Extracellular Vesicles Protein Research Articles

Elevated extracellular vesicular Nm23-H1 subdues the pro-migratory potential of breast cancer cell-derived extracellular vesicles

Metastasis is a key determinant in cancer mortality which is often associated with decreased levels of Nm23-H1, a well-established metastasis suppressor. Despite lacking a secretion signal peptide, Nm23-H1 has been reported to be present in the extracellular space and enclosed within extracellular vesicles (EVs). While the presence of Nm23-H1 proteins in EVs released by cancer cells has been observed through proteomics profiling, the role of vesicular Nm23-H1 remains unclear. Here, we investigated the function of vesicular Nm23-H1 using MDA-MB-231 (highly metastatic, low Nm23-H1) and MCF-7 (low/non-metastatic, high Nm23-H1) breast cancer cell models. Our findings confirm that Nm23-H1 is indeed encapsulated within EVs, and its levels can be manipulated through overexpression and knockdown approaches. Functional assays revealed that EVs derived from MDA-MB-231 cells that contained high levels of Nm23-H1 exhibit impaired pro-migratory properties, suggesting that vesicular Nm23-H1 may act as a metastasis suppressor. Furthermore, EVs with increased levels of Nm23-H1 altered the transcript levels of multiple cancer-related genes in recipient cells and stimulated type I interferon signaling through STAT1 phosphorylation. These results suggest the existence of an unconventional signaling pathway mediated by the uptake of EVs enriched with Nm23-H1, which may contribute to the anti-metastatic effect of Nm23-H1 in the tumor microenvironment. Additionally, our study demonstrates that elevated Nm23-H1 levels can impact the abundance of various other proteins encapsulated within breast cancer cell-derived EVs, such as SUSD2 (Sushi Domain Containing 2) which can also modulate metastasis.

Association between NDUFS1 from urinary extracellular vesicles and decreased differential renal function in children with ureteropelvic junction obstruction

BackgroundUreteropelvic junction obstruction (UPJO) is the most common cause of pediatric congenital hydronephrosis, and continuous kidney function monitoring plays a role in guiding the treatment of UPJO. In this study, we aimed to explore the differentially expressed proteins (DEPs) in the urinary extracellular vesicles(uEVs) of children with UPJO and determine potential biomarkers of uEVs proteins that reflect kidney function changes.MethodsPreoperative urine samples from 6 unilateral UPJO patients were collected and divided into two groups: differential renal function (DRF) ≥ 40% and DRF < 40%.We subsequently used data-independent acquisition (DIA) to identify and quantify uEVs proteins in urine, screened for DEPs between the two groups, and analyzed biofunctional enrichment information. The proteomic data were evaluated by Western blotting and enzyme-linked immunosorbent assay (ELISA) in a new UPJO testing cohort.ResultsAfter one-way ANOVA, a P adj value < 0.05 (P-value corrected by Benjamin–Hochberg) was taken, and the absolute value of the difference multiple was more than 1.5 as the screening basis for obtaining 334 DEPs. After analyzing the enrichment of the DEPs according to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment combined with the protein–protein interaction (PPI) network results, we selected nicotinamide adenine dinucleotide-ubiquinone oxidoreductase core subunit S1 (NDUFS1) for further detection. The expression of NDUFS1 in uEVs was significantly lower in patients with DRF < 40% (1.182 ± 0.437 vs. 1.818 ± 0.489, P < 0.05), and the expression level of NDUFS1 was correlated with the DRF in the affected kidney (r = 0.78, P < 0.05). However, the NDUFS1 concentration in intravesical urine was not necessarily related to the change in DRF (r = 0.28, P = 0.24).ConclusionsReduced expression of NDUFS1 in uEVs might indicate the decline of DRF in children with UPJO.

Plasma-derived extracellular vesicles improve mice embryo development.

