Abstract
Glycosylation is one of the most common posttranslational modifications of proteins and lipids. In the case of tumors, cell transformation accompanied by aberrant glycosylation results in the expression of tumor-associated glycans that promote tumor invasion. As part of the innate immunity, anti-glycan antibodies recognize tumor-associated glycans, and these antibodies can be present in the bloodstream in the early stages of cancer. Recently, anti-glycan antibody profiles have been of interest in various cancer studies. Novel advantages in the field of analytical techniques have simplified the analysis of anti-glycan antibodies and made it easier to have more comprehensive knowledge about their functions. One of the robust approaches for studying anti-glycan antibodies engages in microarray technology. The analysis of glycan microarrays can provide more expanded information to simultaneously specify or suggest the role of antibodies to a wide variety of glycans in the progression of different diseases, therefore making it possible to identify new biomarkers for diagnosing cancer and/or the state of the disease. Thus, in this review, we discuss antibodies to various glycans, their application for diagnosing cancer and one of the most promising tools for the investigation of these molecules, microarrays.
Glycans: structure and functions
Glycosylation is one of the most complex and common posttranslational modifications, representing the enzymatic addition of carbohydrate chains, called glycans, to a protein or a lipid. The functions of glycans are quite diverse; they are involved in cell-cell adhesion, signal transduction, protein folding, receptor activation and endocytosis [1]. Studying glycosylation is one of the main tasks of molecular biology, regarding its importance and lack of a common glycosylation pattern [2].
Human glycan structures consist of 10 basic monosaccharides: glucose, galactose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid, fucose, mannose, xylose, glucuronic acid and iduronic acid [3]. Different combinations of these saccharides in glycans are involved in the formation of bonds between atoms with different anomeric states, branching, length and functional groups, which leads to a large variety of glycans involved in the glycosylation process [4]. Glycosylation occurs in the Golgi apparatus, the endoplasmic reticulum or the cytosol of the cell [5], linking saccharides with N- or O-atoms of amino acids. O- and N-glycosylation are based on the addition of saccharides to the oxygen atom of serine, threonine, hydroxylysine and hydroxyproline and the nitrogen atom of asparagine (N-glycosidic bond), respectively. However, most human proteins are N-glycosylated, and the addition of N-acetylgalactosamine to the oxygen of a serine or threonine residue is observed in more than 85% of all secreted proteins [6].
In addition to O- and N-glycans, glycosaminoglycans and glycosphingolipids are referred to as the main classes of human glycans. Glycosaminoglycans are sulfated linear polysaccharides covalently linked to proteins. They are part of the connective tissue and participate in intercellular interactions. One of the representatives of glycosaminoglycans, heparin, is a highly active anticoagulant [7]. Glycosphingolipids, consisting of a ceramide fragment and a hydrophilic carbohydrate “head” group, are contained in the lipid bilayer of cell membranes [8]. They play an important role in signal transduction, and nervous tissues are especially rich in them; most human brain glycans are glycosphingolipids [9].
In humans, the glycosylation process occurs with the help of special enzymes, glycosyltransferases and glycosidases. Thus, the attachment of a glucose residue to a protein occurs with the involvement of O-glycosyltransferase (POGLUT1), N-acetylglucosamine (GlcNAc)- and O-GlcNAc-transferase (OGT). After forming the first carbohydrate residue, the next residues are attached using other enzymes, such as either glycoprotein-N-acetyl-galactosamine-3-β-galactosyltransferase1 (C1GalT1) or C1GalT1-specific chaperone 1 (COSMC) [10]. N-glycosylation proceeds in the endoplasmic reticulum lumen with the complete assembly of the Glc3Man9GlcNAc2 precursor, catalyzed by the sequential orchestration of several enzymes. In the next stages, the Glc3Man9GlcNAc2 precursor forms Man5GlcNAc2. Then one GlcNAc residue is attached to the outer branch of mannose using UDP-GlcNAc transferase I forming GlNAcMan5GlcNAc2. This glycan is then either extended by GalNAc or sialic acid, or two mannose residues using Golgi mannosidase II are removed, which generates N-linked GlcNAcMan3GlNAc2, the “core” glycan structure. It is the initial building block for all complex N-glycoproteins that represented a wide variety of highly branched structures, and the sequential modification of mannose, galactose, GlcNAc, fucose and sialic acid in N-glycans modulates many different aspects of the biology of these oligosaccharides. [5], [11], [12].
