Autoimmune Mast Cell Activation Test as a Diagnostic Tool in Chronic Spontaneous Urticaria.

Chronic spontaneous urticaria (CSU) is associated with skin mast cell activation, and its triggering mechanisms are not completely elucidated. Evidence suggests an autoimmune component of CSU. Our aim was to assess the usefulness of an autoimmune mast cell activation test (aiMAT) for diagnosing and differentiating CSU into different subtypes. We enrolled 43 patients with active, uncontrolled CSU before starting treatment with omalizumab and 15 controls. Patients were evaluated based on omalizumab response. aiMATs were performed using non-IgE-sensitized (NS) or myeloma IgE-sensitized (S) LAD2 cells, which were then stimulated with CSU/control sera (25 µL and 10 µL). The expression of CD63 was assessed with flow cytometry. CD63 response on NS-LAD2 was significantly increased in CSU patients compared to controls after the stimulation with 25 µL CSU/control sera (p = 0.0007) and with 10 µL CSU/control sera (p = 0.0001). The ROC curve analysis demonstrated an area under the curve (AUC) of 0.82. The cutoff for autoimmune-non-IgE-sensitized-MAT was 40.3% CD63+ LAD2, which resulted in 73.3% sensitivity and 81.4% specificity. CD63 response on S-LAD2 was significantly increased in CSU patients compared to controls after the stimulation with 25 µL CSU/control sera (p = 0.03). The ROC curve analysis demonstrated an AUC of 0.66. The cutoff for the autoimmune-myeloma IgE-sensitized-MAT was 58.4% CD63+ cells, which resulted in 62.8% sensitivity and 66.7% specificity. Overall, 36 out of 43 (84%) patients responded to omalizumab, and 7 (16%) were nonresponders. We found no differences between LAD2 CD63 response and response to omalizumab. In conclusion, aiMAT could represent a new diagnostic tool in CSU. Additional studies are needed to evaluate the potential benefits during omalizumab therapy.

Platinum-perovskite nanocomposite-based Exosensor for specific detection of prostate cancer in clinical settings.

Exosomes, extracellular vesicles (EVs) with an average size of 50-150 nm, transfer various biomolecules and exchange signaling molecules between cells in a paracrine manner. Molecular investigations have revealed that EVs can reflect real-time metabolic changes in normal- and cancer-origin cells and thus harbor valid diagnostic biomarkers. Despite these advantages, the detection of low concentrations of cancer cell EVs in biological fluids is still a great challenge. Here, a new electrochemical Exosensor based on platinum-perovskite is developed for the direct detection of EVs using a biotinylated monoclonal CD63 antibody as a capture element. The label-free method exhibited higher sensitivity with a lower limit of quantification of 2000 EVs/µL with a dynamic linear range (LDR) of 2000 to 14,000 EVs/µL compared with other available methods. To enhance the selectivity of detection, EVs were simultaneously sandwiched between secondary antibodies of PSA (prostate-specific antigen), as an FDA-approved prostate cancer biomarker. Data indicated that this Exosensor can distinguish normal and cancer EVs in samples from healthy individuals and prostate cancer patients. Taken together, this technology offers a unique approach to label-free quantification of EVs and cancer detection in the early stages.

IRTIDP: A simple integrated real-time isolation and detection platform for small extracellular vesicles Glypican-1 in pancreatic cancer patients.

Glypican-1 (GPC-1) protein-positive small extracellular vesicles (GPC-1+-sEV) have been proposed as potential biomarkers for early diagnosis of pancreatic cancer. In this study, we present an integrated real-time isolation and detection platform (IRTIDP) to capture and analyze GPC-1+-sEV directly from sera of pancreatic cancer patients. First, CD63 antibody-modified metal-organic framework (MOF) materials were utilized to enrich sEVs with a capture efficiency of 93.93 %. Second, a SERS probe was constructed by Raman reporter 4-MBA and GPC-1 antibody modified SERS active silver nanoparticles (AgNPs), which formed a sandwich complex structure of "MOFs@GPC-1+-sEV@AgNPs-4-MBA" with MOFs-enriched sEVs. The IRTSDP can complete the capture and detection process within 35 min, with a detection limit for 1 GPC-1+-sEV/µL, and linear range between 105∼109 GPC-1+-sEV/mL. Furthermore, this approach has been applied to quantify serum sEV GPC-1 in clinical pancreatic cancer patients. Based on the SERS intensity analysis, pancreatic cancer patients can be distinguished from pancreatic cystadenoma patients and healthy individuals effectively using this innovative platform that provides highly specific and sensitive means for early diagnosis of pancreatic cancer as well as other tumor types.

