INTRODUCTION:Blood-based biomarkers are essential for early detection, monitoring, and therapeutic development in Alzheimer's disease (AD) and related dementia (ADRD), but current assays lack brain cell specificity and sensitivity to low-abundant proteins. METHOD:We isolated the following brain cell-derived small extracellular vesicles (sEV) from the plasma of individuals with normal cognition (CN), mild cognitive impairment (MCI), or ADRD: neurons (NDE), astrocytes (ADE), microglia (MDE), oligodendrocytes (ODE), pericytes (PDE), and endothelial cells (EDE). Using NULISAseq, we profiled 122 proteins, spanning AD pathology, neurodegeneration, and neuroinflammation. RESULTS:sEV proteomes showed distinct brain cell-type-specific signatures. MCI exhibited early dysregulation of neuroprotective, inflammatory, and vascular markers in NDE, MDE, and ODE. ADRD displayed broader alteration tau, amyloid, neuroinflammation, vascular dysfunction, and synaptic loss across multiple sEV populations. DISCUSSION:Combining NULISAseq with brain cell-derived plasma sEV enables the detection of multicellular molecular changes in ADRD, supporting their use as a minimally invasive platform for biomarker discovery.

OBJECTIVE:Glucose tolerance (GT) is a major effector for adipose tissue (AT) remodeling in obesity, yet its molecular mechanisms remain incompletely defined. We hypothesized that the biophysical and molecular profiles of AT-derived small extracellular vesicles (sEVAT) change in response to glucose availability and differ by GT status. METHODS:sEVAT were isolated from plasma of individuals with normal GT (NGT) and impaired GT (IGT) (n = 5/group) at fasting (0 h) and 1 h post glucose challenge during oral glucose tolerance test (OGTT). sEVAT were characterized for size, concentration, surface expression of insulin receptor-α (INSRα), proteome, and insulin signaling-related miRNAs. C2C12 myotubes were treated with sEVAT for 48 h, followed by quantification of 84 insulin signaling-related genes. RESULT:The size and concentration of sEVAT did not differ between groups. At fasting, INSRα expression on sEVAT was comparable; however, groups exhibited opposite directional changes at 1-h OGTT. LC-MS/MS identified significant proteomic differences between NGT and IGT sEVAT. miR-27a-5p and miR-145a-5p levels in sEVAT also differed significantly by GT status. Notably, treatment with sEVAT (IGT-0 h) significantly downregulated insulin signaling-related genes in myotubes. CONCLUSIONS:Distinct molecular signatures in sEVAT offer a unique insight into AT dysfunction during IGT and offer novel diagnostic and therapeutic targets.

Obesity is associated with adverse changes in brain structure and function, in part, through crosstalk between adipose tissue (AT) and the brain. AT releases small extracellular vesicles (sEV) that can cross the blood-brain barrier (BBB) and modulate multiple pathophysiological pathways, including BBB function; however, this has never been investigated. We characterized circulating adipose tissue-derived sEV (sEVAT) in adults with overweight and obesity and examined their effects on the BBB. The impact of adiposity and weight loss on these outcomes was also examined. sEVAT were isolated from the plasma of 29 adults (79% male; 93% White; mean age 66.2 ± 7.0 years; mean body mass index 36.0 ± 6.8 kg/m2) randomized to cardiac rehabilitation (CR) alone or CR plus a behavioural weight loss intervention (CR+WL). Following characterization of sEVAT size, concentration and total protein content, we assessed their effect on BBB permeability using an in vitro model. hCMEC/D3 cells were treated with sEVAT, and transendothelial electrical resistance (TEER) was measured at 0, 24, 48 and 72 h. Our findings show that sEVAT treatment decreased TEER by 40%, with a significantly lower TEER at 72 h compared with controls (23.138 ± 1.209 vs. 28.724 ± 1.613 Ω cm2, p = 0.012). TEER was also lower in participants with higher body mass index and body fat. However, we found no difference in TEER between the CR and CR+WL groups and no significant intervention effects on sEVAT characteristics or TEER. In conclusion, higher plasma sEVAT concentrations in adults with overweight and obesity are associated with greater adiposity, which might contribute to reductions in BBB function.