As we can see, the complex biochemical mechanisms of the formation of glycan structures depend on many parameters; therefore, glycomics is still far from routine practice.
Glycans in cancer
The study of glycans and their derivatives in the context of personalized medicine is a promising approach for finding new cancer biomarkers, both prognostic and predictive, as glycans are involved in tumor invasion, angiogenesis, progression and metastasis of tumors (Figure 1). Besides that, glycans are perspective immunotherapy targets, and investigations in this direction could be a promising way for cancer diagnosis and treatment.
Examples of glycans employed for cancer diagnosis and prognosis.
Carbohydrate antigens (CA) CA 15-3, CA 125 and CA 19-9 (also known as sialyl-Lewis A [SiaLeA]) are widely used tumor markers. CA 19-9 is known as the most commonly used serum marker for pancreatic cancer. Tests that determine both CA 19-9 and thrombospondin-2, which is a protein involved in the activation of TGF-β, one of the important processes in the initiation of pancreatic cancer, showed promising results for the early diagnosis of pancreatic ductal adenocarcinoma [27]. Other glycosylated serological tumor markers used in clinical practice include prostate-specific antigen (PSA), carcinoembryonic antigen (CEA) and various mucins [28], [29].
One of the possible diagnostic approaches is the analysis of changes in the profiles of N- or O-glycans, which is observed during tumor transformation and can provide enough information about the patient’s pathological condition. During tumor transformation, aberrant glycosylation of proteins and lipids occurs, leading to the expression of tumor-associated glycans that promote tumor invasion [30], [31]. The formation of such glycans reflects changes in the regulation and activity of the corresponding glycosyltransferases and glycosidases, and the pattern of these enzymes also has diagnostic and prognostic significance [32]. Additionally, because of the possibility of glycoproteins and glycosphingolipids being able to activate tyrosine kinases of growth factor receptors, tumor cells are stimulated to proliferate [33].
Tumor-associated glycans, such as sialylated structures, Tn antigens (N-acetylgalactosamine residues linked to a serine or threonine protein by a glycosidic bond) and Lewis antigens (Lewis A, Lewis B, Lewis X and Lewis Y), are often part of membranes or secreted tumor proteins; one example includes mucins, such as mucin 1 (MUC1).
Truncated O-glycans Tn and SiaTn have been considered as prospective targets for anticancer therapy for a long time. Theratope® anti-SiaTn vaccine seemed to be one of the most promising treatments of metastatic breast cancer according to the phase II clinical trial and a row of scientific studies [34], [35]. However, failure of phase III clinical trial possibly due to the unknown SiaTn expression in studied patients has not allowed using this treatment in clinics [36]. Recently, as Tn and SiaTn are often exposed on MUC1, targeting MUC1-Tn using engineered T cells (CAR T-cell therapy) is now considered as potential cancer immunotherapy [37], [38].
In addition, glycans can be attached to membrane lipids that can be observed for gangliosides, including disialoganglioside 1 (GD1) and monosialic ganglioside 2 (GM2). Gangliosides (GD2, GD3, GM2, N-acetyl-GM3, etc.) are often overexpressed in tumor cells. One of the most common immunotherapy targets is GD2. GD2 is targeted by monoclonal antibodies, including dinutuximab β, which showed positive outcomes in patients with SIOPEN high-risk neuroblastoma [39]. Anti-GD2 monoclonal antibodies also target non-small-cell lung cancer [40], sarcoma and melanoma [41]. Anti-GD2/4-1BB CAR T-cell therapy was shown to be a clinically appealing treatment strategy for melanoma patients [42].
Malignant cell transformation is accompanied by changes in gene expression. For example, epigenetic silencing in pancreatic cancer, leading to the hypermethylation of 1-β-galactosyltransferase core 1, a COSMC-specific molecular chaperone, results in impaired O-glycosylation, which directly induces an increase in the proliferation of tumor cells and their invasive activity [43]. In the case of colorectal cancer, silencing of the gene encoding α2,6-sialyltransferase is the main event causing the increased prevalence of monosialic Lewis A antigens over di-derivatives [44].