Intermittent lysis on a single paper-based device to extract exosomal nucleic acid biomarkers from biological samples for downstream analysis.

As the role of exosomes in physiological and pathological processes has been properly perceived, harvesting them and their internal components is critical for subsequent applications. This study is a debut of intermittent lysis, which has been integrated into a simple and easy-to-operate procedure on a single paper-based device to extract exosomal nucleic acid biomarkers for downstream analysis. Exosomes from biological samples were captured by anti-CD63-modified papers before being intermittently lysed by high-temperature, short-time treatment with double-distilled water to release their internal components. Exosomal nucleic acids were finally adsorbed by sol-gel silica for downstream analysis. Empirical trials not only revealed that sporadically dropping 95 °C ddH2O onto the anti-CD63-modified papers every 5 min for 6 times optimized the exosomal nucleic acids extracted by the anti-CD63 paper but also verified that the whole deployed procedure is applicable for point-of-care testing (POCT) in low-resource areas and for both in vitro (culture media) and in vivo (plasma and chronic lesion) samples. Importantly, downstream analysis of exosomal miR-21 extracted by the paper-based procedure integrated with this novel technique discovered that the content of exosomal miR-21 in chronic lesions related to their stages and the levels of exosomal carcinoembryonic antigen originated from colorectal cancer cells correlated to their exosomal miR-21.

Protein profile of purified plasma- and aqueous humor-derived extracellular vesicles from patients with ocular toxoplasmosis.

PURPOSE: To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis. METHODS: Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis. RESULTS: All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases. CONCLUSION: The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.

Urinary Extracellular Vesicles as a Readily Available Biomarker Source: A Simplified Stratification Method.

Urine, a common source of biological markers in biomedical research and clinical diagnosis, has recently generated a new wave of interest. It has recently become a focus of study due to the presence of its content of extracellular vesicles (EVs). These uEVs have been found to reflect physiological and pathological conditions in kidney, urothelial, and prostate tissue and can illustrate further molecular processes, leading to a rapid expansion of research in this field In this work, we present the advantages of an immunoaffinity-based method for uEVs' isolation with respect to the gold standard purification approach performed by differential ultracentrifugation [in terms of purity and antigen presence. The immunoaffinity method was made feasible by combining specific antibodies with a functionalized polymethacrylate polymer. Flow cytometry indicated a significant fluorescence shift, validating the presence of the markers (CD9, CD63, CD81) and confirming the effectiveness of the isolation method. Microscopy evaluations have shown that the morphology of the vesicles remained intact and corresponded to the expected shapes and dimensions of uEVs. The described protocol is inexpensive, fast, easy to process, has good reproducibility, and can be applied to further biological samples.

Urine beyond electrolytes: diagnosis through extracellular vesicles.

BACKGROUND AND OBJECTIVE: Extracellular vesicles (EV) reflect the pathophysiological state of their cells of origin and are a reservoir of renal information accessible in urine. When biopsy is not an option, EV present themselves as sentinels of function and damage, providing a non-invasive approach. However, the analysis of EV in urine requires prior isolation, which slows down and hinders transition into clinical practice. The aim of this study is to show the applicability of the "single particle interferometric reflectance imaging sensor" (SP-IRIS) technology through the ExoView® platform for the direct analysis of urine EV and proteins involved in renal function. MATERIALS AND METHODS: The ExoView® technology enables the quantification and phenotyping of EV present in urine and the quantification of their membrane and internal proteins. We have applied this technology to the quantification of urinary EV and their proteins with renal tubular expression, amnionless (AMN) and secreted frizzled-related protein 1 (SFRP1), using only 5 µl of urine. Tubular expression was confirmed by immunohistochemistry. RESULTS: The mean size of the EV analysed was 59 ± 16 nm for those captured by tetraspanin CD63, 61 ± 16 nm for those captured by tetraspanin CD81, and 59 ± 10 for tetraspanin CD9, with CD63 being the majority EV subpopulation in urine (48.92%). The distribution of AMN and SFRP1 in the three capture tetraspanins turned out to be similar for both proteins, being expressed mainly in CD63 (48.23% for AMN and 52.1% for SFRP1). CONCLUSIONS: This work demonstrates the applicability and advantages of the ExoView® technique for the direct analysis of urine EV and their protein content in relation to the renal tubule. The use of minimum volumes, 5 µl, and the total analysis time not exceeding three hours facilitate the transition of EV into daily clinical practice as sources of diagnostic information.

