CryoET of ß-amyloid and tau within postmortem Alzheimer's disease brain.

A defining pathological feature of most neurodegenerative diseases is the assembly of proteins into amyloid that form disease-specific structures1. In Alzheimer's disease, this is characterized by the deposition of ß-amyloid and tau with disease-specific conformations. The in situ structure of amyloid in the human brain is unknown. Here, using cryo-fluorescence microscopy-targeted cryo-sectioning, cryo-focused ion beam-scanning electron microscopy lift-out and cryo-electron tomography, we determined in-tissue architectures of ß-amyloid and tau pathology in a postmortem Alzheimer's disease donor brain. ß-amyloid plaques contained a mixture of fibrils, some of which were branched, and protofilaments, arranged in parallel arrays and lattice-like structures. Extracellular vesicles and cuboidal particles defined the non-amyloid constituents of ß-amyloid plaques. By contrast, tau inclusions formed parallel clusters of unbranched filaments. Subtomogram averaging a cluster of 136 tau filaments in a single tomogram revealed the polypeptide backbone conformation and filament polarity orientation of paired helical filaments within tissue. Filaments within most clusters were similar to each other, but were different between clusters, showing amyloid heterogeneity that is spatially organized by subcellular location. The in situ structural approaches outlined here for human donor tissues have applications to a broad range of neurodegenerative diseases.

The extracellular vesicle generation paradox: a bacterial point of view.

All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence.

RASSF1C oncogene elicits amoeboid invasion, cancer stemness, and extracellular vesicle release via a SRC/Rho axis.

Cell plasticity is a crucial hallmark leading to cancer metastasis. Upregulation of Rho/ROCK pathway drives actomyosin contractility, protrusive forces, and contributes to the occurrence of highly invasive amoeboid cells in tumors. Cancer stem cells are similarly associated with metastasis, but how these populations arise in tumors is not fully understood. Here, we show that the novel oncogene RASSF1C drives mesenchymal-to-amoeboid transition and stem cell attributes in breast cancer cells. Mechanistically, RASSF1C activates Rho/ROCK via SRC-mediated RhoGDI inhibition, resulting in generation of actomyosin contractility. Moreover, we demonstrate that RASSF1C-induced amoeboid cells display increased expression of cancer stem-like markers such as CD133, ALDH1, and Nanog, and are accompanied by higher invasive potential in vitro and in vivo. Further, RASSF1C-induced amoeboid cells employ extracellular vesicles to transfer the invasive phenotype to target cells and tissue. Importantly, the underlying RASSF1C-driven biological processes concur to explain clinical data: namely, methylation of the RASSF1C promoter correlates with better survival in early-stage breast cancer patients. Therefore, we propose the use of RASSF1 gene promoter methylation status as a biomarker for patient stratification.

Fluorescent, phosphorescent, magnetic resonance contrast and radioactive tracer labelling of extracellular vesicles.

This review focusses on the significance of fluorescent, phosphorescent labelling and tracking of extracellular vesicles (EVs) for unravelling their biology, pathophysiology, and potential diagnostic and therapeutic uses. Various labeling strategies, such as lipid membrane, surface protein, luminal, nucleic acid, radionuclide, quantum dot labels, and metal complex-based stains, are evaluated for visualizing and characterizing EVs. Direct labelling with fluorescent lipophilic dyes is simple but generally lacks specificity, while surface protein labelling offers selectivity but may affect EV-cell interactions. Luminal and nucleic acid labelling strategies have their own advantages and challenges. Each labelling approach has strengths and weaknesses, which require a suitable probe and technique based on research goals, but new tetranuclear polypyridylruthenium(II) complexes as phosphorescent probes have strong phosphorescence, selective staining, and stability. Future research should prioritize the design of novel fluorescent probes and labelling platforms that can significantly enhance the efficiency, accuracy, and specificity of EV labeling, while preserving their composition and functionality. It is crucial to reduce false positive signals and explore the potential of multimodal imaging techniques to gain comprehensive insights into EVs.

Terahertz s-SNOM Imaging of a Single Cell with Nanoscale Resolution.