The decline in mobility with aging is a major health concern, associated with a high risk for disability. Despite the widespread prevalence of gait slowing in elderly adults, this issue has not been adequately addressed. The central nervous system and skeletal muscle system are key regulators of gait speed. However, direct molecular communication along the brain-muscle axis and the role of these interactions in mobility resilience remain poorly studied. Recently, extracellular vesicles (EV), membrane bound vesicles secreted by cells, have emerged as a key player in long distance inter-cellular communication. Nevertheless, the potential of EVs as biological predictor of mobility resilience in older adults has not been yet studied. In the present study, we used serum samples from 23 participants with gait speed >1.0 m/sec (mobility-resilient group) and 22 participants with gait <1.0 m/sec (mobility non-resilient group) from the Health, Aging and Body Composition (Health ABC) study. First, total circulating serum EVs were isolated and characterized for small noncoding RNAs using un-biased small noncoding RNA sequencing. Given the central role of mitochondria in muscle energy metabolism and their emerging link to age-related physical decline, next, muscle-derived EVs (MDE) were isolated and characterized for specific mitochondrial markers (TOM20, mtCox2, PDH, and VDAC) by flow cytometry, the expression of a panel of 13 miRNAs related to mitochondrial function by RT-PCR, and PPAR-γ expression by ELISA. The results showed differential enrichment of various miRNAs, circRNAs, and mitochondrial proteins in total EVs and/or MDE between mobility-resilient and non-resilient groups, highlighting their potential as non-invasive biomarkers for mobility outcomes. Overall, the findings from the present study suggest a role for serum EVs in mediating molecular communication related to functional aging phenotypes and underscores the potential of EV biomarkers in modulating mobility and promoting healthy aging.

Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with limited therapeutic options, a high mortality rate, and poor overall survival, necessitating the development of new therapeutic and diagnostic strategies. This study investigated the potential of plasma-derived small extracellular vesicles (sEVs) as a source of molecular biomarkers associated with the treatment response. Methods: Plasma samples were obtained from patients with locally advanced and borderline resectable PDAC at baseline and following neoadjuvant chemotherapy, either FOLFIRINOX (5-FU [fluorouracil], leucovorin, oxaliplatin, and irinotecan) or GEM-ABRAX ( gemcitabine plus nab-paclitaxel), followed by stereotactic body radiation therapy (SBRT). sEVs were isolated from plasma at baseline, after neoadjuvant chemotherapy, and following SBRT, and were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), nano-flow cytometry, and real-time PCR (RT-PCR). Results: The isolated sEVs exhibited an average size of <200 nm, expressed canonical exosome markers (CD63 and CD9), and exhibited pancreatic cancer (PanC)-associated markers, including cholecystokinin A receptor (CCK-AR) and carbohydrate antigen 19-9 (CA19-9). The sEV cargo included several PanC-associated microRNAs (miRNAs). Notably, the expression profiles of these miRNAs demonstrated interpatient variability, though a subset of miRNAs showed statistically significant changes following treatment. Conclusions: These findings support the feasibility of sEV isolation and molecular profiling from patient plasma and warrant further investigation as a potential source of biomarkers in pancreatic cancer.