To investigate the effect of plasma-derived extracellular vesicles (EVs) or conventional medium in fertilization and early embryo development rate in mice. MII oocytes (matured in vivo or in vitro conditions) were obtained from female mice. The extracellular vesicles were isolated by ultracentrifugation of plasma and were analyzed and measured for size and morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM). By western blotting analysis, the EVs proteins markers such as CD82 protein and heat shock protein 90 (HSP90) were investigated. Incorporating DiI-labeled EVs within the oocyte cytoplasm was visible at 23h in oocyte cytoplasm. Also, the effective proteins in the early reproductive process were determined in isolated EVs by western blotting. These EVs had a positive effect on the fertilization rate (P < 0.05). The early embryo development (8 cell, morula and blastocyst stages) was higher in groups supplemented with EVs (P < 0.01). Our findings showed that supplementing in vitro maturation media with EVs derived- plasma was beneficial for mice's embryo development.

Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p<0.05). Enzyme digestion, compared to bile treatment (I60+bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p<0.05). Nanoparticle tracking analysis shows a significant reduction (p<0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

Macromolecules with predominant β-pleated sheet proteins in extracellular vesicles released from Raphanus sativus L. var. caudatus Alef microgreens induce DNA damage-mediated apoptosis in HCT116 colon cancer cells

Plant-derived bioactive macromolecules (i.e., proteins, lipids, and nucleic acids) were prepared as extracellular vesicles (EVs). Plant-derived EVs are gaining pharmaceutical research interest because of their bioactive components and delivery properties. The spherical nanosized EVs derived from Raphanus sativus L. var. caudatus Alef microgreens previously showed antiproliferative activity in HCT116 colon cancer cells from macromolecular compositions (predominantly proteins). To understand the mechanism of action, the biological activity studies, i.e., antiproliferation, cellular biochemical changes, DNA conformational changes, DNA damage, apoptotic nuclear morphological changes, apoptosis induction, and apoptotic pathways, were determined by neutral red uptake assay, synchrotron radiation-based Fourier transform infrared microspectroscopy, circular dichroism spectroscopy, comet assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, flow cytometry, and caspase activity assay, respectively. EVs inhibited HCT116 cell growth in concentration- and time-dependent manners, with a half-maximal inhibitory concentration of 675.4 ± 33.8 μg/ml at 48 h and a selectivity index of 1.5 ± 0.076. HCT116 treated with EVs mainly changed the cellular biochemical compositions in the nucleic acids and carbohydrates region. The DNA damage caused no changes in DNA conformation. The apoptotic nuclear morphological changes were associated with the increased apoptotic cell population. The apoptotic cell death was induced by both extrinsic and intrinsic pathways. EVs have potential as antiproliferative bioparticles.

Development of a Gaussia luciferase immunoprecipitation assay for detecting Schistosoma japonicum infection

Timely and accurate diagnosis of Schistosoma infection is important to adopt effective strategies for schistosomiasis control. Previously, we demonstrated that Schistosoma japonicum can secret extracellular vesicles and their cargos may serve as a novel type of biomarkers for diagnosing schistosomiasis. Here, we developed a Gaussia luciferase immunoprecipitation assay combined with S. japonicum extracellular vesicle (SjEV) protein to evaluate its potential for diagnosing schistosomiasis. A saposin-like protein (SjSLP) identified from SjEVs was fused to the Gaussia luciferase as the diagnostic antigen. The developed method showed good capability for detecting S. japonicum infection in mice and human patients. We also observed that the method could detect schistosome infection in mice as early as 7 days of post-infection, which showed better sensitivity than that of indirect ELISA method. Overall, the developed method showed a good potential for detecting Schistosoma infection particularly for early stage, which may provide an alternative strategy for identify Schistosoma infection for disease control.

The Deep Proteomics Approach Identified Extracellular Vesicular Proteins Correlated to Extracellular Matrix in Type One and Two Endometrial Cancer.

Among gynecological cancers, endometrial cancer is the most common in developed countries. Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that contain proteins involved in immune response and apoptosis. A deep proteomic approach can help to identify dysregulated extracellular matrix (ECM) proteins in EVs correlated to key pathways for tumor development. In this study, we used a proteomics approach correlating the two acquisitions-data-dependent acquisition (DDA) and data-independent acquisition (DIA)-on EVs from the conditioned medium of four cell lines identifying 428 ECM proteins. After protein quantification and statistical analysis, we found significant changes in the abundance (p < 0.05) of 67 proteins. Our bioinformatic analysis identified 26 pathways associated with the ECM. Western blotting analysis on 13 patients with type 1 and type 2 EC and 13 endometrial samples confirmed an altered abundance of MMP2. Our proteomics analysis identified the dysregulated ECM proteins involved in cancer growth. Our data can open the path to other studies for understanding the interaction among cancer cells and the rearrangement of the ECM.

