Sialyl-Tn serves as a potential therapeutic target for ovarian cancer.

BACKGROUND: Ovarian cancer remains the deadliest of the gynecologic cancers in the United States. There have been limited advances in treatment strategies that have seen marked increases in overall survival. Thus, it is essential to continue developing and validating new treatment strategies and markers to identify patients who would benefit from the new strategy. In this report, we sought to further validate applications for a novel humanized anti-Sialyl Tn antibody-drug conjugate (anti-STn-ADC) in ovarian cancer. METHODS: We aimed to further test a humanized anti-STn-ADC in sialyl-Tn (STn) positive and negative ovarian cancer cell line, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models. Furthermore, we sought to determine whether serum STn levels would reflect STn positivity in the tumor samples enabling us to identify patients that an anti-STn-ADC strategy would best serve. We developed a custom ELISA with high specificity and sensitivity, that was used to assess whether circulating STn levels would correlate with stage, progression-free survival, overall survival, and its value in augmenting CA-125 as a diagnostic. Lastly, we assessed whether the serum levels reflected what was observed via immunohistochemical analysis in a subset of tumor samples. RESULTS: Our in vitro experiments further define the specificity of the anti-STn-ADC. The ovarian cancer PDO, and PDX models provide additional support for an anti-STn-ADC-based strategy for targeting ovarian cancer. The custom serum ELISA was informative in potential triaging of patients with elevated levels of STn. However, it was not sensitive enough to add value to existing CA-125 levels for a diagnostic. While the ELISA identified non-serous ovarian tumors with low CA-125 levels, the sample numbers were too small to provide any confidence the STn ELISA would meaningfully add to CA-125 for diagnosis. CONCLUSIONS: Our preclinical data support the concept that an anti-STn-ADC may be a viable option for treating patients with elevated STn levels. Moreover, our STn-based ELISA could complement IHC in identifying patients with whom an anti-STn-based strategy might be more effective.

Tumor-associated carbohydrate antigens of MUC1 - Implication in cancer development.

Glycosylation of cancerous epithelial MUC1 protein is specifically altered in comparison to that which is presented by healthy cells. One of such changes is appearing tumor-associated carbohydrate antigens (TACAs) which are rare in normal tissues and are highly correlated with poor clinical outcomes and cancer progression. This review summarizes and describes the role of Tn, T antigens, their sialylated forms as well as fucosylated Lewis epitopes in different aspects of tumor development, progression, and metastasis. Finally, applications of MUC1 glycan epitopes as potential targets for therapeutic strategy of cancers are notified. One of the novelties of this review is presentation of TACAs as inherently connected with MUC1 mucin.

Tn antigen promotes breast cancer metastasis via impairment of CASC4.

Breast cancer is one of the most serious and deadly cancers in women worldwide, with distant metastases being the leading cause of death. Tn antigen, a tumor-associated carbohydrate antigen, was frequently detected in breast cancer, but its exact role in breast cancer metastasis has not been well elucidated. Here we investigated the impact of Tn antigen expression on breast cancer metastasis and its underlying mechanisms. The expression of Tn antigen was induced in two breast cancer cell lines by deleting T-synthase or Cosmc, both of which are required for normal O-glycosylation. It showed that Tn-expressing cancer cells promoted epithelial-mesenchymal transition (EMT) and metastatic features as compared to Tn(-) control cells both in vitro and in vivo. Mechanistically, we found that cancer susceptibility candidate 4 (CASC4), a heavily O-glycosylated protein, was significantly downregulated in both Tn(+) cells. Overexpression of CASC4 suppressed Tn-induced activation of EMT and cancer metastasis via inhibition of Cdc42 signaling. Furthermore, we confirmed that O-glycosylation is essential for the functional role of CASC4 because defective O-glycosylated CASC4 (mutant CASC4, which lacks nine O-glycosylation sites) exerted marginal metastatic-suppressing effects in comparison with WT CASC4. Collectively, these data suggest that Tn-mediated aberrant O-glycosylation contributes to breast cancer metastasis via impairment of CASC4 expression and function.