Terahertz scattering scanning near-field optical microscopy is a robust spectral detection technique with a nanoscale resolution. However, there are still major challenges in investigating the heterogeneity of cell membrane components in individual cells. Here, we present a novel and comprehensive analytical approach for detecting and investigating heterogeneity in cell membrane components at the single-cell level. In comparison to the resolution of the topographical atomic force microscopy image, the spatial resolution of the terahertz near-field amplitude image is 3 times that of the former. This ultrafine resolution enables the compositional distribution in the cell membrane, such as the distribution of extracellular vesicles, to be finely characterized. Furthermore, via extraction of the near-field absorption images at specific frequencies, the visualization and compositional difference analysis of cell membrane components can be presented in detail. These findings have significant implications for the intuitive and visual analysis of cell development and disease evolutionary pathways.

A fast and sensitive size-exclusion chromatography method for plasma extracellular vesicle proteomic analysis.

Extracellular vesicles (EVs) carry diverse biomolecules derived from their parental cells, making their components excellent biomarker candidates. However, purifying EVs is a major hurdle in biomarker discovery since current methods require large amounts of samples, are time-consuming and typically have poor reproducibility. Here we describe a simple, fast, and sensitive EV fractionation method using size exclusion chromatography (SEC) on a fast protein liquid chromatography (FPLC) system. Our method uses a Superose 6 Increase 5/150, which has a bed volume of 2.9 mL. The FPLC system and small column size enable reproducible separation of only 50 µL of human plasma in 15 min. To demonstrate the utility of our method, we used longitudinal samples from a group of individuals who underwent intense exercise. A total of 838 proteins were identified, of which, 261 were previously characterized as EV proteins, including classical markers, such as cluster of differentiation (CD)9 and CD81. Quantitative analysis showed low technical variability with correlation coefficients greater than 0.9 between replicates. The analysis captured differences in relevant EV proteins involved in response to physical activity. Our method enables fast and sensitive fractionation of plasma EVs with low variability, which will facilitate biomarker studies in large clinical cohorts.

Delineating Bovine Milk Derived Microvesicles from Exosomes Using Proteomics.

In the work presented herein, a simple serial-pelleting purification strategy combined with a mass spectrometry-based proteomics analysis was developed as a means of discerning differences in extracellular vesicle (EV) populations found in bovine milk samples. A sequence of ultracentrifugation speeds was used to generate changes in the abundances of EV populations, allowing for the identification of associated proteins. A metric was developed to determine the relative abundances of proteins in large EVs (>200 nm) and small EVs (<200 nm). Of the 476 proteins consistently found in this study, 340 are associated with vesicular components. Of these, 156 were heavily enriched in large EVs, 155 shared between large and small EVs, and 29 heavily enriched in small EVs. Additionally, out of 68 proteins annotated as exosome proteins, 32 were enriched in large EVs, 27 shared between large and small EVs, 5 enriched in small EVs, and 7 were found to be nonvesicular contaminant proteins. The top correlated proteins in the small EV group were predominantly membrane-bound proteins, whereas the top correlated proteins in the large EV group were mostly cytosolic enzymes for molecular processing. This method provides a means of assessing the origins of vesicle components and provides new potential marker proteins within discrete vesicle populations.

Proteomic Characterization of Transfusable Blood Components: Fresh Frozen Plasma, Cryoprecipitate, and Derived Extracellular Vesicles via Data-Independent Mass Spectrometry.

Extracellular vesicles (EVs) are a heterogeneous collection of particles that play a crucial role in cell-to-cell communication, primarily due to their ability to transport molecules, such as proteins. Thus, profiling EV-associated proteins offers insight into their biological effects. EVs can be isolated from various biological fluids, including donor blood components such as cryoprecipitate and fresh frozen plasma (FFP). In this study, we conducted a proteomic analysis of five single donor units of cryoprecipitate, FFP, and EVs derived from these blood components using a quantitative mass spectrometry approach. EVs were successfully isolated from both cryoprecipitate and FFP based on community guidelines. We identified and quantified approximately 360 proteins across all sample groups. Principal component analysis and heatmaps revealed that both cryoprecipitate and FFP are similar. Similarly, EVs derived from cryoprecipitate and FFP are comparable. However, they differ between the originating fluids and their derived EVs. Using the R-package MS-DAP, differentially expressed proteins (DEPs) were identified. The DEPs for all comparisons, when submitted for gene enrichment analysis, are involved in the complement and coagulation pathways. The protein profile generated from this study will have important clinical implications in increasing our knowledge of the proteins that are associated with EVs derived from blood components.