The intracellular localization of glycosyltransferases also determines the expression of specific glycans on the cancer cell membrane. For example, N-acetylgalactosaminyltransferase 1 (GALNT1), a glycosyltransferase initiating O-glycosylation of mucin-type proteins that usually occurs in the Golgi apparatus moves into the endoplasmic reticulum and induces an increase in truncated O-glycan structures, thereby favoring the growth of the tumor [45]. Due to tumor heterogeneity, all these processes can coexist as they are spatially separated within the same tumor [46].
Lewis blood group antigens (LeA and LeB) and related carbohydrate structures are used as markers of cell differentiation and embryonic development. Neoplastic transformation is often associated with characteristic changes in the expression of these structures on the cell surface, and their number usually increases with cell malignancy and tumor progression. The sialylated Lewis A antigen is a ligand of selectins, which are intercellular adhesion molecules that promote the attachment of cancer cells to the vascular endothelium, and this results in metastasis and tumor growth [47], [48].
As many glycans are typical antigens of various blood group systems, studies that reveal a link among the blood groups of patients with solid tumors, survival and risk of relapse are also of interest. For example, it has been shown that in women with the blood group O, the incidence of ovarian cancer is less common; however, in these women, relapses develop earlier [49].
Despite the difficulty of determining glycans, approaches based on the analysis of these markers make it possible to more accurately describe the condition and predict the development of the disease in an individual patient. In addition, such approaches can effectively complement the results of genomic tests for stratifying patients in the absence of hereditary changes [50].
Protein glycosylation is proved to have a profound impact on the molecular interactions modulating the immune system, thus reacting upon a row of physicochemical and pharmacokinetic parameters of immunotherapeutic drugs for treating cancer and other pathologies. For example, sialylation on the termini of the N-glycan branches on the immunotherapeutics results in a higher negative charge of the glycoproteins and increases their half-life by masking the penultimate galactose moiety from the hepatocyte asialoglycoprotein receptor and thus preventing endocytosis to prolong circulatory lifetime [51]. Besides, it was shown that Fc hypersialylation of therapeutic antibodies might influence the IgG interaction with the neonatal Fc receptor extending the half-life of IgG by reducing their intracellular degradation [52].
At the same time, the Fc sialylation of therapeutic antibodies downregulates the ability of the Fc to engage FcγR decreasing the antibody-dependent cell-mediated cytotoxicity (ADCC) [53], [54], so the removal of sialic acid can improve ADCC and complement-dependent cytotoxicity (CDC) [55]. The lack or decrease in core fucose leads to increased ADCC [54] and promotes ADCP (antibody-dependent cellular phagocytosis) of therapeutic antibodies [56].
α-Gal (galactose-alpha-1,3-galactose) is a major carbohydrate antigen that is expressed in non-primate mammals and prosimians. The presence of α-Gal on the monoclonal antibodies was shown to be associated with the possibility of hypersensitivity reactions while treating with Cetuximab, an epidermal growth factor receptor inhibitor, used in metastatic colorectal cancer [57]. That is why monitoring of terminal galactose groups is one of the quality control parameters in the production of immunotherapeutics [51].
As can be seen from the aforementioned text, analysis of glycans can be of great value not only for revealing of malignant tumors but also for the development and use of different immunotherapy drugs. Moreover, taking into account glycosylation patterns of the immunotherapeutic drugs is one of the directions to increase the safety and efficiency of cancer immunotherapy.
Anti-glycan antibodies in cancer
Changes in the levels of tumor-associated glycans are reflected in the levels of the corresponding antibodies to them, allowing one to evaluate the glycosylation pattern without using laborious methods of analyzing glycans (separation of common peaks after chromatography, detection of low-intensity peaks in the mass spectrum, removal of the pool of basic glycans or isolation of the glycans themselves). Moreover, when determining antibodies to glycans in sera stored in different conditions, the levels of anti-glycan antibodies did not change significantly, depending on the times and conditions of storage of the sample; this could facilitate the analysis in case of using glycans in the clinic [58].
