Nanoparticle Tracking Analysis Research Articles

Exosomal miR-15a-5p from cardiomyocytes promotes myocardial fibrosis.

The emergence of myofibroblasts is a key step in myocardial fibrosis, but the trigger for the transformation of cardiac fibroblasts into myofibroblasts remains not entirely clear. Exosomes play a key role between cardiomyocytes and cardiac fibroblasts. Here, we not only investigated the relationship between exosomes derived from angiotensin (Ang)-II-treated cardiomyocytes and cardiac fibroblasts, the underlying mechanisms were also explored. Ang-II-treated C57 male mice and mouse cardiac fibroblasts were employed for in vivo and in vitro experiments, respectively. Transmission electron microscopy nanoparticle tracking analysis, and western blot of CD9, CD63, CD81 were performed to identify exosomes; QRT-PCR was performed to detect miR-15a-5p expression; luciferase reporter assay was employed to determine the interaction between miR-15a-5p and dyrk2; western blot was performed to examine the protein levels of fibrosis markers; Counting Kit-8 was performed to determine cell viability; HE and Masson staining were performed to assess the pathological changes of myocardial tissues. MiR-15a-5p expression was found up-regulated in serum of myocardial fibrosis patients, serum and myocardial tissues of Ang-II-treated mice, and Ang-II-treated cardiomyocytes. Mechanically, exosomes from Ang-II-treated cardiomyocytes shuttled miR-15a-5p to cardiac fibroblasts, where miR-15a-5p dephosphorylated NFAT by targeting dyrk2 to promote cell viability and elevated the protein levels of α-smooth muscle actin, collagen type 1 α1 and collagen type 3 α1, thus promoting myocardial fibrosis. This study identified a novel molecular target for anti-fibrotic therapeutic interventions.

The impact of ammonium hydroxide flow rate on iron oxide nanoparticle hydrodynamic size and colloidal stability

Precise control over the synthesis of iron oxide nanoparticles is essential to achieving optimal particle characteristics such as size, hydrodynamic size, polydispersity index, and zeta potential, all of which significantly influence colloidal stability. The commonly used co-precipitation technique often leads to high polydispersity and variable particle sizes, resulting in poor colloidal stability. This study demonstrates that varying the ammonium hydroxide flow rate during co-precipitation significantly affects particle size, hydrodynamic size, aggregation, and colloidal stability. An optimal flow rate of 4.25 mmol/min produced smaller, more uniform, and colloidally stable nanoparticles. Nanoparticle tracking analysis showed that this flow rate resulted in the smallest and most consistent hydrodynamic size of 123.3 ± 0.7 nm, while both higher and lower flow rates yielded larger and more polydisperse particles, up to 155.8 ± 20 nm. Dynamic light scattering confirmed that the 4.25 mmol/min rate produced the lowest polydispersity index of 0.21 ± 0.04, indicating the highest uniformity. Additionally, UV-Vis measurements demonstrated that nanoparticles synthesized at this optimal flow rate exhibited minimal changes in absorbance over 24 h, indicating superior colloidal stability. This study highlights the importance of optimizing the flow rate during the co-precipitation synthesis of IONPs to control particle hydrodynamic size and polydispersity, which are crucial parameters for numerous biomedical, chemical, and environmental applications.

Mesenchymal stromal cell-derived exosomes protect against abdominal aortic aneurysm formation through CD74 modulation of macrophage polarization in mice

BackgroundMesenchymal stromal cell (MSC)-derived exosomes (MSC-Exo) have been recognized for their significant role in regulating macrophage polarization, a process crucial to the pathogenesis of abdominal aortic aneurysm (AAA). However, the therapeutic effects of MSC-Exo on AAA remain largely unexplored. Therefore, this study aimed to investigate the functional and mechanistic aspects of MSC-Exo in the progression of AAA.MethodsThe MSC-derived exosomes were characterized using Transmission Electron Microscopy, Nanoparticle Tracking Analysis, and Western blotting. An experimental mouse model of AAA was established through the administration of angiotensin II (Ang II) in male apoe−/− mice and calcium chloride (CaCl2) in male C57/B6 mice, with subsequent tail vein injection of exosomes to evaluate their efficacy against AAA. Macrophage polarization was assessed using immunofluorescence staining and WB analysis. Mechanistic analysis was performed using 4D Label-free Proteomics analysis.ResultsWe found that intravenous administration of MSC-Exo induced M2 polarization of macrophages within an inflammatory environment, effectively impeding AAA development in Ang II or CaCl2-induced AAA model. The therapeutic efficacy of MSC-Exo treatment was dependent on the presence of macrophages. Mechanistically, MSC-Exo suppressed the levels of cluster of differentiation 74 (CD74), modulating macrophage polarization through the TSC2-mTOR-AKT pathway. These findings highlight the potential of MSC-Exo as a therapeutic strategy for AAA by modulating macrophage polarization.