Defining the Soluble and Extracellular Vesicle Protein Compartments of Plasma Using In-Depth Mass Spectrometry-Based Proteomics.

Plasma-derived extracellular vesicles (pEVs) are a potential source of diseased biomarker proteins. However, characterizing the pEV proteome is challenging due to its relatively low abundance and difficulties in enrichment. This study presents a streamlined workflow to identify EV proteins from cancer patient plasma using minimal sample input. Starting with 400 µL of plasma, we generated a comprehensive pEV proteome using size exclusion chromatography (SEC) combined with HiRIEF prefractionation-based mass spectrometry (MS). First, we compared the performance of HiRIEF and long gradient MS workflows using control pEVs, quantifying 2076 proteins with HiRIEF. In a proof-of-concept study, we applied SEC-HiRIEF-MS to a small cohort (12) of metastatic lung adenocarcinoma (LUAD) and malignant melanoma (MM) patients. We also analyzed plasma samples from the same patients to study the relationship between plasma and pEV proteomes. We identified and quantified 1583 proteins in cancer pEVs and 1468 proteins in plasma across all samples. While there was substantial overlap, the pEV proteome included several unique EV markers and cancer-related proteins. Differential analysis revealed 30 DEPs in LUAD vs the MM group, highlighting the potential of pEVs as biomarkers. This work demonstrates the utility of a prefractionation-based MS for comprehensive pEV proteomics and EV biomarker discovery. Data are available via ProteomeXchange with the identifiers PXD039338 and PXD038528.

Free-Standing DNA Origami Superlattice to Facilitate Cryo-EM Visualization of Membrane Vesicles.

Technological breakthroughs in cryo-electron microscopy (cryo-EM) methods open new perspectives for highly detailed structural characterizations of extracellular vesicles (EVs) and synthetic liposome-protein assemblies. Structural characterizations of these vesicles in solution under a nearly native hydrated state are of great importance to decipher cell-to-cell communication and to improve EVs' application as markers in diagnosis and as drug carriers in disease therapy. However, difficulties in preparing holey carbon cryo-EM grids with low vesicle heterogeneities, at low concentration and with kinetic control of the chemical reactions or assembly processes, have limited cryo-EM use in the EV study. We report a straightforward membrane vesicle cryo-EM sample preparation method that assists in circumventing these limitations by using a free-standing DNA-affinity superlattice for covering holey carbon cryo-EM grids. Our approach uses DNA origami to self-assemble to a solution-stable and micrometer-sized ordered molecular template in which structure and functional properties can be rationally controlled. We engineered the template with cholesterol-binding sites to specifically trap membrane vesicles. The advantages of this DNA-cholesterol-affinity lattice (DCAL) include (1) local enrichment of artificial and biological vesicles at low concentration and (2) isolation of heterogeneous cell-derived membrane vesicles (exosomes) from a prepurified pellet of cell culture conditioned medium on the grid.

Programmable RNA Loading of Extracellular Vesicles with Toehold-Release Purification.

Synthetic nanoparticles as lipid nanoparticles (LNPs) are widely used as drug delivery vesicles. However, they hold several drawbacks, including low biocompatibility and unfavorable immune responses. Naturally occurring extracellular vesicles (EVs) hold the potential as native, safe, and multifunctional nanovesicle carriers. However, loading of EVs with large biomolecules remains a challenge. Here, we present a controlled loading methodology using DNA-mediated and programmed fusion between EVs and messenger RNA (mRNA)-loaded liposomes. The fusion efficiency is characterized at the single-particle level by real-time microscopy through EV surface immobilization via lipidated biotin-DNA handles. Subsequently, fused EV-liposome particles (EVLs) can be collected by employing a DNA strand-replacement reaction. Transferring the fusion reaction to magnetic beads enables us to scale up the production of EVLs one million times. Finally, we demonstrated encapsulation of mCherry mRNA, transfection, and improved translation using the EVLs compared to liposomes or LNPs in HEK293-H cells. We envision this as an important tool for the EV-mediated delivery of RNA therapeutics.

