Nanoparticle Tracking Analysis Research Articles

Tumor-derived extracellular vesicles disrupt the blood–brain barrier endothelium following high-frequency irreversible electroporation

AbstractHigh-frequency irreversible electroporation (H-FIRE), a nonthermal brain tumor ablation therapeutic, generates a central tumor ablation zone while transiently disrupting the peritumoral blood–brain barrier (BBB). We hypothesized that bystander effects of H-FIRE tumor cell ablation, mediated by small tumor-derived extracellular vesicles (sTDEV), disrupt the BBB endothelium. Monolayers of bEnd.3 cerebral endothelial cells were exposed to supernatants of H-FIRE or radiation (RT)-treated LL/2 and F98 cancer cells. Endothelial cell response was evaluated microscopically and via flow cytometry for apoptosis. sTDEV were isolated following H-FIRE and RT, characterized via nanoparticle tracking analysis (NTA) and transmission electron microscopy, and applied to a Transwell BBB endothelium model to quantify permeability changes. Supernatants of H-FIRE-treated tumor cells, but not supernatants of sham- or RT-treated cells, disrupted endothelial cell monolayer integrity while maintaining viability. sTDEV released by glioma cells treated with 3000 V/cm H-FIRE increased permeability of the BBB endothelium model compared to sTDEV released after lower H-FIRE doses and RT. NTA revealed significantly decreased sTDEV release after the 3000 V/cm H-FIRE dose. Our results demonstrate that sTDEV increase permeability of the BBB endothelium after H-FIRE ablation in vitro. sTDEV-mediated mechanisms of BBB disruption may be exploited for drug delivery to infiltrative margins following H-FIRE ablation.

Circulating CCRR serves as potential novel biomarker for predicting acute myocardial infarction

Abstract Objective Cold regions exhibit a high prevalence of cardiovascular disease, particularly acute myocardial infarction (AMI), which is one of the leading causes of death associated with cardiovascular conditions. Cardiovascular disease is closely linked to the abnormal expression of long non-coding RNA (lncRNA). This study investigates whether circulating levels of lncRNA cardiac conduction regulatory RNA (CCRR) could serve as a biomarker for AMI. Materials and methods We measured circulating CCRR from whole blood samples collected from 68 AMI patients and 69 non-AMI subjects. An AMI model was established using C57BL/6 mice. Quantitative reverse transcription PCR (qRT-PCR) was used to assess CCRR expression. Exosomes were isolated from cardiomyocytes, and their characteristics were evaluated using electron microscope and nanoparticle tracking analysis. The exosome inhibitor GW4869 was employed to examine the effect of exosomal CCRR on cardiac function using echocardiography. Protein expression was detected using Western blot and immunofluorescence staining. Results The circulating level of CCRR was significantly higher in AMI patients (1.93 ± 0.13) than in non-AMI subjects (1.00 ± 0.05, P < 0.001). The area under the ROC curve (AUC) of circulating CCRR was 0.821. Similar changes in circulating CCRR levels were consistently observed in an AMI mouse model. Exosomal CCRR derived from hypoxia-induced cardiomyocytes and cardiac tissue after AMI were increased, a change that was reversed by GW4869. Additionally, CCRR-overexpressing exosomes improved cardiac function in AMI. Conclusion Circulating lncRNA CCRR is a potential predictor of AMI. Exosomal CCRR plays a role in the communication between the heart and other organs through circulation.

Abstract A030: Rapid extraction and detection of extracellular vesicle-derived PD-L1 in a microfluidic platform

Abstract Background. Extracellular vesicles (EV) are emerging as new biomarkers for cancer diagnostics and therapeutic monitoring1. For example, in patients that receive PD1/PD-L1 immune checkpoint inhibitors (a key pillar of cancer immunotherapy), circulating EV-derived PD-L1 is gaining attention as a non-invasive biomarker for therapeutic efficacy. Standard methodologies to measure EV-derived PD-L1 requires ultracentrifugation to first separate EVs and then quantify those that express PD-L1 using nano flow cytometry. These are tedious processes that require trained technologists in centralized facilities, which is a key barrier to frequently monitoring EV-based biomarkers. Yet, frequent monitoring is crucial to quickly identify and stop an ineffective therapy while more fruitful options are still available. Method. To bridge the gap, we develop a proof-of-concept digital microfluidic (DMF) device that can separate EVs directly from biofluids and quantify PD-L1+ EVs automatedly. Briefly, DMF device supports automatedly droplet handling, including droplet transport, splitting and mixing, based on electrowetting principles2. Immunomagnetic beads were used to capture EVs from biofluids, and EVs were then detected by electrochemical sensors inserted into the device. These tips of the electrochemical sensors were modified with gold nanoparticle self-assembled layers to enhance target binding, and CD9 antibodies were immobilized on the tips to bind to EVs. We then used anti-PD-L1 as secondary antibodies for target detection. Differential Pulse Voltammetry (DPV)3 were performed to obtain the results. Results. We used the DMF device to extract 109- 1011 #/mL EVs secreted by a human breast cancer cell line (MDA-MB-231). Briefly, 10 mL of cell culture media was loaded into the DMF device, and the sample was mixed and incubated with a 10 mL droplet of immunomagnetic beads to capture EVs. After 5 min, EVs were eluted in 20 mL of elution buffer, and the beads were discarded. We estimated the number of EVs using Nanoparticle Tracking Analysis; the number of EVs extracted on-chip was comparable with those that were extracted using standard in-tube method. We also verified the purity of the EVs extracted on-chip using Western Blot. Our results showed that the electrochemical sensors can detect as low as 104 #/mL PD-L1+ EVs, with a linear range of 104 – 107 #/mL (R2=0.9701). The entire process can be completed within 1 hour. Conclusion. We integrated automated EV extraction and their surface marker detection in a single DMF device. Our next step is to extract and detect EV-based biomarkers from plasma samples. This platform holds promise for the regular monitoring of EV-based biomarkers that are indicators of therapeutic responses in patients that receive cancer immunotherapies.