CRISPR-screen identifies ZIP9 and dysregulated Zn2+ homeostasis as a cause of cancer-associated changes in glycosylation.

INTRODUCTION: In epithelial cancers, truncated O-glycans, such as the Thomson-nouveau antigen (Tn) and its sialylated form (STn), are upregulated on the cell surface and associated with poor prognosis and immunological escape. Recent studies have shown that these carbohydrate epitopes facilitate cancer development and can be targeted therapeutically; however, the mechanism underpinning their expression remains unclear. METHODS: To identify genes directly influencing the expression of cancer-associated O-glycans, we conducted an unbiased, positive-selection, whole-genome CRISPR knockout-screen using monoclonal antibodies against Tn and STn. RESULTS AND CONCLUSIONS: We show that knockout of the Zn2+-transporter SLC39A9 (ZIP9), alongside the well-described targets C1GALT1 (C1GalT1) and its molecular chaperone, C1GALT1C1 (COSMC), results in surface-expression of cancer-associated O-glycans. No other gene perturbations were found to reliably induce O-glycan truncation. We furthermore show that ZIP9 knockout affects N-linked glycosylation, resulting in upregulation of oligo-mannose, hybrid-type, and α2,6-sialylated structures as well as downregulation of tri- and tetra-antennary structures. Finally, we demonstrate that accumulation of Zn2+ in the secretory pathway coincides with cell-surface presentation of truncated O-glycans in cancer tissue, and that over-expression of COSMC mitigates such changes. Collectively, the findings show that dysregulation of ZIP9 and Zn2+ induces cancer-like glycosylation on the cell surface by affecting the glycosylation machinery.

Investigating Two Modes of Cancer-Associated Antigen Heterogeneity in an Agent-Based Model of Chimeric Antigen Receptor T-Cell Therapy.

Chimeric antigen receptor (CAR) T-cell therapy has been successful in treating liquid tumors but has had limited success in solid tumors. This work examines unanswered questions regarding CAR T-cell therapy using computational modeling, such as, what percentage of the tumor must express cancer-associated antigens for treatment to be successful? The model includes cancer cell and vascular and CAR T-cell modules that interact with each other. We compare two different models of antigen expression on tumor cells, binary (in which cancer cells are either susceptible or are immune to CAR T-cell therapy) and gradated (where each cancer cell has a probability of being killed by a CAR T-cell). We vary the antigen expression levels within the tumor and determine how effective each treatment is for the two models. The simulations show that the gradated antigen model eliminates the tumor under more parameter values than the binary model. Under both models, shielding, in which the low/non-antigen-expressing cells protect high antigen-expressing cells, reduced the efficacy of CAR T-cell therapy. One prediction is that a combination of CAR T-cell therapies that targets the general population of cells as well as one that specifically targets cancer stem cells should increase its efficacy.

Sialyl-Tn antigen facilitates extracellular vesicle-mediated transfer of FAK and enhances motility of recipient cells.

Protein glycosylation plays a pivotal role in tumour development by modulating molecular interactions and cellular signals. Sialyl-Tn (sTn) antigen is a tumour-associating carbohydrate epitope whose expression correlates with metastasis and poor prognosis of various cancers; however, its pathophysiological function is poorly understood. Extracellular vesicles (EVs) derived from cancer cells act as a signal mediator amongst tumour microenvironments by transferring cargo molecules. sTn antigen has been found in the glycans of EVs, thereby the functional relevance of sTn antigen to the regulation of tumour microenvironments could be expected. In the present study, we showed that sTn antigen induced TP53 and tumour suppressor-activated pathway 6 (TSAP6) and consequently enhanced EV production. Besides, the genetic attenuation of TSAP6 resulted in the reduction of the EV production in the sTn antigen expressing cells. The enhanced EV production in the sTn antigen-expressing cells consequently augmented the delivery of EVs to recipient cells. The produced EVs selectively and abundantly encased focal adhesion kinase and transferred it to EV-recipient cells, and thus, their cellular motility was enhanced. These findings would contribute to facilitate the elucidation of the pathophysiological significance of the sTn antigen in the tumour microenvironments and tumour development.

CDKN1A (p21 gene) polymorphisms correlates with age in esophageal cancer.

