Exosomal Levels Research Articles

Antitumor activity of bintrafusp alfa in previously treated patients with recurrent or metastatic nasopharyngeal cancer (NPC): A single arm, prospective phase II trial.

e18029 Background: Patients with recurrent or metastatic nasopharyngeal cancer (R/M NPC) who failed platinum-based chemotherapy have poor prognoses. We report the clinical activity and safety of bintrafusp alfa, a first-in-class bifunctional fusion protein composed of the extracellular domain of the transforming growth factor (TGF)-βRII receptor fused to a human IgG1 antibody blocking programmed death-ligand 1 (PD-L1), in patients with heavily pretreated R/M NPC. Tumor and plasma-based biomarkers were investigated in an exploratory analysis. Methods: Eligible patients had histologically confirmed NPC that had recurred at distant sites and were not amenable to curative treatment. All patients received at least one prior line of platinum-based chemotherapy for recurrent disease. Patients were treated with bintrafusp alfa (1200mg every 2 weeks) until disease progression. The sample size was estimated to assume a 40% objective response rate (ORR) to bintrafusp alfa compared with 20% for checkpoint inhibitors. Modified Simon two-stage optimal design was used (power, 80%; a = 0.05; P0 = 0.20; P1 = 0.40; n1 = 18; n = 33 with an additional five patients to allow for ineligibility or other reasons). The primary endpoint was ORR and secondary endpoints included survival and toxicity. Expression of PD-L1 in archived tumors, plasma clearance of Epstein-Barr virus (EBV) DNA, plasma clearance of TGF-β, and exosomal PD-L1 were assessed for a potential correlation with ORR. (NCT 04396886). Results: Out of 43 patients screened, 38 patients were enrolled. After a median follow-up of 14.9 months (range: 1.6-23.3 months), the confirmed ORR was 23.7% (95% CI: 12.4-38.8%) (complete response, n = 1; partial response, n = 8). The median treatment duration was 1.8 months (range: 0.5-14.3 months). 8 patients (21.1%) and 2 patients (5.3%) received bintrafusp alfa for > 6 months and > 12 months respectively. The 1-year overall survival (OS) rate was 57.5% (95% CI, 40.2% to 71.5%) and 1-year progression-free survival rate was 23% (95% CI, 10.1% to 39.4%). ORR was higher in patients with a decreasing trend in EBV-DNA at week 4 (40% vs. 6.3%, p = 0.02), whereas high exosomal PD-L1 levels at week 4 were predictive of worse ORR (5.3% vs. 41.7%, p = 0.012). There were no associations between clinical outcome and tissue PD-L1 expression (p = 0.952) or plasma TGF-β clearance (p = 0.28). 16 patients (42.4%) experienced ≥ grade 3 treatment-related adverse events, most commonly anemia (n = 9, 23.7%) and secondary malignancies (n = 4, 10.5%). Conclusions: Bintrafusp alfa has promising activity in heavily pretreated R/M NPC and a favorable 1-year OS rate, though the observed activity was not as high as the study initially aimed. The biomarker results warrant validation in larger cohorts. Clinical trial information: NCT04396886.

Blood-based miRNA, exosomal PD-L1, and immune profiling analysis as predictive biomarkers for immune checkpoint inhibitor treatment response in urothelial cancer.

e16559 Background: Predicting response to immune checkpoint inhibitor (ICI) is difficult and adequate biomarkers are lacking. PD-L1 expression of tumor cells (TC) and tumor-infiltrating immune cells (IC) has limitations. We hypothesized that peripheral blood analysis would provide systemic insights about the immune responses induced by therapies blocking the PD-1/PD-L1 pathway. Methods: First, plasma samples from urothelial cancers (N = 30) with matched paired samples (N = 27) at baseline, after 1 week of PD-L1 inhibitor administration, and at response evaluation were analyzed. Exosomes were extracted and the expression of plasma-derived exosomal PD-L1 (exo PD-L1) and cytokine levels (IFN-γ, IL-10, TGF-ß, granzyme B) were determined with ELISA. Peripheral blood mononuclear cells (PBMCs) were analyzed with FACS. Samples were stained with anti-CD3, CD8, CD69, and PD1. Secondly, small RNA-sequencing was done using plasma samples from 8 patients and samples from 4 normal ones to identify differentially expressed miRNAs (DE-miRNAs) between treatment responders and non-responders. Expressions of selected DE-miRNAs were validated in a separate patient cohort of 54 patients. Results: In all of patients’ samples tested, exoPD-L1 was detected. Pretreatment level of exoPD-L1 was significantly higher in patients with objective tumor response (P = 0.010). For both clinical responders and non-responders, the level of exoPD-L1 expression increased at treatment day 8. A lower fold change (< 18) in plasma exoPD-L1 was associated with a better rate of response to the anti-PD-L1 therapy (P = 0.032). Compared to non-responder group, the PD1+CD8+T-cell fraction was significantly lower at baseline (p = 0.005) and increased at treatment day 8 in the responder group (p = 0.018). At baseline, the IL-10 level was significantly higher in the responder group (p = 0.005). Decreased levels of immune-suppressive cytokines, TGF-beta1/2 and IL-10, at post-treatment day 8 indicated better response (p = 0.005 and 0.020, respectively). From small RNA sequencing analysis, two upregulated (miR-451a and let-7i-5p) and eight downregulated (miR-1290, miR-151a-3p, miR-10b-5p, miR-148a-3p, miR-10a-5p, miR-1246, miR-34c-3p, let-7b-3p) differentially expressed miRNA in responders were identified. Conclusions: Our findings suggested that the higher expression of exoPD-L1, early peripheral PD1+CD8+T-cell response, and decrease of immunosuppressive cytokines could serve as blood-based predictive biomarkers for responses to ICI treatment and that miRNAs could act as regulators in these processes.