Follicular fluid-derived extracellular vesicles miR-34a-5p regulates granulosa cell glycolysis in polycystic ovary syndrome by targeting LDHA.

Polycystic ovary syndrome (PCOS) is highly prevalent in women of reproductive age worldwide, exhibits highly heterogeneous clinical presentation and biochemical parameters, and has no cure. This study aimed to investigate the role of miR-34a-5p in PCOS, its effect on glycolysis in granulosa cells (GCs), and its potential contribution to follicular dysregulation. Herein, Follicular follicular fluid (FF) samples were collected from six patients with PCOS and six healthy controls undergoing in vitro fertilization-embryo transfer. The isolated extracellular vesicles were characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Additionally, miRNA sequencing was performed to identify differentially expressed microRNAs, and their functions were analyzed through transcriptomics. The In vitro effects of miR-34a-5p on glycolysis, cell proliferation, and apoptosis were assessed in human ovarian granulosa tumour cell line (KGN cells). Targets of miR-34a-5p were identified by dual-luciferase reporter assays, and quantitative real-time polymerase chain reaction and western blotting were performed to determine gene and protein expression. The level of miR-34a-5p in FF-derived extracellular vesicles derived from patients with PCOS was significantly higher than that of the control group. Transcriptomic analysis highlighted miR-34a-5p as a key regulator of glycolysis and apoptosis. Furthermore, in vitro analysis showed that miR-34a-5p targeted lactate dehydrogenase A (LDHA), inhibited glycolysis, reduced energy supply to GCs, and promoted apoptosis of KGN cells. Conversely, miR-34a-5p inhibition restored glycolysis function and cell viability. The findings of this study show that miR-34a-5p mediates GC apoptosis in PCOS by targeting LDHA and inhibiting glycolysis, suggesting its crucial role in PCOS pathophysiology, and offering potential therapeutic targets for improving follicular development and fertility outcomes in patients with PCOS. Further research is needed to explore the clinical implications of miR-34a-5p and its use as a biomarker for early diagnosis and prognosis of PCOS.

Abstract B003: Dogs with cancer have higher plasma exosome concentrations compared with healthy dogs in the COED (canine osteosarcoma early detection) study

Abstract Background: Osteosarcoma a common cancer in dogs that can serve as a translational model for certain aspects of human osteosarcoma. We previously developed a serum exosome gene signature in dogs with osteosarcoma capable of detecting minimal residual disease and predicting prognosis after therapy. Our goal is to continue expanding our understanding of exosomes for liquid biopsy applications in dogs with cancer, and to leverage this information in the design of liquid biopsy tests to address risk, early detection, and management of human cancer patients. For this study, we hypothesized that dogs with cancer would have higher plasma exosome concentrations compared with healthy control dogs. Methods: Plasma exosomes were enriched from 29 dogs with cancer, including 8 dogs with osteosarcoma, and 26 healthy control dogs. Exosome size and concentration in the plasma samples were characterized with nanoparticle tracking analysis. Wilcoxon rank sum tests were performed to assess differences between groups. Results: Dogs with cancer had a higher mean exosome concentration at 1.4 x 1012 (+/- 1.1 x 1012) particles/ml, compared with 5.0 x 1011 (+/- 2.7 x 1011) particles/ml in healthy control dogs (p = 0.0046). There was no difference in exosome size between dogs with cancer (mean diameter = 99.9 nm) and healthy control dogs (107.8 nm; p = 0.16) when all cancer types were included. However, exosomes enriched from healthy control samples were found to be significantly larger when compared to the exosomes enriched from the subset of cancer dogs that had osteosarcoma (mean exosome diameter = 83.9 nm; p = 0.0065). Similarly, when comparing exosome size between dogs with osteosarcoma and dogs with other cancers, plasma exosomes were larger in the dogs with other cancers (107.4 nm; p = 0.023). Conclusions: In this population, dogs with cancer have higher plasma exosome concentrations compared with healthy control dogs. Interestingly, we also found that dogs with osteosarcoma had significantly smaller exosome size than dogs with other cancers and healthy control dogs. Though the size of exosomes from dogs with osteosarcoma was found to be smaller, it is possible that the larger exosome concentration in dogs with osteosarcoma results in a similar total exosomal mass. Ongoing work includes analysis of exosomal cargo to develop a liquid biopsy test capable of assessing risk of osteosarcoma development in dogs, and applying similar methodology to determine exosomal genes associated with prognosis in pediatric osteosarcoma. The higher exosome concentrations in dogs with cancer suggests that this feature, combined with analysis of exosomal cargo, may have utility in development of a liquid biopsy cancer test in dogs. Citation Format: Kelly M. Makielski, Jaron M. Magstadt, Courtney H. Labe, Ali Khammanivong, Meagan Wojtysiak, Kyle Duval, Mitzi Lewellen, Amber Winter, Kelly Reid, Andrea Chehadeh, Michelle Buettner, Caitlin Feiock, Aaron K. Rendahl, Gary R. Cutter, Logan G. Spector, Brenda J. Weigel, Jaime F. Modiano. Dogs with cancer have higher plasma exosome concentrations compared with healthy dogs in the COED (canine osteosarcoma early detection) study [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B003.

