Phospholipid-Anchored Ligand Conjugation on Extracellular Vesicles for Enhanced Cancer Targeting.

Extracellular vesicles (EVs) are recognized as potential candidates for next-generation drug delivery systems. However, the inherent cancer-targeting efficiency is unsatisfactory, necessitating surface modification to attach cell-binding ligands. By utilizing phospholipase D from Streptomyces in combination with maleimide-containing primary alcohol, the authors successfully anchored ligands onto milk-derived EVs (mEVs), overcoming the issues of ligand leakage or functional alteration seen in traditional methods. Quantitative nano-flow cytometry demonstrated that over 90% of mEVs are effectively modified with hundreds to thousands of ligands. The resulting mEV formulations exhibited remarkable long-term stability in conjugation proportion, ligand number, size distribution, and particle concentration, even after months of storage. It is further shown that conjugating transferrin onto mEVs significantly enhanced cellular uptake and induced pronounced cytotoxic effects when loaded with paclitaxel. Overall, this study presents a highly efficient, stable, cost-effective, and scalable ligand conjugation approach, offering a promising strategy for targeted drug delivery of EVs.

Single-particle assessment of six different drug-loading strategies for incorporating doxorubicin into small extracellular vesicles.

Extracellular vesicles (EVs) have emerged as an attractive drug delivery system owing to their natural roles in intercellular communication. On account of the large intrinsic heterogeneity of EVs, it is highly desirable to evaluate not only the encapsulation efficiency but also the alteration of biological functionality after the drug-loading process at the single-particle level. However, the nanoscale size of EVs poses a great challenge. Taking advantage of nano-flow cytometry (nFCM) in the multiparameter analysis of single EVs as small as 40 nm, six commonly used drug-loading strategies (coincubation, electroporation, extrusion, freeze-thawing, sonication, and surfactant treatment) were exploited by employing doxorubicin (Dox) as the model drug. Encapsulation ratio, EV concentration, drug content, and membrane proteins of Dox-loaded EVs were measured at the single-particle level. Our data indicated that coincubation and electroporation outperformed other methods with an encapsulation ratio of approximately 45% and a higher Dox content in single EVs. Interestingly, the labeling ratios of membrane proteins indicated that varying degrees of damage to the surface proteins of EVs occurred upon extrusion, freeze-thawing, sonication, and surfactant treatment. Confocal fluorescence microscopy and flow cytometry analysis revealed that Dox-loaded EVs prepared by electroporation induced the strongest apoptosis followed by coincubation. These results correlated well with their cellular uptake rate and fundamentally with the Dox encapsulation efficiency of single EVs. nFCM provides a rapid and sensitive platform for single-particle assessment of drug-loading strategies for incorporating drugs into EVs.

The Impact of Oncogenic EGFRvIII on the Proteome of Extracellular Vesicles Released from Glioblastoma Cells.

Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvironment. This is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. We observed that EGFRvIII expression alters the expression of EV-regulating genes (vesiculome) and EV properties, including their protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRvIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing glioma cells, and downregulation of exosomal markers (CD81 and CD82) relative to EVs of EGFRvIII-negative cells. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of vesicles contained specific proteins (including EGFRvIII). Notably, cells expressing EGFRvIII released EVs double positive for CD44/BSG, and these proteins also colocalized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome and uptake of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for the development of EV-based cancer biomarkers.

Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity.

Mutational and epigenetic driver events profoundly alter intercellular communication pathways in cancer. This effect includes deregulated release, molecular composition, and biological activity of extracellular vesicles (EVs), membranous cellular fragments ranging from a few microns to less than 100 nm in diameter and filled with bioactive molecular cargo (proteins, lipids, and nucleic acids). While EVs are usually classified on the basis of their physical properties and biogenetic mechanisms, recent analyses of their proteome suggest a larger than expected molecular diversity, a notion that is also supported by multicolour nano-flow cytometry and other emerging technology platforms designed to analyze single EVs. Both protein composition and EV diversity are markedly altered by oncogenic transformation, epithelial to mesenchymal transition, and differentiation of cancer stem cells. Interestingly, only a subset of EVs released from mutant cells may carry oncogenic proteins (e.g., EGFRvIII), hence, these EVs are often referred to as "oncosomes". Indeed, oncogenic transformation alters the repertoire of EV-associated proteins, increases the presence of pro-invasive cargo, and alters the composition of distinct EV populations. Molecular profiling of single EVs may reveal a more intricate effect of transforming events on the architecture of EV populations in cancer and shed new light on their biological role and diagnostic utility.

