Prostate-Specific Membrane Antigen (PSMA)-Positive Extracellular Vesicles in Urine-A Potential Liquid Biopsy Strategy for Prostate Cancer Diagnosis?

All cells release extracellular vesicles (EVs) to communicate with adjacent and distant cells. Consequently, circulating EVs are found in all bodily fluids, providing information applicable for liquid biopsy in early cancer diagnosis. Studies observed an overexpression of the membrane-bound prostate-specific membrane antigen (PSMA) on prostate cancer cells. To investigate whether EVs derived from communicating prostate cells allow for reliable conclusions on prostate cancer development, we isolated PSMA-positive, as well as CD9-positive, EVs from cell-free urine with the use of magnetic beads. These populations of EVs were subsequently compared to CD9-positive EVs isolated from female urine in Western blotting, indicating the successful isolation of prostate-derived and ubiquitous EVs, respectively. Furthermore, we developed a device with an adapted protocol that enables an automated immunomagnetic enrichment of EVs of large sample volumes (up to 10 mL), while simultaneously reducing the overall bead loss and hands-on time. With an in-house spotted antibody microarray, we characterized PSMA as well as other EV surface markers of a prostate cohort of 44 urine samples in a more simplified way. In conclusion, the automated and specific enrichment of EVs from urine has a high potential for future diagnostic applications.

Two-Step Competitive Hybridization Assay: A Method for Analyzing Cancer-Related microRNA Embedded in Extracellular Vesicles.

With an increased understanding of the role of microRNAs (miRNAs) in cancer evolution, there is a growing interest in the use of these non-coding nucleic acids in cancer diagnosis, prognosis, and treatment monitoring. miRNAs embedded in extracellular vesicles (EVs) are of particular interest given that circulating EVs carry cargo that are strongly correlated to their cells of origin such as tumor cells while protecting them from degradation. As such, there is a tremendous interest in new simple-to-operate vesicular microRNA analysis tools for widespread use in performing liquid biopsies. Herein, we present a two-step competitive hybridization assay that is rationally designed to translate low microRNA concentrations to large electrochemical signals as the measured signal is inversely proportional to the microRNA concentration. Using this assay, with a limit-of-detection of 122 aM, we successfully analyzed vesicular miRNA 200b from prostate cancer cell lines and human urine samples, demonstrating the expected lower expression levels of miRNA 200b in the EVs from prostate cancer cells and in the prostate cancer patient's urine samples compared to healthy patients and non-tumorigenic cell lines, validating the suitability of our approach for clinical analysis.

Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations.

Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.

Application of nanotechnology in miniaturized systems and its use for advanced analytics and diagnostics - an updated review.

A combination of Micro-Electro-Mechanical Systems and nanoscale structures allows for the creation of novel miniaturized devices, which broaden the boundaries of the diagnostic approaches. Some materials possess unique properties at the nanolevel, which are different from those in bulk materials. In the last few years these properties became a focus of interest for many researchers, as well as methods of production, design and operation of the nanoobjects. Intensive research and development work resulted in numerous inventions exploiting nanotechnology in miniaturized systems. Modern technical and laboratory equipment allows for the precise control of such devices, making them suitable for sensitive and accurate detection of the analytes. The current review highlights recent patents in the field of nanotechnology in microdevices, applicable for medical, environmental or food analysis. The paper covers the structural and functional basis of such systems and describes specific embodiments in three principal branches: application of nanoparticles, nanofluidics, and nanosensors in the miniaturized systems for advanced analytics and diagnostics. This overview is an update of an earlier review article.

Method for chromatographic analysis of whey protein-dextran glycation products.

A chromatographic method for the analysis of whey protein isolate (WPI)-dextran glycates was developed in this work that is useful for quantification of sample purity and concentration, and as a sample-preparation method for subsequent analysis by gel electrophoresis (SDS-PAGE) and laser-light scattering. Glycation was by the Maillard reaction between WPI and dextran of 3 different sizes. Glycate fractions from each dextran were collected and analyzed by fluorescent and glycoprotein staining of gels, bicinchoinic acid protein assay, and static and dynamic laser light scattering. The weight-average molecular mass of the glycates was 27-34 kDa (from 3.5 kDa dextran), 32-39 kDa (from 10 kDa dextran), and 250-270 kDa (from 150 kDa dextran). The new method was used to characterize the kinetics of the glycation reaction, which followed a reversible pseudo first-order model. The kinetics of decomposition of the purified glycate by hydrolysis was also examined. The new method is rapid (25 min) and quantitative, and is the first chromatographic method for direct analysis of WPI-dextran glycation products.

Application of nanotechnology in miniaturized systems and its use in medical and food analysis.

A combination of Micro-Electro-Mechanical Systems and nanoscale structures allows for the creation of novel miniaturized devices, which broaden the boundaries of the diagnostic approaches. Some materials possess unique properties at the nanolevel, which are different from those in bulk materials. In the last years these properties became a focus of interest for many researchers, as well as methods of production, design and operation of the nanoobjects. Intensive research and development work resulted in numerous inventions, exploiting nanotechnology in miniaturized systems. Modern technical and laboratory equipment allows for the precise control of such devices, making them suitable for sensitive and accurate detection of the analytes. The current review highlights recent patents in the field of nanotechnology in microdevices, applicable for medical and food analysis. The paper covers the structural and functional basis of such systems and describes specific embodiments in three principal branches: application of nanoparticles, nanofluidics, and nanosensors in the miniaturized systems for advanced analytics and diagnostics.

Chromatographic purification and characterization of whey protein-dextran glycation products.

A process for the food-grade preparative-scale production and chromatographic purification of whey protein isolate (WPI)-dextran glycates was developed in this work. The Maillard reaction was used to produce the glycates in aqueous solution. A 5 mL cation exchange column and salt step gradients were utilized to elute the glycated protein at low salt and unreacted protein at high salt. The process was scaled-up 160-fold to an 800 mL column. Glycated products were analyzed by SDS-PAGE, BCA protein assay and glycoprotein carbohydrate estimation kit, MALDI-TOF and static and dynamic light scattering. Glycated protein was relatively pure, containing only traces of beta-lactoglobulin, and it was heterogeneous due to oligo-glycation. It had a molecular mass of 26-35 kDa by static light scattering, and 22-67 kDa by MALDI-TOF. Mono-glycated protein would have been 23.8 kDa from beta-lactoglobulin (18.6 kDa) and dextran (5.2 kDa). This work demonstrated the utility of cation exchange chromatography for the large-scale purification of glycated proteins using food-grade chemicals and procedures.