USP9X-enriched MSC-sEV inhibits LSEC angiogenesis in MASH mice by downregulating the IκBα/NF-κB/Ang-2 pathway.

Pathological angiogenesis of liver sinusoidal endothelial cells (LSEC) plays a crucial role in the progression of metabolic dysfunction-associated steatohepatitis (MASH)-induced liver fibrosis. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) have shown promising therapeutic potential against MASH. This study aimed to investigate the impact of MSC-sEV on LSEC angiogenesis and elucidate the underlying molecular mechanisms. The effects of MSC-sEV on LSEC angiogenesis were evaluated in Tumor Necrosis Factor- alpha (TNF-α)-treated LSECs in vitro and in Methionine and Choline Deficient Diet (MCD)-induced MASH mice in vivo. Herein, we found that MSC-sEV effectively suppressed LSEC angiogenesis by targeting the angiogenesis marker Angiogenin 2 (Ang-2) in both TNF-α-treated LSECs and MASH mice. Gene manipulation experiments revealed that the primary mechanism by which MSC-sEV inhibited LSEC angiogenesis was through the modulation of nuclear factor kappa B inhibitor alpha (IκBα) / nuclear factor kappa B (NF-κB) / Ang-2 pathway. Additionally, mass spectrometry and co-immunoprecipitation (Co-IP) data suggested that MSC-sEV delivered the ubiquitin specific peptidase 9 X-linked (USP9X) protein to LSECs, leading to enhanced IκBα deubiquitination and NF-κB in activation, ultimately resulting in the inhibition of Ang-2-mediated LSEC angiogenesis. Knockdown of USP9X attenuated the regulatory effects of MSC-sEV on Ang-2 expression, LSEC angiogenesis, and the progression of MASH. In conclusion, our findings indicate that USP9X delivered via MSC-sEV can suppress LSEC angiogenesis and alleviate MASH-induced liver fibrosis through the IκBα/NF-κB/Ang-2 signaling pathway.

miRNA packaging into small extracellular vesicles and implications in pain.

Extracellular vesicles (EVs) are a heterogenous group of lipid bilayer bound particles naturally released by cells. These vesicles are classified based on their biogenesis pathway and diameter. The overlap in size of exosomes generated from the exosomal pathway and macrovesicles that are pinched off from the surface of the plasma membrane makes it challenging to isolate pure populations. Hence, isolated vesicles that are less than 200 nm are called small extracellular vesicles (sEVs). Extracellular vesicles transport a variety of cargo molecules, and multiple mechanisms govern the packaging of cargo into sEVs. Here, we discuss the current understanding of how miRNAs are targeted into sEVs, including the role of RNA binding proteins and EXOmotif sequences present in miRNAs in sEV loading. Several studies in human pain disorders and rodent models of pain have reported alterations in sEV cargo, including miRNAs. The sorting mechanisms and target regulation of miR-939, a miRNA altered in individuals with complex regional pain syndrome, is discussed in the context of inflammation. We also provide a broad overview of the therapeutic strategies being pursued to utilize sEVs in the clinic and the work needed to further our understanding of EVs to successfully deploy sEVs as a pain therapeutic.

High-throughput screening identifies ibuprofen as an sEV PD-L1 inhibitor for synergistic cancer immunotherapy.

Programmed death-ligand 1 (PD-L1) on tumor-derived small extracellular vesicles (sEVs) limits therapeutic effectiveness by interacting with the PD-1 receptor on host immune cells. Targeting the secretion of sEV PD-L1 has emerged as a promising strategy to enhance immunotherapy. However, the lack of small-molecule inhibitors poses a challenge for clinical translation. In this study, we developed a target and phenotype dual-driven high-throughput screening strategy that combined virtual screening with nanoflow-based experimental verification. We identified ibuprofen (IBP) as a novel inhibitor that effectively targeted sEV PD-L1 secretion. IBP disrupted the biogenesis and secretion of PD-L1+ sEVs in tumor cells by physically interacting with a critical regulator of sEV biogenesis, hepatocyte growth factor-regulated tyrosine kinase substrate. Notably, the mechanism of action of IBP is distinct from its commonly known targets, cyclooxygenases. Administration of IBP stimulated antitumor immunity and enhanced the efficacy of anti-PD-1 therapy in melanoma and oral squamous cell carcinoma mouse models. To address potential adverse effects, we further developed an IBP gel for topical application, which demonstrated remarkable therapeutic efficacy when combined with anti-PD-1 treatment. The discovery of this specific small inhibitor provides a promising avenue for establishing durable, systemic antitumor immunity.