Exploring cell-derived extracellular vesicles in peripheral blood and bone marrow of B-cell acute lymphoblastic leukemia pediatric patients: proof-of-concept study.

Extracellular vesicles (EVs) are heterogeneous, phospholipid membrane enclosed particles that are secreted by healthy and cancerous cells. EVs are present in diverse biological fluids and have been associated with the severity of diseases, which indicates their potential as biomarkers for diagnosis, prognosis and as therapeutic targets. This study investigated the phenotypic characteristics of EVs derived from peripheral blood (PB) and bone marrow (BM) in pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) during different treatment stages. PB and BM plasma were collected from 20 B-ALL patients at three time points during induction therapy, referred to as: diagnosis baseline (D0), day 15 of induction therapy (D15) and the end of the induction therapy (D35). In addition, PB samples were collected from 10 healthy children at a single time point. The EVs were measured using CytoFLEX S flow cytometer. Calibration beads were employed to ensure accurate size analysis. The following, fluorescent-labeled specific cellular markers were used to label the EVs: Annexin V (phosphatidylserine), CD235a (erythrocyte), CD41a (platelet), CD51 (endothelial cell), CD45 (leukocyte), CD66b (neutrophil), CD14 (monocyte), CD3 (T lymphocyte), CD19, CD34 and CD10 (B lymphoblast/leukemic blast). Our results demonstrate that B-ALL patients had a marked production of EV-CD51/61+, EV-CD10+, EV-CD19+ and EV-CD10+CD19+ (double-positive) with a decrease in EV-CD41a+ on D0. However, the kinetics and signature of production during induction therapy revealed a clear decline in EV-CD10+ and EV-CD19+, with an increase of EV-CD41a+ on D35. Furthermore, B-ALL patients showed a complex biological network, exhibiting distinct profiles on D0 and D35. Interestingly, fold change and ROC curve analysis demonstrated that EV-CD10+CD19+ were associated with B-ALL patients, exhibited excellent clinical performance and standing out as a potential diagnostic biomarker. In conclusion, our data indicate that EVs represent a promising field of investigation in B-ALL, offering the possibility of identifying potential biomarkers and therapeutic targets.

Evaluation of cell disruption technologies on magnetosome chain length and aggregation behaviour from Magnetospirillum gryphiswaldense MSR-1.

Magnetosomes are biologically-derived magnetic nanoparticles (MNPs) naturally produced by magnetotactic bacteria (MTB). Due to their distinctive characteristics, such as narrow size distribution and high biocompatibility, magnetosomes represent an attractive alternative to existing commercially-available chemically-synthesized MNPs. However, to extract magnetosomes from the bacteria, a cell disruption step is required. In this study, a systematic comparison between three disruption techniques (enzymatic treatment, probe sonication and high-pressure homogenization) was carried out to study their effect on the chain length, integrity and aggregation state of magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 cells. Experimental results revealed that all three methodologies show high cell disruption yields (>89%). Transmission electron microscopy (TEM), dynamic light scattering (DLS) and, for the first time, nano-flow cytometry (nFCM) were employed to characterize magnetosome preparations after purification. TEM and DLS showed that high-pressure homogenization resulted in optimal conservation of chain integrity, whereas enzymatic treatment caused higher chain cleavage. The data obtained suggest that nFCM is best suited to characterize single membrane-wrapped magnetosomes, which can be particularly useful for applications that require the use of individual magnetosomes. Magnetosomes were also successfully labelled (>90%) with the fluorescent CellMask™ Deep Red membrane stain and analysed by nFCM, demonstrating the promising capacity of this technique as a rapid analytical tool for magnetosome quality assurance. The results of this work contribute to the future development of a robust magnetosome production platform.

