Aberrant Glycosylation as Immune Therapeutic Targets for Solid Tumors.

Glycosylation occurs at all major types of biomolecules, including proteins, lipids, and RNAs to form glycoproteins, glycolipids, and glycoRNAs in mammalian cells, respectively. The carbohydrate moiety, known as glycans on glycoproteins and glycolipids, is diverse in their compositions and structures. Normal cells have their unique array of glycans or glycome which play pivotal roles in many biological processes. The glycan structures in cancer cells, however, are often altered, some having unique structures which are termed as tumor-associated carbohydrate antigens (TACAs). TACAs as tumor biomarkers are glycan epitopes themselves, or glycoconjugates. Some of those TACAs serve as tumor glyco-biomarkers in clinical practice, while others are the immune therapeutic targets for treatment of cancers. A monoclonal antibody (mAb) to GD2, an intermediate of sialic-acid containing glycosphingolipids, is an example of FDA-approved immune therapy for neuroblastoma indication in young adults and many others. Strategies for targeting the aberrant glycans are currently under development, and some have proceeded to clinical trials. In this review, we summarize the currently established and most promising aberrant glycosylation as therapeutic targets for solid tumors.

Targeting altered glycosylation in secreted tumor glycoproteins for broad cancer detection.

There is an urgent need to develop new tumor biomarkers for early cancer detection, but the variability of tumor-derived antigens has been a limitation. Here we demonstrate a novel anti-Tn antibody microarray platform to detect Tn+ glycoproteins, a near universal antigen in carcinoma-derived glycoproteins, for broad detection of cancer. The platform uses a specific recombinant IgG1 to the Tn antigen (CD175) as a capture reagent and a recombinant IgM to the Tn antigen as a detecting reagent. These reagents were validated by immunohistochemistry in recognizing the Tn antigen using hundreds of human tumor specimens. Using this approach, we could detect Tn+ glycoproteins at subnanogram levels using cell lines and culture media, serum, and stool samples from mice engineered to express the Tn antigen in intestinal epithelial cells. The development of a general cancer detection platform using recombinant antibodies for detection of altered tumor glycoproteins expressing a unique antigen could have a significant impact on cancer detection and monitoring.

Transcriptomic analysis of pancreatic adenocarcinoma specimens obtained from Black and White patients.

In pancreatic cancer clinical trials, Black patients are under-represented while having higher morbidity and mortality rates as compared to other racial groups. Multiple factors, including socioeconomic and lifestyle factors may contribute to this disparity, but genomic contributions remain unclear. In an exploratory project to identify genes that may contribute to differences in survival between Black (n = 8) and White (n = 20) patients with pancreatic cancer, transcriptomic sequencing of over 24,900 genes was performed in human pancreatic tumor and non-tumor tissue obtained from Black and White patients. Over 4,400 genes were differentially expressed in tumor and non-tumor tissue, irrespective of race. To validate these results, the expression of four genes (AGR2, POSTN, TFF1, and CP) reported to be up-regulated in pancreatic tumor tissue as compared to non-tumor tissue were confirmed using quantitative PCR. Transcriptomic analysis that compared pancreatic tumor tissue from Black and White patients revealed differential expression in 1,200 genes, while a comparison of the non-tumor and tumor gene expression differences within each race revealed over 1,500 tumor-specific differentially expressed genes in pancreatic tumor and non-tumor tissue from Black patients. We identified TSPAN8 as a potential tumor-specific gene significantly overexpressed in pancreatic tumor tissue in Black patients as compared to White patients. Using Ingenuity Pathway Analysis software to compare the race-associated gene expression profiles, over 40 canonical pathways were identified to be potentially impacted by the gene expression differences between the races. Heightened expression of TSPAN8 was associated with poor overall survival, suggesting TSPAN8 as one potential genetic factor contributing to the differential outcomes in Black patients with pancreatic cancer, supporting the potential utility of larger genomic studies to further explore the role of TSPAN8 in pancreatic cancer.

SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.

Morphology of Biomaterials Affect O-Glycosylation of HUVECs.