Peptide ligands for the universal purification of exosomes by affinity chromatography.

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.

Expression of the αVβ3 integrin affects prostate cancer sEV cargo and density and promotes sEV pro-tumorigenic activity in vivo through a GPI-anchored receptor, NgR2.

It is known that small extracellular vesicles (sEVs) are released from cancer cells and contribute to cancer progression via crosstalk with recipient cells. We have previously reported that sEVs expressing the αVβ3 integrin, a protein upregulated in aggressive neuroendocrine prostate cancer (NEPrCa), contribute to neuroendocrine differentiation (NED) in recipient cells. Here, we examine the impact of αVβ3 expression on sEV protein content, density and function. sEVs used in this study were isolated by iodixanol density gradients and characterized by nanoparticle tracking analysis, immunoblotting and single vesicle analysis. Our proteomic profile of sEVs containing αVβ3 shows downregulation of typical effectors involved in apoptosis and necrosis and an upregulation of tumour cell survival factors compared to control sEVs. We also show that the expression of αVβ3 in sEVs causes a distinct reposition of EV markers (Alix, CD81, CD9) to a low-density sEV subpopulation. This low-density reposition is independent of extracellular matrix (ECM) protein interactions with sEVs. This sEV subset contains αVβ3 and an αVβ3 downstream effector, NgR2, a novel marker for NEPrCa. We show that sEVs containing αVβ3 are loaded with higher amounts of NgR2 as compared to sEVs that do not express αVβ3. Mechanistically, we demonstrate that sEVs containing NgR2 do not affect the sEV marker profile, but when injected in vivo intratumorally, they promote tumour growth and induce NED. We show that sEVs expressing NgR2 increase the activation of focal adhesion kinase (FAK), a known promoter of cancer cell proliferation, in recipient cells. We also show that NgR2 mimics the effect of sEVs containing αVβ3 since it displays increased growth of NgR2 transfectants in vivo, as compared to control cells. Overall, our results describe the changes that occur in cargo, density and functions of cancer cell-derived sEVs containing the αVβ3 integrin and its effector, NgR2, without affecting the sEV tetraspanin profiles.

Exosomal circ-0100519 promotes breast cancer progression via inducing M2 macrophage polarisation by USP7/NRF2 axis.

Breast cancer (BC) is one of the most prevalent malignant tumours that threatens women health worldwide. It has been reported that circular RNAs (circRNAs) play an important role in regulating tumour progression and tumour microenvironment (TME) remodelling. Differentially expression characteristics and immune correlations of circRNAs in BC were verified using high-throughput sequencing and bioinformatic analysis. Exosomes were characterised by nanoparticle transmission electron microscopy and tracking analysis. The biological function of circ-0100519 in BC development was demonstrated both in vitro and in vivo. Western blotting, RNA pull-down, RNA immunoprecipitation, flow cytometry, and luciferase reporter were conducted to investigate the underlying mechanism. Circ-0100519 was significant abundant in BC tumour tissues and related to poor prognosis. It can be encapsulated into secreted exosomes, thereby promoting BC cell invasion and metastasis via inducing M2-like macrophages polarisation.Mechanistically, circ-0100519 acted as a scaffold to enhance the interaction between the deubiquitinating enzyme ubiquitin-specific protease 7 (USP7) and nuclear factor-like 2 (NRF2) in macrophages, inducing the USP7-mediated deubiquitination of NRF2. Additionally,HIF-1α could function as an upstream effector to enhance circ-0100519 transcription. Our study revealed that exosomal circ-0100519 is a potential biomarker for BC diagnosis and prognosis, and the HIF-1α inhibitor PX-478 may provide a therapeutic target for BC.

Collection of serum albumin aggregate nanoparticles from human plasma by dielectrophoresis.

