Integrated biomarker landscape for the early detection and management of calcific aortic valve disease

Measurable Residual Disease in Extracellular Vesicles from Bone Marrow and Peripheral Blood of Patients with Multiple Myeloma: A Proof-of-Concept Study

DETECT: Development of Technologies for Early HCC Detection

Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumor growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so, they represent an excellent potential source of novel biomarkers for the disease. In this review, we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but many studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insights into the stage of the disease and predict the likely therapeutic outcome. There remain many challenges to the use of EVs as biomarkers, but, through ongoing research and innovation in this exciting field, there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome.

Endothelial activation and alteration during dengue virus (DENV) infection are multifactorial events; however, the role of extracellular vesicles (EVs) in these phenomena is not known. In the present study, we characterized the EVs released by DENV-2 infected U937 macrophage cell line and evaluated the changes in the physiology and integrity of the EA.hy926 endothelial cells exposed to them. U937 macrophages were infected, supernatants were collected, and EVs were purified and characterized. Then, polarized endothelial EA.hy926 cells were exposed to the EVs for 24 h, and the transendothelial electrical resistance (TEER), monolayer permeability, and the expression of tight junction and adhesion proteins and cytokines were evaluated. The isolated EVs from infected macrophages corresponded to exosomes and apoptotic bodies, which contained the viral NS3 protein and different miRs, among other products. Exposure of EA.hy926 cells to EVs induced an increase in TEER, as well as changes in the expression of VE-cadherin and ICAM in addition leads to an increase in TNF-α, IP-10, IL-10, RANTES, and MCP-1 secretion. These results suggest that the EVs of infected macrophages transport proteins and miR that induce early changes in the physiology of the endothelium, leading to its activation and eliciting a defense program against damage during first stages of the disease, even in the absence of the virus.

Size Matters: Identification of Larger Size CD19 Positive Extracellular Vesicles in Chronic Lymphocytic Leukemia That Inhibit Chimeric Antigen Receptor T Cell Functions

Tumor-derived extracellular vesicles (EVs) carry tumor-specific proteins and RNAs, thus becoming prevalent targets for early cancer diagnosis. However, low expression of EV cargos and insufficient diagnostic power of individual biomarkers hindered EVs application in clinical practice. Herein, we propose a multiplex Codetection platform of proteins and RNAs (Co-PAR) for EVs. Co-PAR adopted a pair of antibody-DNA probes to recognize the same target protein, which in turn formed a double-stranded DNA. Thus, the target protein could be quantified by detecting the double-stranded DNA via qPCR. Meanwhile, qRT-PCR simultaneously quantified the target RNAs. Thus, with a regular qPCR instrument, Co-PAR enabled the codetection of multiplex proteins and RNAs, with the sensitivity of 102 EVs/μL (targeting CD63) and 1 EV/μL (targeting snRNA U6). We analyzed the coexpressions of three protein markers (CD63, GPC-1, HER2) and three RNA markers (snRNA U6, GPC-1 mRNA, miR-10b) on EVs from three pancreatic cell lines and 30 human plasma samples using Co-PAR. The diagnostic accuracy of the 6-biomarker combination reached 92.9%, which was at least 6.2% higher than that of 3-biomarker combinations and at least 13.5% higher than that of 6 single biomarkers. Co-PAR, as a multiparameter detection platform for EVs, has great potential in early disease diagnosis.

The production and release of extracellular vesicles (EV) is a property shared by all eukaryotic cells and a phenomenon frequently exacerbated in pathological conditions. The protein cargo of EV, their cell type signature and availability in bodily fluids make them particularly appealing as biomarkers. We recently demonstrated that platelets, among all types of blood cells, contain the highest concentrations of the mutant huntingtin protein (mHtt)-the genetic product of Huntington's disease (HD), a neurodegenerative disorder which manifests in adulthood with a complex combination of motor, cognitive and psychiatric deficits. Herein, we used a cohort of 59 HD patients at all stages of the disease, including individuals in pre-manifest stages, and 54 healthy age- and sex-matched controls, to evaluate the potential of EV derived from platelets as a biomarker. We found that platelets of pre-manifest and manifest HD patients do not release more EV even if they are activated. Importantly, mHtt was not found within EV derived from platelets, despite them containing high levels of this protein. Correlation analyses also failed to reveal an association between the number of platelet-derived EV and the age of the patients, the number of CAG repeats, the Unified Huntington Disease Rating Scale total motor score, the Total Functional Capacity score or the Burden of Disease score. Our data would, therefore, suggest that EV derived from platelets with HD is not a valuable biomarker in HD.