Evaluation of miRNA-21-5p and miRNA-10b-5p levels in serum-derived exosomes of breast cancer patients in different grades

Background & objectivesTumor cells have various effects and dominance over other healthy cells. Cancer cells alter the cell program in healthy cells by secreting exosomes containing microRNAs involved in epithelial-mesenchymal transition (EMT). They can migrate to distant organs and establish a pre-metastatic niche. The purpose of this study was to determine the expression of miRNA-21–5p and miRNA-10b-5p, both of which are involved in EMT, in breast cancer-derived exosomes of various grades in order to identify new biomarkers involved in breast cancer progression. MethodsIn this study, a blood sample was taken from 60 patients with grades I, II, or III breast cancer, as well as twenty healthy individuals as a control group. The exosomes were then purified from serum samples, and their relative expression of miRNA-21–5p and miRNA-10b-5p was determined using the real-time PCR method. ResultsmiRNA-21–5p expression was significantly increased in patients with breast cancer grades I, II, and III compared to the control group (p < 0.01), (p < 0.0001) and (p < 0.0001), respectively, as was miRNA-10b-5p expression in patients with breast cancer grades I, II, and III compared to the control group (p < 0.0001), (p < 0.0001) and (p < 0.0001), respectively. ConclusionOur results show that both microRNAs increase as cells lose their differentiation and become more invasive, which is evidence of cancer progression. Hence, both microRNAs may have the potential to be used alone or in combination with other biomarkers for the diagnosis and prognosis of breast cancer.

Blocking exosomal secretion aggravates 1,4-benzoquinone-induced mitochondrial fission activated by the AMPK/MFF/Drp1 pathway in HL-60 cells.

There is in vivo and in vitro evidence that exposure to benzene or its metabolites could affect the mitochondrial function. However, the underlying molecular mechanism of mitochondrial damage remains to be elucidated. In this study, exposure of human promyelocytic leukemia cells (HL-60) to 1,4-benzoquinone (1,4-BQ; an active metabolite of benzene) increased the intracellular reactive oxygen species levels, decreased the mitochondrial membrane potential, adenosine triphosphate production and mitochondrial DNA (mtDNA) copy number, up-regulated the expression of mitochondrial fission proteins Drp1 and Fis1, and down-regulated the expression of mitochondrial fusion proteins Mfn2 and Opa1. Further study showed that 1,4-BQ mediated mitochondrial fission through activation of the AMP-activated protein kinase/mitochondrial fission factor/dynamin-related protein 1 pathway. Additionally, we also examined the role of exosomal secretion in mitochondrial damage under 1,4-BQ treatment. Results showed that 1,4-BQ increased the total protein level and mtDNA content in exosomes. Upon pre-treatment with the mitochondria-targeted antioxidant SS-31, there was attenuation of the mitochondrial damage induced by 1,4-BQ, accompanied by a change in the exosome release characteristics, while inhibition of exosomal secretion using GW4869 aggravated the 1,4-BQ-mediated mitochondrial fission. We concluded that exosomal secretion may serve as a self-protective mechanism of cells against 1,4-BQ-induced mitochondria damage and mitochondrial dynamics interference.

Circulating exosome miRNA, is it the novel nutrient molecule through cross-kingdom regulation mediated by food chain transmission from microalgae to bivalve?

MicroRNAs (miRNAs) can efficiently regulate gene expression at intracellular and extracellular levels. Plant-derived miRNAs are highly enriched in animal haemolymph and regulate mammalian gene expression. However, evidence for food-derived miRNAs in Mollusca species is lacking. In this study, we fed the microalga Nannochloropsis oculata to the pearl oyster Pinctada fucata martensii and detected dietary miRNAs in exosomes isolated from the haemolymph by RNA-seq. In total, 273 endogenous miRNAs were identified in all biological replicates. We identified 23 microalgae-derived miRNAs in the exosomes of pearl oyster haemolymph. Most microalgae-derived miRNAs showed high expression levels in both exosomes and microalgae and exhibited apparent variation among individuals. These food-derived miRNAs were predicted to participate in endocytosis, apoptosis, signal transduction, energy metabolism, and biomineralization by targeting multiple genes. These findings demonstrated the cross-kingdom transport of miRNAs from microalgae to bivalves and provide insights into novel nutrient transmission through the food chain.

