Differential Ultracentrifugation Research Articles

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

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

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.

Mesenchymal stem cells-derived extracellular vesicles ameliorate lupus nephritis by regulating T and B cell responses

BackgroundHuman umbilical cord mesenchymal stem cells-derived extracellular vesicles (hUCMSC-EVs) have potent immunomodulatory properties similar to parent cells. This study investigated the therapeutic effects and immunomodulatory mechanisms of hUCMSC-EVs in an experimental lupus nephritis model.MethodsThe hUCMSC-EVs were isolated by using differential ultracentrifugation. In vivo, the therapeutic effects of hUCMSC-EVs in lupus-prone MRL/lpr mice were investigated, and the mechanisms of treatment were explored according to the abnormal T and B cell responses among both the spleen and kidney.ResultsMRL/lpr mice treated with hUCMSC-EVs reduced proteinuria extent, serum creatinine and renal pathological damage; decreased splenic index and serum anti-dsDNA IgG level; and improved survival rate. hUCMSC-EVs lowered the percentage of T helper (Th)1 cells, double-negative T (DNT) cells, and plasma cells among splenocytes; inhibited the infiltration of Th17 cells but promoted regulatory T (Treg) cells in the kidney, followed by a reduction in pro-inflammatory cytokine levels(IFN-γ, IL-2, IL-6, IL-21, and IL-17 A). In addition, hUCMSC-EVs inhibited the activation of STAT3 and down-regulated IL-17 A protein levels in the kidney.ConclusionThe results of this study demonstrated that hUCMSC-EVs had therapeutic effects on experimental lupus nephritis (LN) by regulating Th1/Th17/Treg imbalance and inhibiting DNT and plasma cells. Additionally, hUCMSC-EVs inhibited Th17 cell differentiation in kidney by regulating the IL-6/STAT3/IL-17 signal pathway, which might be an important mechanism for alleviating renal injury. Taken together, we demonstrated that hUCMSC-EVs regulating T and B cell immune responses might represent a novel mechanism of hUCMSCs in treating LN, thus providing a new strategy for treating LN.

P-290 Isolation and characterization of extracellular vesicles from human endometrial organoids

Abstract Study question Do human endometrial organoids produce extracellular vesicles (EVs)? Summary answer Endometrial organoids release EVs (ORG-EVs) that we successfully and comprehensively characterize according to guidelines. What is known already EVs play a crucial role in cellular communication, garnering increasing attention in physiological and pathological processes. Although the enormous potential of EVs has been extensively demonstrated across a large variety of research fields, the promise held by EVs derived from human endometrial organoids is still largely unexplored. Indeed, nowadays there is limited research available on the presence, role, and function of EVs derived from organoids of female reproductive tissue samples. This underscores the importance of adopting standardized methods for isolating, characterizing, and reporting findings, which is crucial for advancing in this promising and innovative, yet challenging, research field. Study design, size, duration This study involved establishing organoid cultures from endometrial biopsies of healthy women (n = 10) of reproductive age, who underwent ovarian surgery between January and October 2023. ORG-EVs were isolated using differential ultracentrifugation. Characterization of ORG-EVs, including assessment of their biophysical and biochemical properties, adhered to guidelines established by the International Society for Extracellular Vesicles (ISEV). Participants/materials, setting, methods Endometrial specimens from 10 women were employed to establish organoid cultures. ORG-EVs were isolated from spent medium through differential ultracentrifugation. Morphology, size distribution and concentration analysis were performed using Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA). Western blotting was used to confirm the presence of EVs markers. Main results and the role of chance TEM analysis confirmed the presence of isolated ORG-EVs, elucidating their morphology and structural integrity. NTA revealed that ORG-EVs had an average size of 160.4 ± 6.2 nm (mean ± SD) and a mean concentration of 5.2E+11 ± 2.1E+10 particles/ml (mean ± SD). Western blot analysis further confirmed the vesicular nature of the assessed particles, demonstrating positivity for standard EV markers such as tetraspanins (CD63 and CD9), and soluble proteins (ALIX and TSG101), while excluding the endoplasmic reticulum-resident protein calnexin (negative control). Additionally, ORG-EVs exhibited a positive signal for mucin1 (MUC1), a glycoprotein present on the surface of endometrial epithelial cells, thereby confirming their cellular origin. Limitations, reasons for caution This is a pilot study, characterized by a small sample size requiring larger cohorts to validate results and, at the same time, to undertake deeper exploration of ORG-EVs’ molecular cargo. Emphasizing the critical need for standardized isolation and characterization methods is paramount. Wider implications of the findings Endometrial organoids enhance EVs production, providing a powerful tool for reproductive aspects studies, and improving regenerative medicine and artificial reproductive technology. These initial findings propel further exploration, unveiling of ORG-EVs’ molecular cargo aiming to elucidate their potential role in human female reproductive tissues and mechanism related to embryo implantation process. Trial registration number not applicable