Abstract Background: A major challenge in managing pancreatic cancer (PanC) is late detection and the inability to identify non-responders early. Existing liquid biopsy approaches offer limited capacity to rapidly and noninvasively track the tumor’s evolving molecular landscape, which is essential for overcoming therapy resistance and guiding targeted treatment. Small extracellular vesicles (sEV; <200 nm) circulate widely and carry cargo reflective of their cells of origin. Using our established methods for isolating tissue-specific sEV, we identified pancreas-specific surface markers, isolated circulating pancreas derived sEV (sEVPancreas), and evaluated their potential as a liquid biopsy platform. Methods: We analyzed fresh-frozen PanC tissues (n=12) along with matched healthy samples. sEV were isolated from tissues, subjected to surface protein shaving, and analyzed using LC-MS/MS based on published methods. Proteomic data, combined with the Human Protein Atlas, were used to identify sEVPancreas . They were isolated from archived plasma samples of PanC patients (n=10) and healthy individuals (n=5) using biotin-tagged antibodies and streptavidin-coated magnetic beads. Isolated sEV/sEVPancreas were characterized for size and concentration by nanoparticle tracking analysis (NTA), and for various biomarkers’ expression using nano-flow cytometry, RT-PCR, RNA sequencing, and digital PCR. Results: Prolyl 4-hydroxylase subunit beta (P4HB) and annexin A4 (ANXA4) were identified as pancreas-specific sEV surface markers. Nano-flow cytometry confirmed significantly higher levels of P4HB- and ANXA4-positive sEV in PanC plasma compared to healthy controls (p<0.01). Using these markers, we isolated sEVPancreas from blood plasma samples of PanC patients and controls. NTA confirmed that the isolated vesicles were <200 nm in both groups. Notably, sEVPancreas from PanC patients exhibited significantly higher expression of the PanC biomarker cholecystokinin A receptor (p<0.01) and lower levels of miR-320-5p, a microRNA associated with poor prognosis. RNA sequencing of sEVPancreas revealed several upregulated (e.g., endosulfine-α, MUC12) and downregulated (e.g., DYNC1I2, POM121C) genes in the PanC group. Finally, KRAS copy number and mutation status was reliably assessed in sEVPancreas, highlighting the potential for KRAS mutational profiling.: we reliably characterized wild type and mutated KRAS status (G12D and G12V). Conclusions. We demonstrate that pancreas-derived sEV can be selectively isolated from blood using newly identified pancreas-specific markers. These sEVPancreas harbor distinct molecular signatures and reliably capture KRAS copy number and mutations, supporting their potential as a rapid, minimally invasive liquid biopsy platform. These findings lay the groundwork for sEV-based assays for treatment monitoring and response assessment. Citation Format: Ravi Kumar Paluri, Ashish Kumar, Yixin Su, Gregory L. Kucera, Ashish Manne, Jingyun Lee, Sangeeta Sing, Susy Kim, Cristina Furdui, Gagan Deep. Isolation and characterization of pancreatic cancer-derived small extracellular vesicles as a novel liquid biopsy approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3739.

Preeclampsia is a multisystem hypertensive disorder of pregnancy and a leading cause of maternal morbidity and mortality. Despite its increasing incidence and the debilitating nature of its cerebrovascular complications, the underlying mechanisms remain incompletely understood. The goals of this study were to 1) determine whether middle cerebral artery (MCA) hemodynamics are altered in late gestation (LG) or two months postpartum in transgenic rats with preeclampsia compared to normal pregnant Sprague-Dawley (SD) rats, and 2) evaluate the microRNA (miRNA) profiles of circulating placental-derived extracellular vesicles (EV PD ) using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway framework using NEST-Scoring [Node, Edge, System, Topology], a novel target gene profile network analysis. Our data showed elevated blood pressure in transgenic preeclampsia rats at both LG and two months post-partum (PP-2M) compared to normal pregnant SD rats in the setting of hypertension. The heart rate was also significantly higher in preeclampsia rats at LG. No differences were observed in left MCA (LMCA) velocities between strains or time points. However, LMCA resistance and pulsatility indexes were lower at PP-2M in transgenic preeclampsia rats compared to SD rats. Exposure to preeclampsia differentially altered the miRNA profiles of circulating EV PD in transgenic preeclampsia rats at both LG and PP-2M. The identified miRNA targets were associated with key vascular and cellular regulatory pathways, suggesting a mechanistic link between placental dysfunction and postpartum vascular alterations in this preeclampsia model. Overall, this study suggests that the TGA-PE rat model could be a valuable platform to study the mechanistic link between preeclampsia and cerebrovascular disease.