BACKGROUND: CDKN1A gene encoding p21 is an important tumour supressor involved in the pathogenesis of cancers. A few studies have been devoted to the association between CDKN1A single nucleotide polymorphisms (SNPs) and esophageal cancer (EC) in China, India and Iran. The aim of this case-control study was to investigate the association of CDKN1A polymorphisms with EC risk in the Turkey population for the first time. METHODS: In the present study, CDKN1A SNPs (rs1801270 C > T, rs1059234 C > A and rs3176352 C > G) were genotyped with the use of TaqMan SNP genotyping assays in 102 patients and 119 controls. RESULTS: The genotypes and alleles of CDKN1A SNPs were not significantly different among patients and controls. However, TT-genotype and T-allele of the rs1059234, the rs1801270 CC-genotype and rs3176352 G-allele were significantly associated with EC risk for ≤ 55 age (p < 0.05). In those over 55 age, CC-genotype and C-allele of the rs1059234 was significantly associated with EC (p < 0.05). The rs1059234 T-carriers had a higher risk of high globulin level (p = 0.017) and low albumin/globulin ratio (p = 0.019) when compared to non-T carriers (CC). The rs3176352 CC-genotype carriers had a higher risk of esophageal adenocarcinoma (EAC) subtype when compared to CG-genotype carriers and CG-genotype carriers had a higher risk of squamous cell carcinoma (ESCC) subtype (OR/95% CI = 4.00/1.06-15.08, p = 0.04). The rs3176352 CC-genotype is also a risk factor for the higher BMI (p = 0.04) and the higher CA-19-9 level (p = 0.009). CONCLUSION: Our study suggests that the CDKN1A polymorphisms may play an important role in EC risk in relation to age. Future studies are needed to validate our findings.

Targeting Tn-Antigen-Positive Human Tumors with a Recombinant Human Macrophage Galactose C-Type Lectin.

Alterations in glycosylation cause the emergence of tumor-associated carbohydrate antigens (TACAs) during tumorigenesis. Truncation of O-glycans reveals the Thomsen nouveau (Tn) antigen, an N-acetylgalactosamine (GalNAc) frequently attached to serine or threonine amino acids, that is accessible on the surface of cancer cells but not on healthy cells. Interestingly, GalNac can be recognized by macrophage galactose lectin (MGL), a type C lectin receptor expressed in immune cells. In this study, recombinant MGL fragments were tested in vitro for their cancer cell-targeting efficiency by flow cytometry and confocal microscopy and in vivo after administration of fluorescent MGL to tumor-bearing mice. Our results demonstrate the ability of MGL to target Tn-positive human tumors without inducing toxicity. This outcome makes MGL, a fragment of a normal human protein, the first vector candidate for in vivo diagnosis and imaging of human tumors and, possibly, for therapeutic applications.

Effect of triptolide and chemotherapy on carcinoembryonic and carbohydrate antigens levels and first-line treatment of recurrent nasopharyngeal carcinoma.

To investigate the first-line treatment of recurrent Nasopharyngeal Carcinoma treprimcab combined with chemotherapy. From January 2019 to January 2020, 48 patients with recurrent nasopharyngeal Carcinoma (RNPC) were treated in our hospital. According to the method of the random number, 24 patients were divided into the combined group and the Control Group. The patients in the combined group were given the Combined Treatment of triptolide and chemotherapy. While the Control Group only received chemotherapy. The therapeutic effects and adverse reactions of the two groups were compared, the levels of Carcinoembryonic Antigen (CEA) and carbohydrate Antigen 19-9 (CA19-9) were measured before and after treatment. The total effective rate of the combined group was 79.17% higher than that of the control group (62.50%). The total effective rate of the two groups was statistically significant (P & Lt; 0.05). The incidence of grade i/ii adverse reaction in the control group was lower than that in the combined group, such as nausea and vomiting, oral mucositis, Leukopenia, liver and kidney function damage, central granulocyte count reduction, anaemia adverse reaction. The incidence of grade iii/iv Adr in the control group was higher than that in the combined group. The incidence of grade i/ii Adr in the thrombocytopenia group was higher than that in the combined group, and the incidence of grade iii/iv Adr in the control group was lower than that in the combined group. The side effects of nausea and vomiting and oral mucositis in the control group and the combined group were statistically significant (P & Lt; 0.05). There was no significant difference between the control group and the combined group in the incidence of Leukopenia, liver and kidney injury, neutrophil, anaemia and Thrombocytopenia (P & GT; 0.05). The level of CD4 + / CD8 + in control group and combined group before treatment was higher than that after treatment (P & Lt; 0.05). The quality of life of the combined group was 91.67% higher than that of the control group (70.83%). The quality of life of the control group was significantly higher than that of the combined group (P & Lt; 0.05). The levels of CEA and CA19-9 in the two groups after treatment were lower than those before treatment, and the levels of CEA and CA19-9 in the combined group were lower than those in the control group (P & Lt; 0.05). The first-line treatment of recurrent nasopharyngeal Carcinoma with triprimmab combined with chemotherapy has a good clinical effect and has a broad clinical research prospect.