O-267 Extracellular vesicles as biomarkers of endometriosis

Abstract Study question Can extracellular vesicles serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound? Summary answer Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes. What is known already Endometriosis is characterised by intraperitoneal lesions of endometrial tissue. Although ultrasound detection of severe endometriosis is possible, the diagnosis of peritoneal lesions currently relies on invasive laparoscopy, which leads to a delay of 8-10 years. We have previously shown that peritoneal fluid (PF) of women with endometriosis contains endometriosis specific EVs. Traced in peripheral blood (PB), EVs could serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound. Study design, size, duration Women 18-45 years of age undergoing laparoscopic surgery for endometriosis or unrelated conditions were invited to participate in our study (HREC 20-159A/HREC 20-882A). Following informed consent, we collected PF and PB samples from n = 101 participants (n = 47 controls, n = 54 cases). Exclusion criteria were pregnancy, malignancy, and menopause. Participants/materials, setting, methods We processed PF and PB samples by differential ultracentrifugation and validated the presence of EVs by nanoparticle tracking analysis (NTA), Western blotting and transmission electron microscopy (TEM). EVs were analysed by untargeted, label-based quantitative proteomics using Tandem Mass Tags (TMT). Data analysis was performed using Proteome Discoverer v2.4 (Thermo Fisher) followed by statistical analysis using R. Candidate biomarker proteins were tested by traditional and automated Western blotting (WES, ProteinSimple). Main results and the role of chance We ascertained the identity of EVs by TEM and NTA (mode size 121.8 ± 18.0 nm (blood, n = 5) and 155.9 ± 37.2 (PF, n = 6)), and signals for syntenin and ALIX in immunoblots as well as expression of CD9, CD63 and CD81 in capture bead-flow cytometry. Proteomic analysis identified 9145 protein groups across all sample groups, with 602 significantly regulated between comparisons (adjusted P-value <0.05). In PF, 533 proteins changed significantly in abundance (245 up/288 down), while in blood, only four proteins changed in abundance. We tested the presence/absence of two of these as candidate markers on PB EV protein preparations from n = 32 samples to date; our best marker showed a sensitivity of 67% and a specificity of 90%; upon exclusion of severe endometriosis cases, sensitivity improved to 80%. Limitations, reasons for caution Because of the changes in vesicle composition and amounts with the menstrual cycle, we have to group the samples by menstrual cycle phase and disease severity. This leaves relatively small numbers per group per condition. Another limitation concerns the relatively small amount of sample liquid and thus, EVs available for analysis. Wider implications of the findings Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes. A non-invasive clinical test would allow for earlier diagnosis and thus treatment of endometriosis. It would also allow patients to initiate fertility treatment early. Trial registration number not applicable

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

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

Mechanism study on fig exosome-like nanoparticles inhibiting the progression of bone metastasis in breast cancer.

e12562 Background: Breast cancer has overtaken lung cancer as the most common cancer worldwide. Breast cancer bone metastases can lead to death. How to inhibit the progression of bone metastasis of breast cancer is the key to improve the quality of life of breast cancer patients. In recent years, there have been increasing studies on the therapeutic significance of plant-derived extracellular vesicles (PDEVs), which play an important role in anti-tumor and anti-inflammatory. It is a new potential anti-tumor therapy. According to the Chinese medicine classics, figs have anti-cancer effect, but the main active ingredients are not clear yet. Methods: The exosomes were extracted by differential ultracentrifugation. The breast cancer cells (MDA-MB-231, MCF-7, 4T1, E0771) were treated with 0.1 ug/ml and 0.5 ug/ml fig exosome-like nanoparticles (FELNs) for 48 hours. The proliferation rate was measured by BrdU kit and verified by RT-qPCR. The total RNA from MDA-MB-231 cells treated by FELNs was sequenced to screen the differential genes. Key genes were differentially expressed in MDA-MB-231 and MCF-7 cells and further verified. Animal models of breast cancer in situ and bone metastasis were constructed in mice. FELNs were injected intraperitoneally twice a week, live imaging of small animals was performed on the 14th and 21st day to observe the tumor size and progression of breast cancer, and tibia samples were collected for Trap staining. The bone marrow macrophages of mice were extracted and treated by FELNs. The polarization was detected by RT-qPCR. After the removal of the proteins and nucleic acids in FELNs by protease, DNase and RNase, FELNs intervened in breast cancer cells and macrophages to determine the effective components of the FELNs. Results: After breast cancer cells were treated with FELNs, the proliferation rate was significantly decreased, especially MDA-MB-231 (from 70.3% to 17.7%) and MCF-7 (from 7.32% to 1.63%), and the RNA level of proliferation-related gene were significantly down-regulated. The key gene RN7SL1 was screened by sequencing. Overexpression of RN7SL1 significantly promoted the proliferation of MDA-MB-231 and MCF-7 cells, while the level of proliferation-related gene RNA was significantly up-regulated. FELNs significantly inhibited the tumor size and bone metastasis of breast cancer, and the tibial osteolysis was significantly abated by the treatment of FELNs in tibial Trap staining. After the intervention of FELNs in mouse macrophages, the polarization of M1 macrophages was significantly promoted. The proliferation rate of breast cancer cells increased and the polarization of M1 macrophages disappeared by treatment with protein-free FELNs. Conclusions: The protein components in FELNs inhibit the proliferation of breast cancer cells through RN7SL1, thereby inhibiting the progression of breast cancer bone metastasis.