Enhanced secretion of promyogenic exosomes by quiescent muscle cells.

Signaling interactions are important during skeletal muscle regeneration, where muscle cells in distinct states (quiescent, reactivated, proliferating and differentiated) must coordinate their response to injury. Here, we probed the role of secreted small extracellular vesicles (sEV/exosomes) using a culture model of physiologically relevant cell states seen in muscle regeneration. Unexpectedly, G0 myoblasts exhibited enhanced secretion of sEV (∼150 nm) displaying exosome markers (Alix, TSG101, flotillin-1, and CD9), and increased expression of Kibra, a regulator of exosome biogenesis. Perturbation of Kibra levels confirmed a role in controlling sEV secretion rates. Purified sEVs displayed a common exosome marker-enriched proteome in all muscle cell states, as well as state-specific proteins. Exosomes derived from G0 cells showed an antioxidant signature, and were most strongly internalized by differentiated myotubes. Functionally, donor exosomes from all muscle cell states could activate an integrated Wnt reporter in target cells, but only G0-derived exosomes could induce myogenic differentiation in proliferating cells. Taken together, we provide evidence that quiescence in muscle cells is accompanied by enhanced secretion of exosomes with distinct uptake, cargo and signal activating features. Our study suggests the novel possibility that quiescent muscle stem cells in vivo may play a previously under-appreciated signaling role during muscle homeostasis.

Oncoviral Infections and Small Extracellular Vesicles.

Small extracellular vesicles (sEV) are small membrane-bound nanovesicles with a size range below 200 nm that are released by all types of cells. sEV carry a diverse cargo of proteins, lipids, glycans, and nucleic acids that mimic the content of producer cells. sEV mediate intercellular communication and play a key role in a broad variety of physiological and pathological conditions. Recently, numerous reports have emerged examining the role of sEV in viral infections. A significant number of similarities in the sEV biogenesis pathways and the replication cycles of viruses suggest that sEV might influence the course of viral infections in diverse ways. Besides directly modulating virus propagation by transporting the viral cargo (complete virions, proteins, RNA, and DNA), sEV can also modify the host antiviral response and increase the susceptibility of cells to infection. The network of mutual interactions is particularly complex in the case of oncogenic viruses, deserving special consideration because of its significance in cancer progression. This review summarizes the current knowledge of interactions between sEV and oncogenic viruses, focusing on sEV abilities to modulate the carcinogenic properties of oncoviruses.

MiR-4465-modified mesenchymal stem cell-derived small extracellular vesicles inhibit liver fibrosis development via targeting LOXL2 expression. / MiR-4465修饰的间质干细胞来源的小细胞外囊泡通过靶向LOXL2表达抑制肝纤维化的进展.

Liver fibrosis is a significant health burden, marked by the consistent deposition of collagen. Unfortunately, the currently available treatment approaches for this condition are far from optimal. Lysyl oxidase-like protein 2 (LOXL2) secreted by hepatic stellate cells (HSCs) is a crucial player in the cross-linking of matrix collagen and is a significant target for treating liver fibrosis. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have been proposed as a potential treatment option for chronic liver disorders. Previous studies have found that MSC-sEV can be used for microRNA delivery into target cells or tissues. It is currently unclear whether microRNA-4465 (miR-4465) can target LOXL2 and inhibit HSC activation. Additionally, it is uncertain whether MSC-sEV can be utilized as a gene therapy vector to carry miR-4465 and effectively inhibit the progression of liver fibrosis. This study explored the effect of miR-4465-modified MSC-sEV (MSC-sEVmiR-4465) on LOXL2 expression and liver fibrosis development. The results showed that miR-4465 can bind specifically to the promoter of the LOXL2 gene in HSC. Moreover, MSC-sEVmiR-4465 inhibited HSC activation and collagen expression by downregulating LOXL2 expression in vitro. MSC-sEVmiR-4465 injection could reduce HSC activation and collagen deposition in the CCl4-induced mouse model. MSC-sEVmiR-4465 mediating via LOXL2 also hindered the migration and invasion of HepG2 cells. In conclusion, we found that MSC-sEV can deliver miR-4465 into HSC to alleviate liver fibrosis via altering LOXL2, which might provide a promising therapeutic strategy for liver diseases.