Dielectrophoresis (DEP) is a fast and reliable nanoparticle recovery method that utilizes nonuniform electric fields to manipulate particles based on their material composition and size, enabling recovery of biologically-derived nanoparticles from plasma for diagnostic applications. When applying DEP to undiluted human plasma, collection of endogenous albumin proteins was observed at electric field gradients much lower than predicted by theory to collect molecular proteins. To understand this collection, nanoparticle tracking analysis of bovine serum albumin (BSA) dissolved in 0.5× phosphate-buffered saline was performed and showed that albumin spontaneously formed aggregate nanoparticles with a mean diameter of 237nm. These aggregates experienceda dielectrophoretic force as a function of aggregate radius rather than the diameter of individual protein molecules which contributed to their collection. In high conductance buffer (6.8mS/cm), DEP was able to move these aggregates into regions of high electric field gradient, and in lower conductance buffer (0.68mS/cm), these aggregates could be moved into high or low gradient regions depending on the applied frequency. Disruption of BSA aggregates using a nonionic detergent significantly decreased the particle diameter, resulting in decreased dielectrophoretic collection of albumin which increased the collection consistency of particles of interest. These results provide techniques to manipulate albumin aggregates via DEP, which impacts collection of diagnostic biomarkers.

Exosomes Derived from Rejuvenated Stem Cells Inactivate NLRP3 Inflammasome and Pyroptosis of Nucleus Pulposus Cells via the Transfer of Antioxidants.

Accumulating evidence supports the potential of exosomes as a promising therapeutic approach for intervertebral disc degeneration (IDD). Nevertheless, enhancing the efficiency of exosome treatment remains an urgent concern. This study investigated the impact of quercetin on the characteristics of mesenchymal stem cells (MSCs) and their released exosomes. Exosomes were obtained from quercetin pre-treated MSCs and quantified for the production based on nanoparticle tracking and western blot analysis. The molecules involved in the secretion and cargo sorting of exosomes were investigated using western blot and immunofluorescence analysis. Based on the in vitro biological analysis and in vivo histological analysis, the effects of exosomes derived from conventional or quercetin-treated MSCs on nucleus pulposus (NP) cells were compared. A significant enhancement in the production and transportation efficiency of exosomes was observed in quercetin-treated MSCs. Moreover, the exosomes derived from quercetin-treated MSCs exhibited a greater abundance of antioxidant proteins, specifically superoxide dismutase 1 (SOD1), which inhibit the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome in NP cells. Through in vitro and in vivo experiments, it was elucidated that exosomes derived from quercetin-treated MSCs possessed enhanced anti-inflammatory and antioxidant properties. Collectively, our research underscores an optimized therapeutic strategy for IDD utilizing MSC-derived exosomes, thereby augmenting the efficacy of exosomes in intervertebral disc regeneration.

Nanoparticles in liposomes: a platform for increased antibiotic selectivity in multidrug resistant bacteria in respiratory tract infections.

Antibiotic resistance is a cause of serious illness and death, originating often from insufficient permeability into gram-negative bacteria. Nanoparticles (NP) can increase antibiotic delivery in bacterial cells, however, may as well increase internalization in mammalian cells and toxicity. In this work, NP in liposome (NP-Lip) formulations were used to enhance the selectivity of the antibiotics (3C and tobramycin) and quorum sensing inhibitor (HIPS-1635) towards Pseudomonas aeruginosa by fusing with bacterial outer membranes and reducing uptake in mammalian cells due to their larger size. Poly (lactic-co-glycolic) acid NPs were prepared using emulsion solvent evaporation and incorporated in larger liposomes. Cytotoxicity and uptake studies were conducted on two lung cell lines, Calu-3 and H460. NP-Lip showed lower toxicity and uptake in both cell lines. Then formulations were investigated for suitability for oral inhalation. The deposition of NP and NP-Lip in the lungs was assessed by next generation impactor and corresponded to 75% and 45% deposition in the terminal bronchi and the alveoli respectively. Colloidal stability and mucus-interaction studies were conducted. NP-Lip showed higher diffusion through mucus compared to NPs with the use of nanoparticle tracking analyzer. Moreover, the permeation of delivery systems across a liquid-liquid interface epithelial barrier model of Calu-3 cells indicated that NP-Lip could cause less systemic toxicity upon in-vivo like administration by aerosol deposition. Monoculture and Pseudomonas aeruginosa biofilm with Calu-3 cells co-culture experiments were conducted, NP-Lip achieved highest toxicity towards bacterial biofilms and least toxicity % of the Calu-3 cells. Therefore, the NP- liposomal platform offers a promising approach for enhancing antibiotic selectivity and treating pulmonary infections.

