Narrative review of urinary glycan biomarkers in prostate cancer

Abstract

Prostate cancer (PC) is the second most common cancer in men worldwide. The application of the prostate-specific antigen (PSA) test has improved the diagnosis and treatment of PC. However, the PSA test has become associated with overdiagnosis and overtreatment. Therefore, there is an unmet need for novel diagnostic, prognostic, and predictive biomarkers of PC. Urinary glycoproteins and exosomes are a potential source of PC glycan biomarkers. Urinary glycan profiling can provide noninvasive monitoring of tumor heterogeneity and aggressiveness throughout a treatment course. However, urinary glycan profiling is not popular due to technical disadvantages, such as complicated structural analysis that requires specialized expertise. The technological development of glycan analysis is a rapidly advancing field. A lectin-based microarray can detect aberrant glycoproteins in urine, including PSA glycoforms and exosomes. Glycan enrichment beads can enrich the concentration of N-linked glycans specifically. Capillary electrophoresis, liquid chromatography-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry can detect glycans directory. Many studies suggest potential of urinary glycoproteins, exosomes, and glycosyltransferases as a biomarker of PC. Although further technological challenges remain, urinary glycan analysis is one of the promising approaches for cancer biomarker discovery.

Keywords: Prostate cancer (PC); biomarker; glycan; glycosylation; urinary.

Figure 1

Role and types of glycans. Role of glycans in cell-to-cell communications (A) and types of glycoproteins (B) are shown.

Figure 2

Potential urinary biomarkers for prostate cancer. Urine after prostate massage contains many potential biomarkers for PC, including cells, DNA, RNA, proteins, exosomes, bacteria, virus, and other small molecules.

Figure 3

Search methods for identification of studies. PubMed online database was accessed for research on Aug 10th, 2020. Searches were performed using the keywords: “prostate cancer”, “urine”, and “glycan”. Inclusion criteria were (I) independent cohort, (II) a proper number of samples and controls, (III) clinically meaningful outcomes, and (IV) promising diagnostic/prognostic performance.

Figure 4

Schematic protocol of direct glycan analysis using capillary electrophoresis (Gly-Q). Capillary electrophoresis-LED-induced fluorescence-based Gly-Q N-glycan analysis system (Prozyme, Inc., CA, USA) combined with Gly-X rapid N-glycan preparation method can measure the amount of glycans under controlled automated SweetblotTM (System Instruments, Tokyo, Japan) machinery. Briefly, 1 mg/mL of target protein from the urine and 2 µL of Gly-X denaturant was mixed. Then, 2 µL of N-glycanase working solution was added to the denatured samples. After deglycosylation, 5 µL of InstantPC dye solution was added to the deglycosylated samples. The InstantPC Dye and deglycosyalted sample mixture was then loaded onto prewetted Gly-X cleanup plate and applied vacuum to <5 inHg. Then, 100 µL of Gly-X InstantPC eluent added to each well and collected InstanPC-labeled glycan samples into the Collection Plate using vacuum. Finally, InstantQ is a charged N-glycan dye that facilitates separation of labeled N-glycans on the Gly-Q CE system. Composition and structures of the glycans were analyzed using the Gly-Q Manager software performing automated peak analysis (Relative Fluorescence Unit: RFU and Glucose Unit: GU) and glycan assignments from the glycan library.

Figure 5

Schematic protocol of direct glycan analysis using SweetBlot and MALDI-TOF/MS. Fluid samples are applied to the SweetBlot for glycoblotting. After enzymatic cleavage from serum protein, total serum N-glycans released into the digestion mixture are directly mixed with BlotGlyco H beads to capture N-glycans. After the beads are separated from other molecules by washing, sialic acid is methyl-esterified. These processed N-glycans are then labeled with benzyloxyamine (BOA) and released from BlotGlyco H beads. Mass spectra of BOA-labeled N-glycans are acquired using an Ultraflex III instrument.

Figure 6

Schematic protocol of glycoprotein analysis using lectin-based microarray system. Glycoproteins are added to the wells of the recombinant lectin array chip. After reactions occur, the chip is scanned utilizing evanescent-wave fluorescence microscopy. Briefly, 20 µL of sample was diluted by 80 µL of probing buffer. Diluted sample was added to the well of recombinant lectin array chip (Rexxam Co. Ltd., Osaka, Japan). After 70 mins incubation and washing twice, 100 µL of probing buffer containing 1 µg/mL biotinylated specific antibody for the target protein was added to well. After 60 mins incubation and washing twice, 100 µL of probing buffer containing 1 µg/mL Cy3 labeled streptavidin (GE-Healthcare, Buckinghamshire, UK) was added to the well. After 30 mins incubation and washing twice, the chip was scanned by utilizing Bio-REX Scan 200 evanescent fluorescence scanner (Rexxam Co., Ltd.). Mean fluorescence intensity of lectin reactive glycan carrying Igs was normalized by total Igs level.