Quantitative assessment of lipophilic membrane dye-based labelling of extracellular vesicles by nano-flow cytometry.

Although lipophilic membrane dyes (LMDs) or probes (LMPs) are widely used to label extracellular vesicles (EVs) for detection and purification, their labelling performance has not been systematically characterized. Through concurrent side scattering and fluorescence detection of single EVs as small as 40 nm in diameter by a laboratory-built nano-flow cytometer (nFCM), present study identified that (1) PKH67 and PKH26 could maximally label ∼60%-80% of EVs isolated from the conditioned cell culture medium (purity of ∼88%) and ∼40%-70% of PFP-EVs (purity of ∼73%); (2) excessive PKH26 could cause damage to the EV structure; (3) di-8-ANEPPS and high concentration of DiI could achieve efficient and uniform labelling of EVs with nearly 100% labelling efficiency for di-8-ANEPPS and 70%-100% for DiI; (4) all the four tested LMDs can aggregate and form micelles that exhibit comparable side scatter and fluorescence intensity with those of labelled EVs and thus hardly be differentiate from each other; (5) as the LMD concentration went up, the particle number of self-aggregates increased while the fluorescence intensity of aggregates remained constant; (6) PKH67 and PKH26 tend to form more aggregated micelles than di-8-ANEPPS and DiI, and the effect of LMD self-aggregation can be negligible at optimal staining conditions. (7) All the four tested LMDs can label almost all the very-low-density lipoprotein (VLDL) particles, indicating potential confounding factor in plasma-EV labelling. Besides, it was discovered that DSPE-PEG2000 -biotin can only label ∼50% of plasma-EVs. The number of LMP inserted into the membrane of single EVs was measured for the first time and it was confirmed that membrane labelling by lipophilic dyes did not interfere with the immunophenotyping of EVs. nFCM provides a unique perspective for a better understanding of EV labelling by LMD/LMP.

Characterization of lipid-based nanomedicines at the single-particle level.

Lipid-based nanomedicines (LBNMs), including liposomes, lipid nanoparticles (LNPs) and extracellular vesicles (EVs), are recognized as one of the most clinically acceptable nano-formulations. However, the bench-to-bedside translation efficiency is far from satisfactory, mainly due to the lack of in-depth understanding of their physical and biochemical attributes at the single-particle level. In this review, we first give a brief introduction of LBNMs, highlighting some milestones and related scientific and clinical achievements in the past several decades, as well as the grand challenges in the characterization of LBNMs. Next, we present an overview of each category of LBNMs as well as the core properties that largely dictate their biological characteristics and clinical performance, such as size distribution, particle concentration, morphology, drug encapsulation and surface properties. Then, the recent applications of several analytical techniques including electron microscopy, atomic force microscopy, fluorescence microscopy, Raman microscopy, nanoparticle tracking analysis, tunable resistive pulse sensing and flow cytometry on the single-particle characterization of LBNMs are thoroughly discussed. Particularly, the comparative advantages of the newly developed nano-flow cytometry that enables quantitative analysis of both the physical and biochemical characteristics of LBNMs smaller than 40 nm with high throughput and statistical robustness are emphasized. The overall aim of this review article is to illustrate the importance, challenges and achievements associated with single-particle characterization of LBNMs.

Single-particle analysis of tear fluid reveals abundant presence of tissue factor-exposing extracellular vesicles with strong coagulation activity.

