Biology subjects

Hade, M. D.

Publications and source records attributed to Hade, M. D..

5 recordsLinked to original sources

bioRxiv2026

TriPort tangential flow filtration: a lab-assembled system for EV isolation across diverse cell-culture sources

Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.

cell biology↗

bioRxiv2026

Multidimensional Cellular Micro-Compartments to Model Invasive Lobular Carcinoma Dormancy

Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancers. Despite favorable responses to anti-estrogen therapy, the dissemination of cancer cells and resistance to therapies are significant risks for patients with ILC. Late recurrences are prevalent in ILC, suggesting that disseminated tumor cell dormancy may be a mechanism preceding their late overt growth into metastatic lesions. Herein, we investigated the relationship between anti-estrogen resistance and dormancy through multidimensional in vitro models. The bioengineered platforms recapitulated the morphological characteristics of ILC and highlighted its distinction from invasive ductal carcinoma. Inducing a dormant phenotype revealed epigenetic changes and enhanced chemical and mechanical sensing of anti-estrogen-resistant ILC cells to the substrate surface, with p27Kip1 signaling playing a central role. We propose this platform as a high-throughput method to investigate the propensity of dormancy and its manifestation via a simplified and expedited approach.

bioengineering↗

bioRxiv2025

Human differentiated adipocytes can serve as surrogate mature adipocytes for adipocyte-derived extracellular vesicle analysis

Obesity is a growing global health concern, contributing to diseases such as cancer, autoimmune disorders, and neurodegenerative conditions. Adipose tissue dysfunction, characterized by abnormal adipokine secretion and chronic inflammation, plays a key role in these conditions. Adipose-derived extracellular vesicles (ADEVs) have emerged as critical mediators in obesity-related diseases. However, the study of mature adipocyte-derived EVs (mAdipo-EVs) is limited due to the short lifespan of mature adipocytes in culture, low EV yields, and the low abundance of these EV subpopulations in the circulation. Additionally, most studies rely on rodent models, which have differences in adipose tissue biology compared to humans. To overcome these challenges, we developed a standardized approach for differentiating human preadipocytes (preAdipos) into mature differentiated adipocytes (difAdipos), which produce high-yield, human adipocyte EVs (Adipo-EVs). Using visceral adipose tissue from bariatric surgical patients, we isolated the stromal vascular fraction (SVF) and differentiated preAdipos into difAdipos. Brightfield microscopy revealed that difAdipos exhibited morphological characteristics comparable to mature adipocytes (mAdipos) directly isolated from visceral adipose tissue, confirming their structural similarity. Additionally, qPCR analysis demonstrated decreased preadipocyte markers and increased mature adipocyte markers, further validating successful differentiation. Functionally, difAdipos exhibited lipolytic activity comparable to mAdipos, supporting their functional resemblance to native adipocytes. We then isolated preAdipo-EVs and difAdipo-EVs using tangential flow filtration and characterized them using bulk and single EV analysis. DifAdipo-EVs displayed classical EV and adipocyte-specific markers, with significant differences in biomarker expression compared to preAdipo-EVs. These findings demonstrate that difAdipos serve as a reliable surrogate for mature adipocytes, offering a consistent and scalable source of adipocyte-derived EVs for studying obesity and its associated disorders.

cell biology↗

bioRxiv2025

Integrated Techniques for Extracellular Particle Separation and Single-Particle Multiparametric Characterization to Track Cancer Biomarkers from Tissue to Biofluids

Gastric cancer (GC) remains a formidable global health challenge, with late-stage diagnosis and high recurrence rates resulting in poor patient outcomes. This study explores the potential of advanced technologies, namely Bessel Beam Excitation Separation Technology (BEST) and multiparametric biochip assay (MBA), to track single extracellular vesicle and particle (EVP) cargo from organs of pathology into biofluids such as plasma and saliva. Using GC as a study model, we conducted high throughput, multiparametric analyses of EVPs derived from plasma, saliva, and tissue samples. Our findings demonstrate the feasibility of these techniques in isolating and characterizing EVPs, revealing consistent EVP morphology and size across biofluids. Furthermore, differential expression patterns of the developed and validated salivary GC biomarkers, miR-140-5p and miR-301a-3p, were observed in GC patient biofluids, supporting the diagnostic relevance of EVP cargo. Notably, saliva emerged as the most promising biofluid for GC diagnosis, achieving superior Receiver Operating Characteristic (ROC) curve values compared to plasma and tissue. This study highlights the role of BEST and MBA in advancing single-EVP analysis and elucidating EVP trafficking, paving the way for future diagnostic applications of EVP cargo.

cancer biology↗

bioRxiv2024

Novel multiparametric bulk and single EV pipeline for adipose cell-specific biomarker discovery in paired 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 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 are lacking for specifically isolating and characterizing human ADEVs. To bridge this gap, we have 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 individual 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 exhibited enhanced roles in chromatin remodeling, oxidative stress responses, and metabolic regulation. Notably, adipose tissue-specific proteins such as adiponectin and perilipin were highly enriched in ADEVs and confirmed in circulating plasma EVs. Colocalization of key EV and adipocyte markers, including CD63 and PPARG, were 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.

molecular biology↗