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Background Although basophils are considered to play an important role for maintenance of type 2 inflammation in atopic dermatitis (AD), studies on basophils in AD patients are limited. Some studies have reported the activation status, including CD203c and CD63, of peripheral blood basophils in AD patients. Methods We examined the features of circulating basophils in AD patients, assessed cell surface marker expressions and total serum IgE, and compared basophil responsiveness to stimulation between AD patients and healthy controls (HCs). In addition, the correlations among AD severity, laboratory factors, and features of basophils were examined. Blood samples from 38 AD patients and 21 HCs were analyzed. Basophil response markers CD203c and CD63, and expression of surface-bound IgE and FceRI on basophils were measured. CD203c and CD63 expressions induced by stimulation with anti-IgE and anti-FceRI antibodies were measured. Clinical/laboratory factors including total serum IgE were examined for correlations with these basophil parameters. Results Baseline CD203c and CD63 expression on basophils were significantly higher in AD patients compared with HCs. The CD203c/CD63 response ratio to anti-FceRI stimulation was higher than that to anti-IgE stimulation in AD patients, but not HCs. FceRI expression on basophils was higher in AD patients than in HCs, although surface-bound IgE on basophils was equivalent. Total serum IgE had negative correlations with surface-bound IgE and CD63 responsiveness to anti-IgE stimulation. Conclusions Basophils were spontaneously activated under steady-state conditions in AD patients and responsiveness to anti-IgE stimulation was lower than in HCs. Despite high serum IgE and high basophil FceRI expression, surface-bound IgE on basophils remained relatively low. Basophils might be suppressed or exhausted regarding FceRI signaling via IgE in severe AD.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by germline mutations of PKD1 or PKD2 on one allele and a somatic mutation inactivating the remaining normal allele. However, if and how null ADPKD gene renal epithelial cells affect the biology and function of neighboring cells, including heterozygous renal epithelial cells, fibroblasts and macrophages during cyst initiation and expansion remains unknown. Here we address this question with a “cystic extracellular vesicles/exosomes theory”. We show that cystic cell derived extracellular vesicles and urinary exosomes derived from ADPKD patients promote cyst growth in Pkd1 mutant kidneys and in 3D cultures. This is achieved by: 1) downregulation of Pkd1 gene expression and upregulation of specific miRNAs, resulting in the activation of PKD associated signaling pathways in recipient renal epithelial cells and tissues; 2) the activation of fibroblasts; and 3) the induction of cytokine expression and the recruitment of macrophages to increase renal inflammation in cystic kidneys. Inhibition of exosome biogenesis/release with GW4869 significantly delays cyst growth in aggressive and milder ADPKD mouse models, suggesting that targeting exosome secretion has therapeutic potential for ADPKD., Autosomal dominant polycystic kidney disease is characterized by the formation of cysts in the kidney. Here the authors show that cystic extracellular vesicles/exosomes play a critical role in regulating the biology and function of adjacent cells, including renal epithelial cells, fibroblasts and macrophages, and contribute to renal cyst growth.

Human immunodeficiency virus type 1 (HIV-1) persists lifelong in infected individuals and has evolved unique strategies in order to evade the immune system. One of these strategies is the direct cell-to-cell spread of HIV-1. The formation of a virological synapse (VS) between donor and target cell is important for this process. Tetraspanins are cellular proteins that are actively involved in the formation of a VS. However, the molecular mechanisms of recruiting host proteins for the cell-cell transfer of particles to the VS remains unclear. Our study has mapped the binding site for the transmembrane envelope protein gp41 of HIV-1 within the large extracellular loop (LEL) of CD63 and showed that this interaction occurs predominantly at the VS between T cells where viral particles are transferred. Mutations within the highly conserved CCG motif of the tetraspanin superfamily abrogated recruiting of expressed HIV-1 GFP fused Gag core protein and CD63 to the VS. This demonstrates the biological significance of CD63 for enhanced formation of a VS. Since cell-cell spread of HIV-1 is a major route of persistent infection, these results highlight the central role of CD63 as a member of the tetraspanin superfamily during HIV-1 infection and pathogenesis.

Background Recently, fusion variants of the breast cancer anti-oestrogen-resistance 4 (BCAR4) gene were recurrently discovered in lung adenocarcinoma from the genome-wide studies. However, the functional characterisation of BCAR4 fusion has not been investigated. Methods Based on the analysis of RNA-sequencing data, we identified a fusion transcript of CD63–BCAR4 in a Korean patient with lung adenocarcinoma who did not harbour any known activating mutations in EGFR and KRAS genes. To investigate the oncogenic effect of CD63–BCAR4, in vitro and in vivo animal experiments were performed. Results In vitro experiments showed strongly enhanced cell migration and proliferation by the exogenous expression of CD63–BCAR4 protein in bronchial epithelial cells. Cell migration was notably reduced after knockdown of BCAR4 fusion by small-interfering RNA. The tumorigenic and metastatic capability of the CD63–BCAR4 fusion was confirmed by using the mouse xenograft model. Fusion-overexpressed cells result in metastasis to the liver and lung as well as the primary tumours after subcutaneous injection into mice. Cyclin D1, MMP1, Slug and mesenchymal markers were significantly increased after CD63–BCAR4 overexpression in the in vitro and in vivo experiments. Conclusions Taken together, our results suggest a newly identified fusion gene, CD63–BCAR4 as a potential novel oncogene in lung adenocarcinoma.