Biomaterials have been widely used as substitutes for diseased tissue in surgery and have gained great success and attention. At present, the biocompatibility of biomaterials such as PET woven fabrics is often evaluated both in vitro and in vivo. However, the current experimental methods cannot reveal the relationship between material surfaces and cell adhesion, and few research works have focused on the mechanisms of how the surface morphology of biomaterials affects cell adhesion and proliferation. Thus, it is meaningful to find out how the altered surfaces could affect cell adhesion and growth. In this study, we employed Ar low-temperature plasma treatment technology to create nano-grooves on the warp yarn of PET woven fabrics and seeded human umbellar vein endothelial cells (HUVEC) on these fabrics. We then assessed the O-glycan and N-glycan profiles of the cells grown on different structures of the polyester woven fabrics. The result showed that the surface morphology of polyester woven fabrics could affect the O-glycan profile but not the N-glycan profile of cultured HUVEC. Taken together, the study describes the effects of the surface morphology of biomaterial on the biosynthesis of cellular glycans and may provide new insights into the design and manufacture of biomaterials used as blood vessels based on the expression profiles of O-glycans on cultured cells.

An etanercept O-glycovariant with enhanced potency.

Most therapeutic proteins are glycosylated with N-glycans and/or O-glycans. N-glycans on therapeutic proteins have been extensively studied for their control strategy and impact on drug product quality. However, knowledge of O-glycosylation in therapeutic protein production and its impact on product quality remains elusive. To address this gap, we generated an O-glycoengineered Chinese Hamster Ovary (CHO) cell line platform to modulate O-glycosylation of therapeutic proteins and investigated the impact of O-glycans on the physicochemical and biological properties of etanercept. Our results demonstrate that this CHO cell line platform produces controlled O-glycosylation profiles containing either truncated O-glycans (sialylTn and/or Tn), or sialylCore 3 alone, or sialylCore 1 with sialylTn or sialylCore 3 O-glycans on endogenous and recombinant proteins. Moreover, the platform demonstrated exclusive modulation of O-glycosylation without affecting N-glycosylation. Importantly, certain O-glycans on etanercept enhanced tumor necrosis factor-α binding affinity and consequent potency. This is the first report that describes the systematic establishment of an O-glycoengineered CHO cell line platform with direct evidence that supports the applicability of the platform in the production of engineered proteins with desired O-glycans. This platform is valuable for identifying O-glycosylation as a critical quality attribute of biotherapeutics using the quality by design principle.

Cosmc deficiency causes spontaneous autoimmunity by breaking B cell tolerance.

Factors regulating the induction and development of B cell­mediated autoimmunity are not well understood. Here, we report that targeted deletion in murine B cells of X-linked Cosmc, encoding the chaperone required for expression of core 1 O-glycans, causes the spontaneous development of autoimmune pathologies due to a breakdown of B cell tolerance. BC-CosmcKO mice display multiple phenotypic abnormalities, including severe weight loss, ocular manifestations, lymphadenopathy, and increased female-associated mortality. Disruption of B cell tolerance in BC-CosmcKO mice is manifested as elevated self-reactive IgM and IgG autoantibodies. Cosmc-deficient B cells exhibit enhanced basal activation and responsiveness to stimuli. There is also an elevated frequency of spontaneous germinal center B cells in BC-CosmcKO mice. Mechanistically, loss of Cosmc confers enhanced B cell receptor (BCR) signaling through diminished BCR internalization. The results demonstrate that Cosmc, through control of core 1 O-glycans, is a previously unidentified immune checkpoint gene in maintaining B cell tolerance.

Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo.

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.

Cellular O-Glycome Reporter/Amplification (CORA): Analytical and Preparative Tools to Study Mucin-Type O-Glycans of Living Cells.