Exosomal Metabolic Signatures Are Associated with Differential Response to Neoadjuvant Chemotherapy in Patients with Breast Cancer.

Neoadjuvant chemotherapy (NAC) is commonly used in breast cancer (BC) patients to increase eligibility for breast-conserving surgery. Only 30% of patients with BC show pathologic complete response (pCR) after NAC, and residual disease (RD) is associated with poor long-term prognosis. A critical barrier to improving NAC outcomes in patients with BC is the limited understanding of the mechanisms underlying differential treatment outcomes. In this study, we evaluated the ability of exosomal metabolic profiles to predict NAC response in patients with BC. Exosomes isolated from the plasma of patients after NAC were used for metabolomic analyses to identify exosomal metabolic signatures associated with the NAC response. Among the 16 BC patients who received NAC, eight had a pCR, and eight had RD. Patients with RD had 2.52-fold higher exosome concentration in their plasma than those with pCR and showed significant enrichment of various metabolic pathways, including citrate cycle, urea cycle, porphyrin metabolism, glycolysis, and gluconeogenesis. Additionally, the relative exosomal levels of succinate and lactate were significantly higher in patients with RD than in those with pCR. These data suggest that plasma exosomal metabolic signatures could be associated with differential NAC outcomes in BC patients and provide insight into the metabolic determinants of NAC response in patients with BC.

Human adipose-derived mesenchymal stem cells prevent type 1 diabetes induced by immune checkpoint blockade

Aims/hypothesisImmunomodulators blocking cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) or programmed death-ligand 1 (PD-L1) have improved the treatment of a broad spectrum of cancers. These immune checkpoint inhibitors (ICIs) reactivate the immune system against tumour cells but can also trigger autoimmune side effects, including type 1 diabetes. Mesenchymal stem cell (MSC) therapy is the most prevalent cell therapy, with tissue-regenerating, anti-fibrosis and immunomodulatory functions provided by the secretome of the cells. Here, we examined whether systemic MSC treatment could prevent the development of type 1 diabetes in a NOD mouse model.MethodsThe purified PD-L1 monoclonal antibody was administered to induce diabetes in male NOD mice which normally do not develop diabetes. Human adipose-derived MSCs were administered by tail vein injections. T cells, macrophages and monocyte-derived macrophages expressing C-X-C motif chemokine ligand 9 (CXCL9) in pancreatic sections of NOD mice and a cancer patient who developed diabetes following the ICI treatments were analysed by immunofluorescence. Tissue localisation of the injected MSCs, plasma exosome levels and plasma cytokine profiles were also investigated.ResultsPD-1/PD-L1 blockade induced diabetes in 16 of 25 (64%) NOD mice which received anti-PD-L1 mAb without hMSCs [MSC(−)], whereas MSC administration decreased the incidence to four of 21 (19%) NOD mice which received anti-PD-L1 mAb and hMSCs [MSC(+)]. The PD-1/PD-L1 blockade significantly increased the area of CD3-positive T cells (6.2-fold) and macrophage-2 (Mac-2) antigen (2.5-fold)- and CXCL9 (40.3-fold)-positive macrophages in the islets. MSCs significantly reduced T cell (45%) and CXCL9-positive macrophage (67%) accumulation in the islets and the occurrence of diabetes. The insulin content (1.9-fold) and islet beta cell area (2.7-fold) were also improved by MSCs. T cells and CXCL9-positive macrophages infiltrated into the intricate gaps between the beta cells in the islets by PD-1/PD-L1 blockade. Such immune cell infiltration was largely prevented by MSCs. The most striking difference was observed in the CXCL9-positive macrophages, which normally did not reside in the beta cell region in the islets but abundantly accumulated in this area after PD-1/PD-L1 blockade and were prevented by MSCs. The CXCL9-positive macrophages were also observed in the islets of a cancer patient who developed diabetes following the administration of ICIs but few CXCL9-positive macrophages were observed in a control patient. Mechanistically, the injected MSCs accumulated in the lung but not in the pancreas and strongly increased plasma exosome levels and changed plasma cytokine profiles.Conclusions/interpretationOur results suggest that MSCs can prevent the incidence of diabetes associated with immune checkpoint cancer therapy and may be worth further consideration for new adjuvant cell therapy.Graphical abstract