Background and Aims In recent years there has been a growth in the number of patients with chronic kidney disease (CKD). As it is known, one of the most severe complications of CKD is mineral and bone disorder (MBD). MBD, which develops in childhood, contributes not only to the development of degenerative bone disease, but also to the growth of vascular morbidity and mortality in adulthood. Therefore, adequate control of bone and mineral metabolism is one of the goals in the treatment of children with CKD. Due to the recently discovered FGF-23, a significant role in the pathogenesis of MBD is given to hyperphosphatemia as the initiator of the process. However, changes in value of phosphorus and parathyroid hormone (PTH) are revealed only on the last stages of the disease. FGF-23 is a protein, which produced in bone cells and nowadays it is considered to be the central regulator of mineral-bone metabolism. It increases the loss of phosphorus in the urine due to the blockade of the sodium-phosphorus transporter in the proximal tubule of the nephron. FGF-23 also inhibits 1α-hydroxylase and stimulates 24-hydroxylase, leading to accelerated degradation of the active form of vitamin D. Thus, the aim of our study was to investigate the relation between FGF-23 and other markers of bone metabolism such as phosphorus and parathyroid hormone. Method The study was conducted on 73 children (38 boys and 35 girls) with different stages of CKD. An average age of the patients was 9.89 ± 0.57 years. Exclusion criteria: active inflammatory, bone, infectious, oncological, immunological diseases; taking steroids and vitamin D supplements. We performed further laboratory tests: phosphorus, PTH, vitamin D and FGF-23. Serum concentration of intact FGF-23 was assessed by using the kit for ELISA method (Biomedica Medizinprodukte GmbH, Austria). This study was approved by the local scientific ethics committee of National medical university. Ethical standards and rights of patients were not violated. Descriptive statistics and correlation analysis were performed in MS Excel 2016 and SPSS 18.0. Results The laboratory tests results revealed that the mean value of phosphorus was 1.77±0.04 mmol/l among all patients with different stages of CKD. There were 29 (33.3%) children with hyperphosphatemia. The most of these patients were ESRD and they needed a renal replacement therapy. No patients with CKD 1-3 stages had high level of phosphorus. The values of PTH increase as CKD progresses. The patients with the first and second stages had absolutely normal PTH value and only 2 patients with the third stage had slightly elevated level. Only 1 out of 17 patients on dialysis (both hemodialysis and peritoneal dialysis) had an acceptable PTH value. By contrast, there was a vitamin D deficiency (a mean value was 22.4±1.64 ng/ml). The results of identification FGF-23 by the ELISA kit showed that there was also a gradual increase in its level depending on the stage of the disease. Moreover, there were 14 (19.2%) children with elevated FGF-23 concentration though other markers of bone metabolism were normal. The correlation analysis revealed positively significant associations between FGF-23 and phosphorus (r=0.60, p=0.00), FGF-23 and PTH (r=0.68, p=0.00). Conclusion Overall, our investigation proved that FGF-23 is positively correlated to phosphorus and PTH. Furthermore, in most cases, FGF-23 responds much sooner than other markers of mineral and bone metabolism and its increased value might be an early predictor of mineral and bone disorder. However, more research is required in this area.

Ovarian cancer is the most lethal gynecologic cancer in the world. Most patients are diagnosed at advanced stages with the formation of ascites. Extracellular vesicles (EVs) have been elucidated to play a pivotal role in cancer development and progression, also emerging as promising resources for clinical biomarkers in liquid biopsy. In this study, we aim to identify cancer-specific protein biomarkers in ascites-derived EVs for ovarian cancer diagnosis. Malignant ascites from high grade ovarian cancer patients (HGSOC) and benign peritoneal fluids from female patients with benign gynecologic diseases were collected with informed consent. EVs were isolated from these biofluids using the Exoquick kit. Meanwhile, HGSOC cell lines and normal cell lines were cultured in conditioned media supplemented with EVs-depleted FBS. EVs were isolated from the culture media. Proteins were extracted from EVs and fragmented into peptides. Tandem mass tag (TMT) hyper multiplex quantitation assay was carried out to detect the EV proteomic profiling. EVs isolated from biofluids and cell culture media were identified as double-membrane nanoparticles sizing ranging from 30nm to 200nm. Proteomic analysis of biofluids-derived EVs showed that malignant ascites-derived EVs displayed different proteomic profiling compared to benign peritoneal fluids-derived EVs. The differently expressed proteins between malignant ascites-derived EVs and benign peritoneal fluids-derived EVs were defined as candidate EV protein biomarkers. In addition, proteins that were expressed in at least one cancer cell line-derived EVs but not expressed in normal cell line-derived EVs were also identified as ovarian cancer-specific proteins. A diagnostic model constructed based on the integrative analysis of candidate EV protein biomarkers and ovarian cancer-specific proteins yielded a good discrimination ability between benign and cancer patients.In conclusion, this study demonstrates the distinct proteomic profiling in ascites- and cancer cell-derived EVs in ovarian cancer, which sheds new light on implementing EV proteins as a potential diagnostic strategy in clinical settings. Citation Format: Wenyu Wang, Dohyun Han, Yong Sang Song. Proteomic analysis of ascites- and cancer cell-derived EVs for identifying ovarian cancer diagnostic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5081.

