Abstract
Proteoglycans in bladder tumors are modified with a distinct oncofetal chondroitin sulfate (ofCS) glycosaminoglycan that is normally restricted to placental trophoblast cells. This ofCS-modification can be detected in bladder tumors by the malarial VAR2CSA protein, which in malaria pathogenesis mediates adherence of parasite-infected erythrocytes within the placenta. In bladder cancer, proteoglycans are constantly shed into the urine, and therefore have the potential to be used for detection of disease. In this study we investigated whether recombinant VAR2CSA (rVAR2) protein could be used to detect ofCS-modified proteoglycans (ofCSPGs) in the urine of bladder cancer patients as an indication of disease presence. We show that ofCSPGs in bladder cancer urine can be immobilized on cationic nitrocellulose membranes and subsequently probed for ofCS content by rVAR2 protein in a custom-made dot-blot assay. Patients with high-grade bladder tumors displayed a marked increase in urinary ofCSPGs as compared to healthy individuals. Urine ofCSPGs decreased significantly after complete tumor resection compared to matched urine collected preoperatively from patients with bladder cancer. Moreover, ofCSPGs in urine correlated with tumor size of bladder cancer patients. These findings demonstrate that rVAR2 can be utilized in a simple biochemical assay to detect cancer-specific ofCS-modifications in the urine of bladder cancer patients, which may be further developed as a noninvasive approach to detect and monitor the disease.
Highlights
Bladder cancer is the fifth most common cancer in the world and one of the most expensive cancers to treat on a per-patient basis due to the need for constant surveillance and multiple therapeutic interventions[1,2]
Oncofetal chondroitin sulfate can be detected in urine from bladder cancer patients For optimization of the assay, nitrocellulose membranes were treated with two different concentrations of cationic detergents, cetylpyridinium chloride (CPC) or benzyalkonium chloride (BAC)
When probing immobilized urine samples from high grade (HG) and low grade (LG) bladder cancer patients, as well as healthy individuals (H1-4) with recombinant VAR2CSA (rVAR2), strong reactivity was observed in the HG group (Fig. 1c)
Summary
Bladder cancer is the fifth most common cancer in the world and one of the most expensive cancers to treat on a per-patient basis due to the need for constant surveillance and multiple therapeutic interventions[1,2]. Urinary biomarkers in bladder cancer have been researched for decades with the aim of noninvasive detection of disease and to monitor high-risk patients with nonmuscle invasive bladder cancer (NMIBC)[3,4,5]. CS chains are comprised of linear polysaccharides made up of repeated N-acetyl-Dgalactosamine (GalNAc) and Glucuronic Acid (GlcA) disaccharide units and can vary greatly in length. While the basic structure is simple, an immense structural heterogeneity is achieved through modifications of the carbohydrate backbone, such as sulfation of component hydroxyl groups. The nontemplate driven synthesis ofCS that vary in size and sulfation patterns makes CS
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