Extracellular vesicles (EVs) and their cargo constitute novel biomarkers. EV subpopulations have been defined not only by abundant tetraspanins (e.g., CD9, CD63 and CD81) but also by specific markers derived from their source cells. However, it remains a challenge to robustly isolate and characterize EV subpopulations. Here, we combined affinity isolation with super‐resolution imaging to comprehensively assess EV subpopulations from human plasma. Our Single Extracellular VEsicle Nanoscopy (SEVEN) assay successfully quantified the number of affinity‐isolated EVs, their size, shape, molecular tetraspanin content, and heterogeneity. The number of detected tetraspanin‐enriched EVs positively correlated with sample dilution in a 64‐fold range (for SEC‐enriched plasma) and a 50‐fold range (for crude plasma). Importantly, SEVEN robustly detected EVs from as little as ∼0.1 μL of crude plasma. We further characterized the size, shape and molecular tetraspanin content (with corresponding heterogeneities) for CD9‐, CD63‐ and CD81‐enriched EV subpopulations. Finally, we assessed EVs from the plasma of four pancreatic ductal adenocarcinoma patients with resectable disease. Compared to healthy plasma, CD9‐enriched EVs from patients were smaller while IGF1R‐enriched EVs from patients were larger, rounder and contained more tetraspanin molecules, suggestive of a unique pancreatic cancer‐enriched EV subpopulation. This study provides the method validation and demonstrates that SEVEN could be advanced into a platform for characterizing both disease‐associated and organ‐associated EV subpopulations.

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.

Exosomes are small extracellular vesicles (sEVs) of ~30–150 nm in diameter that are enriched in exosome marker proteins and play important roles in health and disease. To address large unanswered questions regarding exosome biology in vivo, we created the Exomap1 transgenic mouse, which in response to Cre recombinase expresses the most highly enriched exosomal marker protein known, human CD81, fused to mNeonGreen (HsCD81mNG), and prior to Cre expresses a mitochondrial red fluorescent protein. Validation of the exomap1 mouse with eight distinct Cre drivers demonstrated that HsCD81mNG was expressed only in response to Cre, that murine cells correctly localized HsCD81mNG to the plasma membrane, and that this led to the secretion of HsCD81mNG in EVs that had the size (~70–80 nm), topology, and composition of exosomes. Furthermore, cell type-specific activation of the exomap1 transgene allowed us to use quantitative single molecule localization microscopy to calculate the cell type-specific contribution to biofluid exosome populations. Specifically, we show that neurons contribute ~1% to plasma and cerebrospinal fluid exosome populations whereas hepatocytes contribute ~15% to plasma exosome populations, numbers that reflect the known vascular permeabilities of brain and liver. These observations validate the use of Exomap1 mouse models for in vivo studies of exosome biology.

Trastuzumab, the prototype HER2-directed therapy, has markedly improved survival for women with HER2-positive breast cancers. However, only 40–60% of women with HER2-positive breast cancers achieve a complete pathological response to chemotherapy combined with HER2-directed therapy. The current diagnostic assays have poor positive-predictive accuracy in identifying therapy-responsive breast cancers. Here, we deployed quantitative single molecule localization microscopy to assess the molecular features of HER2 in a therapy-responsive setting. Using fluorescently labeled trastuzumab as a probe, we first compared the molecular features of HER2 in trastuzumab-sensitive (BT-474 and SK-BR-3) and trastuzumab-resistant (BT-474^R and JIMT-1) cultured cell lines. Trastuzumab-sensitive cells had significantly higher detected HER2 densities and clustering. We then evaluated HER2 in pre-treatment core biopsies from women with breast cancer undergoing neoadjuvant therapy. A complete pathological response was associated with a high detected HER2 density and significant HER2 clustering. These results established the nano-organization of HER2 as a potential signature of therapy-responsive disease.

Epigallocatechin-gallate (EGCG) is the main polyphenol ingredient of green tea. This compound is a strong antioxidant and oxidizes easily. Numerous studies demonstrated its beneficial effects on the human health, for example its anticancer and anti-inflammatory activity. In the body, EGCG is transported by serum albumin. EGCG easily oxidizes and the interactions of the oxidized form presumably present significant differences. However, the presence of oxidized EGCG is usually neglected in the literature and its effects have not been investigated in detail. Here, we applied the label-free grating coupled interferometry method that performs dual-channel measurements. The measured kinetic signal can be compensated with a signal of a reference channel at each measurement time. By testing both hydrophilic and hydrophobic platforms, we found that EGCG can bind to a wide range of surfaces. Exploiting the dual-channel referencing ability as well as the unique sensitivity and throughput of the employed label-free technique, the experiments revealed the specific interactions between bovine serum albumin (BSA) and EGCG and determined the characteristic dissociation constant (K_d) of the binding equilibrium. The obtained binding constants were compared to literature values, showing reasonable agreement with NMR data. Besides the native EGCG, the oxidized form of EGCG was also examined, whose binding behaviors to serum albumins have never been studied. Overstoichiometric binding obtained; BSA has stronger and weaker binding sites, which could be characterized by two separate K_d values. Furthermore, EGCG oxidization increased the bound amount.