Abstract B017: MicroRNA and proteomic analyses of plasma extracellular vesicles detect diffuse large B-cell lymphoma

Abstract Background: Liquid biopsy offers a non-invasive method for detection of non-Hodgkin lymphoma (NHL). Tissue biopsy, the standard method of NHL detection, causes patient discomfort and increases risk of infection. Extracellular vesicles (EVs) are secreted by cells as particles with cargos of nucleic acids and proteins enclosed in a lipid bilayer. The most common NHL subtype is diffuse large B-cell lymphoma (DLBCL). We analyzed plasma-derived EVs from DLBCL patients and matching healthy controls for microRNA (miRNA) and protein to identify potential liquid biopsy biomarkers. Approach: Plasma samples (1 mL) from DLBCL patients and healthy controls were eluted through Izon size-exclusion chromatography (SEC) to obtain EVs. The size distribution of EVs was characterized with nanoparticle tracking analysis. For miRNA analysis, SEC EVs from three DLBCL patients and three matching healthy controls underwent further selection by immunocapture and release according to NHL antigens CD19, CD20, CD22, and C37, with an EV microfluidic affinity purification (EV-MAP) chip. Western blot confirmed the presence of these NHL antigens in NHL cell lysate and NHL cell culture SEC EVs, with flow cytometry verifying expression on NHL cells. MiRNA from these immunocaptured EVs were then isolated and sequenced with Qiagen kits. MiRNA levels were assessed for fold change (FC) expression between cases and controls. Upregulated miRNAs from DLBCL patients were compiled with the miRNA Enrichment Analysis and Annotation Tool (miEAA). For proteomic analysis, SEC EVs from ten DLBCL patients and ten matching healthy controls were measured with label-free mass spectrometry and a custom peptide library. Protein levels were assessed for FC expression and compiled with the Mann-Whitney U test. Results: The size of EVs ranged from 50–300 nm. Exclusively different upregulated miRNAs (FC>2, p<0.05, FDR p<0.05) were found by selection with different antigens: None unique with CD19, one with CD20, eight with CD22, and one with CD37. MiR-320b was upregulated with CD20 (FC=52, p=0.0002, FDR p=0.02), and miR-3168 with CD37 (FC=906, p=0.0003, FDR p=0.02). With CD22, miR-190b-5p, let-7c-5p, miR-1-3p, miR-125b-5p, miR-133a-3p, miR-153-3p, miR- 184, and miR-206 were upregulated in DLBCL patients (FC>17, p<0.003, FDR p<0.05). The miEAA recognized all the upregulated CD22 miRNAs as overrepresented in B-cell lymphoma, except for miR-190b-5p. Proteomic analysis of EVs found 42 different exo-proteins with AUC>0.90 (p<0.05) and 12 different exo-proteins with AUC>0.95 (p<0.009) upregulated in DLBCL patients. Two upregulated exo-proteins (FC>2.81, p<0.0002, adjusted p<0.004), ADAM metallopeptidase with thrombospondin type 1 motif 13 (ADAMTS13) and von Willebrand Factor (vWF), achieved a perfect AUC=1 (p=0.00001) for all DLBCL patients and healthy controls. Conclusions: These results support the hypothesis that liquid biopsy of plasma EVs detect DLBCL. Upregulated miRNA and proteomic signatures via EVs indicate the presence of DLBCL for non-invasive NHL detection. Citation Format: Arthur A. Lee, Sagar Rayamajhi, Mengjia Hu, Harsh Pathak, Rajni V. Puri, Kamilla Isakova, Mei Han, Claire S. Berquist, Samuel G. Mackintosh, Stephanie D. Byrum, Dennis W. Province, Mohammod M. Rahman, Foyez Ahmmed, Leonidas E. Bantis, Malgorzata A. Witek, Steven A. Soper, Andrew K. Godwin, Haitham Abdelhakim. MicroRNA and proteomic analyses of plasma extracellular vesicles detect diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B017.