Oligodendrocyte precursor cell-derived exosomes combined with cell therapy promote clinical recovery by immunomodulation and gliosis attenuation.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by autoimmune destruction of the myelin sheath, leading to irreversible and progressive functional deficits in patients. Pre-clinical studies involving the use of neural stem cells (NSCs) have already demonstrated their potential in neuronal regeneration and remyelination. However, the exclusive application of cell therapy has not proved sufficient to achieve satisfactory therapeutic levels. Recognizing these limitations, there is a need to combine cell therapy with other adjuvant protocols. In this context, extracellular vesicles (EVs) can contribute to intercellular communication, stimulating the production of proteins and lipids associated with remyelination and providing trophic support to axons. This study aimed to evaluate the therapeutic efficacy of the combination of NSCs and EVs derived from oligodendrocyte precursor cells (OPCs) in an animal model of multiple sclerosis. OPCs were differentiated from NSCs and had their identity confirmed by gene expression analysis and immunocytochemistry. Exosomes were isolated by differential ultracentrifugation and characterized by Western, transmission electron microscopy and nanoparticle tracking analysis. Experimental therapy of C57BL/6 mice induced with experimental autoimmune encephalomyelitis (EAE) were grouped in control, treated with NSCs, treated with OPC-derived EVs and treated with a combination of both. The treatments were evaluated clinically using scores and body weight, microscopically using immunohistochemistry and immunological profile by flow cytometry. The animals showed significant clinical improvement and weight gain with the treatments. However, only the treatments involving EVs led to immune modulation, changing the profile from Th1 to Th2 lymphocytes. Fifteen days after treatment revealed a reduction in reactive microgliosis and astrogliosis in the groups treated with EVs. However, there was no reduction in demyelination. The results indicate the potential therapeutic use of OPC-derived EVs to attenuate inflammation and promote recovery in EAE, especially when combined with cell therapy.

Optimizing of a suitable protocol for isolating tissue-derived extracellular vesicles and profiling small RNA patterns in hepatocellular carcinoma.

Extracellular vesicles (EVs) facilitate cell-cell interactions in the tumour microenvironment. However, standard and efficient methods to isolate tumour tissue-derived EVs are lacking, and their biological functions remain elusive. To determine the optimal method for isolating tissue-derived EVs, we compared the characterization and concentration of EVs obtained by three previously reported methods using transmission electron microscopy, nanoparticle tracking analysis, and nanoflow analysis (Nanoflow). Additionally, the differential content of small RNAs, especially tsRNAs, between hepatocellular carcinoma (HCC) and adjacent normal liver tissues (ANLTs)-derived EVs was identified using Arraystar small RNA microarray. The targets of miRNAs and tsRNAs were predicted, and downstream functional analysis was conducted using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, non-negative matrix factorization and survival prediction analysis. A differential centrifugation-based protocol without cell cultivation (NC protocol) yielded higher EV particles and higher levels of CD9+ and CD63+ EVs compared with other isolation protocols. Interestingly, the NC protocol was also effective for isolating frozen tissue-derived EVs that were indistinguishable from fresh tissue. HCC tissues showed significantly higher EV numbers compared with ANLTs. Furthermore, we identified different types of small RNAs in HCC tissue-derived EVs, forming a unique multidimensional intercellular communication landscape that can differentiate between HCC and ANLTs. ROC analysis further showed that the combination of the top 10 upregulated small RNAs achieved better diagnostic performance (AUC = .950 [.895-1.000]). Importantly, most tsRNAs in HCC tissue-derived EVs were downregulated and mitochondria-derived, mainly involving in lipid-related metabolic reprogramming. The NC protocol was optimal for isolating EVs from HCC, especially from frozen tissues. Our study emphasized the different roles of small-RNA in regulating the HCC ecosystem, providing insights into HCC progression and potential therapeutic targets.