Cross-sectional data from a sample of older adults with obesity was used to determine how peripheral insulin resistance (PIR) and neuronal insulin signaling abnormalities (NISAs) relate to executive function and functional brain network topology. Older adults (n = 71) with obesity but without type 2 diabetes were included. PIR was quantified by HOMA2-IR. NISAs were quantified according to an established neuron-derived small-extracellular-vesicle-based metric, R. An executive function composite score, summed scores to the Auditory Verbal Learning Test (AVLT) trials 1-5, and functional brain networks generated from resting-state functional magnetic resonance imaging data were outcomes in analyses. We used general linear models and a novel regression framework for brain network analysis to identify relationships between insulin-related biomarkers and brain-related outcomes. HOMA2-IR, but not R, was negatively associated with executive function. Neither measure was associated with AVLT score. HOMA2-IR was also related to hippocampal network topology in participants who had undergone functional neuroimaging. Neither HOMA2-IR nor R were significantly related to network topology of the central executive network. This study provides further evidence that PIR is associated with aging brain function. NISAs were not found to be related to PIR, cognition, or functional brain network topology.

BACKGROUND:The current study utilized positron emission tomography (PET) imaging to examine how long-term cocaine self-administration (SA) and time off cocaine affected kappa opioid receptor (KOR) availability in the brain of previously cocaine-naïve monkeys. In addition, neuronally derived small extracellular vesicles (NDEs) were measured from plasma to identify peripheral measures of KORs. METHODS:Female (n = 6) and male (n = 7) cynomolgus monkeys, living in stable same-sex social groups, were trained to self-administer intravenous cocaine. PET imaging with the KOR selective agonist [11C]EKAP occurred after monkeys had self-administered ∼100-mg/kg total cocaine intake and after ∼30 days off cocaine; in a subset of monkeys, a third PET scan was conducted after ∼100 days off cocaine. Blood samples were obtained prior to each PET study, and NDEs from the plasma were isolated by immunocapture method and analyzed for percentage of KOR+. RESULTS:There were significant interactions between condition (100 mg/kg cocaine and 30 days off cocaine), sex, and social rank in KOR availability across 7 of 15 brain regions. More specifically, these interactions were associated with increased KOR availability following cocaine SA and after 30 days off cocaine in dominant females. In a subset of monkeys, no differences were observed in [11C]EKAP binding between 30 and 100 days off cocaine. NDEs showed significant interactions between sex and condition, providing a peripheral measure consistent with the PET results. CONCLUSIONS:These findings extend previous research with socially housed monkeys on KORs and suggest that KOR may be a viable target for pharmacological interventions for cocaine misuse, especially in women.