The tumour-derived extracellular vesicle proteome varies by endometrial cancer histology and is confounded by an obesogenic environment.

Endometrial cancer, the most common gynaecological cancer worldwide, is closely linked to obesity and metabolic diseases, particularly in younger women. New circulating biomarkers have the potential to improve diagnosis and treatment selections, which could significantly improve outcomes. Our approach focuses on extracellular vesicle (EV) biomarker discovery by directly profiling the proteome of EVs enriched from frozen biobanked endometrial tumours. We analysed nine tissue samples to compare three clinical subgroups-low BMI (Body Mass Index) Endometrioid, high BMI Endometrioid, and Serous (any BMI)-identifying proteins related to histological subtype, BMI, and shared secreted proteins. Using collagenase digestion and size exclusion chromatography, we successfully enriched generous quantities of EVs (range 204.8-1291.0µg protein: 1.38×1011-1.10×1012 particles), characterised by their size (∼150nm), expression of EV markers (CD63/81), and proposed endometrial cancer markers (L1CAM, ANXA2). Mass spectrometry-based proteomic profiling identified 2075 proteins present in at least one of the 18 samples. Compared to cell lysates, EVs were successfully depleted for mitochondrial and blood proteins and enriched for common EV markers and large secreted proteins. Further analysis highlighted significant differences in EV protein profiles between the high BMI subgroup and others, underlining the impact of comorbidities on the EV secretome. Interestingly, proteins differentially abundant in tissue subgroups were largely not also differential in matched EVs. This research identified secreted proteins known to be involved in endometrial cancer pathophysiology and proposed novel diagnostic biomarkers (EIF6, MUC16, PROM1, SLC26A2).

Characterization and LC-MS/MS based proteomic analysis of extracellular vesicles separated from blood serum of healthy and dogs naturally infected by Babesia canis. A preliminary study

Canine babesiosis is a rapidly spreading tick-borne disease in Europe, which entails protozoan parasites invading red blood cells. Small extracellular vesicles (EVs) (< 200 nm) were isolated from the serum of 15 healthy and 15 by Babesia canis naturally infected dogs aimed to distinguish EV characteristics and protein profiles. There were no significant differences (P = 0.05) observed in the mean sizes and concentrations of serum EVs between the healthy and canine babesiosis groups. Despite a higher number of Canis lupus proteins detected in EVs from serum of diseased dogs, there were no statistically significant differences (P < 0.05) in the number of protein IDs between the experimental groups. We successfully identified 211 Canis lupus proteins across both experimental groups, of which 147 Canis lupus proteins were validated as being EV-associated. This data set is accessible via the ProteomeXchange PXD047647. EVs isolated from serum of B. canis infected dogs were Cd9+, Cd63+, Cd81+, and Cd82+. Furthermore, 73 Canis lupus proteins were validated as EV-associated and specific for EVs isolated from serum of B. canis-infected dogs. These were predominantly membrane and cytosolic proteins, and innate and adaptive immune system-related proteins, especially those involved in adhesion and proteoglycan mechanisms like integrins. Enrichment was also observed for proteins involved in vascular and cellular responses, including signalling pathways such as VEGF, VEGFR, and the LKB1 network. When only blood-related sites of EV expression were evaluated, the origins of EV proteins were mostly cells of immune system. These were dendritic cells, neutrophils, B cells, monocytes and platelets. In general, proteins were enriched in pathways that collectively regulate various cellular processes, including immune responses, communication, signal transduction, membrane trafficking, and apoptosis. Serum EVs and their protein cargo may have an important role in both the invasion of B. canis and the host's response to the parasitic infection, nevertheless, additional experimental research is warranted. The overall count of identified EV proteins of parasitic origin, meeting cut off criteria of two peptides and 1 % FDR, was relatively low.

Proteomic Identification of Small Extracellular Vesicle Proteins LAMB1 and Histone H4 for Prostate Cancer Diagnosis and Risk Stratification.