EGFR Mutation and TKI Treatment Promote Secretion of Small Extracellular Vesicle PD-L1 and Contribute to Immunosuppression in NSCLC.

In Asian populations with non-small-cell lung cancer (NSCLC), EGFR mutations are highly prevalent, occurring in roughly half of these patients. Studies have revealed that individuals with EGFR mutation typically fare worse with immunotherapy. In patients who received EGFR tyrosine kinase inhibitor (TKI) treatment followed by anti-PD-1 therapy, poor results were observed. The underlying mechanism remains unclear. We used high-resolution flow cytometry and ELISA to detect the circulating level of small extracellular vesicle (sEV) PD-L1 in NSCLC individuals with EGFR mutations before and after receiving TKIs. The secretion amount of sEV PD-L1 of lung cancer cell lines with EGFR mutations under TKI treatment or not were detected using high-resolution flow cytometry and Western blotting. The results revealed that patients harboring EGFR mutations exhibit increased levels of sEV PD-L1 in circulation, which inversely correlated with the presence of CD8+ T cells in tumor tissues. Furthermore, tumor cells carrying EGFR mutations secrete a higher quantity of PD-L1-positive sEVs. TKI treatment appeared to amplify the levels of PD-L1-positive sEVs in the bloodstream. Mutation-induced and TKI-induced sEVs substantially impaired the functionality of CD8+ T cells. Importantly, our findings indicated that EGFR mutations and TKI therapies promote secretion of PD-L1-positive sEVs via distinct molecular mechanisms, namely the HRS and ALIX pathways, respectively. In conclusion, the increased secretion of PD-L1-positive sEVs, prompted by genetic alterations and TKI administration, may contribute to the limited efficacy of immunotherapy observed in EGFR-mutant patients and patients who have received TKI treatment.

Decoding the secrets of small extracellular vesicle communications: exploring the inhibition of vesicle-associated pathways and interception strategies for cancer treatment.

Cancer disease is the second leading cause of death worldwide. In 2023, about 2 million new cancer cases and 609,820 cancer deaths are projected to occur in the United States. The driving forces of cancer progression and metastasis are widely varied and comprise multifactorial events. Although there is significant success in treating cancer, patients still present with tumors at advanced stages. Therefore, the discovery of novel oncologic pathways has been widely developed. Tumor cells communicate with each other through small extracellular vesicles (sEVs), which contribute to tumor-stromal interaction and promote tumor growth and metastasis. sEV-specific inhibitors are being investigated as a next-generation cancer therapy. A literature search was conducted to discuss different options for targeting sEV pathways in cancer cells. However, there are some challenges that need to be addressed in targeting sEVs: i) specificity and toxicity of sEV inhibitor, ii) targeted delivery of sEV inhibitors, iii) combination of sEV inhibitors with current standard chemotherapy to improve patients' clinical outcomes, and iv) data reproducibility and applicability at distinct levels of the disease. Despite these challenges, sEV inhibitors have immense potential for effectively treating cancer patients.

Effects of small extracellular vesicles derived from normoxia- and hypoxia-treated prostate cancer cells on the submandibular salivary gland epithelium in vitro.