Insulin signaling deregulation in the brain is a critical risk factor for Alzheimer's disease (AD); however, molecular changes in this pathway during AD pathogenesis cannot be currently accessed in clinical setting due to lack of brain tissues. Here, we propose small extracellular vesicles (sEV) characterization as a non-invasive approach to assess the status of insulin signaling in the AD brain. In postmortem brain tissues of cognitively normal (CN) and AD (n=5 each) subjects, expression of 84 genes, involved in insulin signaling and resistance was analyzed using pathway specific PCR array. Next, the level of key miRNAs (miR185-5p, miR-210-3p, and miR342-3p), that regulate the expression of insulin signaling genes, was assessed by TaqMan-based qPCR in brain tissues and secreted sEV in their microenvironment. The expression of these miRNA was also analyzed in the neuron-derived sEV (NDE) isolated from the plasma of 28 CN (18 female and 10 male) and 28 dementia (11 female and 17 male) subjects, all with type-2 diabetes. These highly specific NDE were isolated by sequential immunoprecipitation using CD171 and synaptophysin surface markers, and the expression of studied miRNAs was correlated with corresponding clinical measures (MMSE score, Aβ1-40 and Aβ1-42 levels). AD brain tissue revealed significant deregulation of genes involved in insulin signaling and resistance (e.g., AKT2, GSK3β, INSR, KRAS, PIK3R1, IGF1R, PTPN1, BRAF, GCK, UCP1, LDLR, NPY, and RRAS2). We also observed a significant increase in the expression of miRNAs (miR185-5p, miR-210-3p, and miR342-3p) involved in regulation of these genes. Interestingly, the expression of these miRNAs in the brain tissues as well as their secreted sEV showed a high degree of concordance. Most importantly, NDE in the plasma of individuals with dementia also showed a similar change in the expression of miRNAs regulating insulin signaling in the brain, suggesting suitability of sEV application as liquid biopsy for the brain tissue. Lastly, this increased expression of NDE miR-185-5p in dementia showed significant inverse correlation with MMSE Score. NDE in plasma offer critical molecular information about insulin signaling and resistance in the brain, which could be valuable in making diagnostic and treatment decisions in AD.

Gut dysbiosis contributes to multiple pathologies, yet the mechanisms of the gut microbiota-mediated influence on systemic and distant responses remain largely elusive. This study aimed to identify the role of nanosized bacterial extracellular vesicles (bEVs) in mediating allodynia, i.e., pain hypersensitivity, in a diet-induced obesity (DIO) gut dysbiosis model. bEVs were enriched from the feces of lean (bEVLean) and DIO (bEVDIO) mice by an approach combining ultracentrifugation and immunoprecipitation and then extensively analyzed for purity and bacterial characteristics. Next, bEVs were injected, either intraplantarly or intravenously, in mice to assess pain sensitivity. Fluorescence-labeled bEVs were injected in mice by enema to assess biodistribution. The effect of bEV on immune cells and inflammation was analyzed by array, immunophenotyping, microscopy, NF-κB activation, and cellular uptake assays. Results showed that bEVDIO administration in wild-type mice replicated the allodynia phenotype observed in DIO mice for both mechanical and thermal stimuli. Importantly, this effect was compromised in TRPA1/TRPV1 double-knockout mice. Biodistribution analyses showed bEV entry into systemic circulation with subsequent localization at distant sites. Multiple analyses revealed that bEVDIO exposure incited systemic inflammation, primarily through modulating the innate immune system. This inflammatory mechanism involved LPS on the bEV surface, activating TLR2- and TLR4-related pathways, as confirmed using TLR2 and TLR4 inhibitors and shaving bEV surface proteins. Interestingly, the enhanced cellular uptake of bEVDIO was contingent on interactions involving LPS and proteins on bEVs and TLR2/TLR4 on monocytes. These findings illuminate the hitherto unexplored role of bEV as pivotal mediators of allodynia and inflammation linked to gut dysbiosis.