Extracellular vesicles (EVs) in easily accessible body fluids have emerged as a promising source for liquid biopsy. Although tear collection is fast, safe, and noninvasive, EVs of tear fluid are less studied and their involvement in physiological and pathological processes is largely unknown. The aim of present study was to analyze and characterize EVs in tear fluid at the single-particle level to reveal the population heterogeneity. A laboratory-built nano-flow cytometer (nFCM) was used to analyze the purity, size distribution, and particle concentration of EVs isolated from unstimulated tears (basal tears) upon double ultracentrifugation (17 min at 100,000×g, 4 °C) via side scattering detection. The expression of CD9, CD63, CD81, CD47, CD45, CD24, and EpCAM was assessed via immunofluorescent detection. The EV concentration in tear fluid was measured to be 1.1 ± 0.7 × 1011 particles/mL, which is approximately 100-fold higher than that of plasma EVs. In particular, it was identified for the first time that tears have strong coagulant activity owing to the abundant presence of tissue factor (TF) on tear EVs. The concentration of TF-exposing EVs (4.4 ± 3.1 × 1010 particles/mL) was found to be approximately 100-fold higher than their counterparts in saliva (4.5 ± 2.1 × 108 particles/mL). We postulate that TF-exposing vesicles in tears might play a role in host defense by promoting clot formation and thus reducing the risk of pathogen invasion. The coagulant activity of tears triggered by TF-exposing EVs could provide a new research perspective for ophthalmic research.

Analysis of extracellular vesicle DNA at the single-vesicle level by nano-flow cytometry.

It has been demonstrated recently that extracellular vesicles (EVs) carry DNA; however, many fundamental features of DNA in EVs (EV-DNA) remain elusive. In this study, a laboratory-built nano-flow cytometer (nFCM) that can detect single EVs as small as 40 nm in diameter and single DNA fragments of 200 bp upon SYTO 16 staining was used to study EV-DNA at the single-vesicle level. Through simultaneous side-scatter and fluorescence (FL) detection of single particles and with the combination of enzymatic treatment, present study revealed that: (1) naked DNA or DNA associated with non-vesicular entities is abundantly presented in EV samples prepared from cell culture medium by ultracentrifugation; (2) the quantity of EV-DNA in individual EVs exhibits large heterogeneity and the population of DNA positive (DNA+ ) EVs varies from 30% to 80% depending on the cell type; (3) external EV-DNA is mainly localized on relatively small size EVs (e.g. <100 nm for HCT-15 cell line) and the secretion of external DNA+ EVs can be significantly reduced by exosome secretion pathway inhibition; (4) internal EV-DNA is mainly packaged inside the lumen of relatively large EVs (e.g. 80-200 nm for HCT-15 cell line); (5) double-stranded DNA (dsDNA) is the predominant form of both the external and internal EV-DNA; (6) histones (H3) are not found in EVs, and EV-DNA is not associated with histone proteins and (7) genotoxic drug induces an enhanced release of DNA+ EVs, and the number of both external DNA+ EVs and internal DNA+ EVs as well as the DNA content in single EVs increase significantly. This study provides direct and conclusive experimental evidence for an in-depth understanding of how DNA is associated with EVs.

Platelet Activation in Ovarian Cancer Ascites: Assessment of GPIIb/IIIa and PF4 in Small Extracellular Vesicles by Nano-Flow Cytometry Analysis.

In ovarian cancer, ascites represent the microenvironment in which the platelets extravasate to play their role in the disease progression. We aimed to develop an assay to measure ascites' platelet activation. We enriched small extracellular vesicles (EVs) (40-200 nm) from ascites of high-grade epithelial ovarian cancer patients (n = 12) using precipitation with polyethylene glycol, and we conducted single-particle phenotyping analysis by nano-flow cytometry after labelling and ultra-centrifugation. Atomic force microscopy single-particle nanomechanical analysis showed heterogeneous distributions in the size of the precipitated particles and their mechanical stiffness. Samples were fluorescently labelled with antibodies specific to the platelet markers GPIIb/IIIa and PF4, showing 2.6 to 18.16% of all particles stained positive for the biomarkers and, simultaneously, the EV membrane labelling. Single-particle phenotyping analysis allowed us to quantify the total number of non-EV particles, the number of small-EVs and the number of platelet-derived small-EVs, providing a platelet activation assessment independent of the ascites volume. The percentage of platelet-derived small-EVs was positively correlated with platelet distribution width to platelet count in sera (PDW/PLT). Overall, we presented a high-throughput method that can be helpful in future studies to determine the correlation between the extent of platelet activation in ascites and disease status.