Small extracellular vesicles (sEVs) are an important part of intercellular communication. They are phospholipid bilayer particles that carry active biomolecules such as proteins, various nucleic acids, and lipids. In recipient cells, sEVs can alter cellular functions, including cancer development and premetastatic niche formation in distant organs. Moreover, sEVs can carry cancer-specific features, which makes them promising biomarker candidates. However, the interactions of sEVs with biological barriers and consequences thereof, are not clarified yet. The blood-saliva barrier is crucial for preventing the entry of pathogens and (in)organic substances into the bloodstream, as well as molecule filtration from blood to saliva. The effects of brain derived DU145 prostate cancer (PCa) sEVs on a human submandibular salivary gland barrier (SSGB) in vitro were investigated. Small EVs were harvested from normoxic (N, atmospheric O2) or hypoxic (H, 1% O2) conditions, fluorescently labeled with CellTrackerTM Orange and thoroughly characterized. HTB-41 B2 cells were used as SSGB model cultured on 24-well ThinCert® inserts. After model optimization indicating effects of serum and serum-sEVs on barrier properties, PCa sEVs were applied to the basolateral (blood) side in either 10% serum, or serum-free conditions, and barrier integrity was continuously monitored for 40 hours. This study found that H and N PCa sEVs were uptaken by the SSGB in vitro model in similar quantities regardless of the media composition in the basolateral compartment. Permeation of fluorescent PCa sEVs into the apical compartment was not detectable with the applied methods. However, treatment with H and N sEVs under different serum conditions revealed distinct molecular clusters after hierarchical analysis of mRNA data measured by high-throughput qPCR, which were partly reflected at the protein level. For example, serum-reduction dependent decrease of barrier properties was accompanied with the decrease of CDH1 or Claudin-7 expression. Interestingly, the presence of H sEVs significantly increased the number of sEV-sized particles in the apical compartment of the SSGB model compared to basolaterally added N sEVs. This functional effect on the number of particles in the saliva (apical) compartment induced by different sEVs applied in the blood (basolateral) compartment might be a new approach to understand one possible mechanism how differences of salivary EVs might occur which then could be used as biomarker.

Sensitive electrochemical biosensor for rapid detection of sEV-miRNA based turbo-like localized catalytic hairpin assembly.

Small extracellular vesicle-associated microRNAs (sEV-miRNAs) have emerged as critical biomarkers for cancer diagnosis, yet the rapid detection of these low-abundance molecules in clinical samples remains a formidable challenge. Herein, a simple turbo-like localized catalytic hairpin assembly (TL-CHA) was proposed for sEV-miR-1246 measurement. This electrochemical sensor achieves dual localization through the ingeniously use of AuNPs and DNA nanowires, which provides rich sites for CHA cascade amplification, significantly enhancing the effective reaction and amplify the detection response. Leveraging this innovative design, this biosensor demonstrated the ability to detect sEV-miRNA at concentrations as low as 5.24 aM in a time frame of 30 min. The precision of the measurements was validated through reverse transcription quantitative polymerase chain reaction. Furthermore, the sensor was used for analyzing plasma samples from gastric cancer patients yielded AUC values of 0.973 for all stages and 0.945 for early stages. This demonstrates the sensor's robust performance in both the staging diagnosis and early screening of gastric cancer. Therefore, this platform has great potential for the clinical cancer diagnosis.

Analysis of CD20 and PD-L1 levels on small extracellular vesicles (sEV) produced by DLBCL cells and EBV-transformed B cells, and potential role in T cell inhibition.

Increasing evidence supports a role for small extracellular vesicles (sEV, including exosomes) in Diffuse Large B-cell lymphoma (DLBCL) progression and resistance to treatment. CD20 and PD-L1 are found on DLBCL-derived sEV, but little is known about their patient-level heterogeneity. Moreover, the capacity of PD-L1+ sEV to modulate T cells needs to be clarified. Herein we analyzed sEV produced by human DLBCL cell lines and EBV-transformed B cell-lymphoblastoid cell lines (LCLs), a model allowing autologous T cell co-cultures. We determined CD20 and PD-L1 levels on plasma sEV from patient samples vs healthy volunteers (HV). sEV functional relevance was also investigated on CD4+ and CD8+ T cells. sEV derived from all cell lines showed an enrichment of CD20 and a high glycosylated PD-L1 expression when compared to cell lysates. High PD-L1 expression on LCL-derived sEV was associated with higher CD4+ and CD8+ T cell apoptosis. In patients, plasma sEV concentration was higher vs HV. Compared to sEV-CD20 level that seemed higher in patients, PD-L1 level in sEV was not different from those of HV. A high glycosylated PD-L1 level was shown in sEV from both patients and HV plasma samples, that was associated with the same inhibiting effect on activated T cells. We conclude that sEV derived from EBV-transformed B cells realize an immunosuppressive role that involved cell-cell interaction and probably at least PD-L1. Furthermore, our findings suggest the potential of circulating sEV as a source of biomarkers in DLBCL, notably to have information on immunotherapeutic target levels of parental tumor cells.