FC049: Nefecon® (Budesonide) is an Epigenetic Modifier in IGA Nephropathy

Abstract BACKGROUND AND AIMS In recent years, the dysregulated expression of microRNAs (miRs)—small molecules of noncoding RNA that epigenetically fine-tune post-transcriptional gene expression—has been increasingly implicated in the pathogenesis of IgA nephropathy (IgAN). Extracellular miRs, contained within extracellular vesicles such as exosomes and microvesicles, are thought to function as important intercellular and interorgan modes of communication in health and disease. In the current study, next-generation sequencing (NGS) and subsequent real-time quantitative polymerase chain reaction (RT-qPCR) validation were performed to identify serum miRs associated with a high risk of future kidney function decline in IgAN. We then went on to determine whether treatment with Nefecon® 16 mg/day for 9 months was capable of modifying the serum levels of miRs associated with high-risk IgAN. This study was supported by a grant from Calliditas Therapeutics AB. METHOD NGS was performed by Qiagen (Germany) on serum from patients with IgAN (10 at high risk and 10 at low risk of progression) or membranous nephropathy (MN, n=10), as well as healthy individuals (HS, n=10). Sequencing data were validated by RT-qPCR using TaqMan miR PCR assays in independent sets of serum samples (20 per cohort). Using the Total Exosome Isolation (from serum) reagent (Thermo Fisher Scientific, UK), extracellular exosomes were isolated from the serum of participants of the NEFIGAN trial from the placebo group (n=40) and Nefecon® 16 mg/day group (n=40) at the start of treatment (SOT) and end of treatment (EOT). RNA was isolated and levels of miRs-483-5p and -122-5p were measured by RT-qPCR. The NEFIGAN trial (NCT01738035) was a randomized, double-blind, placebo-controlled Phase 2b trial designed to assess the safety and efficacy of Nefecon® a novel targeted-release formulation of budesonide, designed to deliver the drug to the distal ileum in patients with IgAN. The trial comprised a 6-month run-in, a 9-month treatment and a 3-month follow-up phase. A total of 48 patients received Nefecon® 16 mg/day, 51 patients received Nefecon® 8 mg/day and 50 patients received placebo. The key result of the study was that Nefecon® 16 mg/day, added to optimized renin–angiotensin system blockade, reduced proteinuria and stabilized estimated glomerular filtration rate in patients with IgAN. These findings have now been replicated in the NefIgArd study, which released data in 2021. RESULTS miRs-483-5p and -122-5p were significantly overexpressed in the serum of patients with IgAN at high risk of progression compared with those who had stable IgAN or HS, but not compared with patients with MN. Both miRs were also found to be significantly increased in extracellular exosomes in high-risk IgAN compared with low-risk IgAN, HS and MN. Interestingly, levels of exosomal miRs-483-5p and -122-5p did not correlate with total serum IgA, IgA1, galactose-deficient IgA1 or IgA–IgG immune complex levels, suggesting a novel mechanism of action. Treatment with 16 mg/day of Nefecon® led to a significant reduction in the exosomal levels of miR-122-5p, while no change in miR-483-5p levels was observed in the treated group. CONCLUSION These data reveal a dysregulation of systemic exosomal miR expression in IgAN, and demonstrate for the first time that it is possible to pharmacologically modify the miR content of circulating exosomes in IgAN. Treatment with Nefecon® 16 mg/day for 9 months selectively downregulated exosomal miR-122-5p, while having no effect on exosomal miR-483-5p levels, arguing against a generalized ‘anti-miR’ effect of Nefecon®. Studies are ongoing to both precisely delineate the cellular source(s) of these exosomes and determine the downstream impact of these changes on glomerular and tubulointerstitial biology.

Amyloid-β protein and MicroRNA-384 in NCAM-Labeled exosomes from peripheral blood are potential diagnostic markers for Alzheimer's disease.

ObjectiveWe aimed to establish a method to determine whether amyloid‐β (Aβ) protein and miR‐384 in peripheral blood neural cell adhesion molecule (NCAM)/ATP‐binding cassette transporter A1 (ABCA1) dual‐labeled exosomes may serve as diagnostic markers for the diagnosis of Alzheimer's disease (AD).MethodsThis was a multicenter study using a two‐stage design. The subjects included 45 subjective cognitive decline (SCD) patients, 50 amnesic mild cognitive impairment (aMCI) patients, 40 AD patients, and 30 controls in the discovery stage. The results were validated in the verification stage in 47 SCD patients, 45 aMCI patients, 45 AD patients, and 30 controls. NCAM single‐labeled and NCAM/ABCA1 double‐labeled exosomes in the peripheral blood were captured and detected by immunoassay.ResultsThe Aβ42, Aβ42/40, Tau, P‐T181‐tau, and miR‐384 levels in NCAM single‐labeled and NCAM/ABCA1 double‐labeled exosomes of the aMCI and AD groups were significantly higher than those of the SCD, control, and vascular dementia (VaD) groups (all p < 0.05). The Aβ42 and miR‐384 levels in NCAM/ABCA1 dual‐labeled exosomes of the aMCI and AD groups were higher than those of the control and VaD groups (all p < 0.05). The exosomal Aβ42, Aβ42/40, Tau, P‐T181‐tau, and miR‐384 levels in peripheral blood were correlated with those in cerebrospinal fluid (all p < 0.05).ConclusionThis study, for the first time, established a method that sorts specific surface marker exosomes using a two‐step immune capture technology. The plasma NCAM/ABCA1 dual‐labeled exosomal Aβ42/40 and miR‐384 had potential advantages in the diagnosis of SCD.