Fingerprint Profiling of Glycans on Extracellular Vesicles via Lectin-Induced Aggregation Strategy for Precise Cancer Diagnostics.

Extracellular vesicles (EVs) harbor abundant glycans that mediate various functions, such as intercellular communication and disease advancement, which play significant roles in disease progression. However, the presence of EV heterogeneity in body fluids and the complex nature of the glycan structures have posed challenges for the detection of EV glycans. In this study, we provide a streamlined method integrated, membrane-specific separation with lectin-induced aggregation strategy (MESSAGE), for multiplexed profiling of EV glycans. By leveraging a rationally designed lectin-induced aggregation strategy, the expression of EV glycans is converted to size-based signals. With the assistance learning machine algorithms, the MESSAGE strategy with high sensitivity, specificity, and simplicity can be used for early cancer diagnosis and classification, as well as monitoring cancer metastasis via 20 µL plasma sample within 2 h. Furthermore, our platform holds promise for advancing the field of EV-based liquid biopsy for clinical applications, opening new possibilities for the profiling of EV glycan signatures in various disease states.

De Novo Glycan Display on Cell Surfaces Using HaloTag: Visualizing the Effect of the Galectin Lattice on the Lateral Diffusion and Extracellular Vesicle Loading of Glycosylated Membrane Proteins.

Glycans cover the cell surface to form the glycocalyx, which governs a myriad of biological phenomena. However, understanding and regulating glycan functions is extremely challenging due to the large number of heterogeneous glycans that engage in intricate interaction networks with diverse biomolecules. Glycocalyx-editing techniques offer potent tools to probe their functions. In this study, we devised a HaloTag-based technique for glycan manipulation, which enables the introduction of chemically synthesized glycans onto a specific protein (protein of interest, POI) and concurrently incorporates fluorescent units to attach homogeneous, well-defined glycans to the fluorescence-labeled POIs. Leveraging this HaloTag-based glycan-display system, we investigated the influence of the interactions between Gal-3 and various N-glycans on protein dynamics. Our analyses revealed that glycosylation modulates the lateral diffusion of the membrane proteins in a structure-dependent manner through interaction with Gal-3, particularly in the context of the Gal-3-induced formation of the glycan network (galectin lattice). Furthermore, N-glycan attachment was also revealed to have a significant impact on the extracellular vesicle-loading of membrane proteins. Notably, our POI-specific glycan introduction does not disrupt intact glycan structures, thereby enabling a functional analysis of glycans in the presence of native glycan networks. This approach complements conventional glycan-editing methods and provides a means for uncovering the molecular underpinnings of glycan functions on the cell surface.

Fluorogenic Probes of the Mycobacterial Membrane as Reporters of Antibiotic Action.

The genus Mycobacterium includes species such as Mycobacterium tuberculosis, which can cause deadly human diseases. These bacteria have a protective cell envelope that can be remodeled to facilitate their survival in challenging conditions. Understanding how such conditions affect membrane remodeling can facilitate antibiotic discovery and treatment. To this end, we describe an optimized fluorogenic probe, N-QTF, that reports on mycolyltransferase activity, which is vital for cell division and remodeling. N-QTF is a glycolipid probe that can reveal dynamic changes in the mycobacterial cell envelope in both fast- and slow-growing mycobacterial species. Using this probe to monitor the consequences of antibiotic treatment uncovered distinct cellular phenotypes. Even antibiotics that do not directly inhibit cell envelope biosynthesis cause conspicuous phenotypes. For instance, mycobacteria exposed to the RNA polymerase inhibitor rifampicin release fluorescent extracellular vesicles (EVs). While all mycobacteria release EVs, fluorescent EVs were detected only in the presence of RIF, indicating that exposure to the drug alters EV content. Macrophages exposed to the EVs derived from RIF-treated cells released lower levels of cytokines, suggesting the EVs moderate immune responses. These data suggest that antibiotics can alter EV content to impact immunity. Our ability to see such changes in EV constituents directly results from exploiting these chemical probes.