Mucin-type O-glycosylation (O-glycans, O-glycome) is among the most biologically important post-translational modification in glycoproteins but O-glycan structural diversity and expression are poorly understood due to the inadequacy of current analytical methods. We recently developed a new tool termed cellular O-glycome reporter/amplification (CORA), which uses O-glycan precursors, benzyl-α-GalNAc (Bn-α-GalNAc) or azido-Bn-α-GalNAc (N3 -Bn-α-GalNAc), as surrogates of protein O-glycosylation. Living cells metabolically convert these precursors to all types of O-GalNAc glycans representative of the cells' capabilities. The amplification and secretion of the O-glycome products greatly facilitates their analysis and functional studies. Here we describe protocols for analytical and preparative applications. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Cellular O-glycome reporter/amplification for the analysis of mucin-type O-glycans from living cells Basic Protocol 2: Preparation of cellular O-glycans from living cells for functional glycomics and glycan microarrays Basic Protocol 3: Conjugation of cellular O-glycans with a bifunctional fluorescent tag Basic Protocol 4: 2D-HPLC purification and MALDI-TOF/MS identification of individual PYAB-Bn-O-glycan.

Cosmc controls B cell homing.

The molecular mechanisms regulating lymphocyte homing into lymph nodes are only partly understood. Here, we report that B cell-specific deletion of the X-linked gene, Cosmc, and the consequent decrease of protein O-glycosylation, induces developmental blocks of mouse B cells. After transfer into wild-type recipient, Cosmc-null B cells fail to home to lymph nodes as well as non-lymphoid organs. Enzymatic desialylation of wild-type B cells blocks their migration into lymph nodes, indicating a requirement of sialylated O-glycans for proper trafficking. Mechanistically, Cosmc-deficient B cells have normal rolling and firm arrest on high endothelium venules (HEV), thereby attributing their inefficient trafficking to alterations in the subsequent transendothelial migration step. Finally, Cosmc-null B cells have defective chemokine signaling responses. Our results thus demonstrate that Cosmc and its effects on O-glycosylation are important for controlling B cell homing.

An atlas of O-linked glycosylation on peptide hormones reveals diverse biological roles.

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.

O-glycans on death receptors in cells modulate their sensitivity to TRAIL-induced apoptosis through affecting on their stability and oligomerization.

The TNF-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in cells by signaling through the O-glycosylated death receptors (DR4 and DR5), but the sensitivity to TRAIL-induced apoptosis of cells varies, and the attributes of this phenomenon are complex. Human carcinoma cells often express truncated O-glycans, Tn (GalNAcα1-Ser/Thr), and Sialyl-Tn (Siaα2-6GalNAcα1-Ser/Thr, STn) on their surface glycoproteins, yet molecular mechanisms in terms of advantages for tumor cells to have these truncated O-glycans remain elusive. Normal extended O-glycan biosynthesis is regulated by a specific molecular chaperone Cosmc through assisting of the correct folding of Core 1 ß3 Galactosyltransferase (T-synthase). Here, we use tumor cell lines harboring mutations in Cosmc, and therefore expressing Tn and STn antigens to study the role of O-glycans in TRAIL-induced apoptosis. Expression of Tn and STn in tumor cells attenuates their sensitivity to TRAIL treatment; when transfected with wild-type Cosmc, these tumor cells thus express normal extended O-glycans and become more sensitive to TRAIL treatment. Mechanistically, Tn/STn antigens impair homo-oligomerization and stability of DR4 and DR5. These results represent the first mechanistic insight into how O-glycan structures on cell surface modulate their sensitivity to apoptotic stimuli, suggesting expression of Tn/STn may offer tumor cell survival advantages through altering DR4 and/or DR5 activity.

Amplification and Preparation of Cellular O-Glycomes for Functional Glycomics.

Mucin-type O-glycans play key roles in many cellular processes, and they are often altered in human diseases. A major challenge in studying the role of O-glycans through functional O-glycomics is the absence of a complete repertoire of the glycans that comprise the human O-glycome. Here we describe a cellular O-glycome preparation strategy, Preparative Cellular O-Glycome Reporter/Amplification (pCORA), that introduces 4-N3-Bn-GalNAc(Ac)3 as a novel precursor in large-scale cell cultures to generate usable amounts of O-glycans as a potential O-glycome factory. Cultured human non-small cell lung cancer (NSCLC) A549 cells take up the precursor, which is extended by cellular glycosyltransferases to produce 4-N3-Bn-α-O-glycans that are secreted into the culture medium. The O-glycan derivatives can be clicked with a fluorescent bifunctional tag that allows multidimensional HPLC purification and production of a tagged glycan library, representing the O-glycome of the corresponding cells. We obtained ∼5% conversion of precursor to O-glycans and purified a tagged O-glycan library of over 100 O-glycan derivatives, many of which were present in >100 nmol amounts and were sequenced by sequential MS fragmentation (MSn). These O-glycans were successfully printed onto epoxy glass slides as an O-glycome shotgun microarray. We used this novel array to explore binding activity of serum IgM in healthy persons and NSCLC patients at different cancer stages. This novel strategy provides access to complex O-glycans in significant quantities and may offer a new route to discovery of potential diagnostic disease biomarkers.