Development of Alginate Hydrogels Incorporating Essential Oils Loaded in Chitosan Nanoparticles for Biomedical Applications

A hybrid alginate hydrogel–chitosan nanoparticle system suitable for biomedical applications was prepared. Chitosan (CS) was used as a matrix for the encapsulation of lavender (Lavandula angustifolia) essential oil (LEO) and Mentha (Mentha arvensis) essential oil (MEO). An aqueous solution of an acidic Natural Deep Eutectic Solvent (NADES), namely choline chloride/ascorbic acid in a 2:1 molar ratio, was used to achieve the acidic environment for the dissolution of chitosan and also played the role of the ionic gelator for the preparation of the chitosan nanoparticles (CS-NPs). The hydrodynamic diameter of the CS-MEO NPs was 130.7 nm, and the size of the CS-LEO NPs was 143.4 nm (as determined using Nanoparticle Tracking Analysis). The CS-NPs were incorporated into alginate hydrogels crosslinked with CaCl2. The hydrogels showed significant water retention capacity (>80%) even after the swollen sample was kept in the aqueous HCl solution (pH 1.2) for 4 h, indicating a good stability of the network. The hydrogels were tested (a) for their ability to absorb dietary lipids and (b) for their antimicrobial activity against Gram-positive and Gram-negative foodborne pathogens. The antimicrobial activity of the hybrid hydrogels was comparable to that of the widely used food preservative sodium benzoate 5% w/v.

Pancreatic cancer-derived exosomal miR-510 promotes macrophage M2 polarization and facilitates cancer cell aggressive phenotypes.

Extensive tumor microenvironment (TME) and tumor-associated macrophages (TAMs) contribute to the initiation and progression of pancreatic cancer (PC). Cancer cell-derived exosomal miRNAs that stimulate macrophage M2 polarization might play an important role in the process. In the current study, we observed significant upregulation of miR-510 in PC cell lines in comparison to normal HPDE cell line, with PANC-1 exhibiting the highest and MIA PaCa-2 the lowest miR-510 levels. Functional assays demonstrated that miR-510 overexpression enhanced, while its inhibition reduced, PC cell viability, migration, invasion, and EMT. In vivo, miR-510 mimics promoted tumor growth and macrophage M2 polarization, whereas miR-510 inhibition had the opposite effect. Exosomes from PANC-1 and MIA PaCa-2 cells, characterized by nanoparticle tracking analysis and TEM, contained significantly higher miR-510 levels than those from HPDE cells. Macrophages incubated with conditioned media from these PC cells showed increased M2 polarization markers, a process inhibited by the exosome inhibitor GW4869. The delivery of miR-510 via PC cell-derived exosomes facilitated macrophage M2 polarization and regulated the STAT signaling pathway, suggesting that exosomal miR-510 plays a crucial role in the tumor microenvironment of PC by modulating macrophage M2 polarization.

TMIC-23. INVESTIGATING THE FUNCTIONAL ROLE OF SECRETED FERRITIN FROM GLIOBLASTOMA CELL TYPES

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor in adults with a survival rate ~5%. Our laboratory previously showed the inhibitory effect of iron and ferritin-bound iron on migration and spheroid formation in GBM cells. Furthermore, studies have shown the release of extracellular vesicles (EVs) and their iron-related cargoes are regulated by cellular iron concentrations. Building on these findings, this study aims to investigate the impact of EV-associated ferritin on migration in GBM. To achieve this, we examined the influence of iron on EV release from four GBM cell lines, comprising two cancer stem cells (CSCs) and two non-CSCs: T387, T3691, LN229, and T98G. Using Nanoparticle tracking analysis (NTA), we demonstrated that 100 μM of ferric ammonium citrate (FAC) significantly increases EV release in the CSC line after 24 hours (p-value <0.01). Conversely, there was no significant impact on the non-CSC line under the same conditions. Based on these results, the ability of iron to regulate EV release is dependent on CSC versus non-CSC status. Given the functional role of ferritin on migration potential in GBM, we used immunoblotting to examine ferritin release from cells following iron treatment. We observed a reduction in ferritin release from CSCs following FAC treatment compared to the control, while non-CSCs, in contrast, released more ferritin after treatment. Interestingly, ferritin released from CSCs was predominantly within EVs, whereas ferritin released from non-CSCs was not associated with EVs. To assess the functional impact of ferritin release on migration, we utilized a Transwell migration system, and observed that EVs from iron-treated cells reduced migration in the recipients. Together, these findings expand our understanding of differential responses of ferritin in CSC and non-CSC EVs to iron treatment and will allow us to identify new targets and strategies for managing GBM progression.

