Tumor-derived extracellular vesicles (EVs) have attracted significant attention, yet the molecular mechanisms that govern their specific binding to recipient cells remain elusive. Our in vitro study utilizing single-particle tracking demonstrated that integrin heterodimers comprising α6β4 and α6β1 and ganglioside, GM1, are responsible for the binding of small EV (sEV) subtypes to laminin. EVs derived from four distinct tumor cell lines, regardless of size, exhibited high binding affinities for laminin but not for fibronectin, although fibronectin receptors are abundant in EVs and have functional roles in EV-secreting cells. Our findings revealed that integrins in EVs bind to laminin via the conventional molecular interface, facilitated by CD151 rather than by inside-out signaling of talin-1 and kindlin-2. Super-resolution movie observation revealed that sEV integrins bind only to laminin on living recipient cells. Furthermore, sEVs bound to HUVEC and induced cell branching morphogenesis in a laminin-dependent manner. Thus, we demonstrated that EVs predominantly bind to laminin on recipient cells, which is indispensable for cell responses.
© 2025 Isogai et al.

The isolation of extracellular vesicles (EVs) using currently available methods frequently compromises purity and yield to prioritize speed. Here, we present a next-generation aqueous two-phase system (next-gen ATPS) for the isolation of EVs regardless of scale and volume that is superior to conventional methods such as ultracentrifugation (UC) and commercial kits. This is made possible by the two aqueous phases, one rich in polyethylene glycol (PEG) and the other rich in dextran (DEX), whereby fully encapsulated lipid vesicles preferentially migrate to the DEX-rich phase to achieve a local energy minimum for the EVs. Isolated EVs as found in the DEX-rich phase are more amenable to biomarker analysis such as nanoscale flow cytometry (nFC) when using various pre-conjugated antibodies specific for CD9, CD63 and CD81. TRIzol RNA isolation is further enabled by the addition of dextranase, a critical component of this next-gen ATPS method. RNA yield of next-gen ATPS-isolated EVs is superior to UC and other commercial kits. This negates the use of specialized EV RNA extraction kits. The use of dextranase also enables more accurate immunoreactivity of pre-conjugated antibodies for the detection of EVs by nFC. Transcriptomic analysis of EVs isolated using the next-gen ATPS revealed a strong overlap in microRNA (miRNA), circular RNA (circRNA) and small nucleolar RNA (snoRNA) profiles with EV donor cells, as well as EVs isolated by UC and the exoRNeasy kit, while detecting a superior number of circRNAs compared to the kit in human samples. Overall, this next-gen ATPS method stands out as a rapid and highly effective approach to isolate high-quality EVs in high yield, ensuring optimal extraction and analysis of EV-encapsulated nucleic acids.
© 2025 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.

The early detection of neurodegenerative diseases necessitates the identification of specific brain-derived biomolecules in peripheral blood. In this context, our investigation delineates the role of amyloid precursor-like protein 1 (APLP1)-a protein predominantly localized in oligodendrocytes and neurons-as a previously unidentified biomarker in extracellular vesicles (EVs). Through rigorous analysis, APLP1+ EVs from human sera were unequivocally determined to be of cerebral origin. This assertion was corroborated by distinctive small RNA expression patterns of APLP1+ EVs. The miRNAs' putative targets within these EVs manifested pronounced expression in the brain, fortifying their neurospecific provenance. We subjected our findings to stringent validation using Thy-1 GFP M line mice, transgenic models wherein GFP expression is confined to hippocampal neurons. An amalgamation of these results with an exhaustive data analysis accentuates the potential of APLP1+ EVs as cerebrally originated biomarkers. Synthesizing our findings, APLP1+ EVs are postulated not merely as diagnostic markers but as seminal entities shaping the future trajectory of neurodegenerative disease diagnostics.

Extracellular vesicles (EV) have emerged as promising cell-free therapeutics in regenerative medicine. However, translating primary cell line-derived EV to clinical applications requires large-scale manufacturing and several challenges, such as replicative senescence, donor heterogeneity, and genetic instability. To address these limitations, we used a reprogramming approach to generate human induced pluripotent stem cells (hiPSC) from the young source of cord blood mesenchymal stem/stromal cells (CBMSC). Capitalizing on their inexhaustible supply potential, hiPSC offer an attractive EV reservoir. Our approach encompassed an exhaustive characterization of hiPSC-EV, aligning with the rigorous MISEV2023 guidelines. Analyses demonstrated physical features compatible with small EV (sEV) and established their identity and purity. Moreover, the sEV-shuttled non-coding (nc) RNA landscape, focusing on the microRNA and circular RNA cargo, completed the molecular signature. The kinetics of the hiPSC-sEV release and cell internalization assays unveiled robust EV production and consistent uptake by human neurons. Furthermore, hiPSC-sEV demonstrated ex vivo cell tissue-protective properties. Finally, via bioinformatics, the potential involvement of the ncRNA cargo in the hiPSC-sEV biological effects was explored. This study significantly advances the understanding of pluripotent stem cell-derived EV. We propose cord blood MSC-derived hiPSC as a promising source for potentially therapeutic sEV.
© The author(s).

Nasopharyngeal carcinoma (NPC) is characterized by Epstein-Barr virus (EBV) infection and severe immune cell infiltration. How tumor-derived EBV or viral products regulate macrophage polarization within NPC microenvironment remains to be explored. Here, we investigated the exosome-mediated intercellular communications between EBV + NPC cells and tumor-associated macrophages (TAMs). We demonstrate that leukemia inhibitory factor (LIF) expression correlates with poorer metastasis/recurrence-free survival in NPC patients. Immunohistochemical analyses revealed that LIF is highly expressed by both tumor cells and macrophages. The uptake of NPC-derived exosomes by human monocyte-derived macrophages induced an immunosuppressive polarization and enhanced LIF expression, exhibiting a reduced cytotoxicity against primary cancer cells. Blocking LIF suppressed the pro-tumor functions mediated by tumor-derived exosomes, in both in vitro and in vitro zebrafish xenografts model. Furthermore, single-cell transcriptomic analysis (scRNA-seq) suggested that tumor- derived exosomes remodeled the NPC microenvironment by increasing the proportion of a pro-tumoral TAM subtype, CCL18-MΦ. Intercellular analysis revealed that exosome-treated macrophages could modulate T cell cytotoxicity and promoting Treg-mediated immunosuppression. Collectively, EBV shapes the tumor microenvironment by driving macrophage toward an immunosuppressive phenotype via exosome cargos, facilitating a pro-tumoral niche and contribute to NPC progression.