Extracellular vesicles (EVs) are emerging as key factors in maintaining cellular homeostasis, critical mediators of intercellular communication, potential biomarkers, and therapeutic tools. While small EVs have been extensively characterized, the molecular signatures of large EVs (including those generated during regulated cell death pathways) remain poorly defined. Here, we investigated the characteristics of large EVs released during apoptosis and pyroptosis by human monocytic cell lines (THP-1 and U937). Apoptosis was induced by staurosporine and blocked using the pan-caspase inhibitor Q-VD-OPh, whereas pyroptosis was triggered by LPS/nigericin and inhibited with a selective NLRP3 inhibitor. We found that both forms of regulated cell death markedly enhanced the release of large EVs. Both apoptotic and pyroptotic large EVs showed increased Annexin V binding and decreased CD9 expression compared with those released by healthy cells. Large EVs derived from apoptotic and pyroptotic cells exhibited distinct proteomic profiles. Pyroptotic large EVs carried interacting protein networks of RNA-binding proteins and chromatin-associated proteins many of which are known damage-associated molecular patterns or alarmins. In contrast, we found that a subpopulation of apoptotic large EVs was characterized by the presence of dsDNA, and active caspase-3/7. Together, our data shed light on the specific protein cargo of large EVs released by cells during apoptosis and pyroptosis. This study identifies candidate markers of large EVs released by dying cells and may enhance our understanding of the role of EVs in regulated cell death.

Extracellular vesicles (EVs) are central to intercellular communication, yet the mechanisms underlying their biogenesis and diversity remain incompletely understood. Here, we integrate meta-analysis, advanced lipidomic, protein-to-lipid profiling, and super-resolution imaging to define the fundamental principles governing EV heterogeneity. Our meta-analysis of published transmission electron microcrographs across kingdoms reveals a highly conserved 110 nm average diameter and 200 nm upper size limit for intraluminal vesicles (ILVs), which are secreted as exosomes. Besides classical EV populations, we also characterize a distinct nanoparticle class: 167000 xg pellet of non-vesicular extracellular particles (167k-NVEPs), which exhibit a significantly higher protein-to-lipid ratio than 14000 xg pellet of large EVs (14k-lEVs) and 100000 xg pellet of small EVs (100k-sEVs), as measured by both biochemical assays and Raman spectroscopy. Lipid profiling demonstrates that 167k-NVEPs exhibit significant enrichment in cholesteryl esters and triacylglycerols, lipids typically associated with lipid droplets and the endosome/lysosome system. Analysis of lipid carbon-chain lengths reveals distinct signatures: 167k-NVEPs show pronounced enrichment at 16 and 18 carbons, while 100k-sEVs display enrichment at 32 and 34 carbons. This divergence indicates a potential connection to flexible biogenesis pathways. Marker heterogeneity across EV populations, confirmed by confocal and super-resolution microscopy, further underscores the limitations of relying on canonical tetraspanins for EV classification. Notably, 167k-NVEPs (likely exomeres) exhibit enrichment of Arf6 and CD63. Together, our findings provide compositional, biophysical, and molecular evidence supporting the formal recognition of 167k-NVEPs as a distinct class of extracellular particles and enabling exploring in disease biology and therapeutic delivery. Significance Statement: Extracellular vesicles (EVs) are critical mediators of intercellular communication, yet their classification remains clouded by ambiguity in terms of their composition and biogenesis. This study resolves critical uncertainties through a cross-kingdom meta-analysis, establishing a conserved ∼110nm diameter and ∼200 nm upper size limit for intraluminal vesicles (ILVs), the precursors to exosomes. More significantly, we identify non-vesicular extracellular particles (167k-NVEPs) as a distinct class based on their unique sterol-rich lipidome, enrichment in lipids of 16 and 18 carbon chain length, elevated protein-to-lipid ratio, and functional cargo delivery. These features, alongside evidence of non-canonical origin and functional cargo delivery, establish NVEPs as a discrete class of extracellular particles.