Diagnosis and stratification of prostate cancer (PCa) patients using the prostate-specific antigen (PSA) test is challenging. Extracellular vesicles (EVs), as a new star of liquid biopsy, has attracted interest to complement inaccurate PSA screening and invasiveness of tissue biopsy. In this study, a panel of potential small EV (sEV) protein biomarkers is identified from PCa cell lines using label-free LC-MS/MS proteomics. These biomarkers underwent further validation with plasma and urine samples from different PCa stages through parallel reaction monitoring-based targeted proteomics, western blotting, and ELISA. Additionally, a tissue microarray containing cancerous and noncancerous tissues is screened to provide additional evidence of selected sEV proteins associated with cancer origin. Results indicate that sEV protein LAMB1 is highly expressed in human plasma of metastatic PCa patients compared with localised PCa patients and control subjects, while sEV protein Histone H4 is highly expressed in human urine of high-risk PCa patients compared to low-risk PCa patients and control subjects. These two sEV proteins demonstrate higher specificity and sensitivity than the PSA test and show promise for metastatic PCa diagnosis, progression monitoring, and risk stratification.

Surface protein profiling and subtyping of extracellular vesicles in body fluids reveals non-CSF biomarkers of Alzheimer's disease.

Noninvasive and effortless diagnosis of Alzheimer's disease (AD) remains challenging. Here we report the multiplexed profiling of extracellular vesicle (EV) surface proteins at the single EV level in five types of easily accessible body fluids using a proximity barcoding assay (PBA). A total of 183 surface proteins were detected on the EVs from body fluids collected from APP/PS1 transgenic mice and patients with AD. The AD-associated differentially expressed EV proteins could discriminate between the control and AD/AD model samples with high accuracy. Based on machine learning predictive models, urinary EV proteins exhibited the highest diagnostic potential compared to those on other biofluid EVs, both in mice and humans. Single EV analysis further revealed AD-associated EV subpopulations in the tested body fluids, and a urinary EV subpopulation with the signature proteins PLAU, ITGAX and ANXA1 could diagnose patients with AD in blinded datasets with 88% accuracy. Our results suggest that EVs and their subpopulations from noninvasive body fluids, particularly urine, are potential diagnostic biomarkers for AD.

208 Preliminary Results of Novel Method for Identifying Ischemic Stroke Vesicular Protein Signature in Cardiac Surgery Patients

INTRODUCTION: Acute ischemic stroke (AIS) treatment depends on timely diagnosis. Identifying an efficient, blood-based protein signature for AIS has been limited by the lack of a pre-ischemia control group. Here we report the preliminary results of a model for AIS protein signature discovery using cardiac surgery patients and novel mass spectrometry techniques. METHODS: Twenty patients undergoing aortic arch repair with deep hypothermic circulatory arrest (n = 17) or coronary artery bypass graft (n = 3) were enrolled. Blood samples were obtained immediately pre- and postoperatively. Magnetic resonance imaging (MRI) of the brain was obtained within 72 hours of surgery. Presence of infarct and total ischemic volume was determined. A novel protein enrichment and mass spectrometry analysis technique (MAG-Net) was used to characterize the pre- and post-surgical proteomes present in extracellular vesicles. Analysis of variance (ANOVA) (false discovery rate &lt; 0.05) was used to analyze protein signatures associated with infarction. RESULTS: Post-surgical infarct occurred in 15 patients (75%), with a median volume of 108mm3. A total of 5,376 proteins were identified, 1,125 of which showed a significant difference between paired pre- and post-operative concentrations. Of these 1,125 proteins, 524 proteins were unique to patients without infarction, 340 proteins had a significant change in both populations, and 261 proteins were unique to patients with infarcts. There were no significant proteins for the interaction term comparing pre- and post-operative differences between the infarct/no infarct groups. CONCLUSIONS: This clinical model for AIS protein signature development relies on the high rate of ischemia during cardiac surgery to allow for pre-stroke sample collection. The MAG-Net technique enables efficient processing of biologically relevant extracellular vesicle proteins and analysis of an unbiased protein signature. The preliminary results demonstrate the feasibility of combining these innovations for AIS protein signature discovery.