Identification of Tn antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies.

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.

Synthesis and Characterization of Versatile O-Glycan Precursors for Cellular O-Glycomics.

Protein O-glycosylation is a universal post-translational modification and plays essential roles in many biological processes. Recently we reported a technology termed cellular O-glycome reporter/amplification (CORA) to amplify and profile mucin-type O-glycans of living cells growing in the presence of peracetylated Benzyl-α-GalNAc (Ac3GalNAc-α-Bn). However, the application and development of the CORA method are limited by the properties of the precursor benzyl aglycone, which is relatively inert to further chemical modifications. Here we described a rapid parallel microwave-assisted synthesis of Ac3GalNAc-α-Bn derivatives to identify versatile precursors for cellular O-glycomics. In total, 26 derivatives, including fluorescent and bioorthogonal reactive ones, were successfully synthesized. The precursors were evaluated for their activity as acceptors for T-synthase and for their ability to function as CORA precursors. Several of the precursors possessing useful functional groups were more efficient than Ac3GalNAc-α-Bn as T-synthase acceptors and cellular O-glycome reporters. These precursors will advance the CORA technology for studies of functional O-glycomics.

Differential effects of bioreactor process variables and purification on the human recombinant lysosomal enzyme ß-glucuronidase produced from Chinese hamster ovary cells.

ß-Glucuronidase is a lysosomal enzyme and a molecular model of a class of therapeutics approved as enzyme replacement therapies for lysosomal storage diseases. Understanding the effect of bioreactor process variables on the production and quality of the biologics is critical for maintaining quality and efficacy of the biotherapeutics. Here, we have investigated the effect of three process variables, in a head-to-head comparison using a parallel bioreactor system (n = 8), namely 0.25 mM butyrate addition, a temperature shift (from 37 to 32 °C), and a pH shift (from 7.0 to 6.7) along with a control (pH 7, temperature 37 °C, and no additive) on the production and quality of human recombinant ß-glucuronidase (GUS) by a Chinese hamster ovary (CHO) cell line. The study was performed as two independent runs (2 bioreactors per treatment per run; n ≤ 4). Although statistically not significant, protein production slightly increased with either 0.25 mM butyrate addition (13%) or pH shift (7%), whereas temperature shift decreased production (12%, not significant). Further characterization of the purified GUS samples showed that purification selectively enriched the mannose-6-phosphate (M6P)-containing GUS protein. Noticeably, a variation observed for the critical quality attribute (CQA) of the enzyme, namely M6P content, decreased after purification, across treatment replicates and, more so, across different treatments. The dimer content in the purified samples was comparable (~25%), and no significant discrepancy was observed in terms of GUS charge variants by capillary electrophoresis analysis. MALDI-TOF/TOF analysis of released N-glycans from GUS showed a minor variation in glycoforms among the treatment groups. Temperature shift resulted in a slightly increased sialylated glycan content (21.6%) when compared to control (15.5%). These results suggest that bioreactor processes have a differential effect, and better control is required for achieving improved production of GUS enzyme in CHO cells without affecting drastically its CQAs. However, the purification method allowed for enrichment of GUS with similar CQA profiles, regardless of the upstream treatments, indicating for the first time that the effect of slight alterations in upstream process parameters on the CQA profile can be offset with an effective and robust purification method downstream to maintain drug substance uniformity.

A validated collection of mouse monoclonal antibodies to human glycosyltransferases functioning in mucin-type O-glycosylation.