During the pathological process, N-glycans are fucosylated and sialylated and form highly ordered branched structures on the surface of tumor cells, by increasing the expression of GlcNAc transferases [59]. It was also shown that the shortening of carbohydrate chains and the rapid increase in short, di- and trisaccharide repeats are often observed during tumor transformation for O-glycans. During the progression of neuroblastoma and melanoma, the level of glycosphingolipids increases [60]. All of the aforementioned processes result in changes in the levels of antibodies that specifically bind to glycans.
Studies of the repertoires of anti-glycan antibodies conducted on the sera of healthy donors and patients with cancer allow for differentiating three groups of anti-glycan antibodies [61]. The first group includes conservative antibodies that are present in all (or almost all) healthy donors and the variation in their content, affinity and epitope specificity is low.
The second group includes alloantibodies and antibodies to group-specific blood antigens that are absent in a significant part of donors (for example, anti-A antibodies in individuals with blood group A); their levels vary considerably, even in individuals with the same blood group.
The third group of antibodies includes both conservative and group-specific antibodies; and the levels markedly change in noninfectious diseases and some temporary conditions, including inflammation. Importantly, antibodies of this group are markers of a number of diseases, primarily oncological states.
Detection of antibodies to cancer-associated antigens is a promising approach for the development of cancer diagnostics as these antibodies are detected in the blood in the early stages of cancer [62], [63]. Numerous studies of antibodies to cancer-associated glycans revealed that their unusual level (below or above the normal level) may be associated with oncological disease. Many of the identified anti-glycan antibodies resulted in being directed toward an inaccessible inner (core) part of the glycan that is directly linked with the cell membrane lipids or proteins.
Changes in the glycome that are associated with the oncological process can be reliably characterized using antibodies against specific glycan structures. Therefore, the study of anti-glycan antibodies can be of value during some pathological processes. For example, in patients with carcinomas, a correlation was found among the expression of T (Galβ1-3GalNAc) and Tn (GalNAc) antigens, the level of serum antibodies to them, and the prognosis of the disease [64].
Different isotypes of anti-glycan antibodies
The repertoire of anti-glycan antibodies varies, depending on the isotype of immunoglobulins G, A or M. These differences may reflect characteristics and the physiological state of the population and healthy individuals and the stage and localization of the disease in cancer patients. Antibody levels also change in response to vaccination or pathogenic infection and can increase more than 10 times. Moreover, there may be competition between antibodies of different isotypes for antigen binding, the result of which depends on the specificity, affinity, concentration and isotype itself. During the development of an immune response, when produced by lymphocyte immunoglobulin type changes, IgG and IgA appear to have a higher affinity than that of IgM [65]. Often, in cases of detecting IgM, the pool of all IgGs is depleted to reduce the proportion of false-negative IgM results and vice versa. In some cases, it is desirable as IgM, which is pentameric, has a greater avidity than IgG and IgA, and this also affects the efficiency of binding to glycans and complicates the detection of IgG and IgA to glycans. However, the levels of IgG, IgA and IgM to glycans are interrelated. For example, a decrease in the level of IgM to a glycan is associated with an increase in the levels of IgG and/or IgA associated with it [66]. This advantage in the binding of IgM to glycans compared with isotypes G and A can be explained by the avidity, the large absolute amount of specific IgM antibodies to the same glycan, and the faster rate of formation of the antigen-antibody complex. The repertoire of anti-glycan antibodies of different isotypes is often stable for an extended period of time in the same healthy person [67], thus confirming the hypothesis that anti-glycan antibodies belong to a subpopulation of natural antibodies produced by B1-positive cells [68].