Intrinsic CO2 nanobubbles in alkaline aqueous solutions

Nanobubbles (NBs) and their derivative, bulk nanobubbles (BNBs), have been widely studied due to their potential wide range of applications. However, the stable existence of intrinsic NBs and BNBs in solutions is still controversial. This study investigates the existence and behaviour of unknown nano-entities in dilute alkaline solutions using Flow Field Fractionation Multi-Angle Light Scattering (FFF-MALS), resulting in the first report of the presence, size distribution, and size distribution changes under different treatments. The results suggest that neutral and acidic pure solutions of salts at concentrations below the solubility product do not contain detectable nano entities. Alkaline solutions, on the other hand, such as carbonate buffer and sodium hydroxide, do contain nano scatterers in the range of ~100nm diameter that cannot be removed by boiling or filtration. Reducing the pH of the solutions or freezing reduces their number densities. The freeze-thaw procedure strongly suggests that these nano entities are BNBs. Zeta Nanoparticle Tracking Analysis (ZNTA) shows the zeta potential and the concentration in agreement with previous literature. It can be concluded that intrinsic BNBs in alkaline solutions are an integral part of the total nano-entity population. This study highlights the importance of considering intrinsic BNBs when investigating nanoparticles in alkaline solutions with analytical methods such as Static Light Scattering (SLS), which can detect low number densities in the original liquid state that may not be plausible by other detection methods due to their detection thresholds or acquisition of sample preparation methods of drying or freezing that do not allow real-time observations.

Isolation, characterization, and immunomodulatory effects of extracellular vesicles isolated from fish pathogenic Aeromonas hydrophila

Bacterial extracellular vesicles (BEVs) are natural nanocarriers that have shown great potential for biomedical applications such as biomarkers, cancer therapy, immunomodulators, vaccines, wound healing, tissue engineering, and drug carriers. In the present study, BEVs were isolated from the gram-negative bacterium, Aeromonas hydrophila using the ultracentrifugation method and denoted as AhEVs. Using transmission electron microscopy imaging, we confirmed the ultrastructure and spherical shape morphology of AhEVs. Nanoparticle-tracking analysis results showed a mean particle size of 105.5 ± 2.0 nm for AhEVs. Moreover, the particle concentration of AhEVs was 2.34 ± 0.12 × 1011 particles/mL of bacterial supernatant. AhEV-treated fathead minnow (FHM) cells did not show cytotoxicity effects up to 50 μg/mL with no significant decrease in cells. Moreover, no mortality was observed in larval zebrafish up to 50 μg/mL which indicates that the AhEVs are biocompatible at this concentration. Furthermore, fluorescent-labeled AhEVs were internalized into FHM cells. Results of qRT-PCR analysis in FHM cells revealed that cellular pro-inflammatory cytokines such as nuclear factor (NF)-κB, interferon (Ifn), Irf7, interleukin (Il) 8, and Il11 were upregulated while downregulating the expression of anti-inflammatory Il10 in a concentration-dependent manner. AhEV-treated adult zebrafish (5 μg/fish) induced toll-like receptor (tlr) 2 and tlr4; tumor necrosis factor-alpha (tnfα); heat shock protein (hsp) 70; and il10, il6, and il1β in kidney. Protein expression of NF-κB p65 and Tnfα presented amplified levels in the spleen of AhEVs-treated zebrafish. Based on the collective findings, we conclude that AhEVs exhibited morphological and physicochemical characteristics to known EVs of gram (−)ve bacteria. At biocompatible concentrations, the immunomodulatory activity of AhEVs was demonstrated by inducing different immune response genes in FHM cells and zebrafish. Hence, we suggest that AhEVs could be a novel vaccine candidate in fish medicine due to their ability to elicit strong immune responses.

Olig2-enriched exosomes: A novel therapeutic approach for cuprizone-induced demyelination

The research aims to study the therapeutic impact of HEK293-XPack-Olig2 cell-derived exosomes on remyelination of the corpus callosum in a cuprizone-induced demyelinating disease model. A lentiviral vector expressing Olig2 was constructed using XPack technology. The highly abundant Olig2 exosomes (ExoOs) were isolated by centrifugation for subsequent experiments. Western blot, nanoparticle tracking analysis (NTA), and electron microscopy showed no significant difference in particle size and morphology between Exos and ExoOs, and a high level of Olig2 expression could be detected in ExoOs, indicating that exosome modification by XPack technology was successful. The Black Gold/Fluromyelin staining analysis showed that the ExoOs group significantly reduced the demyelination area in the corpus callosum compared to the PBS and Exos groups. Additionally, the PDGFRα/APC staining of the demyelinating region revealed an increase in APC+ oligodendrocytes and a decrease in PDGFRα+ oligodendrocyte progenitor cells (OPCs) in the ExoOs group. Furthermore, there was evident myelin regeneration in the demyelinated areas after ExoOs treatment, with better g-ratio and a higher number of intact myelin compared to the other treatment groups. The level of Sox10 expression in the brain tissue of the ExoOs group were higher compared to those of the PBS and Exos groups. The demyelination process can be significantly slowed down by the XPack-modified exosomes, the differentiation of OPCs promoted, and myelin regeneration accelerated under pathological conditions. This process is presumed to be achieved by changing the expression level of intracellular differentiation-related genes after exosomes transport Olig2 enriched into oligodendrocyte progenitors.