Exosomal miR-4466 from nicotine-activated neutrophils promotes tumor cell stemness and metabolism in lung cancer metastasis

Smoking is associated with lung cancer and has a profound impact on tumor immunity. Nicotine, the addictive and non-carcinogenic smoke component, influences various brain cells and the immune system. However, how long-term use of nicotine affects brain metastases is poorly understood. We, therefore, examined the mechanism by which nicotine promotes lung cancer brain metastasis. In this study, we conducted a retrospective analysis of 810 lung cancer patients with smoking history and assessed brain metastasis. We found that current smoker’s lung cancer patients have significantly higher brain metastatic incidence compared to the never smokers. We also found that chronic nicotine exposure recruited STAT3-activated N2-neutrophils within the brain pre-metastatic niche and secreted exosomal miR-4466 which promoted stemness and metabolic switching via SKI/SOX2/CPT1A axis in the tumor cells in the brain thereby enabling metastasis. Importantly, exosomal miR-4466 levels were found to be elevated in serum/urine of cancer-free subjects with a smoking history and promote tumor growth in vivo, suggesting that exosomal miR-4466 may serve as a promising prognostic biomarker for predicting increased risk of metastatic disease among smoker(s). Our findings suggest a novel pro-metastatic role of nicotine-induced N2-neutrophils in the progression of brain metastasis. We also demonstrated that inhibiting nicotine-induced STAT3-mediated neutrophil polarization effectively abrogated brain metastasis in vivo. Our results revealed a novel mechanistic insight on how chronic nicotine exposure contributes to worse clinical outcome of metastatic lung cancer and implicated the risk of using nicotine gateway for smoking cessation in cancer patients.

Bu Shen Yi Sui Capsules Promote Remyelination by Regulating MicroRNA-219 and MicroRNA-338 in Exosomes to Promote Oligodendrocyte Precursor Cell Differentiation.

Remyelination is a refractory feature of demyelinating diseases such as multiple sclerosis (MS). Studies have shown that promoting oligodendrocyte precursor cell (OPC) differentiation, which cannot be achieved by currently available therapeutic agents, is the key to enhancing remyelination. Bu Shen Yi Sui capsule (BSYSC) is a traditional Chinese herbal medicine over many years of clinical practice. We have found that BSYSC can effectively treat MS. In this study, the effects of BSYSC in promoting OPCs differentiation and remyelination were assessed using an experimental autoimmune encephalomyelitis (EAE) model in vivo and cultured OPCs in vitro. The results showed that BSYSC reduced clinical function scores and increased neuroprotection. The expression of platelet-derived growth factor receptor α (PDGFR-α) was decreased and the level of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) was increased in the brains and spinal cords of mice as well as in OPCs after treatment with BSYSC. We further found that BSYSC elevated the expression of miR-219 or miR-338 in the serum exosomes of mice with EAE, thereby suppressing the expression of Sox6, Lingo1, and Hes5, which negatively regulate OPCs differentiation. Therefore, serum exosomes of BSYSC-treated mice (exos-BSYSC) were extracted and administered to OPCs in which miR-219 or miR-338 expression was knocked down by adenovirus, and the results showed that Sox6, Lingo1, and Hes5 expression was downregulated, MBP expression was upregulated, OPCs differentiation was increased, and the ability of OPCs to wrap around neuronal axons was improved. In conclusion, BSYSC may exert clinically relevant effects by regulating microRNA (miR) levels in exosomes and thus promoting the differentiation and maturation of OPCs.

Paper-Based Devices for Capturing Exosomes and Exosomal Nucleic Acids From Biological Samples.

Exosomes, nanovesicles derived from cells, contain a variety of biomolecules that can be considered biomarkers for disease diagnosis, including microRNAs (miRNAs). Given knowledge and demand, inexpensive, robust, and easy-to-use tools that are compatible with downstream nucleic acid detection should be developed to replace traditional methodologies for point-of-care testing (POCT) applications. This study deploys a paper-based extraction kit for exosome and exosomal miRNA analytical system with some quantifying methods to serve as an easy sample preparation for a possible POCT process. Exosomes concentrated from HCT116 cell cultures were arrested on paper-based immunoaffinity devices, which were produced by immobilizing anti-CD63 antibodies on Whatman filter paper, before being subjected to paper-based silica devices for nucleic acids to be trapped by silica nanoparticles adsorbed onto Whatman filter paper. Concentrations of captured exosomes were quantified by enzyme-linked immunosorbent assay (ELISA), demonstrating that paper-based immunoaffinity devices succeeded in capturing and determining exosome levels from cells cultured in both neutral and acidic microenvironments, whereas microRNA 21 (miR-21), a biomarker for various types of cancers and among the nucleic acids absorbed onto the silica devices, was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) to prove that paper-based silica devices were capable of trapping exosomal nucleic acids. The developed paper-based kit and the devised procedure was successfully exploited to isolate exosomes and exosomal nucleic acids from different biological samples (platelet-poor plasma and lesion fluid) as clinical applications.

The Value of Serum Exosomal miR-184 in the Diagnosis of NSCLC.