Simple SummaryWe define the transcriptome and secretome of primary LSECs during the progression of cirrhosis, revealing specific molecular signatures, novel biomarkers and therapeutic targets for new disease-modifying treatments for patients with advanced chronic liver disease.The poor prognosis of chronic liver disease (CLD) generates the need to investigate the evolving mechanisms of disease progression, thus disclosing therapeutic targets before development of clinical complications. Considering the central role of liver sinusoidal endothelial cells (LSECs) in pre-neoplastic advanced CLD, the present study aimed at investigating the progression of CLD from an endothelial holistic perspective. RNAseq defined the transcriptome of primary LSECs isolated from three pre-clinical models of advanced CLD, during the progression of the disease, and from fresh human cirrhotic tissue. At each stage of the disease, the effects of LSECs secretome on neighboring cells and proteomic analysis of LSECs-derived extracellular vesicles (EVs) were also determined. CLD was associated with deep common modifications in the transcriptome of LSECs in the pre-clinical models. Pathway enrichment analysis showed predominance of genes related with pro-oncogenic, cellular communication processes, and EVs biogenesis during CLD progression. Crosstalk experiments revealed endothelial EVs as potent angiocrine effectors. The proteome of LSECs EVs showed stage-specific signatures, including over-expression of tropomyosin-1. Proof-of-principle experiments treating cirrhotic HSCs with recombinant tropomyosin-1 suggested de-activating effects. Our data provide the basis for discovering novel biomarkers and therapeutic targets for new disease-modifying treatments for patients with advanced CLD.

Recent findings indicate that urinary extracellular vesicles (EVs) might reflect the pathophysiological state of urinary system; and that EVs-induced ciliary signaling is a possible mechanism of intercellular communication within the tract. Here, we aimed to analyze the protein expression of urinary EVs during autosomal dominant polycystic kidney disease (ADPKD). EVs were isolated from pooled urine samples of healthy control and ADPKD patients at two different stages of the disease and under tolvaptan treatment using the double-cushion ultracentrifugation method. Proteins were identified and quantified by iTRAQ and multidimensional protein identification technology (MudPIT)-based quantitative proteomics. Quantitative analyses identified 83 differentially expressed EV proteins. Many of these have apical membrane origin and are involved in signal transduction pathways of primary cilia, Ca(2+) -activated signaling, cell-cycle regulation, and cell differentiation. The reduced AQP-2 and the increased APO-A1 levels observed in all ADPKD-affected groups may reflects the impaired renal concentrating capability of these patients and correlated with estimated glomerular filtration rate decline. The levels of some upregulated proteins involved in Ca(2+) -activated signaling declined upon tolvaptan treatment. The results obtained suggest that the quantitative proteomics of urinary EVs might be useful to monitor proteins difficult to access noninvasively, and thus advance our understanding of urinary tract physiology and pathology.