Active cargo loading into extracellular vesicles: Highlights the heterogeneous encapsulation behaviour.

Extracellular vesicles (EVs) have demonstrated unique advantages in serving as nanocarriers for drug delivery, yet the cargo encapsulation efficiency is far from expectation, especially for hydrophilic chemotherapeutic drugs. Besides, the intrinsic heterogeneity of EVs renders it difficult to evaluate drug encapsulation behaviour. Inspired by the active drug loading strategy of liposomal nanomedicines, here we report the development of a method, named "Sonication and Extrusion-assisted Active Loading" (SEAL), for effective and stable drug encapsulation of EVs. Using doxorubicin-loaded milk-derived EVs (Dox-mEVs) as the model system, sonication was applied to temporarily permeabilize the membrane, facilitating the influx of ammonium sulfate solution into the lumen to establish the transmembrane ion gradient essential for active loading. Along with extrusion to downsize large mEVs, homogenize particle size and reshape the nonspherical or multilamellar vesicles, SEAL showed around 10-fold enhancement of drug encapsulation efficiency compared with passive loading. Single-particle analysis by nano-flow cytometry was further employed to reveal the heterogeneous encapsulation behaviour of Dox-mEVs which would otherwise be overlooked by bulk-based approaches. Correlation analysis between doxorubicin auto-fluorescence and the fluorescence of a lipophilic dye DiD suggested that only the lipid-enclosed particles were actively loadable. Meanwhile, immunofluorescence analysis revealed that more than 85% of the casein positive particles was doxorubicin free. These findings further inspired the development of the lipid-probe- and immuno-mediated magnetic isolation techniques to selectively remove the contaminants of non-lipid enclosed particles and casein assemblies, respectively. Finally, the intracellular assessments confirmed the superior performance of SEAL-prepared mEV formulations, and demonstrated the impact of encapsulation heterogeneity on therapeutic outcome. The as-developed cargo-loading approach and nano-flow cytometry-based characterization method will provide an instructive insight in the development of EV-based delivery systems.

Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry.

Extracellular vesicles (EVs) have sparked tremendous interest owing to their prominent potential in diagnostics and therapeutics. Isolation of EVs from complex biological fluids with high purity is essential to the accurate analysis of EV cargo. Unfortunately, generally used isolation techniques do not offer good separation of EVs from non-EV contaminants. Hence, it is important to have a standardized method to characterise the properties of EV preparations, including size distribution, particle concentration, purity and phenotype. Employing a laboratory-built nano-flow cytometer (nFCM) that enables multiparameter analysis of single EVs as small as 40 nm, here we report a new benchmark to the quality and efficiency assessment of EVs isolated from plasma, one of the most difficult body fluids to work with. The performance of five widely used commercial isolation kits was examined and compared with the traditional differential ultracentrifugation (UC). Two to four orders of magnitude higher particle concentrations were observed for EV preparations from platelet-free plasma (PFP) by kits when compared with the EV preparation by UC, yet the purity was much lower. Meanwhile, the particle size distribution profiles of EV preparations by kits closely resembled those of PFP whereas the EV preparation by UC showed a broader size distribution at relatively large particle size. When these kits were used to isolate EVs from vesicle-depleted PFP (VD-PFP), comparable particle counts were obtained with their corresponding EV preparations from PFP, which confirmed again the isolation of a large quantity of non-vesicular contaminants. As CD9, CD63 and CD81 also exist in the plasma matrix, single-particle phenotyping of EVs offers distinct advantage in the validation of EVs compared with ensemble-averaged approaches, such as Western blot analysis. nFCM allows us to compare different isolation techniques without prejudice.

Rapid quantification of pathogenic Salmonella Typhimurium and total bacteria in eggs by nano-flow cytometry.