EXTH-38. EVALUATION OF STORAGE CONDITIONS ON SERUM-DERIVED EXTRACELLULAR VESICLES IN BRAIN TUMOR SAMPLES

Abstract Tumor-derived Extracellular Vesicles (EVs) are emerging as potential liquid biopsy tool in the field of cancer diagnosis and therapeutic targets. The functionality of EVs depends on its diverse cargo, which is known to alter host-receipient cells. Nevertheless, extracting EVs while preserving its integrity and functionality of its contents is challenging, specifically in Low-middle Income countries (LMIC) countries. Our study focussed on extraction of serum derived EVs analysis from brain-tumor samples encompassing Oligodendrogliomas, Astrocytomas and Glioblastomas. The stability of EVs were examined in serum stored for 2 years at -80°C to understand the effect of preservation and storage in different conditions, that were left exposed to room temperature for variable periods of time before freezing. To explore the optimal suitable conditions, EVs were isolated from serum samples using IZON column size-exclusion based-method, and evaluated for structural integrity and stability through nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) technology. Interestingly, the majority of isolated vesicles exhibited round, intact structure with bilayer membrane of size varying between 30 - 250nm. Moreover, protein content of EVs positive markers (CD63, CD81 and CD9) was also detected with minimal contaminations evidenced by weak bands for negative marker (ApoB), as determined by Western blot analysis. Further, column-based EV-derived RNA demonstrated optimal yield and purity was confirmed by RNA integrity number (RIN) using Qubit. While cryopreservation had minimum detrimental effects on mRNA sample quality, leaving it exposed to room temperature before freezing had a significant effect on EV isolation, as samples were often haemolyzed. This will provide insights in optimizing pre-analytical and analytical procedures for more effective outcomes in the clinical setting

Adipose-derived stem cell exosomal miR-21-5p enhances angiogenesis in endothelial progenitor cells to promote bone repair via the NOTCH1/DLL4/VEGFA signaling pathway

BackgroundAngiogenesis is essential for repairing critical-sized bone defects. Although adipose-derived stem cell (ADSC)-derived exosomes have been shown to enhance the angiogenesis of endothelial progenitor cells (EPCs), the underlying mechanisms remain unclear. This study aims to explore the effects and mechanisms of ADSC-derived exosomes in enhancing bone repair by promoting EPC angiogenesis.MethodsTransmission electron microscopy, nanoparticle tracking analysis, and Dil reagent kit were employed to identify ADSC-derived exosomes and their internalization by EPCs. Micro-CT analysis, H&E staining, and Masson staining were used to assess bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), as well as the pathological changes and fibrosis at defect sites. Cell viability, migration, invasion, and tube formation of EPCs were evaluated using CCK-8, wound healing, Transwell, and tube formation assays. Immunohistochemical staining, RT-PCR, and Western blotting were utilized to measure the gene and protein expression of markers such as CD31, VEGFA, OCN, RUNX2, NOTCH1, and DLL4. Gene sequencing and bioinformatics analyses were conducted to identify the most highly expressed miRNA in exosomes, while miRDB and dual-luciferase reporter assays were used to explore the interaction between miR-21-5p and NOTCH1.ResultsThe ADSC-derived exosomes, averaging 126 nm in diameter, were internalized by EPCs. In vivo, these exosomes promoted new bone formation, increased BMD, BV/TV, Tb.Th, and Tb.N, reduced pathological damage to cranial defect tissues, enhanced vascular and bone tissue regeneration, and upregulated OCN and RUNX2 expression. In vitro, ADSC-derived exosomes enhanced EPC viability, migration, invasion, and tube formation. Both in vivo and in vitro experiments demonstrated that ADSC-derived exosomes upregulated CD31 and VEGFA expression. miR-21-5p, the most highly expressed miRNA in ADSC-derived exosomes, was found to target NOTCH1. Overexpression of miR-21-5p in these exosomes facilitated EPC migration, tube formation, and VEGFA expression while downregulating NOTCH1 and DLL4 expression. Inhibition of miR-21-5p produced opposite effects on EPCs.ConclusionsThese findings indicate that miR-21-5p in ADSC-derived exosomes promotes angiogenesis in EPCs to accelerate bone repair by targeting the NOTCH1/DLL4/VEGFA signaling pathway, offering a potential therapeutic strategy for bone defect treatment.Graphical

Differential Effects of Extracellular Vesicles from Two Different Glioblastomas on Normal Human Brain Cells

Background/Objectives: Glioblastomas (GBMs) are dreadful brain tumors with abysmal survival outcomes. GBM extracellular vesicles (EVs) dramatically affect normal brain cells (largely astrocytes) constituting the tumor microenvironment (TME). We asked if EVs from different GBM patient-derived spheroid lines would differentially alter recipient brain cell phenotypes. This turned out to be the case, with the net outcome of treatment with GBM EVs nonetheless converging on increased tumorigenicity. Methods: GBM spheroids and brain slices were derived from neurosurgical patient tissues following informed consent. Astrocytes were commercially obtained. EVs were isolated from conditioned culture media by ultrafiltration, concentration, and ultracentrifugation. EVs were characterized by nanoparticle tracking analysis, electron microscopy, biochemical markers, and proteomics. Astrocytes/brain tissues were treated with GBM EVs before downstream analyses. Results: EVs from different GBMs induced brain cells to alter secretomes with pro-inflammatory or TME-modifying (proteolytic) effects. Astrocyte responses ranged from anti-viral gene/protein expression and cytokine release to altered extracellular signal-regulated protein kinase (ERK1/2) signaling pathways, and conditioned media from EV-treated cells increased GBM cell proliferation. Conclusions: Astrocytes/brain slices treated with different GBM EVs underwent non-identical changes in various omics readouts and other assays, indicating “personalized” tumor-specific GBM EV effects on the TME. This raises concern regarding reliance on “model” systems as a sole basis for translational direction. Nonetheless, net downstream impacts from differential cellular and TME effects still led to increased tumorigenic capacities for the different GBMs.