Single-sEV profiling identifies the TACSTD2 + sEV subpopulation as a factor of tumor susceptibility in the elderly.

BACKGROUND: Aging is a very complex physiological phenomenon, and sEVs are involved in the regulation of this mechanism. Serum samples from healthy individuals under 30 and over 60 years of age were collected to analyze differences in sEVs proteomics. RESULTS: Based on PBA analysis, we found that sEVs from the serum of elderly individuals highly express TACSTD2 and identified a subpopulation marked by TACSTD2. Using ELISA, we verified the upregulation of TACSTD2 in serum from elderly human and aged mouse. In addition, we discovered that TACSTD2 was significantly increased in samples from tumor patients and had better diagnostic value than CEA. Specifically, 9 of the 13 tumor groups exhibited elevated TACSTD2, particularly for cervical cancer, colon cancer, esophageal carcinoma, liver cancer and thyroid carcinoma. Moreover, we found that serum sEVs from the elderly (especially those with high TACSTD2 levels) promoted tumor cell (SW480, HuCCT1 and HeLa) proliferation and migration. CONCLUSION: TACSTD2 was upregulated in the serum of elderly individuals and patients with tumors, and could serve as a dual biomarker for aging and tumors.

Keratinocyte-derived small extracellular vesicles supply antigens for CD1a-resticted T cells and promote their type 2 bias in the context of filaggrin insufficiency.

Introduction: Exosome-enriched small extracellular vesicles (sEVs) are nanosized organelles known to participate in long distance communication between cells, including in the skin. Atopic dermatitis (AD) is a chronic inflammatory skin disease for which filaggrin (FLG) gene mutations are the strongest genetic risk factor. Filaggrin insufficiency affects multiple cellular function, but it is unclear if sEV-mediated cellular communication originating from the affected keratinocytes is also altered, and if this influences peptide and lipid antigen presentation to T cells in the skin. Methods: Available mRNA and protein expression datasets from filaggrin-insufficient keratinocytes (shFLG), organotypic models and AD skin were used for gene ontology analysis with FunRich tool. sEVs secreted by shFLG and control shC cells were isolated from conditioned media by differential centrifugation. Mass spectrometry was carried out for lipidomic and proteomic profiling of the cells and sEVs. T cell responses to protein, peptide, CD1a lipid antigens, as well as phospholipase A2-digested or intact sEVs were measured by ELISpot and ELISA. Results: Data analysis revealed extensive remodeling of the sEV compartment in filaggrin insufficient keratinocytes, 3D models and the AD skin. Lipidomic profiles of shFLGsEV showed a reduction in the long chain (LCFAs) and polyunsaturated fatty acids (PUFAs; permissive CD1a ligands) and increased content of the bulky headgroup sphingolipids (non-permissive ligands). This resulted in a reduction of CD1a-mediated interferon-γ T cell responses to the lipids liberated from shFLG-generated sEVs in comparison to those induced by sEVs from control cells, and an increase in interleukin 13 secretion. The altered sEV lipidome reflected a generalized alteration in the cellular lipidome in filaggrin-insufficient cells and the skin of AD patients, resulting from a downregulation of key enzymes implicated in fatty acid elongation and desaturation, i.e., enzymes of the ACSL, ELOVL and FADS family. Discussion: We determined that sEVs constitute a source of antigens suitable for CD1a-mediated presentation to T cells. Lipids enclosed within the sEVs secreted on the background of filaggrin insufficiency contribute to allergic inflammation by reducing type 1 responses and inducing a type 2 bias from CD1a-restricted T cells, thus likely perpetuating allergic inflammation in the skin.

circFTO from M2 macrophage-derived small extracellular vesicles (sEV) enhances NSCLC malignancy by regulation miR-148a-3pPDK4 axis.

