Multiparametric Bulk and Single Extracellular Vesicle Pipeline for Identifying Adipose-Specific Signatures in Matched Human Biospecimens

Obesity remains a growing and global public health burden across a broad spectrum of metabolic, systemic, and neurodegenerative diseases. Previously considered merely a fat storage depot, adipose tissue is now recognized as an active endocrine organ crucial for the metabolic and systemic regulation of local and distant organs. A burgeoning line of investigation centers on adipose-derived extracellular vesicles (ADEVs) and their pivotal role in obesity-associated pathobiology. However, robust methodologies for specifically isolating and characterizing human ADEVs are lacking. To bridge this gap, we developed a robust multiparametric framework incorporating bulk and single EV characterization, proteomics, and mRNA phenotyping. EVs from matched human visceral adipose tissue, mature adipocyte-conditioned media, and plasma collected from the same bariatric surgical patients were analyzed and subjected to bottom-up proteomics analysis. This framework integrates bulk EV proteomics for cell-specific marker identification and subsequent single EV interrogation with single-particle interferometric reflectance imaging (SP-IRIS) and total internal reflection fluorescence (TIRF) microscopy. Our proteomics analysis revealed 76 unique proteins from adipose tissue-derived EVs (ATEVs), 512 unique proteins from adipocyte EVs (aEVs), and 1003 shared proteins. Prominent pathways enriched in ATEVs included lipid metabolism, extracellular matrix organization, and immune modulation, while aEVs were enriched in chromatin remodeling, oxidative stress responses, and metabolic regulation pathways. Notably, we identified obesity-associated and adipose-specific proteins, such as TMEM120A and SACM1L, highly enriched in ATEVs, and histone variants, including MACROH2A1, H3-3A, H2AC20, H2BC12, and H3-7, uniquely upregulated in aEVs. Additionally, classical adipose-associated proteins such as adiponectin and perilipin were highly enriched in ADEVs and were confirmed in circulating plasma EVs. Colocalization of key EV and adipocyte markers, including CD63 and PPARG, was validated in circulating plasma EVs. In summary, our study paves the way toward a tissue and cell-specific, multiparametric framework for an 'adiposity EV signature' in obesity-driven diseases.