High-Performance Gel-Free and Label-Free Size Fractionation of Extracellular Vesicles with Two-Dimensional Electrophoresis in a Microfluidic Artificial Sieve.

Extracellular vesicles (EVs) are cell-derived particles that exhibit diverse sizes, molecular contents, and clinical implications for various diseases depending on their specific subpopulations. However, fractionation of EV subpopulations with high resolution, efficiency, purity, and yield remains an elusive goal due to their diminutive sizes. In this study, we introduce a novel strategy that effectively separates EV subpopulations in a gel-free and label-free manner, using two-dimensional (2D) electrophoresis in a microfluidic artificial sieve. The microfabricated artificial sieve consists of periodically arranged micro-slit-well structures in a 2D array and generates an anisotropic electric field pattern to size fractionate EVs into discrete streams and steer the subpopulations into designated outlets for collection within a minute. Along with fractionating EV subpopulations, contaminants such as free proteins and short nucleic acids can be simultaneously directed to waste outlets, thus accomplishing both size fractionation and purification of EVs with high performance. Our platform offers a simple, rapid, and versatile solution for EV subpopulation isolation, which can potentially facilitate the discovery of biomarkers for specific EV subtypes and the development of EV-based therapeutics.

Label-Free Light Scattering Imaging with Purified Brownian Motion Differentiates Small Extracellular Vesicles in Cell Microenvironments.

Small extracellular vesicles (sEVs) are heterogeneous biological nanoparticles (NPs) with wide biomedicine applications. Tracking individual nanoscale sEVs can reveal information that conventional microscopic methods may lack, especially in cellular microenvironments. This usually requires biolabeling to identify single sEVs. Here, we developed a light scattering imaging method based on dark-field technology for label-free nanoparticle diffusion analysis (NDA). Compared with nanoparticle tracking analysis (NTA), our method was shown to determine the diffusion probabilities of a single NP. It was demonstrated that accurate size determination of NPs of 41 and 120 nm in diameter is achieved by purified Brownian motion (pBM), without or within the cell microenvironments. Our pBM method was also shown to obtain a consistent size estimation of the normal and cancerous plasma-derived sEVs without and within cell microenvironments, while cancerous plasma-derived sEVs are statistically smaller than normal ones. Moreover, we showed that the velocity and diffusion coefficient are key parameters for determining the diffusion types of the NPs and sEVs in a cancerous cell microenvironment. Our light scattering-based NDA and pBM methods can be used for size determination of NPs, even in cell microenvironments, and also provide a tool that may be used to analyze sEVs for many biomedical applications.

Magnetic Nanoparticle-Based Microfluidic Platform for Automated Enrichment of High-Purity Extracellular Vesicles.

Extracellular vesicles (EVs) are available in various biological fluids and have highly heterogeneous sizes, origins, contents, and functions. Rapid enrichment of high-purity EVs remains crucial for enhancing research on EVs in tumors. In this work, we present a magnetic nanoparticle-based microfluidic platform (ExoCPR) for on-chip isolation, purification, and mild recovery of EVs from cell culture supernatant and plasma within 29 min. The ExoCPR chip integrates bubble-driven micromixers and immiscible filtration assisted by surface tension (IFAST) technology. The bubble-driven micromixer enhances the mixing between immunomagnetic beads and EVs, eliminating the need for manual pipetting or off-chip oscillatory incubation. The high-purity EVs were obtained after passing through the immiscible phase interface where hydrophilic or hydrophobic impurities nonspecifically bound to SIMI were removed. The ExoCPR chip had a capture efficiency of 75.8% and a release efficiency of 62.7% for model EVs. We also demonstrated the powerful performance of the ExoCPR in isolating EVs from biological samples (>90% purity). This chip was further employed in clinical plasma samples and showed that the number of GPC3-positive EVs isolated from hepatocellular carcinoma patients was significantly higher than that of healthy individuals. This ExoCPR chip may provide a promising tool for EV-based liquid biopsy and other fundamental research.