Central nervous system (CNS) dysregulated insulin and peripheral hyperinsulinemia has been associated with AD. However, analyzing CNS insulin resistance in living subjects and its implication on cognitive impairment/ AD is difficult to establish due to inaccessibility of brain tissue. In this study we isolated and characterized plasma neuron-derived small extracellular vesicles (NDE), and adopted multi-omics approaches to discover novel biomarkers of AD and CNS insulin resistance and suggested their possible association. Plasma samples were obtained from Look AHEAD-MIND study with 28 cognitively normal (18 female and 10 male) and 28 individuals with probable dementia (11 female and 17 male) based on central adjudication by an expert panel. These individuals had established type 2 diabetes (T2D) and overweight/obesity when enrolled in a randomized controlled clinical trial of a 10-year behavioral intervention. NDE were enriched from plasma sequentially using CD171 and synaptophysin surface markers by immunoprecipitation and characterized for non-coding RNAs by small RNA-sequencing and proteomics by mass spectrometry. The small RNA-sequencing analysis identified the expression of over 100 miRNAs in NDE from both groups. The Comparison of differentially expressed miRNAs revealed 42 miRNAs to be downregulated, while 23 miRNAs were upregulated in individuals with dementia. Importantly, all these miRNAs (both up- and –downregulated) were found to be over-represented in AD- and insulin signaling-related pathways. Also, the targets of these miRNAs were associated with pathways like protein phosphorylation, autophagy, cell cycle and nervous system development. Alongside, the mass spectrometry-based proteomics analysis identified 3 proteins (haptoglobin, complement component C6, and alpha-2-macroglobulin) to be significantly downregulated and 1 protein (desmocolin-1) to be upregulated in the NDE of individuals with dementia. Interestingly, NDE showed upregulated expression of miR-185-5p, with concomitant downregulation of one of its potential targets complement protein C6, both known to be associated with AD and insulin signaling. The study demonstrated multi-omics approach to identify novel plasma NDE biomarkers for dementia pathogenesis and its association with CNS insulin resistance in individuals with T2D.

Age-related bone loss is a highly prevalent condition that contributes substantially to morbidity, mortality, and healthcare costs worldwide. Dysregulated osteoclast activity characterizes aging bone; however, current diagnostic measurements lack specificity, sensitivity, and early detection ability. Small extracellular vesicles (sEV) derived from specific cell types offer a promising non-invasive liquid biopsy approach for assessing hard-to-access tissues, like bone. This proof-of-concept study aimed to isolate osteoclast-specific sEV (sEV-Osteoclast) from blood, laying the foundation for a sensitive, specific, and repeatable method to evaluate osteoclast physiology and molecular status of bone in clinical studies. Four osteoclast-specific surface markers (tartrate-resistant acid phosphatase (TRAP), receptor activator of nuclear factor kappa-Β (RANK), integrin alpha V (CD51) and dendritic cell-specific transmembrane protein (DCStamp)) were identified through extensive literature review and validated using the Human Protein Atlas and UniProt databases. Next, total sEV were isolated from human plasma samples (n = 11; age: 70.0±2.76 years) and characterized for size (99.65±2.49nm) and concentration (3.58E+11±3.13E+10particle/ml) by nanoparticle tracking analysis. Next, the percentage of sEVOsteoclast markers in the total sEV was ascertained by flow cytometry, revealing that 6.70±0.80% of plasma-total sEV were TRAP positive (+), 2.54±0.27% were RANK+, 7.36±0.64% were CD51+, and 3.63±0.27% were DCStamp+. As any specific sEV population can be reliably isolated from plasma if the percentage positivity is ≥ 2% in total sEV, these preliminary findings demonstrate feasibility of sEV-Osteoclast isolation from plasma. Future work aims to measure sEV-Osteoclast in a NIA-funded clinical trial of weight loss in older adults (NCT05764733), as a biomarker of early risk assessment and real-time intervention monitoring.

INTRODUCTION:Brain insulin resistance (bIR) is a risk factor for Alzheimer's disease (AD). However, the association between bIR and peripheral insulin resistance and their effects on cognition remains unclear. METHODS:Here, we analyzed the expression of key genes (n = 84) involved in insulin signaling in brain tissue collected from healthy and AD subjects, as well as regulatory microRNAs (miRNAs) in the brain tissue and tissue-derived small extracellular vesicles (sEV). Subsequently, miRNA expression was analyzed in plasma neuron-derived sEV (NDE) of a second cohort consisting of cognitively normal and adjudicated mixed dementia (aMD) subjects, all with type 2 diabetes. RESULTS:Analysis of miRNAs in brain tissue and their sEV revealed significant and concordant dysregulation. NDE demonstrated similar changes in specific miRNA expression, with significant upregulation exclusively in male aMD subjects, and showed correlation with cognition and plasma β-amyloid (Aβ) 1-40 and Aβ1-42. DISCUSSION:NDE may serve as a liquid biopsy to determine sex-specific bIR and cognitive impairment. HIGHLIGHTS:Insulin signaling is disrupted in brain tissue with Alzheimer's disease (AD). microRNAs (miRNAs) regulate insulin signaling and insulin resistance. miRNAs in neuron-derived small extracellular vesicles (sEV) could serve as biomarkers for brain insulin signaling. Brain insulin signaling biomarkers in neuron-derived sEV (NDE) could predict cognitive impairment. Sex-specific differences exist in brain insulin resistance biomarkers.