The proteomic landscape of extracellular vesicles derived from human intervertebral disc cells.

Extracellular vesicles (EVs) function as biomarkers and are crucial in cell communication and regulation, with therapeutic potential for intervertebral disc (IVD)-related low back pain (LBP). EV cargo is often affected by tissue health, which may affect the therapeutic potential. There is currently limited knowledge of how the cargo of IVD cell-derived EVs varies with tissue health and how differences in proteomic profile affect the predicted biological functions. Our study purified EVs from human IVD cell conditioned media by size-exclusion chromatography. Nanoparticle tracking analysis was conducted to measure EV size and concentration. Transmission electron microscopy and Western blot were performed to examine EV structure and markers. Tandem mass tag-mass spectrometry was conducted to determine protein cargo. Most EVs were exosomes and intermediate microvesicles with an increasing amount linked to disease progression. Of the proteins detected, 88.6% were shared across the non-degenerate, mildly-degenerate, and degenerate samples. GO and KEGG analyses revealed that cargo from the mildly-degenerate samples was the most distinct, with the proteins in high abundance strongly associated with extracellular matrix (ECM) organization and structure. Shared proteins, highly expressed in the non-degenerate and degenerate samples, showed strong associations with cell adhesion, ECM-receptor interaction, and vesicle-mediated transport, respectively. Our findings indicate that EVs from IVD cells from tissue with different degrees of degeneration share a majority of the cargo proteins. However, the level of expression differs with degeneration grade. Cargo from the mildly-degenerate samples exhibits the most differences. A better understanding of changes in EV cargo in the degenerative process may provide novel information related to molecular mechanisms underlying IVD degeneration and suggest new potential treatment modalities for IVD-related LBP.

Extracellular Vesicles from Lung Adenocarcinoma Cells Induce Activation of Different Cancer-Associated Fibroblast Subtypes

Background: Lung cancer, including the major subtype lung adenocarcinoma (LUAD), is the leading cause of cancer deaths worldwide, largely due to metastasis. Improving survival rates requires new treatment strategies and a deeper understanding of the mechanisms that drive tumor progression within the tumor microenvironment (TME). This study investigated the impact of extracellular vesicles (EVs) derived from LUAD cells on lung fibroblasts. Methods: EVs were isolated from LUAD cell lines via ultracentrifugation and characterized using nanoparticle tracking analysis and Western blotting. Lung fibroblasts were treated with PBS, TGFβ, or EVs, and their activation was assessed through protein (Western blotting) and RNA analyses (RNA seq and RT-qPCR). Results: The results confirmed the TGFβ induced activation and showed that LUAD EVs could also activate fibroblasts, increasing cancer-associated fibroblast (CAF) markers. While EV-induced CAF activation displayed unique features, like an increase in proliferation-related genes, the EV and TGFβ treatments also shared some differentially expressed genes. The EV groups induced a higher expression of ECM remodeling and EMT-associated genes, but some of those genes were also upregulated in the TGFβ group. Mesenchymal genes POSTN and SPOCK1 were significantly upregulated in TGFβ- and EV-treated fibroblasts. Their secretion as proteins from the TGFβ- and EV-induced CAFs was not significant, confirmed through ELISA. Conclusions: These findings suggest that LUAD EVs play a role in CAF activation through both shared and distinct pathways compared to canonical TGFβ activation, potentially identifying novel gene expressions involved in CAF activation. Additionally, optimal protein secretion conditions of confirmed CAF-upregulated genes need to be established to determine their contribution to the TME.

Isolation of Small Extracellular Vesicles (sEVs) from the Apoplastic Wash Fluid of Nicotiana benthamiana Leaves.

Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration-1-positive (PEN1), and EXPO vesicles derived from exocyst-positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves.

Leukocyte Platelet-Rich Plasma-Derived Exosomes Restrained Macrophages Viability and Induced Apoptosis, NO Generation, and M1 Polarization.