[Regulatory effect of Diwu Yanggan Decoction on lysoglycerophospholipids in circulating exosomes in a mouse model of nonalcoholic fatty liver disease].

OBJECTIVE: To evaluate the regulatory effect of Diwu Yanggan (DWYG) Decoction on lysoglycerophospholipids (Lyso-GPLs) in circulating exosomes in a mouse model of nonalcoholic fatty liver disease (NAFLD). METHODS: Circulating exosomes isolated from mouse serum by size exclusion chromatography were morphologically characterized using transmission electron microscope and examined for surface markers CD9, CD63 and TSG101 using Western blotting. Twenty-four male Kunming mice were randomized into 3 groups for normal feeding (control, n=8) or high-fat diet feeding for 1 week to induce NAFLD, after which the latter mice were given DWYG decoction (treatment group, n=8) or normal saline (model group, n=8) by gavage for 4 weeks. After the last treatment, blood samples were collected from the mice for testing serum TC, HDL-C, LDL-C, ALT and AST levels and isolating circulating exosomes. Using multivariate statistical analysis based on targeted metabolomics strategy, the potential biomarkers for Lyso-GPLs in the exosomes were screened. RESULTS: The isolated exosomes about 100 nm in size had a typical saucer-like structure with distinct double-layer membranes and a mean particle size of 137.5 nm and expressed the specific surface marker proteins CD9, CD63 and TSG101. The mouse models of NAFLD had significantly increased serum levels of TC, HDL-C, LDL-C and AST and lowered serum ALT level. A total of 43 Lyso-GPLs with significant reduction after DWYG Decoction treatment were identified in NAFLD mice. CONCLUSION: DWYG Decoction can regulate Lyso-GPLs in circulating exosomes in NAFLD mice, which provides a new clue for studying the therapeutic mechanism of DWYG Decoction for liver disease.

Quality control and validation of extracellular vesicles isolated from cultured human breast cancer cells.

OBJECTIVE: Extracellular vesicles (EVs) have been shown to play a critical role in promoting tumorigenesis. As EV research grows, it is of importance to have standardization of isolation, quality control, characterization and validation methods across studies along with reliable references to explore troubleshooting solutions. Therefore, our objective with this Research Note was to isolate EVs from multiple breast cancer cell lines and to describe and perform protocols for validation as outlined by the list of minimal information for studies of EVs (MISEV) from the International Society for Extracellular Vesicles. RESULTS: To isolate EVs, two techniques were employed: ultracentrifugation and size exclusion chromatography. Ultracentrifugation yielded better recovery of EVs in our hands and was therefore used for further validation. In order to satisfy the MISEV requirements, protein quantification, immunoblotting of positive (CD9, CD63, TSG101) and negative (TGFß1, ß-tubulin) markers, nanoflow cytometry and electron microscopy was performed. With these experiments, we demonstrate that yield of validated EVs varied between different breast cancer cell lines. Protocols were optimized to accommodate for low levels of EVs, and various technical and troubleshooting suggestions are included for potential application to other cell types that may provide benefit to investigators interested in future EV studies.

Mast cell secretory granule fusion with amphisomes coordinates their homotypic fusion and release of exosomes.

Secretory granule (SG) fusion is an intermediate step in SG biogenesis. However, the precise mechanism of this process is not completely understood. We show that Golgi-derived mast cell (MC) SGs enlarge through a mechanism that is dependent on phosphoinositide (PI) remodeling and fusion with LC3+ late endosomes (amphisomes), which serve as hubs for the fusion of multiple individual SGs. Amphisome formation is regulated by the tyrosine phosphatase PTPN9, while the subsequent SG fusion event is additionally regulated by the tetraspanin protein CD63 and by PI4K. We also demonstrate that fusion with amphisomes imparts to SGs their capacity of regulated release of exosomes. Finally, we show that conversion of PI(3,4,5)P3 to PI(4,5)P2 and the subsequent recruitment of dynamin stimulate SG fission. Our data unveil a key role for lipid-regulated interactions with the endocytic and autophagic systems in controlling the size and number of SGs and their capacity to release exosomes.

Extracellular vesicle isolation methods identify distinct HIV-1 particles released from chronically infected T-cells.

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.

Co-crosslinking strategy for dual functionalization of small magnetic nanoparticles with redox probes and biological probes.