Systems Approaches to Cell Culture-Derived Extracellular Vesicles for Acute Kidney Injury Therapy: Prospects and Challenges.

Acute kidney injury (AKI) is a heterogeneous syndrome, comprising diverse etiologies of kidney insults that result in high mortality and morbidity if not well managed. Although great efforts have been made to investigate underlying pathogenic mechanisms of AKI, there are limited therapeutic strategies available. Extracellular vesicles (EV) are membrane-bound vesicles secreted by various cell types, which can serve as cell-free therapy through transfer of bioactive molecules. In this review, we first overview the AKI syndrome and EV biology, with a particular focus on the technical aspects and therapeutic application of cell culture-derived EVs. Second, we illustrate how multi-omic approaches to EV miRNA, protein, and genomic cargo analysis can yield new insights into their mechanisms of action and address unresolved questions in the field. We then summarize major experimental evidence regarding the therapeutic potential of EVs in AKI, which we subdivide into stem cell and non-stem cell-derived EVs. Finally, we highlight the challenges and opportunities related to the clinical translation of animal studies into human patients.

Delivering synaptic protein mRNAs via extracellular vesicles ameliorates cognitive impairment in a mouse model of Alzheimer’s disease

BackgroundSynaptic dysfunction with reduced synaptic protein levels is a core feature of Alzheimer’s disease (AD). Synaptic proteins play a central role in memory processing, learning, and AD pathogenesis. Evidence suggests that synaptic proteins in plasma neuronal-derived extracellular vesicles (EVs) are reduced in patients with AD. However, it remains unclear whether levels of synaptic proteins in EVs are associated with hippocampal atrophy of AD and whether upregulating the expression of these synaptic proteins has a beneficial effect on AD.MethodsIn this study, we included 57 patients with AD and 56 healthy controls. We evaluated their brain atrophy through magnetic resonance imaging using the medial temporal lobe atrophy score. We measured the levels of four synaptic proteins, including synaptosome-associated protein 25 (SNAP25), growth-associated protein 43 (GAP43), neurogranin, and synaptotagmin 1 in both plasma neuronal-derived EVs and cerebrospinal fluid (CSF). We further examined the association of synaptic protein levels with brain atrophy. We also evaluated the levels of these synaptic proteins in the brains of 5×FAD mice. Then, we loaded rabies virus glycoprotein-engineered EVs with messenger RNAs (mRNAs) encoding GAP43 and SNAP25 and administered these EVs to 5×FAD mice. After treatment, synaptic proteins, dendritic density, and cognitive function were evaluated.ResultsThe results showed that GAP43, SNAP25, neurogranin, and synaptotagmin 1 were decreased in neuronal-derived EVs but increased in CSF in patients with AD, and the changes corresponded to the severity of brain atrophy. GAP43 and SNAP25 were decreased in the brains of 5×FAD mice. The engineered EVs efficiently and stably delivered these synaptic proteins to the brain, where synaptic protein levels were markedly upregulated. Upregulation of synaptic protein expression could ameliorate cognitive impairment in AD by promoting dendritic density. This marks the first successful delivery of synaptic protein mRNAs via EVs in AD mice, yielding remarkable therapeutic effects.ConclusionsSynaptic proteins are closely related to AD processes. Delivery of synaptic protein mRNAs via EVs stands as a promising effective precision treatment strategy for AD, which significantly advances the current understanding of therapeutic approaches for the disease.

Abstract 1038: Identification of diagnostic biomarkers for colon adenocarcinoma by analyzing proteins in human-derived extracellular vesicles and particles