Background Nano‐sized extracellular vesicles secreted by cells play key roles in intercellular crosstalk, and appear to be an excellent biocompatible material as therapeutic cargoes in vivo. Previously, we have demonstrated that miR‐204‐5p is a key tumor suppressor that could inhibit tumor growth, metastasis and chemoresistance. Methods A HEK293T cell line stably expressing miR‐204‐5p (293T‐miR‐204) was constructed by lentivirus transduction. Fluorescence real‐time quantitative PCR (qPCR) was applied to measure the expression of miR‐204‐5p. CCK‐8 and colony formation assays were used to evaluate the in vitro anticancer effects, and the flow cytometry was used to detect apoptosis. The in vivo therapeutic effects of exosomal miR‐204‐5p were evaluated using a xenograft mouse model. Western blots were used to detect the protein levels of CD63, Flotillin‐2, RAB22A and Bcl2. The protein levels of RAB22A and Bcl2 in tumor tissues were measured by immunohistochemistry staining. Results MiR‐204‐5p was clearly upregulated in CRC cells after coculturing with 293T‐miR‐204 cell‐derived conditioned medium (CM) or exosomes. CCK‐8 and colony formation assays showed that the cell proliferation ability of CRC cells was clearly inhibited by 293T‐miR‐204 cell‐derived CM or exosomes. The inhibitory effects of exosomal miR‐204‐5p on cell proliferation were further confirmed in other types of cancers. Exosomal miR‐204‐5p could induce apoptosis and increase the sensitivity of cancer cells to the chemotherapeutic drug—5‐fluorourcil. In addition, exosomal miR‐204‐5p inhibited the tumor growth in mice. Western blot assay and IHC staining showed that the protein levels of miR‐204‐5p targets were clearly decreased in cancer cells or xenograft tissues treated with exosomal miR‐204‐5p. Conclusions In this study, we confirmed that exosomal miR‐204‐5p could efficiently inhibit cancer cell proliferation, induce apoptosis and increase chemosensitivity by specifically suppressing the target genes of miR‐204‐5p in human cancer cells., In this study, we confirmed that exosomal miR‐204‐5p could efficiently inhibit cancer cell proliferation, induce apoptosis, and increase chemosensitivity by specifically suppressing the target genes of miR‐204‐5p in human cancer cells.

Extracellular vesicles (EVs) derived from different parts of the male reproductive tract can be internalized by human spermatozoa affecting their maturation and regulating their functions. Here we demonstrate that EVs derived from the female tract can be uptaken by sperm and affect their competence. Primary endometrial cells release EVs with a diameter between 50 and 350 nm and bear the standard vesicle and exosome marker proteins CD63, CD9, TSG101 and ALIX. The uptake of dye-labelled endometrial cell-derived EVs by spermatozoa, quantified as fluorescence intensity, was significantly higher when EVs were derived from cells in the proliferative phase. Vital, motile fluorescent sperm could be appreciated after a 48-hour co-incubation with endometrial cells previously labelled with the Vybrant™ DiO dye. EV internalization by sperm was blocked at 4 °C and by incubation with filipin, suggesting an energy-dependent process probably attributable to the lipid-raft domain mediated-endocytosis. Sperm ability to undergo capacitation and acrosome reaction was stimulated by endometrial cell-derived EVs as manifested by the increased protein tyrosine phosphorylation and evident reactivity when stimulated with a calcium ionophore. Based on these findings, EVs exchange may be suggested as an emerging way through which female reproductive tract cells can interact with the passing spermatozoa.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high morbidity that is endemic in South East Asia and northern Australia. An unusual feature of the bacterium is its ability to induce multinucleated giant cell formation (MNGC), which appears to be related to bacterial pathogenicity. The mechanism of MNGC formation is not fully understood, but host cell factors as well as known bacterial virulence determinants are likely to contribute. Since members of the tetraspanin family of membrane proteins are involved in various types of cell:cell fusion, their role in MNGC formation induced by Burkholderia thailandensis, a mildly pathogenic species closely related to B. pseudomallei, was investigated. The effect of antibodies to tetraspanins CD9, CD81, and CD63 in MNGC formation induced by B. thailandensis in infected mouse J774.2 and RAW macrophage cell lines was assessed along with that of recombinant proteins corresponding to the large extracellular domain (EC2) of the tetraspanins. B. thailandensis-induced fusion was also examined in macrophages derived from CD9 null and corresponding WT mice, and in J774.2 macrophages over-expressing CD9. Antibodies to CD9 and CD81 promoted MNGC formation induced by B. thailandensis, whereas EC2 proteins of CD9, CD81, and CD63 inhibited MNGC formation. Enhanced MNGC formation was observed in CD9 null macrophages, whereas a decrease in MNGC formation was associated with overexpression of CD9. Overall our findings show that tetraspanins are involved in MNGC formation induced by B. thailandensis and by implication, B. pseudomallei, with CD9 and CD81 acting as negative regulators of this process.

Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader–follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes., A prior live-cell exosome reporter showed dim fluorescence and could not be expressed stably, limiting its usefulness. Here the authors stabilise the reporter to allow long-term tracking of exosomes, and incorporate a second fluorophore to visualise the entire exosome lifecycle.