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells

BackgroundCanine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis.MethodsTo identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs.ResultsProteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs.ConclusionThis study elucidates AGR2’s pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

Triple-Negative Breast Cancer EVs Modulate Growth and Migration of Normal Epithelial Lung Cells.

Breast cancer is the most common cancer amongst women worldwide. Recently, owing to screening programs and new technologies, the survival rate has increased significantly. Breast cancer can potentially develop metastases, and, despite them, lung metastases generally occur within five years of breast cancer diagnosis. In this study, the objective was to analyze the effect of breast cancer-derived EVs on a lung epithelial cell line. BEAS-2B cells were treated with extracellular vesicles (EVs) derived from triple-negative breast cancer cells (TNBCs), e.g., MDA-MB-231 and HS578T, separated using differential ultracentrifugation. We observed an increased growth, migration, and invasiveness of normal epithelial lung cells over time in the presence of TNBC EVs compared to the control. Therefore, these data suggest that EVs released by tumor cells contain biological molecules capable of influencing the pro-tumorigenic activity of normal cells. Exploring the role of EVs in oncology research and their potential cargo may be novel biomarkers for early cancer detection and further diagnosis.

#1219 Extracellular vesicles from patients with AMR-induced reprogramming of human podocytes into senescent cells: role of SGLT2i

Abstract Background and Aims Extracellular vesicles (EVs) are organelles released by different cells that deliver biological information into the extracellular space and contribute to several diseases. However, their role in patients with antibody-mediated allograft rejection (AMR) remains uncertain. Our aim was to test the influence of plasma-derived EVs on human conditional immortalized podocytes (PODO) by investigating the possible role of dapagliflozin (Dapa) as a pharmacological agent. Methods Twenty-eight Rtx patients were included in the study, 14 with AMR, and 14 control TX patients (RTX). (CTRL). Plasma-derived Evs were used to stimulate PODO for 24 h, followed by exposure to 100 nM Dapa for 24 h. Differential ultracentrifugation at 110,000 g was applied, followed by counting with Nanosight and characterization by high-resolution flow cytometry according to the MISEV2018 criteria. PKH26 dye was used for the EVs-PODO incorporation study using Z-stack microscopy image acquisition. Total RNA and protein were extracted for gene expression and protein analysis, and the cells were fixed for immunostaining evaluation. Results We first found that upon activation with EV, PODO incorporated Es and shared them between cells; moreover, AMR-EVs led to an increased expression of tetraspanin marker of cell activation CD9 (p = 0.028) compared with CTRL. Importantly, the incubation of PODO with RTX-EVs induced a senescence-associated secretory phenotype (TNF alpha p = 0.0006, MCP1 p = 0.014); with cytoskeletal dysregulation and increase in gene expression and proteins involved in the epithelial to mesenchymal transition such as VIM (p = 0.026), senescence P21 (p = 0.04), inflammation CD44 (p = 0.034) with downregulation of Klotho (p = 0.002). Interestingly, incubation with Dapagliflozin significantly counteract PODO activation with reduction in P21 expression (p = 0.039) and an increase in Klotho (p = 0.047) abrogating inflammaging. Conclusions Our findings demonstrate that plasma EVs of AMR induced cellular senescence in PODO, reprogramming cellular processes such as cytoskeleton shape and composition, secretion of inflammatory cytokines, and decrease of the nephroprotective protein Klotho. This pathogenic process can be efficiently counteracted by SGLT2i.