Abstract There is an unmet medical need for circulating biomarkers to improve early cancer detection in colon adenocarcinoma (COAD). We investigated the proteomic profile of extracellular vesicles and particles (EVPs) from the colon tissue explants and plasmas in COAD. The research aims to overcome the limitations of traditional serum Carcinoembryonic Antigen (CEA) testing and invasive procedures like colonoscopy. We analyzed EVPs sourced from tumor and adjacent non-tumor tissues and preoperative and postoperative plasma in COAD patients to identify potential EVP-based tumor diagnostic markers. In the Discovery phase with 50 COAD patients, we conducted a proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS), identifying approximately 50 markers each from tissue explants and plasma. These markers were validated in an independent cohort of 104 participants, including 84 COAD patients and 20 healthy controls. We utilized ELISA to quantify 11 selected EVP proteins. Notably, seven of these proteins exhibited significant differential expression in preoperative and postoperative plasma, highlighting their diagnostic relevance. These seven proteins also showed marked elevation in preoperative plasma compared to healthy controls, reinforcing their potential as diagnostic markers. The study extended to evaluate the diagnostic accuracy of these seven proteins, referred to as "EV-7 levels", across various stages of COAD. Their performance was compared with serum CEA levels and plasma cfDNA mutation rates, as detected by an FDA-approved liquid biopsy assay. The EV-7 levels demonstrated superior detection rates in the early stages of COAD, particularly in Stages I and II, in contrast to the serum CEA method. Among the 84 patients, 73 (86.9%) exhibited EV-7 levels exceeding the established cut-off. In the validation cohort, EV-7 levels were more accurate across different COAD stages than serum CEA levels and cfDNA mutation rates. CEA levels above 5 ng/ml were 27%, 45%, 42%, and 64% for Stages I through IV. cfDNA somatic mutation rates were 60%, 80%, 75%, and 75%. EV-7 levels exceeding the cut-off were 73%, 100%, 88%, and 84%, respectively, with Stages I and II having the highest detection rate among the three methods. ROC curve analysis for EV-7 levels in Stages I to IV showed statistically significant, with AUC values of 0.913, 1.000, 0.985, and 0.984, respectively. Our findings indicate that the seven EVP proteins are significantly present in the plasma of early-stage COAD patients. Integrating these markers with current diagnostic approaches could improve early cancer detection, showing promise for practical application in clinical settings. Citation Format: Yura Seo, Yoon Dae Han, Linda Bojmar, Kyung-A Kim, Yurin Seo, David Lyden, Han Sang Kim. Identification of diagnostic biomarkers for colon adenocarcinoma by analyzing proteins in human-derived extracellular vesicles and particles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1038.

Abstract 1843: Integrating machine learning with microfluidic technologies for proteomic profiling of extracellular vesicles in triple-negative breast cancer

Abstract Accurate diagnosis of breast cancer using circulating biomarkers present in plasma remains an important challenge. In particular, protein changes in tumor-derived extracellular vesicles (tdEVs) have emerged as potential biomarkers for breast cancer diagnosis because they accurately reflect dynamic changes in tumors. In this study, we compared the proteomes of extracellular vesicle (EV) isolated from the plasma of 100 breast cancer patients and 30 healthy individuals who visited Severance Hospital between Mar 2010 and Dec 2015. Microfluidic chip-based protocol that facilitates the removal of contaminants such as albumin and immunoglobulins was used for the extraction of enriched tdEVs with small amounts of plasma. Comparative analysis of the proteomes identified 26 significant biomarkers that could indicate differences between breast cancer patients and healthy individuals. Using the LsBoost-CNN-SVM hybrid machine learning algorithm, we especially identified key EV protein biomarkers for detecting triple-negative breast cancer (TNBC), a breast cancer subtype with a poor prognosis and a high recurrence rate, as well as lacking therapeutic and diagnostic targets. Remarkably, a signature consisting of three EV proteins, specifically extracellular matrix protein 1 (ECM1), mannose-binding lectin 2 (MBL2), and biotinidase (BTD), effectively distinguished TNBC patients from healthy individuals. In our proteomic analysis set (n=73), this signature exhibited impressive performance, achieving a sensitivity of 93.3% and specificity of 93% in the accurate discrimination of TNBC from the control group. The validation set (n=40) confirmed these findings, with 100% sensitivity and 80% specificity. This signature not only served as a diagnostic tool but also provided valuable insights into the risk of recurrence and patient prognosis. We found a novel diagnostic approach that holds the potential to revolutionize breast cancer diagnostics by enhancing the reliability of tumor-related information obtained from blood samples. Citation Format: Min Woo Kim, Jee Ye Kim, Young Kim, Suji Lee, Sol Moon, Ju-yong Hyon, Kyung-A Hyun, Yeji Yang, Seongmin Ha, Sunyoung Park, Hogyeong Gawk, Haeji Lee, Eun Hee Han, Jin Young Kim, Hyo-Il Jung, Young-Ho Chung, Seung Il Kim. Integrating machine learning with microfluidic technologies for proteomic profiling of extracellular vesicles in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1843.