Exosomes are secreted extracellular vesicles with lipid bilayer membranes. They are emerging as a new category of messengers that facilitate cross-talk between cells, tissues, and organs. Thus, a critical demand arises for the development of a sensitive and non-invasive tracking system for endogenous exosomes. We have generated a genetic mouse model that meets this goal. The Nano-luciferase (NanoLuc) reporter was fused with the exosome surface marker CD63 for exosome labeling. The cardiomyocyte-specific αMHC promoter followed by the loxP-STOP-loxP cassette was engineered for temporal and spatial labeling of exosomes originated from cardiomyocytes. The transgenic mouse was bred with a tamoxifen-inducible Cre mouse (Rosa26Cre-ERT2) to achieve inducible expression of CD63NanoLuc reporter. The specific labeling and tissue distribution of endogenous exosomes released from cardiomyocytes were demonstrated by luciferase assay and non-invasive bioluminescent live imaging. This endogenous exosome tracking mouse provides a useful tool for a range of research applications., Using nano-luciferase-CD63, Weijia Luo et al. develop transgenic mice where cardiac exosomes can be tracked with non-invasive bioluminescent live imaging in a tamoxifen-inducible manner. These mice serve as a valuable tool that allow researchers to track cardiac exosomes in a defined time window.

BACKGROUND: Alcohol hypersensitivity (AH), an exacerbation of respiratory symptoms in response to alcohol consumption, is common in Aspirin Exacerbated Respiratory Disease (AERD) and other forms of chronic rhinosinusitis (CRS). We speculated that these reactions relate to the activation of innate immune cells including basophils and, in particular, by platelet-adherent basophils by polyphenolic compounds contained within eliciting alcoholic beverages. OBJECTIVES: We investigated the absolute numbers of these cells in patients with AH and the ability of relevant polyphenolic compounds to cause cellular activation. METHODS: Data were collected from 412 consecutive adults presenting to a tertiary care sinonasal clinic in whom the presence of AH was elicited. CRS phenotype was determined and results from complete blood count and differential were analyzed. A subset of patients were invited to donate blood samples that were used to explore the ability of relevant compounds associated with alcohol consumption to activate platelet non-adherent and platelet-adherent basophils. Activation was quantified by flow cytometry as upregulated expression of CD63 and as secretion of lipid metabolites. RESULTS: Of the 412 patients enrolled, 69 (16.7%) endorsed having AH. Significantly higher platelet counts were seen in patients reporting AH. Red wine extract and several polyphenolic compounds produced basophil activation and this was primarily observed amongst platelet-adherent basophils. Platelet activation was further established as the release of TxB2. CONCLUSION. The presence of AH is associated with significantly higher platelet levels and compounds present in alcoholic beverages can directly mediate both their activation as well as the activation of platelet-adherent basophils.

Understanding molecular mechanisms of intervertebral disc degeneration (IDD) and providing a novel target for the treatment of IDD have important implications. We sought to explore a new promising gene target for the treatment of IDD. This study integrated 19,678 genes of 38 IDD patients from two gene datasets. Differentially Expressed Genes (DEGs) of annulus fibrosus were analyzed in groups with mild disc degeneration (MDD) and severe disc degeneration (SDD). We screened the hub gene through biological information technology (bioinformatic) methods. Then, we further validated the hub gene using annulus fibrosus and nucleus pulposus tissues from 12 patients with qRT-PCR. In addition, we explored its underlying molecular mechanism with GO, KEGG and GSEA. Through multiple screening bioinformatics methods, the hub gene CD63 was identified. The qRT-PCR explored that CD63 decreased significantly in SDD group compared to that in MDD group (P

Nucleofection (NF) is a safe, non-viral transfection method, compatible with Good Manufacturing Practice guidelines. Such a technique is useful to improve therapeutic effectiveness of adipose tissue mesenchymal stem cells (ASC) in clinical settings, but improvement of NF efficiency is mandatory. Supernatant rich in growth factors (SRGF) is a clinical-grade medium additive for ASC expansion. We showed a dramatically increased NF efficiency and post-transfection viability in ASC expanded in presence of SRGF (vs. fetal bovine serum). SRGF expanded ASC were characterized by increased vesicle endocytosis but lower phagocytosis properties. SRGF increased n-6/n-3 ratio, reduced membrane lipid raft occurrence, and lowered intracellular actin content in ASC. A statistical correlation between NF efficiency and lipid raft availability on cell membranes was shown, even though a direct relationship could not be demonstrated: attempts to selectively modulate lipid rafts levels were, in fact, limited by technical constraints. In conclusion, we reported for the first time that tuning clinical-grade compatible cell culture conditions can significantly improve ASC transfection efficiency by a non-viral and safe approach. A deep mechanistic characterization is extremely complex, but we can hypothesize that integrated changes in membrane structure and intracellular actin content could contribute to explain SRGF impact on ASC NF efficiency.