Hypertension and cardiac hypertrophy initiate molecular pathways that can result in heart failure (HF). Despite the importance of cardiac hypertrophy as a risk factor for the development of HF, not all hypertrophied hearts will develop HF. Possibly, HF‐prone hearts are molecularly defined in the early stages of the disease, before the transition to HF. HF diagnosis is most often made at an advanced stage of the disease. This determines the need of markers at the earliest possible stage of the disease. Circulating microRNAs have been considered novel candidate biomarkers for HF. The aim of the present study was to identify if plasma extracellular vesicles (EVs) microRNAs could be used as biomarkers during the transition of compensated cardiac hypertrophy to HF in rats submitted to abdominal aorta constriction. Previous published data from our group showed that at 60‐days post‐surgery (dps) there was 2 groups: controls and operated animals with compensated cardiac hypertrophy and preserved systolic function. At 90 dps, the operated group was subdivided into 2 groups: animals with hypertrophic hearts (resistance to heart failure, R‐HF), around 70% of the animals, and animals with hypertrophic + dilated hearts (HF), around 30%. Since at 60 dps there was no difference among operated animals by echocardiography and there is no signal which animal will develop HF, a retrospective study was performed: plasma microRNAs from animals at 90 dps (HF‐90dps and R‐HF‐90dps) were compared to the plasma from the same animals earlier at 60 dps (HF‐60dps and R‐HF‐60dps) to identify possible microRNAs that could be used as early biomarker of HF. Plasma was collected before surgery and at 60 and 90 dps. Ejection fraction (EF) was evaluated by echocardiography. Plasma circulating microparticles concentration and size were characterized by Nanoparticle tracking analysis. Relative levels of 312 rat miRNAs were evaluated by RT‐qPCR at 60 dps. Data were considered significant when p<0.05. There is no difference in the EF among sham (61.85±6.62%), R‐HF‐60dps (59.50±8.05%) and 60dps (62.07±8.91%) and R‐HF‐90dps (56.33±7.76%). There is no difference in the particle concentration among sham (3.24x1011/ml), R‐HF‐60dps (5.48x1011/ml) and HF‐60dps (3.54x1011/ml). The same was seen in relation to particles size: sham (104.98 nm), R‐HF‐60dps (104.88 nm) and HF‐60dps (107.25 nm). The microRNA profiling detected a total of 191 miRNAs present in sham, R‐HF‐60dps and HF‐60dps. Of these, a total of 42 miRNA were upregulated in the group HF‐60dps when compared to R‐HF‐60dps (let‐7b‐5p; let‐7d‐3p; 125a‐5p; 127‐3p; 130a‐3p; 149‐5p; 30d‐5p; 30e‐5p; 31b; 34a‐3p; 423‐5p; 499‐5p; 145‐5p; 146a‐3p; 15a‐3p; 181d‐5p; 182, 185‐5p; 193b‐5p; 211‐5p; 322‐3p; 375‐5p; 351‐5p; 500‐3p; 92b‐3p; 212‐3p; 22‐3p; 21‐5p; 221‐3p; 27b‐3p; 21‐5p; 221‐3p; 27b‐3p; 301a‐3p; 28‐3p; 301b‐3p; 30a‐5p; 486; 423‐3p; 497‐5p; 671 and 99a‐5p). Further studies at 90 dps should be performed. These 41 upregulated miRNAs need to be compared to the miRNA profiling at 90 dps. If any of these miRNAs are still upregulated at 90 dps, the predicted pathways regulated by these miRNAs can be analyzed and these miRNAs could be used as a biomarker of HF in these animals.

MicroRNAs (miRNAs) in extracellular vesicles (EVs) play essential roles in cancer initiation and progression. Quantitative measurements of EV miRNAs are critical for cancer diagnosis and longitudinal monitoring. Traditional PCR‐based methods, however, require multi‐step procedures and remain as bulk analysis. Here, the authors introduce an amplification‐free and extraction‐free EV miRNA detection method using a CRISPR/Cas13a sensing system. CRISPR/Cas13a sensing components are encapsulated in liposomes and delivered them into EVs through liposome‐EV fusion. This allows for accurately quantify specific miRNA‐positive EV counts using 1 × 108 EVs. The authors show that miR‐21‐5p‐positive EV counts are in the range of 2%–10% in ovarian cancer EVs, which is significantly higher than the positive EV counts from the benign cells (<0.65%). The result show an excellent correlation between bulk analysis with the gold‐standard method, RT‐qPCR. The authors also demonstrate multiplexed protein‐miRNA analysis in tumor‐derived EVs by capturing EpCAM‐positive EVs and quantifying miR‐21‐5p‐positive ones in the subpopulation, which show significantly higher counts in the plasma of cancer patients than healthy controls. The developed EV miRNA sensing system provides the specific miRNA detection method in intact EVs without RNA extraction and opens up the possibility of multiplexed single EV analysis for protein and RNA markers.

Matrix metalloproteinases are elevated in the urine of patients with endometriosis

A magnetic bead-mediated selective adsorption strategy for extracellular vesicle separation and purification