Cosmc transfection decreases malignant behavior of Tn+ cells and enhances sensitivity to apoptosis when induced by Apo2L/TRAIL via alteration of O-glycan structure.

Cosmc mutations may cause abnormal O-glycosylation and result in Tn antigen expression. In the current study, it was discovered that proliferation and migration of Tn+ cells (Jurkat T and LS174T-Tn+ cells) with mutant Cosmc decreased after transfected Cosmc, and their sensitivity to apoptosis induced by Apo2L/TRAIL increased. Core 1-, 2-, and 3-derived O-glycans were absent in Tn+ cells. After Cosmc transfection, normal extended core 1-derived O-glycans appeared and were accompanied by increased T-synthase activity. Core 2-derived O-glycans appeared in transfected LS174T-Tn+ cells, and their structural types and levels were lower than those in LS174T-Tn- cells. Core 3-derived O-glycans were present only in LS174T-Tn- cells. The activity of C3GnT in LS174T-Tn+ cells was lower than that in LS174T-Tn- cells, and it was absent in Jurkat T cells. Cosmc transfection did not alter C3GnT activity or core 3-derived O-glycans in Jurkat T and LS174T-Tn+ cells. The results demonstrated that the composition and structure of O-glycans were different among various Tn+ cells, which not only affected cell malignant behavior but also modulated sensitivity to apoptotic stimuli. Thus, Cosmc transfection may effectively decrease the malignant behavior of Tn+ tumor cells and enhance their sensitivity to apoptosis when induced by Apo2L/TRAIL through modification of O-glycans.

The application of CA72-4 in the diagnosis, prognosis, and treatment of gastric cancer.

The role of conventional serum tumor marker, carbohydrate antigen 72-4 (CA72-4), in assisting diagnosis, monitoring dynamic progression, and evaluating the prognosis of gastric cancer (GC) should not be ignored, especially in the Chinese population. Even though CA72-4 has been used in clinical practice for decades, its modest positivity rate, sensitivity, and specificity did not meet the high demand of the clinical application. However, over the years, some progress in the functions of CA72-4 has been achieved, suggesting that CA72-4 can still be considered a promising marker in oncology. As a biomarker, CA72-4 can achieve improved sensitivity (SEN) and specificity (SPE) when combined with other biomarkers, selecting suitable reference values, improving detection techniques, and identifying the risk threshold. As a predictor, elevated serum CA72-4 levels were found to be significantly associated with prognostic risk factors, further assessing therapeutic validity and resectability. Recently, an effective method to reduce the toxicity of CA72-4 targeted therapy has been developed. Moreover, CA72-4 could induce novel aptamers to react with tumor cells and enhance the efficacy of trastuzumab in HER2-positive GC. Therefore, in this review, we discuss the most recent application of CA72-4 in the diagnosis, prognosis, and treatment of GC.

Mapping of truncated O-glycans in cancers of epithelial and non-epithelial origin.