CD63 is one of the tetraspanin protein family members that is ubiquitously expressed on exosomes and is involved in the signal transduction of various types of immune cells. It may thus contribute to immunometabolic mechanisms of cellular and organ dysfunction in sepsis. Nonetheless, the association of exosomal CD63 with the severity and mortality of sepsis is not well known. Therefore, in the present study, the overall levels of exosomal CD63 were evaluated to ascertain whether they were associated with organ failure and mortality in patients with sepsis. Exosomal CD63 was measured from prospectively enrolled critically-ill patients with sepsis (n = 217) and healthy control (n = 20). To detect and quantify exosomes in plasma, a commercially available enzyme-linked immunosorbent assay kit was used according to the manufacturer’s protocol. The total number of exosomal CD63 was determined by quantifying the immunoreactive CD63. The association between plasma levels of exosomal CD63 and sequential organ failure assessment (SOFA) score was assessed by a linear regression method. The best cut-off level of exosomal CD63 for 28-day mortality prediction was determined by Youden’s index. Among 217 patients with sepsis, 143 (66%) patients were diagnosed with septic shock. Trends of increased exosomal CD63 levels were observed in control, sepsis, and septic-shock groups (6.6 µg/mL vs. 42 µg/mL vs. 90 µg/mL, p r value = 0.35). When patients were divided into two groups according to the best cut-off level, the group with higher exosomal CD63 levels (more than 126 µg/mL) was significantly associated with 28-day and in-hospital mortality. Moreover, the Kaplan–Meier survival method showed a significant difference in 90-day survival between patients with high- and low-exosomal CD63 levels (log-rank p = 0.005). Elevated levels of exosomal CD63 were associated with the severity of organ failure and predictive of mortality in critically ill patients with sepsis.

A hallmark of malignant solid tumor is extracellular acidification coupled with metabolic switch to aerobic glycolysis. Using the human MCF10A progression model of breast cancer, we show that glycolytic switch and extracellular acidosis in aggressive cancer cells correlate with increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), known to induce intracellular signal transduction through the interaction with its cell surface receptor CD63, independent of its metalloproteinase inhibitory function. We found that, in aggressive breast carcinoma, the TIMP-1–CD63 signaling axis induced a metabolic switch by upregulating the rate of aerobic glycolysis, lowering mitochondrial respiration, preventing intracellular acidification, and inducing extracellular acidosis. Carbonic anhydrase IX (CAIX), a regulator of cellular pH through the hydration of metabolically released pericellular CO2, was identified as a downstream mediator of the TIMP-1–CD63 signaling axis responsible for extracellular acidosis. Consistently with our previous study, the TIMP-1–CD63 signaling promoted survival of breast cancer cells. Interestingly, breast carcinoma cell survival was drastically reduced upon shRNA-mediated knockdown of CAIX expression, demonstrating the significance of CAIX-regulated pH in the TIMP-1–CD63-mediated cancer cell survival. Taken together, the present study demonstrates the functional significance of TIMP-1–CD63–CAXI signaling axis in the regulation of tumor metabolism, extracellular acidosis, and survival of breast carcinoma. We propose that this axis may serve as a novel therapeutic target.

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies, which, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in an LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments., Author’s note Concerns have emerged about the immunoelectron microscopy results originally reported in the article by Majumdar and colleagues [1]. In addition, errors were made in the scale bars reported in Figs 2H and 3D of the same article. Accordingly, this article has been retracted. We are grateful for the opportunity to republish a version of this article in which the electron microscopy data have been removed. None of the major conclusions attained in the original article are affected by the removal of the contentious data. We sincerely apologize to PLOS Biology and the scientific community at large for this occurrence.

Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.

Bone metastasis of breast cancer makes patients suffer from pain, fractures, spinal cord compression, and hypercalcemia, and is almost incurable. Although the mechanisms of bone metastasis in breast cancers have been studied intensively, novel specific target will be helpful to the development of new therapeutic strategy of breast cancer. Herein, we focused on the microRNA of tumor cell‐derived exosomes to investigate the communication between the bone microenvironment and tumor cells. The expression of miR‐20a‐5p in the primary murine bone marrow macrophages (BMMs), MCF‐10A, MCF‐7, and MDA‐MB‐231 cell lines, as well as the cell‐derived exosomes were assessed by qRT‐PCR. Transwell assays were used to evaluate the effects of miR‐20a‐5p on tumor cell migration and invasion. The expression of exosomes marker including CD63and TSG101 was detected by Western Blot. Cell cycle distribution of BMMs was analyzed by flow cytometry. 3‐UTR luciferase reporter assays were used to validate the putative binding between miR‐20a‐5p and SRCIN1. MiR‐20a‐5p was highly expressed in breast tumor tissues and the exosomes of MDA‐MB‐231 cells. MiR‐20a‐5p promoted migration and invasion in MDA‐MB‐231 cells, and the proliferation and differentiation of osteoclasts. MDA‐MB‐231 cell‐derived exosomes transferred miR‐20a‐5p to BMMs and facilitated the osteoclastogenesis via targeting SRCIN1. The present work provides evidence that miR‐20a‐5p transferred from breast cancer cell‐derived exosomes promotes the proliferation and differentiation of osteoclasts by targeting SRCIN1, providing scientific foundations for the development of exosome or miR‐20a‐5p targeted therapeutic intervention in breast cancer progression., Highlight：(a) MiR‐20a‐5p promotes migration and invasion in MDA‐MB‐231 cells. (b) MDA‐MB‐231 cell‐derived exosomes transferred miR‐20a‐5p to BMMs and facilitated the osteoclastogenesis via targeting SRCIN1. (c) The study provides evidence that miR‐20a‐5p transferred from breast cancer cell‐derived exosomes promotes the proliferation and differentiation of osteoclasts by targeting SRCIN1.

