchers will delve deeper into the specific mechanisms by which glycan chains influence CSCs. This comprehensive exploration could unveil the vulnerability of CSCs and yield new strategies for the early diagnosis and precise treatment of cancer. Furthermore, a promising new direction involves combining glycan markers with immunotherapy to enhance the effectiveness of treatments targeting CSCs. This approach seeks to bolster the ability of the immune system to recognize and eliminate CSCs, ultimately paving the way for improved therapeutic outcomes and cancer management.

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Figure 3

The glyco-code of CSCs holds great promise as a candidate for clinical diagnostic markers and therapeutic targets. During tumor progression, the glycan structures of CSCs differ from those of non-stem cancer cells. These abnormal glycan structures play crucial roles in immune evasion and metastasis, as well as in maintaining the properties of CSCs and enabling the selection of CSCs through lectin binding. Targeted therapeutic strategies, such as shRNA or pharmacologic inhibitors, can target pathways associated with abnormal glycan structures, providing new strategies for CSC therapy and potential methods to enhance cancer immunotherapy (figure created with BioRender.com).

Author contributions

Conceived and designed the analysis: Yuanyan Wei, Jianhai Jiang.

Wrote the paper: Yuanyan Wei, Anning Wei, Yirong Li, Yuerong Yang, Yu Si, Yi Li, Zhijun Fan, Jianhai Jiang.