Chronic refractory wounds refer to wounds that cannot be repaired timely. Platelet-rich plasma (PRP) has significant potential in chronic wound healing therapy. The exosomes isolated from PRP were proved to exhibit more effectiveness than PRP. However, the therapeutic potential of exosomes from PRP on chronic refractory wounds remained elusive. Hence, this study aimed to clarify the action of exosomes from PRP on chronic refractory wounds by evaluating the response of macrophages to exosomes. Pure platelet-rich plasma (P-PRP) and leukocyte platelet-rich plasma (L-PRP) were prepared from the fasting venous blood of healthy volunteers. Exosomes were extracted from P-PRP and L-PRP using ultracentrifugation and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. Macrophages were obtained by inducing THP-1 cells with phorbol-12-myristate-13 acetate (PMA). The internalization of exosomes into macrophages was observed utilizing confocal laser scanning microscopy after being labeled with PKH67. Cell viability was determined by CCK-8 assay. Cell apoptosis was measured utilizing a flow cytometer. The polarization status of M1 and M2 macrophages were evaluated by detecting their markers. Nitric oxide (NO) detection was conducted using the commercial kit. Exosomes from P-PRP and L-PRP were absorbed by macrophages. Exosomes from L-PRP restrained viability and induced apoptosis of macrophages. Besides, exosomes from P-PRP promoted M2 polarization, and exosomes from L-PRP promoted M1 polarization. Furthermore, exosomes from L-PRP promoted NO generation of macrophages. Exosomes from L-PRP restrained viability, induced apoptosis and NO generation of macrophages, and promoted M1 polarization, while exosomes from P-PRP increased M2 polarization. The exosomes from L-PRP presented a more effective effect on macrophages than that from P-PRP, making it a promising strategy for chronic refractory wound management.

Effect of extracellular vesicles derived from inflammatory H9C2 cardiomyocytes on the polarization of macrophages and its mechanism

Abstract Objective The aim of this study was to investigate the effect of Extracellular vesicles (EVs) derived from inflammatory cardiomyocytes on the polarization of macrophages and its related mechanism. Methods EVs derived from the inflammatory and normal cardiomyocyte cell line H9C2 were extracted by differential centrifugation respectively. The extracted EVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot analysis (WB). The RAW264.7 macrophages in the inflammatory environment were treated with EVs, the proliferative activities were detected by CCK8, the expression of inflammatory factors were detected by qRT-PCR, and the differentiation of RAW264.7 macrophages into M1 and M2 was analyzed by flow cytometry. In vivo experiments, the animal model of viral myocarditis was established in Balb/c mice with intraperitoneal injection of CVB3. EVs derived from inflammatory H9C2 cardiomyocytes and medium were injected into mice by tail vein injection, respectively. The mice myocardium was stained by immunohistochemistry, and the total RNA of myocardial tissue was extracted for the detection of inflammatory factors by RT-PCR. In terms of mechanism research, mRNA expression profiles of RAW264.7 cells after EVs treatment were analyzed by RNA sequencing for functional annotation and mechanism study. At the same time, the extracted EVs derived from H9C2 normal group and the inflammatory group were subjected to 4D-Lable Free technology for quantitative proteomic analysis, and the results were analyzed bioinformatics, and then by WB analysis.Results We found that EVs derived from inflammatory H9C2 cardiomyocytes decreased the proliferative activity of RAW264.7 macrophages, the expression of pro-inflammatory cytokines IL-6, IL-1β, TNF-ɑ, and the proportion of M1 macrophages in the inflammatory state, while the proportion of M2 macrophages, which mainly inhibit inflammation, increased, and the expression levels of anti-inflammatory cytokines IL-10 and CD-206 increased. Results of immunohistochemical staining and RT-PCR showed that the EVs of inflammatory H9C2 regulated the heterogeneity of infiltrating macrophages and the expression of several inflammatory cytokines in mouse myocardium inflammatory tissue. Meanwhile, in vitro studies have found that the regulatory effect of EVs derived from inflammatory H9C2 cardiomyocytes on macrophage heterogeneity is mediated by MAPK signaling pathway. Conclusion EVs derived from inflammatory H9C2 cardiomyocytes can regulate the polarization of macrophages, change the expression of inflammatory factors, and regulate the myocardial inflammatory microenvironment, which may be achieved by mediating the p38 MAPK pathway through the delivery of PP2A.

Sex-specific differences in the severity of pulmonary hypoplasia in experimental congenital diaphragmatic hernia and implications for extracellular vesicle-based therapy.

Amniotic fluid stem cell extracellular vesicles (AFSC-EVs) hold regenerative potential to treat hypoplastic lungs secondary to congenital diaphragmatic hernia (CDH). This study aims to investigate sex-specific differences in pulmonary hypoplasia severity and responses to AFSC-EV administration in an experimental CDH mouse model. C57BL/6J dams were fed with nitrofen + bisdiamine (left-sided CDH) or olive oil only (control) at embryonic day (E) 8.5. Lungs were dissected (E18.5), grown ex vivo and treated with medium ± AFSC-EVs that were collected via ultracentrifugation and characterized (nanoparticle tracking analysis, electron microscopy, Western blotting). Pulmonary hypoplasia was assessed via mean linear intercept (MLI). Gene and protein expression changes (Cd31, Enos, Il1b, TNFa) were measured via RT-qPCR and immunofluorescence. Pups were genotyped for Sry. Experimental CDH showed a male predominance without sex differences for pulmonary hypoplasia severity, fetal lung vascularization, and inflammation. AFSC-EV administration led to improved lung growth (decreased MLI), improved fetal lung vascularization (increased Cd31 and Enos), and decreased fetal lung inflammation (Il1b, TNFa). There was no sex-specific response to AFSC-EV administration. This study shows sex-independent impaired lung growth, vascularization and fetal lung inflammation in a CDH mouse model. Antenatal administration of AFSC-EVs reverses aspects of pulmonary hypoplasia secondary to CDH independent of the biological sex.