Gut microbiota plays a pivotal role in not only the gastrointestinal (GI) immune system but also GI motility and metabolism. Antibiotic treatments are likely to affect the gut flora and GI immune system, subsequently disturbing GI motility and body metabolism. In the present study, we investigated antibiotic‑induced alterations of body metabolism and GI motility in association with the macrophage profile in the colon. Specific pathogen‑free (SPF) mice (ICR; 6 weeks old; female) were orally administered vancomycin (0.2 mg/ml) in drinking water for 5 weeks, and subsequent changes in pathophysiology were observed. The expression of CD80 and CD163 was examined by immunohistochemistry and the expression of cytokines in colonic tissues was evaluated by reverse transcription‑quantitative polymerase chain reaction. The gastrointestinal transit time (GITT) was measured by administration of carmine red (6% w/v) solution. In the vancomycin‑treated SPF mice, significant increases in body weight, cecum weight and GITT were observed compared with the controls. The number of CD80‑positive M1 macrophages and the expression of interferon‑γ and interleukin‑12 were significantly increased, whereas, the numbers of CD163‑positive M2 macrophages in the mucosal and muscular layers were decreased in the colon of vancomycin‑treated mice. GITT was positively correlated with the number of CD80‑positive M1 macrophages in the colonic mucosa; however, was negatively correlated with the number of CD163‑positive M2 macrophages in the mucosal and muscular layers. Therefore, it was suggested that antibiotic treatment affects body metabolism and GI motility, accompanied by alterations in macrophage polarization and cytokine profiles in the colon.

Molecular profiling of circulating extracellular vesicles (EVs) provides a promising noninvasive means to diagnose, monitor, and predict the course of metastatic breast cancer (MBC). However, the analysis of EV protein markers has been confounded by the presence of soluble protein counterparts in peripheral blood. Here we use a rapid, sensitive, and low-cost thermophoretic aptasensor (TAS) to profile cancer-associated protein profiles of plasma EVs without the interference of soluble proteins. We show that the EV signature (a weighted sum of eight EV protein markers) has a high accuracy (91.1 %) for discrimination of MBC, non-metastatic breast cancer (NMBC), and healthy donors (HD). For MBC patients undergoing therapies, the EV signature can accurately monitor the treatment response across the training, validation, and prospective cohorts, and serve as an independent prognostic factor for progression free survival in MBC patients. Together, this work highlights the potential clinical utility of EVs in management of MBC., A thermophoretic aptasensor can be used to profile cancer-associated proteins of extracellular vesicles (EVs) in patients’ plasma. Here, the authors use this technique to develop an EV-signature able to discriminate metastatic breast cancer, monitor treatment response, and predict patients’ progression-free survival.

Aristotelia chilensis (Mol.) Stuntz, also known as maqui, is a plant native to Chile without chemical characterization and quantification of the bioactive compounds present in it. HPLC-UV and HPLC-MS/MS studies have shown the presence, at different concentrations, of phenolic and anthocyanin compounds in fruit and leave extracts of the domesticated maqui clones Luna Nueva, Morena, and Perla Negra. The extracts from leaves and unripe fruits of Luna Nueva and Morena clones significantly inhibit platelet aggregation induced by several agonists; the extracts inhibit platelet granule secretion by decreasing the exposure of P-selectin and CD63 at the platelet membrane. Reactive oxygen species formation in platelets is lower in the presence of maqui extracts. Statistical Pearson analysis supports the levels of phenolic and anthocyanin compounds being responsible for the antiaggregant maqui effects. This work is the first evidence of antiplatelet activity from Aristotelia chilensis giving added value to the use of leaves and unripe fruits from this species.

The Mas-related G-protein-coupled receptor X2 (MRGPRX2) is prominently expressed by mast cells and induces degranulation upon binding by different ligands. Its activation has been linked to various mast cell-related diseases, such as chronic spontaneous urticaria, atopic dermatitis and asthma. Therefore, inhibition of MRGPRX2 activity represents a therapeutic target for these conditions. However, the exact pathophysiology of this receptor is still unknown. In vitro research with mast cells is often hampered by the technical limitations of available cell lines. The human mast cell types LAD2 and HuMC (human mast cells cultured from CD34+ progenitor cells) most closely resemble mature human mast cells, yet have a very slow growth rate. A fast proliferating alternative is the human mast cell line HMC1, but they are considered unsuitable for degranulation assays due to their immature phenotype. Moreover, the expression and functionality of MRGPRX2 on HMC1 is controversial. Here, we describe the MRGPRX2 expression and functionality in HMC1 cells, and compare these with LAD2 and HuMC. We also propose a model to render HMC1 suitable for degranulation assays by pre-incubating them with latrunculin-B (Lat-B). Expression of MRGPRX2 by HMC1 was proven by RQ-PCR and flowcytometry, although at lower levels compared with LAD2 and HuMC. Pre-incubation of HMC1 cells with Lat-B significantly increased the overall degranulation capacity, without significantly changing their MRGPRX2 expression, phenotype or morphology. The MRGPRX2 specific compound 48/80 (C48/80) effectively induced degranulation of HMC1 as measured by CD63 membrane expression and β-hexosaminidase release, albeit in lower levels than for LAD2 or HuMC. HMC1, LAD2 and HuMC each had different degranulation kinetics upon stimulation with C48/80. Incubation with the MRGPRX2 specific inhibitor QWF inhibited C48/80-induced degranulation, confirming the functionality of MRGPRX2 on HMC1. In conclusion, HMC1 cells have lower levels of MRGPRX2 expression than LAD2 or HuMC, but are attractive for in vitro research because of their high growth rate and stable phenotype. HMC1 can be used to study MRGPRX2-mediated degranulation after pre-incubation with Lat-B, which provides the opportunity to explore MPRGRX2 biology in mast cells in a feasible way.