BACKGROUND: Novel immunotherapies targeting cancer-associated truncated O-glycans Tn (GalNAcα-Ser/Thr) and STn (Neu5Acα2-6GalNacα-Ser/Thr) are promising strategies for cancer treatment. However, no comprehensive, antibody-based mapping of truncated O-glycans in tumours exist to guide drug development. METHODS: We used monoclonal antibodies to map the expression of truncated O-glycans in >700 tissue cores representing healthy and tumour tissues originating from breast, colon, lung, pancreas, skin, CNS and mesenchymal tissue. Patient-derived xenografts were used to evaluate Tn expression upon tumour engraftment. RESULTS: The Tn-antigen was highly expressed in breast (57%, n = 64), colorectal (51%, n = 140) and pancreatic (53%, n = 108) tumours, while STn was mainly observed in colorectal (80%, n = 140) and pancreatic (56%, n = 108) tumours. We observed no truncated O-glycans in mesenchymal tumours (n = 32) and low expression of Tn (5%, n = 87) and STn (1%, n = 75) in CNS tumours. No Tn-antigen was found in normal tissue (n = 124) while STn was occasionally observed in healthy gastrointestinal tissue. Surface expression of Tn-antigen was identified across several cancers. Tn and STn expression decreased with tumour grade, but not with cancer stage. Numerous xenografts maintained Tn expression. CONCLUSIONS: Surface expression of truncated O-glycans is limited to cancers of epithelial origin, making Tn and STn attractive immunological targets in the treatment of human carcinomas.

Combined Effect of Anti-SSEA4 and Anti-Globo H Antibodies on Breast Cancer Cells.

The globo-series glycosphingolipids (SSEA3, SSEA4, and Globo H) were shown to express in many cancers selectively, and a combination of anti-SSEA4 and anti-Globo H antibodies was able to suppress tumor growth in mice inoculated with breast cancer cell lines. To further understand the effect, we focused on the combined effect of the two antibodies in target binding and antibody-dependent cellular cytotoxicity (ADCC) in vitro. Here, we report that the binding of anti-Globo H antibody (VK9) to MDA-MB231 breast cancer cells was influenced by anti-SSEA4 antibody (MC813-70), and a combination of both antibodies induced a similar effect as did anti-SSEA4 antibodies alone in a reporter-based ADCC assay, indicating that SSEA4 is a major target in breast cancer due to its higher expression than Globo H. Furthermore, we showed that a homogeneous anti-SSEA4 antibody (chMC813-70-SCT) designed to maximize the ADCC activity can be used to isolate a subpopulation of natural killer (NK) cells that exhibit an ∼23% increase in killing the target cells as compared to the unseparated NK cells. These findings can be used to predict a therapy outcome based on the expression levels of antigens and evaluate therapeutic antibody development.

CEA, CA19-9, circulating DNA and circulating tumour cell kinetics in patients treated for metastatic colorectal cancer (mCRC).

BACKGROUND: We previously reported that CEA kinetics are a marker of progressive disease (PD) in metastatic colorectal cancer (mCRC). This study was specifically designed to confirm CEA kinetics for predicting PD and to evaluate CA19-9, cell-free DNA (cfDNA), circulating tumour DNA (ctDNA) and circulating tumour cell (CTC) kinetics. METHODS: Patients starting a chemotherapy (CT) with pre-treatment CEA > 5 ng/mL and/or CA19.9 > 30 UI/mL were prospectively included. Samples were collected from baseline to cycle 4 for CEA and CA19-9 and at baseline and the sixth week for other markers. CEA kinetics were calculated from the first to the third or fourth CT cycle. RESULTS: A total of 192 mCRC patients were included. CEA kinetics based on the previously identified >0.05 threshold was significantly associated with PD (p < 0.0001). By dichotomising by the median value, cfDNA, ctDNA and CA19-9 were associated with PD, PFS and OS in multivariate analysis. A circulating scoring system (CSS) combining CEA kinetics and baseline CA19-9 and cfDNA values classified patients based on high (n = 58) and low risk (n = 113) of PD and was independently associated with PD (ORa = 4.6, p < 0.0001), PFS (HRa = 2.07, p < 0.0001) and OS (HRa = 2.55, p < 0.0001). CONCLUSIONS: CEA kinetics alone or combined with baseline CA19-9 and cfDNA are clinically relevant for predicting outcomes in mCRC. TRIAL REGISTRATION NUMBER: NCT01212510.