Rapid quantification of pathogenic Salmonella Typhimurium (S. Typhimurium) and total bacteria in eggs is highly desired for food safety control. However, the complexity of egg matrix presents a significant challenge for sensitive detection of bacteria. In this study, a sample pretreatment protocol, including dilution, fat dissolution, protein degradation, filtration, and washing was developed to circumvent this challenge. A laboratory-built nano-flow cytometer (nFCM) that is hundreds of fold more sensitive than the conventional flow cytometer was employed to analyze individual bacteria upon nucleic acid and immunofluorescent staining. Eggs spiked with pathogenic S. Typhimurium and harmless Escherichia coli K12 (E. coli K12) were used as the model system to optimize the sample pretreatment protocol. S. Typhimurium and total bacteria in eggs can be quantified without cultural enrichment, and the whole process of sample pretreatment, staining, and instrument analysis can be accomplished within 1.5 h. The bacterial recovery rate upon sample pretreatment, detection limit, and dynamic range for S. Typhimurium in eggs were 92%, 2 × 103 cells/mL, and from 2 × 103 to 4 × 108 cells/mL, respectively. The as-developed approach can specifically distinguish S. Typhimurium from other bacteria and successful application to bacterial detection in eggs freshly purchased from supermarket and spoiled eggs upon inappropriate storage was demonstrated.

Detection and Sorting of Extracellular Vesicles and Viruses Using nanoFACS.

Extracellular vesicles (EVs) are sub-micron-sized membranous spheres secreted by cells. EVs play a functional role as intercellular communicators and are associated with a number of diseases. Research into EVs is an area of growing interest due their many potential uses as therapeutic agents, as diagnostic and theranostic biomarkers, and as regulators of cellular biology. Flow cytometry is a popular method for enumerating and phenotyping EVs, even though the majority of EVs are below the detection sensitivity of most commercially available flow cytometers. Here, we present optimized protocols for EV labeling that increase the signal-to-noise ratio of EVs by removing residual antibody. Protocols for alignment of high-resolution jet-in-air flow cytometers are also provided. Published 2020. U.S. Government. Basic Protocol 1: Bulk EV staining with CFSE protein binding dye Basic Protocol 2: Antigen-specific staining of EV markers with fluorochrome-conjugated antibodies Basic Protocol 3: Astrios EQ instrument setup and sample acquisition Basic Protocol 4: Counting particles and EVs on Astrios EQ with spike-in reference beads.

Mapping Subpopulations of Cancer Cell-Derived Extracellular Vesicles and Particles by Nano-Flow Cytometry.

The elusive complexity of membranous extracellular vesicle (EV) and membrane-less extracellular particle (EP) populations released from various cellular sources contains clues as to their biological functions and diagnostic utility. In this study, we employed optimized multicolor nano-flow cytometry, structured illumination (SIM), and atomic force microscopy (AFM) to bridge sensitive detection at the single EV/EP level and high-throughput analysis of cancer cell secretomes. We applied these approaches to particles released from intact cells driven by several different transforming mechanisms or to cells under therapeutic stress imposed by pharmacological inhibition of their oncogenic drivers, such as epidermal growth factor receptor (EGFR). We demonstrate a highly heterogeneous distribution of biologically relevant elements of the EV/EP cargo, including oncoproteins (EGFR), clotting factors (tissue factor), pro-metastatic integrins (ITGA6, ITGA4), tetraspanins (CD63), and genomic DNA across the entire particulate secretome of cancer cells. We observed that targeting EGFR activity with irreversible kinase inhibitors (dacomitinib) triggers emission of DNA containing EP/EV subpopulations, including particles (chromatimeres) harboring both EGFR and DNase-resistant chromatin. While nano-flow cytometry enables quantification of these changes across the entire particular secretome, SIM reveals individual molecular topography of EV/EP subsets and AFM exposes some of their physical properties, including the presence of nanofilaments and other substructures. We describe differential uptake rates of distinct EV subsets, resulting in preferential internalization of exosome-like small EVs by cancer cells to the exclusion of larger EVs. Thus, our study illustrates the potential of nano-flow cytometry coupled with high-resolution microscopy to explore the cancer-related EV/EP landscape.