Transient delivery of CRISPR‐based genome editing effectors is important to reduce off‐target effects and immune responses. Recently extracellular vesicles (EVs) have been explored for Cas9 ribonucleoprotein (RNP) delivery. However, lack of mechanisms to enrich RNPs into EVs limited the efficiency of EVs as a RNP delivery vehicle. Here we describe a mechanism to actively enrich RNPs into EVs. We used the specific interaction between RNA aptamer and aptamer‐binding protein (ABP) to enrich RNPs into EVs. We inserted RNA aptamer com into single guide RNA (sgRNA), and fused com‐binding ABP Com to both termini of tetraspan protein CD63 that is abundant in exosomes. We found that the Com/com interaction enriched Cas9 and adenine base editor (ABE) RNPs into EVs, via forming a three‐component complex including CD63‐Com fusion protein, com‐modified sgRNA and Cas9 or ABE. The RNP enriched EVs are efficient in genome editing and transiently expressed. The system is capable of delivering RNPs targeting multiple loci for multiplex genome editing. In addition, Cas9 from different species can be used together. The EV‐delivered RNPs are active in vivo. The data show that the aptamer and ABP interactions can be utilized to actively enrich RNPs into EVs for improved genome editing efficiency and safety.

Aging-associated inflammation is characterized by senescent cell-mediated secretion of high levels of inflammatory mediators, such as microRNA (miR)-146a. Moreover, a rise of circulating cell-free DNA (cfDNA) is also related to systemic inflammation and frailty in the elderly. Exosome-mediated cell-to-cell communication is fundamental in cellular senescence and aging. The plasma changes in exercise-promoted miR-146a-5p, cfDNA, and exosome release could be the key to facilitate intercellular communication and systemic adaptations to exercise in aging. Thirty-eight elderly subjects (28 trained and 10 controls) volunteered in an 8-week resistance training protocol. The levels of plasma miR-146a-5p, cfDNA, and exosome markers (CD9, CD14, CD63, CD81, Flotillin [Flot]-1, and VDAC1) were measured prior to and following training. Results showed no changes in plasma miR-146a-5p and cfDNA levels with training. The levels of exosome markers (Flot-1, CD9, and CD81) as well as exosome-carried proteins (CD14 and VDAC1) remained unchanged, whereas an attenuated CD63 response was found in the trained group compared to the controls. These findings might partially support the anti-inflammatory effect of resistance training in the elderly as evidenced by the diminishment of exosome CD63 protein expression, without modification of plasma miR-146a-5p and cfDNA.

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins, however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.

CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (Kd~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.

Similar to growth-limited human primary cultures of mesenchymal stroma/stem-like cells (MSC), the continuously proliferating human MSC544 cell line produced extracellular vesicles as characterized by expression of the tetraspanin molecules CD9, CD63, and CD81. Release of these particles was predominantly detectable during continuous cell growth of MSC544 in contrast to confluency-mediated transient growth arrest. For therapeutic use, these particles were isolated from proliferating MSC544 after taxol treatment and applied to different cancer cell cultures. A pronounced cytotoxicity of lung, ovarian, and breast cancer cells was observed primarily with taxol-loaded exosomes, similar to the effects displayed by application of taxol substance. While these findings suggested pronounced cancer cell targeting of MSC544 exosomes, a tumor therapeutic approach was performed using a mouse in vivo breast cancer model. Thus, intravenous injection of taxol-loaded MSC544 exosomes displayed superior tumor-reducing capabilities as compared to application of taxol exosomes by oral gavage. To broaden this therapeutic spectrum, epirubicin was applied to MSC544, and the derived exosomes likewise exhibited significant cytotoxic effects in different cancer cell cultures. These findings suggest an unlimited source for large-scale exosome production with reproducible quality to enable variable drug targeting of tumors or other diseases.

Extracellular vesicles (EV) are abundant, lipid-enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type-specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR-Cas9-mediated genome editing to generate mice bearing a CD63-emGFP(loxP/stop/loxP) knock-in cassette that enables the specific labeling of circulating CD63(+) vesicles from any cell type when crossed with lineage-specific Cre recombinase driver mice. As proof-of-principle, we have crossed these mice with Cdh5-Cre(ERT2) mice to generate CD63(emGFP+) vasculature. Using these mice we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma-purified EVs contain a subpopulation of emGFP(+) vesicles that are derived from the endothelium, co-express additional EV (e.g. CD9 and CD81) and endothelial cell (e.g. CD105) markers, and they harbor specific miRNAs (e.g. miR-126, miR-30c, and miR-125b). This new mouse strain should be a useful genetic tool for generating cell type-specific, CD63(+) EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.