Abstract 1070: Overcoming deficiencies in the early detection of high grade serous ovarian cancer: Evaluating exosomal proteins as novel biomarkers of disease

Abstract Introduction: High-grade serous ovarian cancer (HGSOC) accounts for over 75% of all epithelial ovarian cancer and has a high mortality rate due to a lack of early detection methods. Many biomarkers have been proposed; however, none have been shown to improve detection at an early stage in this patient population. The purpose of this study is to assess whether there are unique extracellular vesicle (EV) protein signatures of early-stage HGSOC that could serve as early detection biomarkers. Methods: 250 serum samples were obtained from a multi-institutional retrospective cohort of patients with all stages of HGSOC and healthy controls. LC-MS/MS and PEA of serum samples from patients with early-stage HGSOC identified differentially expressed EVs proteins in HGSOC versus controls. Tob candidate proteins are validated by ELISA. Results: We have identified the top 10 EVs candidate proteins based on their fold change and statistical significance. When comparing early-stage HGSOC and controls using ELISA, CFH, PCP, and CCNE1 exhibited the highest Area Under the Curve (AUC) values of 0.94, 0.91, and 0.83, respectively. In contrast, MUC16 (also known as CA-125) exhibited an AUC of only 0.42 in whole serum. Furthermore, MUC16 distinguished between controls and both early and advanced-stage HGSOC in EVs, while in whole serum, it was only differentially expressed between controls and advanced-stage disease. Our results demonstrate that the predictive performance of the combined biomarkers surpasses that of any individual biomarker. Further, we integrated the ten aforementioned biomarkers, deriving the optimal combination biomarker by maximizing the corresponding Sum of Harmonic Means (SHUM) function by utilizing data from all 70 patients, results showed the optimal coefficients of the biomarkers, which maximize the diagnostic accuracy for early-stage HGSOC. The overall true positive rate (TPR) of 0.943, false positive rate (FPR) of 0.000, and Mathew's correlation coefficient (MCC) were presented for both the combined biomarker approach and individual biomarkers. The results demonstrated that the predictive performance of the combined biomarkers surpassed that of any individual biomarker. Conclusion: Expression of EV-based protein biomarkers is significantly different in early-stage HGSOC compared to both control and late-stage HGSOC. This work can be readily translated to clinical use and potentially improve the early detection of HGSOC substantially. Citation Format: Michelle Lightfoot, Kalpana Deepa Priya Dorayappan, Lianbo Yu, Colin Hisey, Takahiko Sakaue, Muralidharan Anbalagan, Casey M. Cosgrove, Larry G. Maxwell, Premal Thaker, Beth Y. Karlan, David O’Malley, Raphael E. Pollock, David E. Cohn, Rajan Gogna, Selvendiran Karuppaiyah. Overcoming deficiencies in the early detection of high grade serous ovarian cancer: Evaluating exosomal proteins as novel biomarkers of disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1070.

Abstract 3746: Identification of cancer specific extracellular vesical biomarkers to aid in diagnosis of cancer