The levels of different isotypes of anti-glycan antibodies are also associated with age, race, sex and blood group. The most common examples are IgG and IgM to glycoproteins and glycolipids on the surface of erythrocytes and other blood cells, in which these antibody levels correlate strongly with the blood group. The approach, which includes the determination of antibodies to LeA and LeB, is widely used in transplantation to determine the compatibility of the donor and recipient. Additionally, anti-glycan antibodies and blood type are associated with the survival of patients with various solid tumors [69]. It was shown that the overall level of antibodies and levels of IgM to glycans decrease with age, and this is not observed for the levels of IgG to glycans. Moreover, the relationship between IgG antibodies to N-acetyllactosamine-containing glycans and race has been demonstrated [70]. In addition, most of the anti-glycan antibodies studied in [70] did not differ in healthy people and did not correlate with the population characteristics. Apparently, the obtained relationships are not related to the overall level of antibodies. These results may be important in clinical studies for selecting homogeneous cohorts of patients and reducing systematic errors in the formation of samplings.
Methods for analysis of anti-glycan antibodies for cancer diagnosis
In recent decades, there have been active developments of analytical techniques and various bioinformatics approaches in the analysis of glycomes of various living beings. A particularly important step in such studies was the use of high-performance methods, including microarray technology [71], high-performance liquid chromatography [72] and mass spectrometry [73] (Figure 2).
Methods for analysis of glycans and anti-glycan antibodies.
These approaches enable the analysis of a large number of potential markers in biological samples in a relatively short time. Maximum automation at most stages increases the reproducibility of experiments and reduces the final variability, and this is an important parameter in quantitative studies. Several microliters of the sample (most often blood) are used for the analysis; thus, these minimally invasive methods are more commonly used now than before in research and, in some cases, in clinical practice, especially in the study of cancer [106].
Changes in the levels of glycans and anti-glycan antibodies can be detected by methods of immunochemical analysis and those that account for the physicochemical properties of glycans. Methods based on several combined techniques can also be used. For example, Balog et al. [98] thoroughly analyzed glycans as markers of colorectal cancer and studied the degree of glycosylation during the process of malignancy using high-performance liquid chromatography (HPLC) with fluorescence detection and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Using this method, one can recognize a large number of glycans with high accuracy; however, this approach takes quite a long time to analyze one sample and has troubles with separating two molecules with similar physicochemical properties.
With the use of suspension arrays (beads), glycan levels may be analyzed. Hydrazide-coated silica particles are used for the isolation of glycans with further analysis with MALDI-TOF MS [93], [94]. At the same time, analysis of anti-glycan antibodies can be held using micro- and suspension arrays. Examples of anti-glycan analysis for different types of cancer are shown in Table 1.
Anti-glycan antibody profiles in diagnosing cancer.
| Year | Method of detection | Isotype | Glycans | Cancer | Role | Ref. |
|---|---|---|---|---|---|---|
| 2006 | Microarrays + ELISA | M | Tn | Hodgkin’s lymphoma | Diagnostic | [78] |
| 2008 | Microarrays | G, M | Globo H | Breast | Diagnostic | [18] |
| 2010 | Microarrays | G | Tn, SiaTn, T, core 3 | Breast, ovarian, prostate | Diagnostic | [74] |
| 2011 | Microarrays | G+M+A | Neu5Acα2-3Galβ1-4Glcβ and Glcα1-4Glcβ | Mesothelioma | Diagnostic | [83] |
| 2011 | Microarrays | G | Neu5Gcα2-6Tn | Carcinomas | Diagnostic | [84] |
| 2011 | Microarrays | G, A | MUC1 and MUC4 containing Tn and SiaTn | Colorectal | Diagnostic | [81] |
| 2011 | Microarrays | G | SiaTn and core 3 | Breast | Prognostic* (see Ref. [107]) | [82] |
| 2012 | Microarrays | G+M+A | P1 | Ovarian | Diagnostic | [77] |
| 2012 | Microarrays | G | Man9 and multi-antennary type II chains | Prostate | Diagnostic | [79] |
| 2013 | ELISA | G | TF, Tn, αGal | Gastrointestinal | Prognostic | [86] |