Exosomes are cell-derived extracellular vesicles that have great potential in the field of nano-medicine. However, a fundamental challenge in the engineering of exosomes is the design of biocompatible molecular scaffolds on their surface to enable cell targeting and therapeutic functions. CD63 is a hallmark protein of natural exosomes that is highly enriched on the external surface of the membrane. We have already described the engineering of CD63 as a molecular scaffold in order to introduce cell-targeting feature to the exosome surface. Despite this initial success, the restrictive M-shaped topology of full-length CD63 may hinder specific applications that require N- or C-terminal display of cell-targeting moieties on the outer surface of the exosome. In this study, we describe new and topologically distinct CD63 scaffolds that enable robust and flexible surface engineering of the exosome. In particular, we conducted sequential deletions of the transmembrane helix of CD63 to generate a series of CD63 truncates, each genetically-fused to a fluorescent protein. Molecular and cellular characterization studies showed truncates of CD63 harboring the transmembrane helix 3 (TM3) were correctly targeted and anchored to the exosome membrane and exhibited distinct n-, N-, Ω-, or I-shaped membrane topologies in the exosomal membrane. We further established that these truncates retained robust membrane-anchoring and exosome-targeting activities when stably expressed in the HEK293 cells. Moreover, HEK293 cells produced engineered exosomes in similar quantities to cells that expressed full-length CD63. On the basis of the results of our systematic sequential deletion studies, we propose a model to understand molecular mechanisms that underlie membrane-anchoring and exosome targeting features of CD63. In summary, we have established new and topologically distinct scaffolds based on engineering of CD63 that enable flexible engineering of the exosome surface for applications in disease-targeted drug delivery and therapy.

The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100&ndash, 250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by overproduction and altered protein content. The EVs released by renal cyst epithelia in TSC complex may serve as a tool to discover the mechanism of TSC cystogenesis and in developing potential therapeutic strategies.

Extracellular vesicles (EVs) are known to transport DNA, but their implications in embryonic implantation are unknown. The aim of this study was to investigate EVs production and secretion by preimplantation embryos and assess their DNA cargo. Murine oocytes and embryos were obtained from six- to eight-week-old females, cultured until E4.5 and analyzed using transmission electron microscopy to examine EVs production. EVs were isolated from E4.5-day conditioned media and quantified by nanoparticle tracking analysis, characterized by immunogold, and their DNA cargo sequenced. Multivesicular bodies were observed in murine oocytes and preimplantation embryos together with the secretion of EVs to the blastocoel cavity and blastocyst spent medium. Embryo-derived EVs showed variable electron-densities and sizes (20&ndash, 500 nm) and total concentrations of 1.74 &times, 107 &plusmn, 2.60 &times, 106 particles/mL. Embryo secreted EVs were positive for CD63 and ARF6. DNA cargo sequencing demonstrated no differences in DNA between apoptotic bodies or smaller EVs, although they showed significant gene enrichment compared to control medium. The analysis of sequences uniquely mapping the murine genome revealed that DNA contained in EVs showed higher representation of embryo genome than vesicle-free DNA. Murine blastocysts secrete EVs containing genome-wide sequences of DNA to the medium, reinforcing the relevance of studying these vesicles and their cargo in the preimplantation moment, where secreted DNA may help the assessment of the embryo previous to implantation.

ALIX recruits ESCRT-III onto endosomes in a process that does not depend on other ESCRTs but requires the late endosome–specific lipid LBPA in vivo and in vitro. This novel pathway of ILV formation promotes tetraspanin sorting and delivery to exosomes., The intraluminal vesicles (ILVs) of endosomes mediate the delivery of activated signaling receptors and other proteins to lysosomes for degradation, but they also modulate intercellular communication when secreted as exosomes. The formation of ILVs requires four complexes, ESCRT-0, -I, -II, and -III, with ESCRT-0, -I, and -II presumably involved in cargo sorting and ESCRT-III in membrane deformation and fission. Here, we report that an active form of the ESCRT-associated protein ALIX efficiently recruits ESCRT-III proteins to endosomes. This recruitment occurs independently of other ESCRTs but requires lysobisphosphatidic acid (LBPA) in vivo, and can be reconstituted on supported bilayers in vitro. Our data indicate that this ALIX- and ESCRT-III–dependent pathway promotes the sorting and delivery of tetraspanins to exosomes. We conclude that ALIX provides an additional pathway of ILV formation, secondary to the canonical pathway, and that this pathway controls the targeting of exosomal proteins., Graphical Abstract