Lung cancer has the highest morbidity rate (11.6%) and mortality rate (18.4%) among all current tumors. The morbidity rate in China accounts for approximately one-third, and it is still rising. Nonsmall cell lung cancer is the most common type of lung cancer, accounting for 80%–85% of all lung cancers, and approximately 57% of patients with advanced nonsmall cell lung cancer have distant metastases at the time of diagnosis. To explore the expression changes in microRNA-184 (miR-184) and its clinical value in serum exosomes of patients with nonsmall cell lung cancer (NSCLC). This study adopted a case-control study method, selecting 88 patients (NSCLC group) from June 2015 to June 2017 in our hospital who are confirmed to have NSCLC by fiber-optic bronchoscopy, and 90 patients who are confirmed to have benign lung diseases by pathological examination during the same period (control group). Fluorescence quantitative PCR technology is used to detect the levels of miR-184 in serum exosomes of the two groups, and the differences in the levels of miR-184 in serum exosomes of NSCLC patients with different pathological characteristics are analyzed. According to the results of the 3-year follow-up, the miR-184 levels in serum exosomes of NSCLC patients are grouped and compared. The expression level of miR-184 in serum exosomes in the NSCLC group is significantly higher than that in the control group, and the difference between the two groups is statistically significant (p < 0.05). The ROC curve is drawn with the expression level of miR-184 in serum exosomes of the two groups of patients. The results showed that the area under the ROC curve for the differential diagnosis of NSCLC and benign lung tumors with the expression level of miR-184 in serum exosomes is 0.927, and the sensitivity is 87.61%, while the specificity is 84.02%. The expression levels of miR-184 in serum exosomes of NSCLC patients with different pathological characteristics, in different TNM stages [(I+II) vs. (III+IV)], lymph node metastasis (yes vs. no), and different degrees of differentiation [(High + Medium) vs. Poorly differentiated] are compared and showed statistical significance (p < 0.05). In 88 NSCLC patients, after 3 years of follow-up, 33 survived, and 55 died, with a survival rate of 37.50%. The expression of miR-184 in serum exosomes of the 33 surviving patients is significantly lower than that of the nonsurviving group (p < 0.05). The expression level of miR-184 in serum exosomes of NSCLC patients is significantly increased, which has a certain value for the differential diagnosis of the nature of benign and malignant lung diseases and is closely related to the prognosis of patients.

Exosomal miR-4639 and miR-210 in Plasma and Urine as Biomarkers in IgA Nephropathy

Background: It has been widely recognized that exosomal miRNAs can participate in the pathogenesis of different renal disorders and serve as disease biomarkers. Although kidney biopsy is still the gold standard for diagnosing and monitoring immunoglobulin A nephropathy (IgAN), it is highly required to identify new and effective noninvasive biomarkers for IgAN, the most frequently detected primary glomerulonephritis worldwide. Methods: Plasma and urinary exosomes were extracted by PEG precipitation. Size and morphological characteristics of plasma and urinary exosomes were observed by transmission electron microscopy and nanoparticle tracking analysis. The levels of plasma and urinary exosomes were revealed by Western blotting. The expressions of target miRNAs were revealed by in situ hybridization and qRT-PCR. Results: The levels of plasma and urinary exosomes were remarkably enhanced in IgAN patients compared with healthy controls (HCs). The expressions of miR-4639 and miR-210 in IgAN patients were significantly higher in contrast to the individuals with membranous nephropathy, minimal change nephrosis, diabetic nephropathy, or HC. These also played a valuable role in assessing the kidney function and the level of proteinuria. Furthermore, plasma and urinary exosomal miR-4639 expression was associated with more serious and active histological activity (mesangial hypercellularity, crescent, and C3 complement deposition). With an average follow-up of 8 months, miR-4639 and miR-210 expressions in plasma and urinary exosomes were higher in patients with progressive IgAN. Plasma exosomal miR-4639 and miR-210 were better than proteinuria (g/24 h) to estimate renal outcomes. Conclusion: Exosomal miR-4639 and miR-210 could be used as valid biomarkers to assist in diagnosis, evaluate severity, and assess disease development of IgAN.

Clinical study on the relationship between the exosomes in bronchoalveolar lavage fluid and plasma and the severity of lung injury and outcome in early acute respiratory distress syndrome patients

Objective: To investigate the relationship between the levels of exosomes in bronchoalveolar lavage fluid (BALF) and plasma and the severity of lung injury and its outcome in patients with acute respiratory distress syndrome (ARDS). Methods: Patients who were admitted to the Department of Critical Care Medicine, Zhongda Hospital Affiliated to Southeast University and received invasive mechanical ventilation were selected from August 2020 to April 2021, and they were divided into ARDS group and non-ARDS group. Finally, 33 ARDS patients were included, including 18 males and 15 females, aged (65.5±15.5) years; 10 non-ARDS patients, 8 males and 2 females, aged (57.2±15.3) years. The BALF and plasma of the two groups of patients were collected within 24 hours after enrollment, and the total exosomes of the samples were collected by ultracentrifugation. Nanoparticle tracking analysis (NTA) was used to detect and compare the differences in exosome content between the two groups. Correlation of content with the severity and prognosis of lung injury in ARDS patients. Results: There was no significant difference in gender and age between ARDS group and non-ARDS group (both P>0.05). The exosome in plasma of ARDS group was significantly higher than that of non-ARDS group [(25.3±1.2)/ml vs (24.2±1.6)/ml, P=0.031], while the exosomes in BALF of ARDS group was also higher than that of non-ARDS group [(26.5±1.6)/ml vs (24.6±1.1)/ml, P=0.001]. The exosomes in BALF of patients with ARDS caused by intrapulmonary causes was higher than that in ARDS group caused by extrapulmonary causes [(26.9±1.5)/ml vs (25.2±0.9)/ml, P=0.01], and the infection caused by bacterial shows that the highest exosome level in BALF. The exosomes in the BALF of the mild ARDS group was significantly lower than that of the severe ARDS group [(25.7±1.3)/ml vs (27.2±1.5)/ml, P=0.038]; the exosomes in BALF of ARDS patients was negatively correlated with P/F ratio (r=-0.38, P=0.03); and it was positively correlated with Murray lung injury score (r=0.47, P=0.01). However, the static compliance levels, length of hospital stay, duration of mechanical ventilation, and 28-day outcome were not associated with the exosomes in BALF. Conclusion: Compared with non-ARDS patients, ARDS patients have significantly higher levels of exosomes in BALF and plasma, there is a certain correlation between exosomes derived from BALF and the severity of lung injury in ARDS.