Complex carbohydrates serve a wide range of biological functions in cells and tissues, and their biosynthesis involves more than 200 distinct glycosyltransferases (GTfs) in human cells. The kinetic properties, cellular expression patterns and subcellular topology of the GTfs direct the glycosylation capacity of a cell. Most GTfs are ER or Golgi resident enzymes, and their specific subcellular localization is believed to be distributed in the secretory pathway according to their sequential role in the glycosylation process, although detailed knowledge for individual enzymes is still highly fragmented. Progress in quantitative transcriptome and proteome analyses has greatly advanced our understanding of the cellular expression of this class of enzymes, but availability of appropriate antibodies for in situ monitoring of expression and subcellular topology have generally been limited. We have previously used catalytically active GTfs produced as recombinant truncated secreted proteins in insect cells for generation of mouse monoclonal antibodies (mAbs) to human enzymes primarily involved in mucin-type O-glycosylation. These mAbs can be used to probe subcellular topology of active GTfs in cells and tissues as well as their presence in body fluids. Here, we present several new mAbs to human GTfs and provide a summary of our entire collection of mAbs, available to the community. Moreover, we present validation of specificity for many of our mAbs using human cell lines with CRISPR/Cas9 or zinc finger nuclease (ZFN) knockout and knockin of relevant GTfs.

Comprehensive analysis of N-glycans in IgG purified from ferrets with or without influenza A virus infection.

Influenza viruses cause contagious respiratory infections, resulting in significant economic burdens to communities. Production of influenza-specific Igs, specifically IgGs, is one of the major protective immune mechanisms against influenza viruses. In humans, N-glycosylation of IgGs plays a critical role in antigen binding and effector functions. The ferret is the most commonly used animal model for studying influenza pathogenesis, virus transmission, and vaccine development, but its IgG structure and functions remain largely undefined. Here we show that ferret IgGs are N-glycosylated and that their N-glycan structures are diverse. Using a comprehensive strategy based on MS and ultra-HPLC analyses in combination with exoglycosidase digestions, we assigned 42 N-glycan structures in ferret IgGs. We observed that N-glycans of ferret IgGs consist mainly of complex-type glycans, including some high-mannose and hybrid glycans, similar to those observed in human IgG. The complex-type glycans of ferret IgGs were primarily core-fucosylated. Furthermore, a fraction of N-glycans carried bisecting GlcNAc. Ferret IgGs also had a minor fraction of glycans carrying α2-6Neu5Ac(s). We noted that, unlike human IgG, ferret IgGs have αGal epitopes on some N-glycans. Interestingly, influenza A infection caused prominent changes in the N-glycans of ferret IgG, mainly because of an increase in bisecting GlcNAc and F1A2G0 and a corresponding decrease in F1A2G1. This suggests that the glycosylation of virus-specific IgG may play a role in its functionality. Our study highlights the need to further elucidate the structure-function relationships of IgGs in universal influenza vaccine development.

Differential expression of Cosmc, T-synthase and mucins in Tn-positive colorectal cancers.

BACKGROUND: The Tn neoantigen (GalNAcα1-O-Ser/Thr) is an O-glycan expressed in various types of human cancers. Studies in several Tn-expressing cancer cell lines and pancreatic tumors have identified loss of Cosmc expression caused by either mutations or promoter hypermethylation. In this study, we explored the mechanism(s) for Tn expression in human colorectal cancers (CRC). METHODS: Tn-expressing cell populations were isolated from CRC cell lines by Fluorescence-associated cell sorting (FACS). The expression of the Tn and sialylated Tn (STn) antigens, Cosmc, T-synthase, and mucins was characterized in paired specimens with CRC and in CRC cell lines by immunostaining, western blot, and qPCR. RESULTS: Using well-defined monoclonal antibodies, we confirmed prevalent Tn/STn expression in CRC samples. However, a majority of these tumors had elevated T-synthase activity and expression of both Cosmc and T-synthase proteins. Meanwhile, Tn antigen expression was not caused by mucin overproduction. In addition, we found that Tn-expressing CRC cell lines had either loss-of-function mutations in Cosmc or reversible Tn antigen expression, which was not caused by the deficiency of T-synthase activity. CONCLUSIONS: Our results demonstrate multiple mechanisms for Tn expression in CRCs.