Novel multiparametric bulk and single EV pipeline for adipose cell-specific biomarker discovery in paired human biospecimens.

Obesity is a global health crisis that contributes to morbidity and mortality worldwide. Obesity's comorbid association with a variety of diseases, from metabolic syndrome to neurodegenerative disease, underscores the critical need to better understand the pathobiology of obesity. Adipose tissue, once seen as an inert storage depot, is now recognized as an active endocrine organ, regulating metabolic and systemic homeostasis. Recent studies spotlight the theranostic utility of extracellular vesicles (EVs) as novel biomarkers and drivers of disease, including obesity-related complications. Adipose-derived EVs (ADEVs) have garnered increased interest for their roles in diverse diseases, however robust isolation and characterization protocols for human, cell-specific EV subsets are limited. Herein, we directly address this technical challenge by establishing a multiparametric analysis framework that leverages bulk and single EV characterization, mRNA phenotyping and proteomics of human ADEVs directly from paired visceral adipose tissue, cultured mature adipocyte conditioned media, and plasma from obese subjects undergoing bariatric surgery. Importantly, rigorous EV phenotyping at the tissue and cell-specific level identified top 'adipose liquid biopsy' candidates that were validated in circulating plasma EVs from the same patient. In summary, our study paves the way toward a tissue and cell-specific, multiparametric framework for studying tissue and circulating adipose EVs in obesity-driven disease.

Engineering a tunable micropattern-array assay to sort single extracellular vesicles and particles to detect RNA and protein in situ.

The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization. Herein, we present a single-EV and particle (siEVP) protein and RNA assay (siEVP PRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. The siEVP PRA immobilizes and sorts particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting EVPs at a single-particle resolution, the siEVP PRA outperformed the sensitivities of bulk-analysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, the siEVP PRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. The siEVP PRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.