Altered amyloid-β and tau proteins in neural-derived plasma exosomes in obstructive sleep apnea

ObjectiveThe purpose of our study was to investigate the correlation between neural-derived plasma exosomal amyloid-β (Aβ)42, total tau (T-tau) and tau phosphorylated at threonine 181 (P-T181-tau) protein levels and cognitive impairment in patients with obstructive sleep apnea (OSA). MethodsThere were 122 subjects without dementia included in the study: 27 patients with OSA and mild cognitive impairment (MCI), 52 OSA patients without MCI, and 43 subjects diagnosed with simple snoring but not MCI as the control group. Neuronal-derived exosomal proteins were measured by ELISA kits for Aβ42, T-tau and P-T181-tau. The cognitive function was evaluated by a Chinese version of the Montreal Cognitive Assessment (MoCA) questionnaire, and a normal cognitive score was ≥26. ResultsThe exosomal Aβ42, T-tau and P-T181-tau levels in the OSA with MCI group were higher than those in the OSA group. The Aβ42, T-tau, and P-T181-tau levels in the plasma neuronal-derived exosomes were associated with an increased risk of cognitive impairment in OSA patients after additional adjustment for age, gender, education, vascular risk factors, apnea–hypopnea index (AHI) or oxygen reduction index (ODI). Furthermore, there were also significant associations between Aβ42, T-tau, and P-T181-tau in neural-derived plasma exosomes and Epworth Sleepiness Scale, AHI, and ODI in OSA patients. After 1 year of continuous positive airway pressure (CPAP) intervention, the neuronal-derived exosome levels of Aβ42, T-tau, and P-T181-tau were significantly lower than those at baseline (P = 0.001, P = 0.012, and P = 0.034). ConclusionsThese findings indicate that peripheral blood levels of neuronal-derived exosomal Aβ and tau proteins were increased in OSA patients with cognitive impairment. CPAP interventions could possibly improve cognitive function and be associated with decreased levels of exosomal Aβ and tau proteins.

Exosomal hsa-miR-129-2 and hsa-miR-889 from a 6-microRNA Signature Might be a Potential Biomarker for Predicting the Prognosis of Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) is a subtype of thyroid cancer with increasing incidence over time. This study aimed to build a risk score (RS) system for PTC patients. PTC microRNA (miRNA) and messenger RNA (mRNA) expression data were extracted from The Cancer Genome Atlas (TCGA) database. The 491 PTC samples were randomly divided into training and validation sets. Using the limma software package, differentially expressed mRNAs (DEGs) and miRNAs (DEMs) between the tumor and control groups were screened. In order to construct an RS system, a survival package was used to select independent miRNAs related to prognosis. Enrichment analysis was performed, and a miRNA-mRNA co-expression network was constructed. High-throughput sequencing was also used to verify the prognostic miRNAs in exosomes. We found 1363 DEGs and 171 DEMs between the tumor and control groups. After identifying 26 DEMs significantly related to prognosis, 6 independent prognosis-associated miRNAs were selected to build an RS system. The areas under the curves of the overall survival rates of the training, validation, and entire sets were 0.847, 0.772, and 0.819, respectively. By conducting pathway analysis using the miRNA-mRNA co-expression network, one overlapping factor and five overlapping pathways were obtained. In addition, high-throughput sequencing revealed that the hsa-miR-129-2, hsa-miR-548j, hsa-miR-6734, and hsa-miR-889 expression levels in TCGA tumor tissues and exosomes were consistent, and those of hsa-miR-129-2 and hsa-miR- 889 between patients and controls were significantly different in exosomes. The six-miRNA RS system in exosomes may comprise independent signatures for predicting PTC patient prognosis.