| 2014 | ELISA | M, A | TF, SiaTF | Gastric | Prognostic | [87] |
| 2016 | Microarrays | G+M+A | Tn, TF, SiaLeA, and Manβ1-4GlcNAcβ | Colorectal | Diagnostic | [85] |
| 2016 | Beads | M | SiaTn and 6-OSulfo-TF | Ovarian | Diagnostic | [91] |
| 2016 | ELISA | G | TF, Tn, αGal | Breast | Prognostic | [88] |
| 2017 | Beads | G, M | Globo H | Ovarian | Diagnostic | [90] |
| 2017 | Microarrays | G, M | SSEA-3, GD2, GHC, anti-LeY, SiaLeX, NeuAc, (NeuAcα2-8)2, (NeuAcα2-8)3, 6GlcNAc-HSO3-SiaLeX, α2-6 sialylated diantennary N-glycan | Oral | Diagnostic | [80] |
| 2018 | Microarrays | A | GlcNAcα1-4Galβ1-4GlcNAc | Pancreatic | Diagnostic | [76] |
| 2018 | Beads | G | Gal-α3-Gal-β4-Glc, Gal-α3-Gal-β3-GlcNAc, blood group-A trisaccharide | Ovarian | Prognostic | [92] |
| 2018 | ELISA | G, M, A | TF | Colon | Diagnostic, Prognostic | [89] |
| 2019 | Microarrays | G, M | 3ʹ-sialyl-TF and 3ʹ -O-su-LeA | Colorectal | Diagnostic | [75] |
| 2020 | Beads | G | Blood group H, Lewis, Ganglio, Isoglobo, lacto and sialylated tetrarose | Cervical | Prognostic | [95] |
In the last decade, successful attempts to evaluate anti-glycan antibodies as biomarkers of malignant tumors have been made in conjunction with the development of new glycan microarrays [74]. With improvements in the validation and normalization, these approaches may soon become a reality for routine practice [108].
Anti-glycan antibodies have been studied for the diagnosis of serous ovarian cancer of a high degree of malignancy [91]. This tumor is characterized by high mortality rates compared with other gynecological cancers, and the overall survival does not exceed 20%. In this approach, the authors did not use glycan microarrays but rather a suspension matrix with microspheres coated with synthesized glycans. In a small sampling, it was shown that the median levels of antibodies to negatively charged sialylated and sulfated glycans distinguished the groups of patients and healthy donors. This can be explained by the fact that the sialylation of glycans as components of membrane proteins is one of the main stages of tumor transformation, and sulfonation is associated with the process of metastasis. The five selected antibodies to glycans (gangliosides, Tn, TF and Lewis group antigens) were both IgM and IgG isotypes. In addition, it was shown that IgG to the glycosphingolipid Globo H, which is expressed on the surface of many cells in epithelial cancer, can distinguish patients with serous ovarian cancer and healthy donors [90].
In 2018, 961 serum samples from patients with ovarian cancer were analyzed by multiplex analysis based on microspheres with immobilized glycans [92]. Several new potential biomarkers of ovarian cancer were found. However, in routine practice, the use of flow cytometry to analyze microspheres can often be time-consuming and expensive.
A small number of tumor-associated glycans, including O-glycans, Tn, TF, SiaTn, Lewis antigens, gangliosides and Globo H, are often studied by different research groups. Antibodies to the aforementioned tumor-associated glycans, along with others, are determined in the patients’ serum. The median levels and confidence intervals are compared in samples from patients and healthy donors. In case the median levels of anti-glycan antibodies are significantly different in the two groups, after analyzing the sensitivity and specificity of the method, it can be determined if the antibodies can be used as biomarkers for diagnosis or prognosis [109]. Moreover, not only an individual antibody but also a combination of antibodies can act as a biomarker, increasing the diagnostic accuracy of the determination. Such antibody signatures for diagnosing or controlling the development of the solid tumor are widely known and may be an alternative to the search for a “golden bullet”, the one and only universal biomarker.
For example, a group of American researchers applied the CancerSEEK test, which determines the levels of circulating proteins and extracellular DNA to detect eight common types of malignant tumors in more than a thousand nonmetastatic patients. With a sensitivity range of 69%–98%, depending on the type of cancer, the specificity of the determination was 99%. These encouraging results once again confirm the usefulness of the aforementioned approach of implementing the identification of not one but several markers, if possible, in the bloodstream; this increases the attractiveness of this analysis for patients and reduces the trauma due to the low invasiveness of the procedure [110].