BACKGROUND: Accumulation studies found that tumor-associated macrophages (TAMs) are a predominant cell in tumor microenvironment (TME), which function essentially during tumor progression. By releasing bioactive molecules, including circRNA, small extracellular vesicles (sEV) modulate immune cell functions in the TME, thereby affecting non-small cell lung cancer (NSCLC) progression. Nevertheless, biology functions and molecular mechanisms of M2 macrophage-derived sEV circRNAs in NSCLC are unclear. METHODS: Cellular experiments were conducted to verify the M2 macrophage-derived sEV (M2-EV) roles in NSCLC. Differential circRNA expression in M0 and M2-EV was validated by RNA sequencing. circFTO expression in NSCLC patients and cells was investigated via real-time PCR and FISH. The biological mechanism of circFTO in NSCLC was validated by experiments. Our team isolated sEV from M2 macrophages (M2Ms) and found that M2-EV treatment promoted NSCLC CP, migration, and glycolysis. RESULTS: High-throughput sequencing found that circFTO was highly enriched in M2-EV. FISH and RT-qPCR confirmed that circFTO expression incremented in NSCLC tissues and cell lines. Clinical studies confirmed that high circFTO expression correlated negatively with NSCLC patient survival. Luciferase reporter analysis confirmed that miR-148a-3p and PDK4 were downstream targets of circFTO. circFTO knockdown inhibited NSCLC cell growth and metastasis in in vivo experiments. Downregulating miR-148a-3p or overexpressing PDK4 restored the malignancy of NSCLC, including proliferation, migration, and aerobic glycolysis after circFTO silencing. CONCLUSION: The study found that circFTO from M2-EV promoted NSCLC cell progression and glycolysis through miR-148a-3p/PDK4 axis. circFTO is a promising prognostic and diagnostic NSCLC biomarker and has the potential to be a candidate NSCLC therapy target.

Sendai virus is robust and consistent in delivering genes into human pancreatic cancer cells.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly intratumorally heterogeneous disease that includes several subtypes and is highly plastic. Effective gene delivery to all PDAC cells is essential for modulating gene expression and identifying potential gene-based therapeutic targets in PDAC. Most current gene delivery systems for pancreatic cells are optimized for islet or acinar cells. Lentiviral vectors are the current main gene delivery vectors for PDAC, but their transduction efficiencies vary depending on pancreatic cell type, and are especially poor for the classical subtype of PDAC cells from both primary tumors and cell lines. Methods: We systemically compare transduction efficiencies of glycoprotein G of vesicular stomatitis virus (VSV-G)-pseudotyped lentiviral and Sendai viral vectors in human normal pancreatic ductal and PDAC cells. Results: We find that the Sendai viral vector gives the most robust gene delivery efficiency regardless of PDAC cell type. Therefore, we propose using Sendai viral vectors to transduce ectopic genes into PDAC cells.

Detecting Respiratory Viruses Using a Portable NIR Spectrometer-A Preliminary Exploration with a Data Driven Approach.