Extracellular vesicles in fresh frozen plasma and cryoprecipitate: Impact on in vitro endothelial cell viability.

Transfusion-related acute lung injury (TRALI) remains a major contributor to transfusion-associated mortality. While the pathogenesis of TRALI remains unclear, there is evidence of a role for blood components. We therefore investigated the potential effects of fresh frozen plasma (FFP), cryoprecipitate, and extracellular vesicles (EVs) derived from these blood components, on the viability of human lung microvascular endothelial cells (HLMVECs) in vitro. EVs were isolated from FFP and cryoprecipitate using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscopy. The potential effects of these blood components and their EVs on HLMVEC viability (determined by trypan blue exclusion) were examined in the presence and absence of neutrophils, either with or without prior treatment of HLMVECs with LPS. EVs isolated from FFP and cryoprecipitate displayed morphological and biochemical properties conforming to latest international criteria. While FFP, cryoprecipitate, and EVs derived from FFP, each reduced HLMVEC viability, no effect was observed for EVs derived from cryoprecipitate. Our findings demonstrate clear differences in the effects of FFP, cryoprecipitate, and their respective EVs on HLMVEC viability in vitro. Examination of the mechanisms underlying these differences may lead to an improved understanding of the factors that promote development of TRALI.

Exploring Canine Mammary Cancer through Liquid Biopsy: Proteomic Profiling of Small Extracellular Vesicles.

(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.

Extracellular Membrane Vesicles of Escherichia coli Induce Apoptosis of CT26 Colon Carcinoma Cells.

Escherichia coli (E. coli) is commonly utilized as a vehicle for anti-tumor therapy due to its unique tumor-targeting capabilities and ease of engineering modification. To further explore the role of E. coli in tumor treatment, we consider that E. coli outer membrane vesicles (E. coli-OMVs) play a crucial role in the therapeutic process. Firstly, E. coli-OMVs were isolated and partially purified by filtration and ultracentrifugation, and were characterized using techniques such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western Blot (WB). The obtained extracellular nanoparticles, containing OMVs, were found to inhibited the growth of CT26 tumor in mice, while the expression of Bax protein was increased and the expression of Bcl-2 protein decreased. In vitro experiments showed that E. coli-OMVs entered CT26 cells and inhibited cell proliferation, invasion and migration. In addition, in the presence of E. coli-OMVs, we observed an increase in apoptosis rate and a decrease in the ratio of Bcl-2/Bax. These data indicate that E. coli-OMVs inhibits the growth of CT26 colon cancer by inducing apoptosis of CT26 cells. These findings propose E. coli-OMVs as a promising therapeutic drug for colorectal cancer (CRC), providing robust support for further research in related fields.

FGF2 is Secreted in Extracellular Vesicles from Lung Cells.

The 18-kD isoform of basic fibroblast growth factor (bFGF/FGF2) lacks a conventional signal peptide sequence and is exported by a novel membrane-associated transport pathway. Extracellular vesicles (EVs) are increasingly recognized as mediators of intercellular communication in the lung, and our prior work demonstrates that EVs carry cargo that contribute to hyperoxic lung injury and are biomarkers for bronchopulmonary dysplasia. We used primary human bronchial epithelial (HBE), pulmonary artery endothelial (HPAE) and fibroblast (HNF) cells to determine if FGF2 was secreted in EVs. EVs were isolated by ultracentrifugation from HBE, HPAE, and HNF exposed to either normoxia or hyperoxia, followed by nanoparticle tracking analysis and electron microscopy. Hyperoxia exposure increased total EV number. All three cell types released FGF2-18kDa both directly into the extracellular environment (secretome), as well as in EVs. HBE released more FGF2-18kDa in EVs during hyperoxia, and these were internalized and localized to both nuclei and cytoplasm of recipient cells. By co-immunoprecipitation, we identified potential binding partners of FGF2-18kDa in the nuclei, including histone 1.2 (H1.2) binding protein, that may mediate downstream effects that do not involve FGF2 binding to cell surface receptors. FGF2-18kDa interaction with H1.2 binding protein may indicate a mechanism by which FGF2 secreted in EVs modulates cellular processes. FGF2 was also found to increase angiogenesis by Matrigel assay. Further studies are necessary to determine the biological relevance of the FGF2 in EVs as modulators of lung injury and disease.

Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma.

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.

Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response.