O-glycoprofiling of Serum Apolipoprotein C-III in Colorectal Cancer.

BACKGROUND: Aberrant glycosylation is a hallmark of cancer and thereby has an excellent potential for the discovery of novel biomarkers. Impairments in the glycan composition of lipoproteins impact their functional properties and can be associated with various diseases, including cancer. This research is still in its infancy; however, it can lead to the development of new diagnostic and disease stratification approaches as well as therapeutic strategies. Therefore, we aimed to evaluate anomalies in O-glycosylation of apolipoprotein C-III (apoC-III) in colorectal carcinoma (CRC) patients' sera, in comparison with sera from healthy individuals, and assess the disparities of O-glycoforms on apoC-III in CRC. METHODS: The choice of patients (n = 42) was based on the same tumor type (adenocarcinoma) and tumor size (T3), without or with inconsiderable lymph node infiltration. Patients with comorbidities were excluded from the study. The control healthy individuals (n = 40) were age- and sex-matched with patients. We used an approach based on the MALDI-TOF MS in linear positive ion mode, allowing simple analysis of O-glycosylation on intact apoC-III molecules in the serum samples directly, without the need for specific protein isolation. This approach enables relatively simple and high-throughput analysis. RESULTS: In CRC patients' sera samples, we observed significantly elevated apoC-III sialylation. Fully sialylated (disialylated) O-glycans had 1.26 times higher relative abundance in CRC samples compared to controls with a p-value of Mann-Whitney U test of 0.0021. CONCLUSIONS: We found altered O-glycosylation of apoC-III in the serum of CRC patients. However, it can be non-specific as it may be associated with another process such as ongoing inflammation. Therefore, to establish it as a potential novel non-invasive biomarker for CRC in suspected patients, further studies interrogating the changes in apoC-III O-glycosylation and the robustness of this biomarker need to be performed and evaluated.

Amplified suspension magnetic bead-based assay for sensitive detection of anti-glycan antibodies as potential cancer biomarkers.

The development of a novel SUspension Magnetic-Bead-based Assay (SUMBA) for the detection of antibodies against aberrant glycans (AGA) as potential cancer biomarkers is presented here. The SUMBA method was extensively optimised by choosing proper commercially available AGA able to specifically, and with high affinity, recognise aberrant glycans, which were attached to the protein backbone working as a molecular scaffold (a glycoconjugate). The whole SUMBA was optimised using several analytical techniques such as Surface Plasmon Resonance and Energy Dispersive X-ray Spectroscopy. Additionally, the SUMBA method was extensively optimised for signal enhancement. With all steps optimised, we were able to detect AGA ultrasensitively with a limit of detection of 0.45 pM. Moreover, AGA could be detected in serum samples with a recovery index in the range of 98%-104%.

A Graphene-Based Glycan Biosensor for Electrochemical Label-Free Detection of a Tumor-Associated Antibody.

The study describes development of a glycan biosensor for detection of a tumor-associated antibody. The glycan biosensor is built on an electrochemically activated/oxidized graphene screen-printed electrode (GSPE). Oxygen functionalities were subsequently applied for covalent immobilization of human serum albumin (HSA) as a natural nanoscaffold for covalent immobilization of Thomsen-nouvelle (Tn) antigen (GalNAc-O-Ser/Thr) to be fully available for affinity interaction with its analyte-a tumor-associated antibody. The step by step building process of glycan biosensor development was comprehensively characterized using a battery of techniques (scanning electron microscopy, atomic force microscopy, contact angle measurements, secondary ion mass spectrometry, surface plasmon resonance, Raman and energy-dispersive X-ray spectroscopy). Results suggest that electrochemical oxidation of graphene SPE preferentially oxidizes only the surface of graphene flakes within the graphene SPE. Optimization studies revealed the following optimal parameters: activation potential of +1.5 V vs. Ag/AgCl/3 M KCl, activation time of 60 s and concentration of HSA of 0.1 g L-1. Finally, the glycan biosensor was built up able to selectively and sensitively detect its analyte down to low aM concentration. The binding preference of the glycan biosensor was in an agreement with independent surface plasmon resonance analysis.

Antibodies against aberrant glycans as cancer biomarkers.

Introduction: The review provides a comprehensive overview about applicability of serological detection of autoantibodies against aberrant glycans as cancer biomarkers.Areas covered: Clinical usefulness of autoantibodies as cancer biomarkers is discussed for seven types of cancers with sensitivity and specificity of such biomarkers provided. Moreover, an option of using serological antibodies against a non-natural form of sialic acid - N-glycolylneuraminic acid (Neu5Gc), which is taken into our bodies together with red meat, as a potential cancer biomarker is discussed shortly as well.Expert opinion: In the final part of the review, we discuss what measures need to be applied for selective implementation of autoantibody assays into a clinical practice. Moreover, we discuss key challenges ahead for reliable and robust detection of autoantibodies against aberrant glycans as biomarkers for disease diagnostics and for stratification of cancer patients.

Optimization of the Small Glycan Presentation for Binding a Tumor-Associated Antibody: Application to the Construction of an Ultrasensitive Glycan Biosensor.

The main aim of the study was to optimize the interfacial presentation of a small antigen-a Tn antigen (N-acetylgalactosamine)-for binding to its analyte anti-Tn antibody. Three different methods for the interfacial display of a small glycan are compared here, including two methods based on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding with better kinetics of binding (t50% = 137 s, t50% = the time needed to attain 50% of a steady-state signal) compared to the 2D biosensor configuration with t50% = 354 s. The 3D glycan biosensor was finally applied for the analysis of a human serum sample spiked with an analyte.