2073 Background: Glioblastoma (GBM) remains the most common and aggressive primary malignant brain tumor, and afflicted patients have limited options and poor overall survival. Diagnosis, prognosis, and assessment of treatment effectiveness is hampered by the absence of readily available sensitive, tumor specific, non-invasive blood-based assays which could be followed serially. Small extracellular vesicles (sEV) are nano-sized (≤200 nm) membrane bound bodies secreted by all cells, encapsulating cargo reflective of their parent cells. sEV have emerged as promising molecular indicator of a disease condition. Here, we report the feasibility of isolating glioma specific sEV (sEVglioma) from the plasma and characterizing those for GBM specific molecular biomarkers. Methods: Plasma samples were collected from patients (n=31) diagnosed with adult gliomas (including grade 3 and 4 GBM, grade 2 and 3 astrocytoma) and healthy individuals (n=9). Total sEV (TE) population was isolated from plasma by a modified precipitation (ExoQuick) method. Next, sEVglioma were isolated from the TE by an immunoprecipitation approach employing specific surface markers targeting cellular origin of gliomas, including astrocyte (GLAST and EAAT2), oligodendrocyte precursor cell (OSP and MOG), and neural stem cell (CD133). sEVglioma were characterized for size and concentration by nanoparticle tracking analyses; surface expression of glioma specific biomarkers by nano-flow cytometry and confocal microscopy; and the expression of a panel of specific miRNAs by RT-PCR. Lastly, sEVglioma were assessed by digital PCR for IDH1 status (wild type or mutated). Results: The average size of isolated sEVglioma was less than 200nm. Importantly, compared to TE, sEVglioma showed significant enrichment for glioma specific biomarkers such as ephrin type-A receptor 2 (14.7-fold), tenascin C (22.7-fold), and glial fibrillary acidic protein (8.4-fold) by nano-flow cytometry. Similarly, sEVglioma, but not TE, demonstrated high expression of EGFRvIII (3.6-fold), a known biomarker for GBM. These results were confirmed by confocal microscopy. Interestingly, expression of specific miRNAs (miR-9a-5p, miR-16-5p, miR-21-5p) in sEVglioma was higher in glioma patients with shorter survival (<12 months) compared to patients with longer survival (>20 months). Lastly, we successfully detected the existence of wild type IDH1 and absence of mutated IDH1 R132H in sEVglioma from GBM patients. This approach was validated in sEV isolated from the conditioned media of IDH1-wild type GBM cell lines (A172 and T98G) and a IDH1-mutated cell line (BT54). Conclusions: We present a novel approach to isolate sEVglioma from blood that could serve as a liquid biopsy, offering valuable molecular and genetic information. This approach promises early detection, potential to distinguish pseudo-progression, and assessment of treatment effectiveness with remarkable precision.