Microarrays for the detection of anti-glycan antibodies in cancer
Currently, the main and one of the most promising approaches for the analysis of glycans and anti-glycan antibodies is glycan microarrays. Glycan microarrays containing hundreds and thousands of different saccharides that are immobilized on a substrate allow for studying glycan-specific interactions with antibodies and lectins. The use of trace amounts of glycans and small volumes of a sample for the analysis and the ability to adequately cover the glycosylation spectrum of proteins are advantages of this method. Analysis of glycan microarrays allows for clarifying or suggesting the role of structures, including glycans, in the development of various conditions; therefore, this method makes it possible to identify new biomarkers for cancer, infectious and autoimmune diseases.
For these experiments, glycans are most often chemically or enzymatically synthesized. After modifying bovine serum albumin, polyacrylamide or another reagent with glycans, these reagents are covalently bound through an amino group or noncovalently immobilized on a substrate [111], [112]. The substrate may be plastic or glass and treated with unsaturated or heterocyclic compounds, such as N-hydroxysuccinimide or epoxides, for the effective covalent interaction with glycoconjugates. The results are visualized after analyzing biological samples using fluorescently labeled anti-glycan antibodies.
Wang et al. [18] reported the use of a glycan microarray for the determination of antibodies to Globo H and related structures in patients with breast cancer. Antibody levels to the glycan Globo H were significantly higher in patients with breast cancer (n=58) than in healthy donors (n=47) (p<0.0001).
Using an O-glycopeptide microarray, Wandall et al. [74] demonstrated higher levels of antibodies to aberrant O-glycosylated MUC1 in patients with breast cancer (n=26), ovarian cancer (n=20) and prostate cancer (n=10) than in healthy donors (n=39). Elevated levels of IgG antibodies were detected against the glycans Tn-MUC1 (GalNAc-MUC1), STn-MUC1 (NeuAcα2-6GalNAc-MUC1) and truncated glycan core 3 (GlcNAcβ1-3GalNAc-MUC1). In addition, the levels of these antibodies were statistically different for different types of cancer. The authors also compared the levels of antibodies in the serum of patients with CRC (n=58) and healthy donors (n=53) and identified IgG and IgA antibodies to the set of aberrant glycoproteins, MUC1 and MUC4, which are associated with the tumor process [81]. The detection specificity was 92%. Antibodies to tumor-associated Tn and STn glycans most effectively distinguished the two groups.
In 2011, a group of authors published a work with the analysis of the IgG levels to specific MUC1 glycoforms, which were found to be significantly higher in patients with early breast cancer (n=395) than in patients with benign tumors (n=108) and healthy donors (n=99) [82]. Moreover, these IgG autoantibodies were not detected in patients with advanced cancer. In addition, the authors found that levels of IgG autoantibodies to MUC1 glycopeptides containing core 3 glycans and sialylated Tn antigens are associated with a decrease in the incidence of metastases, which indicates that autoantibodies can serve as prognostic markers. However, validation of the source data is necessary to confirm that the correlations are not random, especially those with a limited number of patients. Thus, in a 2013 study, it was reported that antibodies to the MUC1 glycopeptide could not be used for screening breast, ovarian, lung or pancreatic cancer [107].
In another study, the authors determined the levels of anti-glycan antibodies in patients with mesothelioma (n=50) and in patients exposed to asbestos (n=65) [83]. The authors demonstrated the possibility of using glycan microarrays for the diagnosis and prognosis of mesothelioma. The best diagnostic efficiency was noted for levels of antibodies to Neu5Acα2-3Galβ1-4Glcβ (p>0.00005), and the best prediction rate was recorded for levels of antibodies to Glcα1-4Glcβ (p>0.005). The high diagnostic efficiency of antibodies to Neu5Acα2-3Galβ1-4Glcβ can be explained by the fact that Neu5Acα2-3Galβ1-4Glcβ, the glycan part of GM3, has an altered expression in some malignant tumors.