Abstract The limitations of serial tumor biopsies are well known, thus liquid biopsies which allow for repeated plasma-based biomarker analysis are urgently needed. Extracellular vesicles (EVs) represent an extremely stable and emerging proteomic substrate which allows for deep enrichment of biologically relevant signals from plasma, and broad analysis of tumor associated and host response proteins. To identify novel biomarkers, we performed proteomics analysis on EVs purified using size exclusion chromatography and a proprietary buffer system which increases EV and corona protein recovery. Initial DIA mass spectrometry (MS) analysis was performed on EVs from 125 patients (25 each from CRC, breast, NSCLC, ovarian, and normal patients). A subset of 25 normal and 25 ovarian was subjected to a second deeper analysis on a much more powerful DIA platform. Initial analysis identified 204 unique proteins differentially expressed in ovarian cancer patients (&amp;gt;2-fold change; p&amp;lt;0.05). Repeat analysis of the ovarian vs normal cohorts on the Biognosys TrueDiscovery™ platform identified 645 differentially expressed, a significant increase in depth of analysis. These cancer exosome associated proteins represent a broad range of host response signals. Taking ~20 of the top proteins, we extended our analysis using ELISAs to determine if the results by MS were reproducible in a simpler format. The ELISA analysis recapitulated the MS results. This confirms that the TrueDiscovery™ platform can easily be translated into a simple, quick and distributable ELISA platform. Extending these results, a blinded cohort of ovarian and normal EVs was analyzed using a predefined cutoff for a host immune response biomarker. This blinded analysis demonstrated sensitivity of 1.0 and specificity of 0.9, exceptionally high values for a single analyte assay, underscoring the potential value of analyzing the host/immune response proteins in the context of the EV microenvironmentKey biomarkers from cancer patient EVs suggests it is possible to track host immunosuppressive environment via antigen presenting cell and check point antagonist biomarkers. In both cases, highly significant differences are seen comparing EVs from normal patient plasma versus cancer patient plasma. Interestingly, when these biomarkers are evaluated in pre-EV enriched plasma, no significant differences are seen. This underscores the unique nature of the EV microenvironment, and suggests that host immune response and tumor microenvironment may be recapitulated in the EV/host response proteome using our unique methods. These data confirm that the EV microenvironment represents a unique oncological ecosystem with highly specific and sensitive disease biomarkers which are not dysregulated in complete plasma. This novel approach to assessing cancer proteomics has great promise for real time, longitudinal analysis of cancer patient tumor/host immune biology. Citation Format: Todd A. Hembrough, Zachary Opheim, Cheryl Bandoski, Issa Isaac, Martin Mehnert, Alan Ezrin. Identification of cancer specific extracellular vesical biomarkers to aid in diagnosis of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3746.

Extracellular Vesicle Protein Expression in Doped Bioactive Glasses: Further Insights Applying Anomaly Detection.

Proteomic analysis of extracellular vesicles presents several challenges due to the unique nature of these small membrane-bound structures. Alternative analyses could reveal outcomes hidden from standard statistics to explore and develop potential new biological hypotheses that may have been overlooked during the initial evaluation of the data. An analysis sequence focusing on deviating protein expressions from donors' primary cells was performed, leveraging machine-learning techniques to analyze small datasets, and it has been applied to evaluate extracellular vesicles' protein content gathered from mesenchymal stem cells cultured on bioactive glass discs doped or not with metal ions. The goal was to provide additional opportunities for detecting details between experimental conditions that are not entirely revealed with classic statistical inference, offering further insights regarding the experimental design and assisting the researchers in interpreting the outcomes. The methodology extracted a set of EV-related proteins whose differences between conditions could be partially explainable with statistics, suggesting the presence of other factors involved in the bioactive glasses' interactions with tissues. Outlier identification of extracellular vesicles' protein expression levels related to biomaterial preparation was instrumental in improving the interpretation of the experimental outcomes.

Proteomic characterization of hUC-MSC extracellular vesicles and evaluation of its therapeutic potential to treat Alzheimer’s disease

In recent years, human umbilical cord mesenchymal stem cell (hUC-MSC) extracellular vesicles (EVs) have been used as a cell replacement therapy and have been shown to effectively overcome some of the disadvantages of cell therapy. However, the specific mechanism of action of EVs is still unclear, and there is no appropriate system for characterizing the differences in the molecular active substances of EVs produced by cells in different physiological states. We used a data-independent acquisition (DIA) quantitative proteomics method to identify and quantify the protein composition of two generations EVs from three different donors and analysed the function and possible mechanism of action of the proteins in EVs of hUC-MSCs via bioinformatics. By comparative proteomic analysis, we characterized the different passages EVs. Furthermore, we found that adaptor-related protein complex 2 subunit alpha 1 (AP2A1) and adaptor-related protein complex 2 subunit beta 1 (AP2B1) in hUC-MSC-derived EVs may play a significant role in the treatment of Alzheimer's disease (AD) by regulating the synaptic vesicle cycle signalling pathway. Our work provides a direction for batch-to-batch quality control of hUC-MSC-derived EVs and their application in AD treatment.