Respiratory viruses' detection is vitally important in coping with pandemics such as COVID-19. Conventional methods typically require laboratory-based, high-cost equipment. An emerging alternative method is Near-Infrared (NIR) spectroscopy, especially a portable one of the type that has the benefits of low cost, portability, rapidity, ease of use, and mass deployability in both clinical and field settings. One obstacle to its effective application lies in its common limitations, which include relatively low specificity and general quality. Characteristically, the spectra curves show an interweaving feature for the virus-present and virus-absent samples. This then provokes the idea of using machine learning methods to overcome the difficulty. While a subsequent obstacle coincides with the fact that a direct deployment of the machine learning approaches leads to inadequate accuracy of the modelling results. This paper presents a data-driven study on the detection of two common respiratory viruses, the respiratory syncytial virus (RSV) and the Sendai virus (SEV), using a portable NIR spectrometer supported by a machine learning solution enhanced by an algorithm of variable selection via the Variable Importance in Projection (VIP) scores and its Quantile value, along with variable truncation processing, to overcome the obstacles to a certain extent. We conducted extensive experiments with the aid of the specifically developed algorithm of variable selection, using a total of four datasets, achieving classification accuracy of: (1) 0.88, 0.94, and 0.93 for RSV, SEV, and RSV + SEV, respectively, averaged over multiple runs, for the neural network modelling of taking in turn 3 sessions of data for training and the remaining one session of an 'unknown' dataset for testing. (2) the average accuracy of 0.94 (RSV), 0.97 (SEV), and 0.97 (RSV + SEV) for model validation and 0.90 (RSV), 0.93 (SEV), and 0.91 (RSV + SEV) for model testing, using two of the datasets for model training, one for model validation and the other for model testing. These results demonstrate the feasibility of using portable NIR spectroscopy coupled with machine learning to detect respiratory viruses with good accuracy, and the approach could be a viable solution for population screening.

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

Blast-Derived Small Extracellular Vesicles in the Plasma of Patients with Acute Myeloid Leukemia Predict Responses to Chemotherapy.

The small extracellular vesicles (sEV) accumulating in acute myeloid leukemia (AML) patients' plasma are mixtures of vesicles produced by leukemic and non-malignant cells. sEV originating from leukemia blasts could serve as potential non-invasive biomarkers of AML response to therapy. To isolate blast-derived sEV from patients' plasma, we developed a bioprinted microarray-based immunoassay using monoclonal antibodies (mAbs) specific for leukemia-associated antigens (LAAs) and mAbs specific for a mix of tetraspanins (CD9, CD63, and CD81). We determined the proportion of LAA+ sEV relative to total plasma sEV (the LAA+/total sEV ratio) in serially collected samples of newly diagnosed AML patients prior to, during, and after chemotherapy. At AML diagnosis, the LAA+/total sEV ratio was significantly higher in patients than in healthy donors (HDs). In patients who achieved complete remission (CR) after induction chemotherapy, the LAA+/total sEV ratios significantly decreased after each chemotherapy cycle to levels seen in HDs. In contrast, the LAA+/total sEV ratios in AML patients with persistent leukemia after therapy remained elevated during and after therapy, as did the percentage of leukemic blasts in these patients' bone marrows. The LAA+/total sEV ratio emerges as a promising non-invasive biomarker of leukemia response to therapy.

Polarized Desmosome and Hemidesmosome Shedding via Small Extracellular Vesicles is an Early Indicator of Outer Blood-Retina Barrier Dysfunction.

The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD).

Jagged1 contained in MSC-derived small extracellular vesicles promotes squamous differentiation of cervical cancer by activating NOTCH pathway.

PURPOSE: Cervical cancer is the fourth most common cancer in women and poses a major threat to women's health, urgently requiring new treatment methods. METHODS: This study first successfully extracted and identified small extracellular vesicles secreted by human umbilical cord-derived mesenchymal stem cells. We studied the effects of MSC-sEV on the squamous differentiation levels of cervical cancer CaSki cells in vitro, and explored the effects of MSC-sEV on the NOTCH pathway, the growth, proliferation, migration abilities and squamous differentiation levels of cervical cancer cells. The roles of MSC-sEV were also verified in human keratinocyte HaCaT cells. RESULTS: The results showed that Jagged1 protein on MSC-sEV can bind to NOTCH1 on cervical cancer cells, activate NOTCH signaling, and promote squamous differentiation levels in CaSki cells, thus inhibiting the growth, proliferation and migration abilities of CaSki cells. MSC-sEV can also activate the NOTCH pathway in HaCaT cells, but promote the viability of HaCaT cells. CONCLUSION: MSC-sEV can activate the NOTCH pathway to promote squamous differentiation of CaSki cells and inhibit the growth proliferation and migration abilities of CaSki cells which may be a new mechanism for cervical cancer treatment.