extracellular-vesicle-uptake-assay-via-confocal-microscope-imaging

Extracellular Vesicle Uptake Assay <em>via</em> Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

Johns Hopkins University School of Medicine

Ulsan National Institute of Science and Technology (UNIST)

One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co-separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density-gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co-separate contaminants when the non-EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non-EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios.

Comprehensive evaluation of methods for small extracellular vesicles separation from human plasma, urine and cell culture medium.

Extracellular vesicles (EVs) are nano-sized lipid bilayer encapsulated particles with a molecular cargo that appears to play important roles within the human body, such as in cell-to-cell communication. Unraveling the composition of EV cargos remains one of the most fundamental steps toward understanding the role of EVs in intercellular communication and the discovery of new biomarkers. One of the unmet needs in this field is the lack of a robust, sensitive, and multiplexed method for EV mRNA profiling. We established a new protocol using the NanoString low RNA input nCounter assay by which the targeted mRNA transcripts in EVs can be efficiently and specifically amplified and then assayed for 770 mRNAs in one reaction. Prostate cancer cells with epithelial (PC3-Epi) or mesenchymal (PC3-EMT) phenotypes and their progeny EVs were analyzed by the same panel. Among these mRNAs, 157 were detected in PC3-Epi EVs and 564 were detected in PC3-EMT EVs. NOTCH1 was the most significantly abundant mRNA transcripts in PC3-EMT EVs compared to PC3-Epi EVs. Our results demonstrated that when cells undergo epithelial-to-mesenchymal transition (EMT), a more active loading of cancer progression-related mRNA transcripts may occur. The mRNA cargos of EVs derived from mesenchymal prostate cancer cells may contribute to the pro-EMT function. We found that mRNA transcripts are different in progeny EVs compared to parental cells. EV cargos are not completely reflective of their cell origin, and the underlying mechanism of cargo sorting is complicated and needs to be further elucidated.

High-Throughput Simultaneous mRNA Profiling Using nCounter Technology Demonstrates That Extracellular Vesicles Contain Different mRNA Transcripts Than Their Parental Prostate Cancer Cells.

Liquid biopsy has emerged as a complement to invasive tissue biopsy to guide cancer diagnosis and treatment. The common liquid biopsy biomarkers are circulating tumor cells (CTCs), extracellular vesicles (EVs), and circulating tumor DNA (ctDNA). Each biomarker provides specific information based on its intrinsic characteristics. Prostate cancer is the second most common cancer in males worldwide. In men with low-grade localized prostate cancer, the disease can often be managed by active surveillance. For men who require treatment, the 5-year survival rate of localized prostate cancer is the highest among all cancer types, but the metastatic disease remains incurable. Metastatic prostate cancer invariably progresses to involve multiple bone sites and develops into a castration-resistant disease that leads to cancer death. The need to appropriately diagnose and guide the serial treatment of men with prostate cancer has led to the implementation of many studies to apply liquid biopsies to prostate cancer management. This review describes recent advancements in isolation and detection technology and the strength and weaknesses of the three circulating biomarkers. The clinical studies based on liquid biopsy results are summarized to depict the future perspective in the role of liquid biopsy on prostate cancer management.

Chi-Ju Kim

The Brady Urological Institute

Johns Hopkins University School of Medicine

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Chi-Ju Kim

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Brady Urological Institute

Johns Hopkins University School of Medicine

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

The Brady Urological Institute