Serum exosomal long non-coding RNA H19 as a theragnostic target for atrial fibrillation

Abstract Background/Introduction Exosomes are membrane vesicles of 30- to 150-nm secreted by most cell types that can mediate intercellular communication by transmitting various forms of RNAs. Long non-coding RNAs (lncRNAs, ≥ 200 nucleotides length) have been reported to play important roles in the pathophysiological processes that promote the development and progression of many diseases. Interestingly, recent studies have shown that exosomal lncRNAs exhibit different expression profiles in various diseases and are being extensively studied in the medical field as an ideal target for the diagnosis and treatment. However, there is still little known about their role in atrial fibrillation (AF). Purpose The aim of this study was to identify a novel theragnostic target exosomal lncRNA for AF. Methods First, serum exosomes from controls and patients with persistent AF were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Next, serum exosomal lncRNA expression profiles were explored using RNA-sequencing analysis. In addition, the expression levels of candidate exosomal lncRNAs were confirmed using qRT-PCR (n = 50 per group). Finally, AF-related pathophysiological mechanisms of exosomal lncRNA were investigated in angiotensin II (Ang II)-treated human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-atrial CMs). Results After RNA-sequencing analysis, we identified 27 differentially expressed lncRNAs (i.e., 4 upregulated and 23 downregulated lncRNAs with a |fold change| ≥ 2 and p &amp;lt; 0.05) in serum exosomes from patients with persistent AF compared with the controls. In fact, qRT-PCR reveled that exosomal lncRNA H19 was consistently downregulated in the serum of patients with persistent AF compared with the controls (p &amp;lt; 0.01). In addition, exosomal lncRNA H19 exhibited significant diagnostic validity for AF. Notably, we confirmed that exosomal lncRNA H19 was involved in AF-related pathophysiological mechanisms. In Ang II-treated iPSC-atrial CMs, inhibition of lncRNA H19 significantly aggravated Ang II-induced increases in levels of hypertrophic markers (ANP, BNP and β-MHC), cell surface area and inflammation (p &amp;lt; 0.05). Mechanistically, we found that loss of lncRNA H19 weakens the inhibition of its direct target microRNAs, miR-141-3p and miR-200a-3p, leading to downregulation of their target PTEN, thereby promoting the atrial remodeling and the consequent AF. Conclusion In conclusion, serum-derived exosomal lncRNA H19 could serve as a potential target for the diagnosis and treatment of AF.

Human coronary blood single-cell sequencing combined with exosome proteomic profiling reveals the molecular mechanism of recurrent in-stent restenosis (R-ISR)

Abstract Background Percutaneous coronary intervention (PCI) is recognized as the core treatment for symptomatic myocardial ischemia and acute coronary syndrome (ACS). However, the complex pathogenesis of recurrent in-stent restenosis (R-ISR) after stent implantation is still insufficiently understood. Purpose This study reports the first single-cell RNA sequencing and proteomic profiling to characterize pathogenesis of R-ISR in humans. Methods To reveal the pathophysiology and molecular mechanism of R-ISR, we performed single-cell RNA sequencing to identify RISR-induced changes in transcriptional landscape in coronary blood mononuclear cell composition. Besides, proteomic profiling of human coronary plasma-derived exosomes was used to further screen different proteins. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis were performed to identify exosomes. In vitro cell biology experiments were used to validate the discoveries of bioinformatics analysis. Results Compared with normal subjects, the percentage of FCGR3B high monocytes, memory T cells and plasmacytoid dendritic cells (DCs) was increased in R-ISR. Furthermore, we observed a marked increase in the expression of JUN, FOS, CXCL8, S100A8 enriched in the IL-17 signaling pathway that is known to play a crucial role in activating inflammation, mediating immune response, and even maintaining inflammation to chronicity. According to in vitro experiments, the co-cultured system demonstrated that FCGR3B high monocytes isolated from RISR patients activated the expression of inflammatory factors in endothelial cells (ECs), such as IL-1β, IL-6 and TNF-α. Inflammatory activated endothelial cells could release a large amount of exosomes, proteomic profiling of exosomes isolated from ECs observed a significant upregulation of MYLK expression, which could promote proliferation and migration of vascular smooth muscle cells (VSMCs) by activating VEGFR-2 signaling pathway. Conclusions Our study reveals a novel subset of FCGR3B high monocytes in R-ISR patients that could activate ECs inflammation via IL-17 signaling pathway. Activated ECs release exosomes rich in MYLK, which promote proliferation and migration of VSMCs, ultimately leading to repeated restenosis.