Molecular Stratification and Treatment Monitoring of Lung Cancer Using a Small Extracellular Vesicle-Activated Nanocavity Architecture.

Development of molecular diagnostics for lung cancer stratification and monitoring is crucial for the rational planning and timely adjustment of treatments to improve clinical outcomes. In this regard, we propose a nanocavity architecture to sensitively profile the protein signature on small extracellular vesicles (sEVs) to enable accurate, noninvasive staging and treatment monitoring of lung cancer. The nanocavity architecture is formed by molecular recognition through the binding of sEVs with the nanobox-based core-shell surface-enhanced Raman scattering (SERS) barcodes and mirrorlike, asymmetric gold microelectrodes. By imposing an alternating current on the gold microelectrodes, a nanofluidic shear force was stimulated that supported the binding of sEVs and the efficient assembly of the nanoboxes. The binding of sEVs further induced a nanocavity between the nanobox and the gold microelectrode that significantly amplified the electromagnetic field to enable the simultaneous enhancement of Raman signals from four SERS barcodes and generate patient-specific molecular sEV signatures. Importantly, evaluated on a cohort of clinical samples (n = 76) on the nanocavity architecture, the acquired patient-specific sEV molecular signatures achieved accurate identification, stratification, and treatment monitoring of lung cancer patients, highlighting its potential for transition to clinical utility.

Endogenously Gated DNA Walking Machine for Prescreened MicroRNA Detection in Extracellular Vesicles.

Tumor-derived extracellular vesicle (T-EV) microRNAs have been investigated as promising biomarkers in clinical diagnosis as well as disease progression monitoring. However, the expression profiles of microRNA in different tissues vary widely, the precise monitoring of microRNA levels in EVs originating from diseased tissues is susceptible to background interference, thus remains a challenge. Conventional assays require extensive processing, such as EV isolation and even sample lysis, which is both slow and laborious, and the cumbersome pretreatment could spoil the downstream analysis. To address this issue, we developed a generalizable strategy for T-EVs-selective activation and therefore specific amplified microRNA imaging. The conditional signal amplification is established by integrating a traditional DNA walker system with endogenously activated motif to achieve sensitized microRNA imaging in T-EVs. The preorganized endogenous activation with additional sensing criteria narrowed the scope against the complex specimens, and the amplified sensing with reduced off-target signals was supposed to be sensitive to monitor the tiny changes of microRNA expression during the disease course, which holds great potential for accurate diagnosis and prognosis.

Heterogeneous Analysis of Extracellular Vesicles for Osteosarcoma Diagnosis.

Osteosarcoma (OS) is the most prevalent primary tumor of bones, often diagnosed late with a poor prognosis. Currently, few effective biomarkers or diagnostic methods have been developed for early OS detection with high confidence, especially for metastatic OS. Tumor-derived extracellular vesicles (EVs) are emerging as promising biomarkers for early cancer diagnosis through liquid biopsy. Here, we report a plasmonic imaging-based biosensing technique, termed subpopulation protein analysis by single EV counting (SPASEC), for size-dependent EV subpopulation analysis. In our SPASEC platform, EVs are accurately sized and counted on plasmonic sensor chips coated with OS-specific antibodies. Subsequently, EVs are categorized into distinct subpopulations based on their sizes, and the membrane proteins of each size-dependent subpopulation are profiled. We measured the heterogeneous expression levels of the EV markers (CD63, BMP2, GD2, and N-cadherin) in each of the EV subsets from both OS cell lines and clinical plasma samples. Using the linear discriminant analysis (LDA) model, the combination of four markers is applied to classify the healthy donors (n = 37), nonmetastatic OS patients (n = 13), and metastatic patients (n = 12) with an area under the curve of 0.95, 0.92, and 0.99, respectively. SPASEC provides accurate EV sensing technology for early OS diagnosis.