Detection of exosomal miR-18a and miR-222 levels in Egyptian patients with hepatic cirrhosis and hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is known to be the second leading cause of cancer-related mortality worldwide. For improving the prognosis as well as reducing the rate of mortality, early diagnosis of HCC is a must.AimsThis study was conducted to assess the ability of the serum expression of exosomal miR-18a and miR-222 to differentiate and diagnose patients with HCC, patients with liver cirrhosis, and healthy controls.MethodsThis study included 51 patients with liver cirrhosis, 51 patients with HCC on top of hepatitis C virus (HCV) infection, and 50 healthy controls.ResultsmiR-18a and miR-222 were assessed using reverse transcription-polymerase chain reaction. MiR-18a and miR-222 levels were significantly higher in the liver cirrhosis and HCC groups than the control group (p ˂ 0.001). However, no statistically significant difference was found between patients with HCC and liver cirrhosis (p = 0.4 for miR-18a and p = 0.1 for miR-222). ROC curve analyses to evaluate the diagnostic performances of the two miRNAs as important noninvasive diagnostic markers revealed a best cutoff value of 2 for miR-18a to differentiate between liver cirrhosis, HCC, and healthy controls. And for mir-222, a cutoff value of 1.7 and 1.9 showed the highest specificity for discrimination between liver cirrhosis, HCC, and healthy controls, respectively. Moreover, logistic regression model revealed that miR-18a expression was independent predictive factor in HCC patients (p = 0.004), while miR-222 expression was independent predictive factor in liver cirrhosis patients (p < 0.001).ConclusionmiR-18a and miR-222 were significantly discriminative markers between patients with liver cirrhosis and HCC and healthy individuals. Therefore, they have a prognostic rather than a diagnostic value. Moreover, miR-18a and miR-222 could be useful in identifying liver injuries, including fibrosis and cirrhosis.

Exosomes as Potential Biomarkers for Erectile Dysfunction, Varicocele, and Testicular Injury.

Optimal male reproductive health is dependent upon critical mediators of cell-cell communication: exosomes or extracellular vesicles. These vesicles are nano-sized particles released into a variety of bodily fluids, such as blood and semen. Exosomes are highly stable and can carry genetic and other molecules, including DNA, RNA, and proteins, which provide information about their origin cells. To identify exosomes as potential biomarkers or therapeutic mediators in male sexual and reproductive disorders like erectile dysfunction (ED), varicocele, and testicular injury. A PubMed search was performed to highlight all articles available relating to exosomes and extracellular vesicles in the pathogenesis of different male sexual and reproductive disorders, and their importance in clinical use as both diagnostic markers and potential therapeutic mediators. Various male reproductive system disorders, such as ED, varicocele, and testicular injury, are linked to increased or decreased levels of exosomes. Exosomes have a higher number of molecules such as DNA, RNA, and proteins, which can give a more precise and comprehensive result when compared to other biomarkers. Exosomes can be considered as plausible diagnostic biomarkers for male sexual and reproductive diseases, with considerable advantages over other diagnostic procedures such as invasive tissue biopsy. Exosomes can carry cargo such certain drugs and therapeutic molecules making them a promising therapeutic approach. Several studies have begun to test treating various male sexual reproductive disorders with exosomes. Exosomes deliver many components that can regulate gene expression and target signaling pathways. Understanding how extracellular vesicles can be utilized as biomarkers in diagnosing men, particularly those with idiopathic erectile dysfunction, will not only aid in diagnosis but also help with making therapeutic targets. Khodamoradi K, Golan R, Dullea A, et al. Exosomes as Potential Biomarkers for Erectile Dysfunction, Varicocele, and Testicular Injury. Sex Med Rev 2022;10:311-322.

EXOSOMAL CAVEOLIN-1 AS A BIOMARKER OF BLADDER CANCER IN EGYPT

Back ground: Cystoscopic examination and histological evaluation of bladder tissues are considered the gold standard for initial diagnosis of bladder cancer. For decades, researchers explore novel, non-invasive, specific and sensitive biomarkers for preliminary diagnosis and surveillance of bladder carcinoma. Exosomes are nano-sized vesicles present in various biological fluids and encapsulate huge number of biomarkers such as proteins, mRNAs and miRNAs. Caveolin-1(Cav-1) is a major protein of caveolae structure that expressed in a variety of cells and has a fundamental regulatory role in cancer development. Therefore, determination ofexosomal Cav-1 expression in different stages of bladder cancer is a critical concern for clinical diagnosis and prognosis. Methods: Exosomes Cav-1 was isolated and extracted from urine and serum samples of 79 patients of bladder cancer at different stages (T0:T3) and 12 healthy controls.Exosomal Cav-1 expression levels were determined by using Rt-qPCR technique. Results:Exosomal Cav-1expression levels at different stagesof bladder cancer patients were significant in both urine and serum samples. The current study showed upregulation ofCav-1 amongserum (4.03, 9.20, 14.25, 26.92folds) and urine samples (2.55, 3.64, 8.33, 19.37folds) comparative to tumor invasiveness (T0-T3).Serum Cav-1 is more sensitive (96.2%) than that in urine (86.1%) although urine Cav-1 is more specific(94.3%). Conclusion: Exosomal Cav-1 expression could be used as a crucialnon-invasive biomarker in diagnosis and prognosis of bladder cancer in serum and urine.