Background Staining of exteriorized basophil granule matrix by fluorescent avidin might be a reliable technique to monitor basophil degranulation. This study compares the avidin‐based technique with the upregulation of CD203c and appearance of CD63 in response to various stimuli. Methods Fourteen individuals responsive to anti‐IgE, nine healthy controls, and five birch pollen‐allergic patients, and five nonresponders were studied. Activation experiments included anti‐IgE, fMLP, interleukin‐(IL)‐3, and birch pollen allergen. Basophil activation/degranulation was analyzed by flow cytometry and microscopy using anti‐CD63, anti‐CD203c, and avidin. Results Stimulation with anti‐IgE, fMLP, and relevant allergen results in upregulation of CD203c, CD63 appearance, and an increase in avidin binding. In response to anti‐IgE and allergen, upregulation of CD203c peaks within 10 min, CD63 and avidin binding reach a plateau after 10–20 min. CD63 staining leads to a bimodal distribution, avidin staining causes a unimodal shift with a less clear discrimination between degranulating and nondegranulating cells. In response to fMLP, upregulation of CD203c and CD63 and avidin binding are maximal after 2.5 min. Following incubation with anti‐IgE and fMLP, percentages of CD203c+ cells are higher than those of CD63+ and avidin+ cells, pointing to a dissociation between activation and degranulation. Percentages of CD63+ cells are systemically higher than those of avidin+ cells. Incubated with IL‐3 only upregulates CD203c, while no CD63 or avidin binding is observed. Conclusions Staining of exteriorized proteoglycans by avidin is a reliable technique to quantify basophil degranulation but offers no added value when compared to traditional assays that use CD63 as a readout.

Gasdermin D (GSDMD) induces pyroptosis via the pore-forming activity of its N-terminal domain, cleaved by activated caspases associated with the release of IL-1β. Here, we report a nonpyroptotic role of full-length GSDMD in guiding the release of IL-1β–containing small extracellular vesicles (sEVs) from intestinal epithelial cells (IECs). In response to caspase-8 inflammasome activation, GSDMD, chaperoned by Cdc37/Hsp90, recruits the E3 ligase, NEDD4, to catalyze polyubiquitination of pro–IL-1β, serving as a signal for cargo loading into secretory vesicles. GSDMD and IL-1β colocalize with the exosome markers CD63 and ALIX intracellularly, and GSDMD and NEDD4 are required for release of CD63(+) sEVs containing IL-1β, GSDMD, NEDD4, and caspase-8. Importantly, increased expression of epithelial-derived GSDMD is observed both in patients with inflammatory bowel disease (IBD) and those with experimental colitis. While GSDMD-dependent release of IL-1β–containing sEVs is detected in cultured colonic explants from colitic mice, GSDMD deficiency substantially attenuates disease severity, implicating GSDMD-mediated release of IL-1β sEVs in the pathogenesis of intestinal inflammation, such as that observed in IBD.

Hypoxia is the most common characteristic of solid tumours driving cancer metastasis. Cancer cells release exosomes with various functions into the tumour microenvironment during cancer progression. However, the roles and associated mechanisms of hypoxic colorectal cancer (CRC) cell-derived exosomes remain poorly understood. Here, we found that exosomes secreted by hypoxic CRC cells promoted the migration and invasion abilities of normoxic CRC cells. Inhibition of exosome secretion by GW4869 reduced hypoxic exosome-mediated migration and invasion of normoxic CRC cells. Furthermore, we found that these hypoxic exosomes contained Wnt4 depending on HIF1α. Exosomal Wnt4 mediated hypoxic exosome-mediated migration and invasion of normoxic CRC cells. Moreover, exosomal Wnt4 enhanced β-catenin translocation to the nucleus in normoxic CRC cells. The activation of β-catenin signalling was important for the migration and invasion of normoxic CRC cells, which was eliminated by treatment with the β-catenin inhibitor ICG-001. Taken together, the results of our study indicate that hypoxia may stimulate tumour cells to release Wnt4-rich exosomes that are delivered to normoxic cells to enhance prometastatic behaviours, which might provide new targets for CRC treatment.

Objective The aim of the present study was to analyze the expression of the CD63, S100A6, and GNB2L1genes, which participate in mechanisms related to the complex pathophysiology of endometriosis. Methods A case-control study was conducted with 40 women who were diagnosed with endometriosis, and 15 fertile and healthy women. Paired samples of eutopic endometrium and endometriotic lesions (peritoneal and ovarian endometriotic implants) were obtained from the women with endometriosis in the proliferative (n = 20) or secretory phases (n = 20) of the menstrual cycle. As controls, paired endometrial biopsy samples were collected from the healthy women in the proliferative (n = 15) and secretory (n = 15) phases of the same menstrual cycle. We analyzed the expression levels of the CD63, S100A6, and GNB2L1 genes by real-time polymerase chain reaction. Results An increase in CD63, S100A6, and GNB2L1 gene transcript levels was observed in the ectopic implants compared with the eutopic endometrium of the women with and without endometriosis, regardless of the phase of the menstrual cycle. Conclusion These findings suggest that the CD63, S100A6, and GNB2L1 genes may be involved in the pathogenesis of endometriosis, since they participate in mechanisms such as inhibition of apoptosis, angiogenesis and cell proliferation, which lead to the loss of cell homeostasis in the ectopic endometrium, thus contributing to the implantation and survival of the tissue in the extrauterine environment.

The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary extracellular vesicles. We developed a SWATH-MS workflow with a curated spectral library of 1,145 targets. Application of the workflow across nine replicates of three sample types (exosome-like vesicles (ELVs), microvesicles (MVs) and urine proteins (UPs)) resulted in the quantitation of 888 proteins at FDR