Age-associated loss of muscle mass and function and subsequent mobility decline define poor health outcomes, reduced quality of life, and mortality risk. The rate and extent of aging-related muscle loss varies across older adults. It is challenging to understand the molecular pathogenesis of mobility decline, as anthropometric and imaging techniques, primarily used in muscle function assessment, do not offer much molecular information. Small extracellular vesicles (sEV) are lipid membrane-bound, nano-sized (≤ 200 nm) vesicles which carry a wide array of biomolecules as their cargo. sEV contain cell/tissue-specific signatures on their surface and can be isolated from biofluids. These properties pose sEV as a minimally invasive means to monitor the functional and biological health of difficult-to-access tissues, establishing them as a promising liquid biopsy tool. Here, we first isolated skeletal muscle-derived sEV (sEVSKM) from the serum of vervet monkeys (16 to < 25 years old) using alpha sarcoglycan (SGCA) as a muscle-specific sEV surface marker. sEVSKM were extensively characterized for size, concentration, purity, and specificity. Further, sEVSKM isolated from young (11–15 years) and old (25–29 years) monkeys’ serum were characterized for oxidized proteins by mass spectrometry and miRNAs by small-RNAseq. Notably, the analysis of oxidized proteins indicated perturbation of metabolic pathways, actin cytoskeleton, muscle cytoskeleton regulation, and HIF-1 signaling in older monkeys. Furthermore, small-RNAseq analysis identified differential expression of several miRNAs regulating metabolic pathways, inflammation, and stress signaling. Altogether, these results suggest that it is feasible to isolate sEVSKM and use them to identify molecular biomarkers that reflect the physiological state of muscle tissue.

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. Circulating EVs are protected from degradation, making them attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labeling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g. APPswe,PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with A deposition. Genotype, age, and Aβ deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse- EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD.

685 Background: Currently, pancreatic cancer (PanC) is one of the serious gastrointestinal diseases, and more than 90% of the patients die within five years of diagnosis. Therefore, to improve PanC-related mortality, new therapeutic and diagnostic/prognostic measures are urgently needed. In this substudy, we examined the usefulness of plasma small extracellular vesicles (sEV) to discover molecular biomarkers associated with treatment response and overall survival from the plasma samples collected on a prospective clinical trial assessing the efficacy of SBRT following chemotherapy in pancreatic cancers (NCT03600623). Methods: PanCpatients (n=22) with locally advanced and borderline inoperable disease were recruited at the University of Alabama Comprehensive Cancer Center. They were administered either FOLFIRINOX (5-FU, folinic acid, oxaliplatin, and irinotecan) or paclitaxel (gemcitabine-nab-paclitaxel) for 2 months followed by SBRT (33 Gray in 5 fractions). The primary objective of this single-center pilot study was to evaluate the safety and tolerability of this treatment regimen. Further, blood was collected at baseline and, at the end of chemotherapy and radiotherapy. sEV were isolated from archived plasma samples by an established ultracentrifugation method and characterized for size and concentration by nanoparticle tracking analyses (NTA), shape and size by transmission electron microscope (TEM), and surface expression of exosomal tetraspanin markers (CD63, CD9, and CD81) and a PanC marker (CA19-9) by nano-flow cytometry. Lastly, sEV in longitudinal plasma samples were characterized for the expression of specific PanC-related miRNAs (miR196a-5p, miR155-5, miR194-5p, miR301-3p, miR21-5p, miR1246, and miR34a-5p) by real time-PCR (RT-PCR). Results: Neoadjuvant FOLFIRINOX and gemcitabine-nab-paclitaxel followed by SBRT were safe and well tolerated by most patients. NTA data showed that the ultracentrifugation method yielded highly pure sEV (with average diameter of <200 nm) from archived baseline and longitudinal plasma samples. TEM analysis further confirmed the shape and size of the isolated sEV. Nano-flow cytometry showed the expression of exosomal markers, CD63, CD9, and CD81, as well as PanC marker CA19-9 on the surfaces of sEV. The expression of various PanC-related miRNAs in sEV was heterogenous and correlated with corresponding clinical parameters, including treatment response and overall survival. Conclusions: Neoadjuvant chemotherapy in combination with SBRT is safe and tolerable regimen to treat patients with locally advanced and borderline PanC. Further, sEV in the plasma of PanC patients could serve as useful prognostic and predictive markers.