Autoimmune Pancreatitis: A Critical Analysis of the Surgical Experience in an Era of Modern Diagnostics.

OBJECTIVE: The aim of this study was to critically analyze the surgical experience of managing autoimmune pancreatitis (AIP) in an era of modern diagnostics and compare these patients with those who were managed conservatively. METHODS: Two prospectively maintained databases were used to retrospectively identify patients with AIP who were either managed conservatively or underwent pancreatectomy. RESULTS: Eighty-eight patients were included in the study, of which 56 (63.6%) underwent resection and 32 (36.4%) were managed conservatively. Patients who underwent resection were more likely to present with jaundice (64.3% vs 18.1%, P < 0.001) and weight loss (53.6% vs 15.6%, P = 0.005). The cohort who underwent resection had a significantly higher median carbohydrate antigen 19-9 (40.0 vs 18.6 U/mL, P = 0.034) and was less likely to have elevated immunoglobulin G4 (26.1% vs 50.0%, P < 0.001). The most frequent initial diagnosis in the cohort who underwent resection was ductal adenocarcinoma (82.1%). Nine patients (28.1%) in the conservatively managed cohort experienced AIP relapse compared with 6 patients (10.7%) in the cohort who underwent resection. CONCLUSIONS: The most frequent reason for surgical resection of AIP is concern for malignancy. Carbohydrate antigen 19-9 elevations were more common than immunoglobulin G4 in our cohort, suggesting that this laboratory profile is suboptimal for this population.

Crystal Structure of the Carbohydrate Recognition Domain of the Human Macrophage Galactose C-Type Lectin Bound to GalNAc and the Tumor-Associated Tn Antigen.

The human macrophage galactose lectin (MGL) is an endocytic type II transmembrane receptor expressed on immature monocyte-derived dendritic cells and activated macrophages and plays a role in modulating the immune system in response to infections and cancer. MGL contains an extracellular calcium-dependent (C-type) carbohydrate recognition domain (CRD) that specifically binds terminal N-acetylgalactosamine glycan residues such as the Tn and sialyl-Tn antigens found on tumor cells, as well as other N- and O-glycans displayed on certain viruses and parasites. Even though the glycan specificity of MGL is known and several binding glycoproteins have been identified, the molecular basis for substrate recognition has remained elusive due to the lack of high-resolution structures. Here we present crystal structures of the MGL CRD at near endosomal pH and in several complexes, which reveal details of the interactions with the natural ligand, GalNAc, the cancer-associated Tn-Ser antigen, and a synthetic GalNAc mimetic ligand. Like the asialoglycoprotein receptor, additional calcium atoms are present and contribute to stabilization of the MGL CRD fold. The structure provides the molecular basis for preferential binding of N-acetylgalactosamine over galactose and prompted the re-evaluation of the binding modes previously proposed in solution. Saturation transfer difference nuclear magnetic resonance data acquired using the MGL CRD and interpreted using the crystal structure indicate a single binding mode for GalNAc in solution. Models of MGL1 and MGL2, the mouse homologues of MGL, explain how these proteins might recognize LewisX and GalNAc, respectively.

Glycan chip based on structure-switchable DNA linker for on-chip biosynthesis of cancer-associated complex glycans.

On-chip glycan biosynthesis is an effective strategy for preparing useful complex glycan sources and for preparing glycan-involved applications simultaneously. However, current methods have some limitations when analyzing biosynthesized glycans and optimizing enzymatic reactions, which could result in undefined glycan structures on a surface, leading to unequal and unreliable results. In this work, a glycan chip is developed by introducing a pH-responsive i-motif DNA linker to control the immobilization and isolation of glycans on chip surfaces in a pH-dependent manner. On-chip enzymatic glycosylations are optimized for uniform biosynthesis of cancer-associated Globo H hexasaccharide and its related complex glycans through stepwise quantitative analyses of isolated products from the surface. Successful interaction analyses of the anti-Globo H antibody and MCF-7 breast cancer cells with on-chip biosynthesized Globo H-related glycans demonstrate the feasibility of the structure-switchable DNA linker-based glycan chip platform for on-chip complex glycan biosynthesis and glycan-involved applications.

