Biofluid‐accessible extracellular vesicles (EVs) may represent a new means to improve the sensitivity and specificity of detecting disease. However, current methods to isolate EVs encounter challenges when they are used to select specific populations. Moreover, it has been difficult to comprehensively characterize heterogeneous EV populations at the single vesicle level. Here, we robustly assessed heterogeneous EV populations from cultured cell lines via nanoparticle tracking analysis, proteomics, transcriptomics, transmission electron microscopy, and quantitative single molecule localization microscopy (qSMLM). Using qSMLM, we quantified the size and biomarker content of individual EVs. We applied qSMLM to patient plasma samples and identified a pancreatic cancer‐enriched EV population. Our goal is to advance single molecule characterization of EVs for early disease detection.Abbreviations: EV: Extracellular Vesicle; qSMLM: quantitative Single Molecule Localization Microscopy; PDAC: Pancreatic Ductal Adenocarcinoma; EGFR: epidermal growth factor receptor 1; CA19‐9: carbohydrate antigen 19‐9; SEC: size exclusion chromatography; WGA: wheat germ agglutinin; AF647: Alexa Fluor 647; Ab: antibody; HPDEC: Healthy Pancreatic Ductal Epithelial Cell; TEM: Transmission Electron Microscopy.

Quantitative single molecule localization microscopy (qSMLM) is a powerful approach to study in situ protein organization. However, uncertainty regarding the photophysical properties of fluorescent reporters can bias the interpretation of detected localizations and subsequent quantification. Furthermore, strategies to efficiently detect endogenous proteins are often constrained by label heterogeneity and reporter size. Here, a new surface assay for molecular isolation (SAMI) was developed for qSMLM and used to characterize photophysical properties of fluorescent proteins and dyes. SAMI-qSMLM afforded robust quantification. To efficiently detect endogenous proteins, we used fluorescent ligands that bind to a specific site on engineered antibody fragments. Both the density and nano-organization of membrane-bound epidermal growth factor receptors (EGFR, HER2, and HER3) were determined by a combination of SAMI, antibody engineering, and pair-correlation analysis. In breast cancer cell lines, we detected distinct differences in receptor density and nano-organization upon treatment with therapeutic agents. This new platform can improve molecular quantification and can be developed to study the local protein environment of intact cells.

Vascular smooth muscle cell (VSMC) diversification drives atherosclerotic coronary artery disease (CAD), but the mechanisms governing these cell state transitions remain unclear. We applied multiomic single-cell profiling, epitope mapping, and spatial transcriptomics across 27 human coronary arteries, identifying fibroblast activation protein (FAP) as a marker of modulated VSMCs. Lineage tracing in mice indicated that FAP ⁺ cells originate from Myh11 ⁺ VSMCs, and FAP positron emission tomography imaging in CAD patients showed plaque uptake. FAP ⁺ cell states resided in the macrophage-rich neo-intima. Therapeutically, we developed an anti-FAP bispecific T cell engager, which reduced plaque burden and remodeled the stromal–immune microenvironment through T cell clonal expansion. Our study delivers a single-cell and spatial atlas of human CAD, establishes FAP as a marker of modulated VSMCs, and highlights immunotherapy for lipid-independent targets.

Quantitative phase imaging (QPI) is an ideal method to non-invasively monitor cell populations and provide label-free imaging and analysis. QPI offers enhanced sample characterization and cell counting compared to conventional label-free techniques. We demonstrate this in the current study through a comparison of cell counting data from digital phase contrast (DPC) imaging and from QPI using a system based on Fourier ptychographic microscopy (FPM). Our FPM system offers multi-well, parallel imaging and a QPI-specific cell segmentation method to establish automated and reliable cell counting. Three cell types were studied and FPM showed improvement in the ability to resolve fine details and thin cells, despite limitations of the FPM system incurred by imaging artifacts. Relative to manually counted fluorescence ground-truth, cell counting results after automated segmentation showed improved accuracy with QPI over DPC.

The complex spatiotemporal organization of proteins and lipids in the plasma membrane is an important determinant of receptor function. Certain substances, such as ethanol, can penetrate into the hydrophobic regions of the plasma membrane. By altering protein–lipid and protein–protein interactions, these substances can modify the dynamic lateral organization and the function of plasma membrane receptors. To assess changes in plasma membrane receptor organization, we used photoactivated localization microscopy (PALM). This single molecule localization microscopy technique was employed to quantitatively characterize the effects of pharmacologically relevant concentrations of ethanol and naltrexone (an opioid receptor antagonist and medication used to treat alcohol use disorders) on the lateral nano-organization of mu and kappa opioid receptors (MOR and KOR, respectively). Ethanol affected the lateral organization of MOR and KOR similarly: It reduced the size and occupancy of opioid receptor nanodomains and increased the fraction of opioid receptors residing outside of nanodomains. In contrast, naltrexone affected MOR and KOR lateral organization differently. It significantly increased KOR surface density, nanodomain size, and the occupancy of KOR nanodomains. However, naltrexone marginally affected these parameters for MOR. Pretreatment with naltrexone largely protected against ethanol-induced changes in MOR and KOR lateral organization. Based on these data, we propose a putative mechanism of naltrexone action that operates in addition to its canonical antagonistic effect on MOR- and KOR-mediated signaling.

In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain β2-microglobulin. A series of pH-dependent internalization studies using a model antibody demonstrated proper function of the human FcRn complex. We then applied our cellular assays to assess the contribution of FcRn and non-specific interactions in the cellular turnover for a panel of 8 clinically relevant mAbs exhibiting variable human pharmacokinetic behavior. Our results demonstrate that non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of therapeutic monoclonal antibodies. The predictive capacity of our assay approach was highlighted by successful identification of all mAbs within our panel possessing clearance in humans greater than 5 mL/day/kg. These results demonstrate that a combination of cell-based in vitro assays can properly resolve individual mechanisms underlying the overall in vivo recycling efficiency and non-target mediated clearance of therapeutic mAbs.