Surface functionalization strategy is becoming a crucial bridge from magnetic nanoparticles (MNPs) to their broad bio-application. To realize the multiple functions of MNPs such as magnetic manipulation, target capture, and signal amplification in their use of electrochemical biosensing, co-crosslinking strategy was proposed here to construct dual-functionalized MNPs by combining ultra-sensitive redox moieties and specific biological probes. In this work, MNPs with a TEM size of 10 nm were synthesized by co-precipitation for amination and PEGylation to maintain colloid stability once dispersed in high-ionic-strength buffer (such as phosphate-buffered saline). Then, MNPs@IgG were prepared via the bis(sulfosuccinimidyl) suberate (BS3) cross-linker to conjugate these IgG onto the MNP surface, with a binding efficiency of 73%. To construct dual-functionalized MNPs, these redox probes of ferrocene-NHS (Fc) were co-crosslinked onto the MNP surface, together with IgG, by using BS3. The developed MNPs@Redox@IgG were characterized by SDS‒PAGE to identify IgG binding and by square wave voltammetry (SWV) to validate the redox signal. Additionally, the anti-CD63 antibodies were selected for the development of MNPs@anti-CD63 for use in the bio-testing of exosome sample capture. Therefore, co-crosslinking strategy paved a way to develop dual-functionalized MNPs that can be an aid of their potential utilization in diagnostic assay or electrochemical methods.

An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma.

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.

Accurate and highly sensitive detection of Alzheimer's disease-related extracellular vesicles via förster resonance energy transfer.

Alzheimer's disease (AD) is the most common neurodegenerative disease in the world and poses a huge challenge to global healthcare. Early and accurate detection of amyloid-ß (1-42) (Aß42), a key biomarker of AD, is crucial for effective diagnosis and intervention of AD. Specific or overexpressed proteins on extracellular vesicles (EVs) describe a close correlation with the occurrence and development of diseases. EVs are a very promising non-invasive biomarker for the diagnosis of AD and other diseases. As a sensitive, simple and rapid analytical method, fluorescence resonance energy transfer (FRET) has been widely applied in the detection of EVs. Herein, we developed a dual labelling strategy for simultaneously detecting EV membrane proteins of Aß42 and CD63 based on FRET pair consisting of Au nanoclusters (AuNCs) and polydopamine nanospheres (PDANSs). The constructed nanoprobe, termed EVMPFAP assay, could specifically measure the Aß42 and CD63 on EVs with excellent sensitivity, high specificity and satisfactory accuracy. The limit of detection of EVMPFAP assay was 1.4 × 103 particles mL-1 and the linear range was from 104 to 108 particles mL-1. EVMPFAP assay was successfully used to analyze plasma EVs to distinguish AD and healthy mice. We expect that EVMPFAP assay can be routinely applied for early diagnosis and development-monitoring of AD, thus facilitating the fight against AD.

Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

CD63 + tumor-associated macrophages drive the progression of hepatocellular carcinoma through the induction of epithelial-mesenchymal transition and lipid reprogramming.

BACKGROUND: Tumor-associated macrophages (TAMs) constitute a substantial part of human hepatocellular carcinoma (HCC). The present study was devised to explore TAM diversity and their roles in HCC progression. METHODS: Through the integration of multiple 10 × single-cell transcriptomic data derived from HCC samples and the use of consensus nonnegative matrix factorization (an unsupervised clustering algorithm), TAM molecular subtypes and expression programs were evaluated in detail. The roles played by these TAM subtypes in HCC were further probed through pseudotime, enrichment, and intercellular communication analyses. Lastly, vitro experiments were performed to validate the relationship between CD63, which is an inflammatory TAM expression program marker, and tumor cell lines. RESULTS: We found that the inflammatory expression program in TAMs had a more obvious interaction with HCC cells, and CD63, as a marker gene of the inflammatory expression program, was associated with poor prognosis of HCC patients. Both bulk RNA-seq and vitro experiments confirmed that higher TAM CD63 expression was associated with the growth of HCC cells as well as their epithelial-mesenchymal transition, metastasis, invasion, and the reprogramming of lipid metabolism. CONCLUSIONS: These analyses revealed that the TAM inflammatory expression program in HCC is closely associated with malignant tumor cells, with the hub gene CD63 thus representing an ideal target for therapeutic intervention in this cancer type.

A proximity ligation hybridization triggered structure-switching based signal amplification strategy for sensitive and accurate exosome detection.