Imaging Mass Spectrometry and Lectin Analysis of N-Linked Glycans in Carbohydrate Antigen-Defined Pancreatic Cancer Tissues.

The early detection of pancreatic ductal adenocarcinoma (PDAC) is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers carbohydrate antigen 19-9 (CA19-9) and sialylated tumor-related antigen (sTRA) are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and noncancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry (IMS) approach was used to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of patients with PDAC represented by tissue microarrays and whole-tissue sections. Orthogonally, these same tissues were characterized by multiround immunofluorescence that defined expression of CA19-9 and sTRA as well as other lectins toward carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated biantennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9-expressing tissues tended to be biantennary, triantennary, and tetra-antennary structures with both core and terminal fucose residues and bisecting GlcNAc. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored triantennary and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-immunohistochemistry and lectin-IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

Calorimetric Analysis of the Interplay between Synthetic Tn Antigen-Presenting MUC1 Glycopeptides and Human Macrophage Galactose-Type Lectin.

Human macrophage galactose-type lectin (hMGL, HML, CD301, CLEC10A), a C-type lectin expressed by dendritic cells and macrophages, is a receptor for N-acetylgalactosamine α-linked to serine/threonine residues (Tn antigen, CD175) and its α2,6-sialylated derivative (sTn, CD175s). Because these two epitopes are among malignant cell glycan displays, particularly when presented by mucin-1 (MUC1), assessing the influence of the site and frequency of glycosylation on lectin recognition will identify determinants governing this interplay. Thus, chemical synthesis of the tandem-repeat O-glycan acceptor region of MUC1 and site-specific threonine glycosylation in all permutations were carried out. Isothermal titration calorimetry (ITC) analysis of the binding of hMGL to this library of MUC1 glycopeptides revealed an enthalpy-driven process and an affinity enhancement of an order of magnitude with an increasing glycan count from 6-8 µM for monoglycosylated peptides to 0.6 µM for triglycosylated peptide. ITC measurements performed in D2O permitted further exploration of the solvation dynamics during binding. A shift in enthalpy-entropy compensation and contact position-specific effects with the likely involvement of the peptide surroundings were detected. KinITC analysis revealed a prolonged lifetime of the lectin-glycan complex with increasing glycan valency and with a change in the solvent to D2O.

Target Score-A Proteomics Data Selection Tool Applied to Esophageal Cancer Identifies GLUT1-Sialyl Tn Glycoforms as Biomarkers of Cancer Aggressiveness.

Esophageal cancer (EC) is a life-threatening disease, demanding the discovery of new biomarkers and molecular targets for precision oncology. Aberrantly glycosylated proteins hold tremendous potential towards this objective. In the current study, a series of esophageal squamous cell carcinomas (ESCC) and EC-derived circulating tumor cells (CTCs) were screened by immunoassays for the sialyl-Tn (STn) antigen, a glycan rarely expressed in healthy tissues and widely observed in aggressive gastrointestinal cancers. An ESCC cell model was glycoengineered to express STn and characterized in relation to cell proliferation and invasion in vitro. STn was found to be widely present in ESCC (70% of tumors) and in CTCs in 20% of patients, being associated with general recurrence and reduced survival. Furthermore, STn expression in ESCC cells increased invasion in vitro, while reducing cancer cells proliferation. In parallel, an ESCC mass spectrometry-based proteomics dataset, obtained from the PRIDE database, was comprehensively interrogated for abnormally glycosylated proteins. Data integration with the Target Score, an algorithm developed in-house, pinpointed the glucose transporter type 1 (GLUT1) as a biomarker of poor prognosis. GLUT1-STn glycoproteoforms were latter identified in tumor tissues in patients facing worst prognosis. Furthermore, healthy human tissues analysis suggested that STn glycosylation provided cancer specificity to GLUT1. In conclusion, STn is a biomarker of worst prognosis in EC and GLUT1-STn glycoforms may be used to increase its specificity on the stratification and targeting of aggressive ESCC forms.