The increased consumption of red meat from animals, which contains glycans such as N-glycolylneuraminic acid (Neu5Gc), is also associated with the development of malignant tumors [105]. Thus, antibodies to the xenoantigen Neu5Gcα2-6Tn were increased in patients with various carcinomas [84]. These xeno-autoantibodies (autoantibodies against human antigens that are included in the glycan structure of cell surfaces) mediate the destruction of Neu5Gcα2-6Tn-positive tumors by complement- or antibody-dependent cytotoxicity. However, they have a dose-dependent effect, promoting the progression of tumors at low concentrations and inhibiting the progression at high concentrations [113]. Therefore, antibodies against Neu5Gc can probably serve as diagnostic and prognostic biomarkers or as immunotherapeutic agents for various tumors.
In the work of Butvilovskaya et al. [85], an approach for the simultaneous determination of serological cancer markers, anti-glycan antibodies and levels of circulating immunoglobulins of various classes using hydrogel microarrays was proposed. Of the most promising candidates, a signature that detects colorectal cancer with high sensitivity and specificity was developed. Later, while extending research on the use of anti-glycan antibodies for diagnosing colorectal cancer, diagnostically effective IgG and IgM antibodies to a number of glycans were found, and an IgM antibody to glycan 3′-O-su-LeA was shown to distinguish patients with the absence and presence of regional metastases [75].
Anti-glycan antibodies can be determined in blood and other liquids. Moreover, sometimes only a certain isotype of anti-glycan antibodies, but not the entire pool, is of value. For example, a group of researchers from the University of South Alabama, USA, used gastrointestinal lavage fluid (fluid after washing the intestines) as a surrogate for pancreatic juice, which can contain up to 50% IgA of a total protein in patients with pancreatic ductal adenocarcinoma. Although the sampling of patients was small and amounted to only 20 volunteers, the authors were able to identify a group of glycans, and the levels of antibodies to these glycans were increased in patients with pancreatic cancer. Most of these glycans contained the terminal motif of GlcNAcα1-4Galβ1-4GlcNAc, which is a component of the lipopolysaccharide from Proteus mirabilis, a potentially pathogenic bacterium often found in the intestine [76]. This may be due to the link among inflammation caused by this bacterium, the production of interleukin 1 β, changes in the microbiome and carcinogenesis [114]. Determining the expression profiles of anti-glycan antibodies in the various liquids seems to be a promising approach, especially considering the influence of the microbiome and the close relationship between glycosylation and the immune response to pathogens.
Anti-glycan antibodies are most often studied as diagnostic and prognostic markers for ovarian cancer. This malignant neoplasm is the main tumor among gynecological cancers, and the high mortality rate from this disease is associated with a late diagnosis. Using glycan microarrays in work [77], the authors discovered a set of anti-glycan antibodies that make it possible to distinguish patients with nonmucinous cancer, borderline ovarian tumors and healthy donors. However, statistically significant differences were also observed for the CA 125 glycoprotein level, which, as a rule, does not allow differentiation of these groups. This indicates some specificity of the sample under consideration. In comparison with CA 125, several antibodies have shown comparable specificities and sensitivities, whereas antibodies to glycan P1 and to an antigen from the P blood group system appeared to be more sensitive and specific. After adding five more markers to the aforementioned set, the diagnostic efficiency of the analysis increased; however, the specificity and sensitivity did not manage to exceed 84%, which may be explained by the heterogeneity of the populations and tumors, as well as cross-reactivity of antibodies or their low titers in biological samples.
However, monitoring the changes in glycosylation directly or through the levels of different anti-glycan antibodies has limitations, and certain difficulties are observed, especially when analyzing samples of patients with early stages of cancer. Due to the escape of tumors from immunological surveillance and their heterogeneity, the pool of glycans and antibodies to them often differs within the same stage and histological type, which leads to a decrease in the accuracy and an increase in the ratio of false-positive results in diagnostic tests. Therefore, only a small number of biomarkers have entered clinical practice [115].
Conclusions
Antibodies to tumor-associated glycans are novel and currently important potential biomarkers of oncological diseases. The variety of existing glycans and corresponding specific antibodies do not allow one to draw a conclusion that the conducted studies are exhaustive. Therefore, research in this direction is still a compelling task.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: Authors state no conflict of interest.
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