Exosomes have significant functions in intercellular communication, as well as in tumor migration and invasion. Nevertheless, the precise identification of exosomes poses a significant obstacle due to their low abundance in biofluids and potential disruption caused by free protein molecules, such as CD63 protein. In this study, we have developed a signal amplification method for precise detection of exosomes using a proximity ligation hybridization triggered structure-switching approach. The method involves the dual-recognition of exosomes by two probes: an aptamer probe that recognizes the exosomal surface protein CD63 (L1 probe), and a cholesterol probe that targets the biolipid layer of the exosomes (L2 probe). Based on the dual-recognition of exosomes, we have successfully developed an accurate and sensitive approach that integrates the proximity ligation hybridization technique with a structure-switching based signal cycle. This approach allows for the simultaneous analysis of two biomarkers, enabling both quantification and tracing of exosomes without the need for enzymes. Eventually, the proposed method exhibits a wide detection range of 5 orders of magnitude and a low limit of detection of 36 particles per µL, making it suitable for a wide range of applications in the fields of biological science, biomedical engineering, and personalized medicine.

CD63 sorts cholesterol into endosomes for storage and distribution via exosomes.

Extracellular vesicles such as exosomes are now recognized as key players in intercellular communication. Their role is influenced by the specific repertoires of proteins and lipids, which are enriched when they are generated as intraluminal vesicles (ILVs) in multivesicular endosomes. Here we report that a key component of small extracellular vesicles, the tetraspanin CD63, sorts cholesterol to ILVs, generating a pool that can be mobilized by the NPC1/2 complex, and exported via exosomes to recipient cells. In the absence of CD63, cholesterol is retrieved from the endosomes by actin-dependent vesicular transport, placing CD63 and cholesterol at the centre of a balance between inward and outward budding of endomembranes. These results establish CD63 as a lipid-sorting mechanism within endosomes, and show that ILVs and exosomes are alternative providers of cholesterol.

A cavity induced mode hybridization plasmonic sensor for portable detection of exosomes.

Exosomes have been considered as promising biomarkers for cancer diagnosis due to their abundant information from originating cells. However, sensitive and reliable detection of exosomes is still facing technically challenges due to the lack of a sensing platform with high sensitivity and reproducibility. To address the challenges, here we propose a portable surface plasmon resonance (SPR) sensing of exosomes with a three-layer Au mirror/SiO2 spacer/Au nanohole sensor, fabricated by an economical polystyrene nanosphere self-assembly method. The SiO2 spacer can act as an optical cavity and induce mode hybridization, leading to excellent optimization of both sensitivity and full width at half maximum compared with normal single layer Au nanohole sensors. When modified with CD63 or EpCAM aptamers, a detection of limit (LOD) of as low as 600 particles/µL was achieved. The sensors showed good capability to distinguish between non-tumor derived L02 exosomes and tumor derived HepG2 exosomes. Additionally, high reproducibility was also achieved in detection of artificial serum samples with RSD as low as 2%, making it feasible for clinical applications. This mode hybridization plasmonic sensor provides an effective approach to optimize the detection sensitivity of exosomes, pushing SPR sensing one step further towards cancer diagnosis.

The impact of exosomes derived from B-cell acute lymphoblastic leukemia as a growth factor on bone marrow mesenchymal stromal cells.

Background The incidence of various types of cancers, including leukemia, is on the rise and many challenges in both drug resistance and complications related to chemotherapy appeared. Recently, the development and application of extracellular vesicles (EV) such as exosomes in the management of cancers, especially leukemia, holds great significance. In this article, we extracted exosomes from NALM6 cells and assessed their regulatory effects on proliferation and apoptosis in mesenchymal stem cells (MSCs). Method and result We first verified the exosomes using various techniques, including flow cytometry, transient electron microscopy, dynamic light scattering (DLS), and BCA protein assay. Then MTT analysis and flowcytometry (apoptosis and cell cycle assay) besides gene expressions were employed to determine the state of MSC proliferations. The results indicated that exosome-specific pan markers like CD9, CD63, and CD81 were present. Through DLS, we found out that the mean size of the exosomes was 89.68 nm. The protein content was determined to be 956.292 µg/ml. Analysis of MTT, flow cytometry (cell cycle and apoptosis assay), and RT-qPCR showed that in the dose of 50 µg/ml the proliferation of MSCs was increased significantly (p-value < 0.05). Conclusion All these data showed that exosomes use several signaling pathways to increase the MSCs' proliferation and drug resistance, ultimately leading to high mortalities and morbidities of acute lymphoblastic leukemia.