Small extracellular vesicles (sEVs) are 30–200 nm nanovesicles enriched with unique cargoes of nucleic acids, lipids, and proteins. sEVs are released by all cell types and have emerged as a critical mediator of cell‐to‐cell communication. Although many studies have dealt with the role of sEVs in health and disease, the exact mechanism of sEVs biogenesis and uptake remain unexplored due to the lack of suitable imaging technologies. For sEVs functional studies, imaging has long relied on conventional fluorescence microscopy that has only 200–300 nm resolution, thereby generating blurred images. To break this resolution limit, recent developments in super‐resolution microscopy techniques, specifically single‐molecule localization microscopy (SMLM), expanded the understanding of subcellular details at the few nanometer level. SMLM success relies on the use of appropriate fluorophores with excellent blinking properties. In this review, the basic principle of SMLM is highlighted and the state of the art of SMLM use in sEV biology is summarized. Next, how SMLM techniques implemented for cell imaging can be translated to sEV imaging is discussed by applying different labeling strategies to study sEV biogenesis and their biomolecular interaction with the distant recipient cells.

Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell–cell communication in cancer.

Extracellular vesicles (EVs) play a key role in cell‐to‐cell communication and have drawn significant attention due to their potential clinical applications. However, much remains to be understood about the biology of EV‐associated DNA (EV‐DNA). EV‐DNA is actively released by both normal and malignant cells and consists of diverse fragments with varying structures. Because EV‐DNA spans the entire genome of cells from which it originates, it continues to be attractive as a biomarker for cancer diagnosis and monitoring. Further, EV‐DNA delivery can alter the function of recipient cells by interfering with cytoplasmic DNA sensor pathways. This review explores the biology and significance of EV‐DNA, including its topology and fragmentomics features, modality of association with EVs, packaging mechanisms, and potential functions. It also emphasizes the specificity of vesicular DNA in identifying genetic and epigenetic changes in cancer. Additionally, it delves into the impact of EV‐DNA on cellular behaviour and its potential use as a therapeutic target in cancer. The review discusses new insights into EV‐DNA biology and provides perspectives and alternatives to address the challenges and concerns for future EV‐DNA studies.

Small extracellular vesicles (sEVs) released by acute myeloid leukaemia (AML) cells have been reported to influence the trilineage differentiation of bone marrow‐derived mesenchymal stem cells (BM‐MSCs). However, it remains elusive which biological cargo from AML‐sEVs is responsible for this effect. In this study, sEVs were isolated from cell‐conditioned media and blood plasma using size‐exclusion chromatography and ultrafiltration and characterized according to MISEV2018 guidelines. Our results demonstrated that AML‐sEVs increased the proliferation of BM‐MSCs. Conversely, key proteins that are important for normal haematopoiesis were downregulated in BM‐MSCs. Additionally, we revealed that AML‐sEVs significantly reduced the differentiation of BM‐MSCs to osteoblasts without affecting adipogenic or chondrogenic differentiation. Next, LC‐MS/MS proteomics elucidated that various proteins, including Y‐box‐binding protein 1 (YBX1), were upregulated in both AML‐sEVs and BM‐MSCs treated with AML‐sEVs. Clinically relevant, we found that YBX1 is considerably upregulated in most paediatric AML patient‐derived sEVs compared to healthy controls. Interestingly, sEVs isolated after the downregulation of YBX1 in AML cells remarkably rescued the osteoblastic differentiation of BM‐MSCs. Altogether, our data demonstrate for the first time that YBX1 containing AML‐sEVs is one of the key players that disrupt the normal function of bone marrow microenvironment by reducing the osteogenic differentiation of BM‐MSCs.

The understanding of medulloblastoma (MB) progression is limited by the lack of minimally invasive monitoring methods. Extracellular vesicles (EVs) carrying disease-specific signatures are promising for liquid biopsies, but clinical translation is hindered by inconsistent isolation techniques. This study compares small EVs (sEVs) and their proteomes from blood plasma (BP) and cerebrospinal fluid (CSF) in MB. Using ultrafiltration and size exclusion chromatography (UF-SEC), we isolated sEVs from pediatric patient samples. sEV proteins from matched CSF-BP samples from MB patients (MBCSF/MBBP), healthy BP controls (HCBP), and MB cell lines (MBCL) were analyzed by liquid chromatography-tandem mass spectrometry, subjected to Gene Ontology and Cytoscape analyses, and compared to published MB, CSF, and EV datasets. By optimizing UF-SEC for small volumes, we found that CSF-sEVs are smaller and elute in later SEC fractions. Proteins linked to the extracellular matrix (ECM) were enriched in MBCSF and MBCL, while integrin binding showed inconsistent patterns between MBCSF and MBBP. MBBP and HCBP showed no significant differences. Fourteen proteins from MB datasets were identified in our analysis and primarily enriched in CSF. These findings support CSF-sEVs as more informative than BP-sEVs for MB diagnosis and monitoring, emphasize the need for fluid-specific sEV isolation, and suggest that ECM components and integrins may mediate MB progression.

Background: Small extracellular vesicles (sEVs) are critical mediators of intercellular communication in both physiological and pathological contexts, including cancer, by transporting key biomolecules between cells. However, the biogenesis, packaging, and functional roles of DNA associated with sEVs (EV-DNA) remain poorly understood, largely due to the lack of efficient EV-DNA labeling dyes compatible with super-resolution imaging techniques. Results: Here, we employed BODIPY, a green-emitting, buffer-independent blinking fluorophore, to label EV-DNA cargo and applied single-molecule localization microscopy (SMLM) for the first time under physiological conditions to achieve nanoscale imaging of EV-DNA in recipient cells. This approach overcomes conventional fluorophore limitations, enabling high-resolution dual-color imaging without quenching artifacts. We further developed a co-labeling strategy combining click chemistry (EV-DNA) and nanobody-based immunostaining (CD63-GFP⁺-sEVs), achieving precise multi-target labeling with a calculated linkage error of ~ 2 nm. Dual-color SMLM imaging revealed limited co-localization between EV-DNA and CD63-GFP⁺-sEVs, suggesting that EV-DNA may associate with distinct vesicle populations. Additionally, dual-color SMLM combined with cluster analysis indicated partial spatial proximity between EV-DNA and the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS), suggesting potential but limited functional interactions. Conclusions: The SMLM-based imaging approach established in this study provides a powerful platform for investigating the packaging and subcellular fate of EV-DNA at nanometer resolution. Our results uncover new aspects of EV-DNA biology, including limited association with CD63⁺ vesicles and partial proximity to cGAS, suggesting alternative intracellular pathways. This versatile approach will enable detailed exploration of EV-DNA dynamics and its functional roles in health and disease.