#1841 Trimethylamine N-oxide (TMAO) induces the formation of endothelial extracellular vesicles

Abstract Background and Aims Endothelial dysfunction caused by the accumulation of uremic toxins is an important contributor to the development of cardiovascular disease in patients with chronic kidney disease (CKD). The uremic toxin trimethylamine n-oxide (TMAO) is associated with oxidative stress, vascular calcification, and inflammation, triggering the release of several proinflammatory molecules and Extracellular Vesicles (EVs). Clinical studies have shown that patients with CKD have high levels of circulating EVs, however, their role is still poorly understood. In this dysfunctional scenario, EVs can be used as damage biomarkers for CKD, as they carry information from their cells of origin. Here, we aim to elucidate the formation of EVs in endothelial cells exposed to TMAO. Method Human endothelial cells (EA.hy926, ATCC CRL-2922) were exposed for 24 h with TMAO at uremic concentrations (7.49 mg/L). EVs were isolated by differential ultracentrifugation. Protein concentration was analyzed by Pierce™ BCA Protein Assay Kit, and particle size and particle concentration were analyzed by NanoParticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), and Scanning Electron Microscopy (SEM). EVs were stained with calcein and their internalization in endothelial cells was analyzed with Fluorescence Confocal Microscopy. Results TMAO treatment at uremic concentration induced the formation of EVs with a size of 202.8 ± 3.1 nm (image 1B) and 1.09 × 109 ± 2.68 × 107 particles/mL and a total protein concentration of 88.57 µg/mL. The morphological features of EVs are shown in images 1C-1F. TMAO-induced EVs were incorporated by endothelial cells, being dispersed in the cytoplasm or perinuclear regions (images 1G and end 1H). Conclusion TMAO was able to induce the formation of EVs in endothelial cells, with characteristics morphological features. When compared to control EVs (image 1A), TMAO-EVs have a more heterogeneous size, which indicate a change in the size pattern, and thus the nature of the EVs induced by TMAO. TMAO-EVs were able to be internalized by human endothelial cells. This uptake can be closely related to the effects of EVs in endothelial cells, but more experiments need to be performed. Taken together, these data could contribute to understanding how the formation and uptake of TMAO-EVs by the endothelium occurs, relating their effects to the cardiovascular complications of CKD.

Exosomal HMGB3 released by glioma cells confers the activation of NLRP3 inflammasome and pyroptosis in tumor-associated macrophages

BackgroundPrevious evidences has highlighted the pivotal role of NOD-like receptor family pyrin domain-containing 3 (NLRP3)-mediated inflammasomes and pyroptosis activation in driving tumor malignancy and shaping the tumor microenvironment. Herein, we aimed to elucidate the impact of high-mobility group box 3 (HMGB3) released in glioma-derived exosomes on macrophage infiltration in gliomas, NLRP3 inflammasome activation and polarization. MethodsTranscripts and protein levels of HMGB3, and cytokines associated with macrophage phenotypes and pyroptosis were assessed in glioma tissues and cell lines (U251, LN229, T98G, A172) using qRT-PCR and/or Western blot analysis. Exosomes secreted from LN229 and NHA cells were isolated via differential ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and analysis of exosome-related markers. PKH67 staining was employed to examine exosomes uptake by THP-1 differentiated macrophages. Flow cytometry was utilized to assess macrophage pyroptotic rates and polarization-related markers. ResultsHMGB3 expression was elevated in glioma tissues, serum samples and tumor cell lines. Kaplan-Meier curves revealed a positive correlation between higher HMGB3 expression and poor overall survival and recurrence-free survival. Moreover, glioma tissues with increased HMGB3 expression exhibited significant upregulation of M2 macrophages markers (CD68, CD206, Arg1) and NLRP3 inflammasome components (NLRP3, IL-1β, ASC), suggesting that HMGB3 was closely associated with macrophage infiltration and NLRP3 inflammasome activation. Notably, HMGB3 was found to be enriched in glioma cell- secreted exosomes and could be internalized by macrophages. Knockdown of HMGB3 in glioma cell exosomes could restrain M2 macrophage polarization, NLRP3 inflammasome activation and pyroptosis. ConclusionThese findings suggested that glioma cells secreted exosomal HMGB3 could facilitate macrophage M2 polarization, pyroptosis and inflammatory infiltration, indicating HMGB3 might be a poor prognosis factor for glioma.

Abstract PO1-25-01: LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes

Abstract Background: Breast cancer (BC) is a leading cause of cancer death and despite numerous treatment advancements, metastatic BC is incurable. Extracellular vesicles (EVs) are lipid-delimited particles released from cells which contain bioactive protein and nucleic acid cargo. Numerous studies have shown their importance in facilitating communication between cancer cells and the tumour microenvironment, implicating them as mediators of many aspects of carcinogenesis. EV-mediated communication between BC and the innate immune system plays a crucial role in BC progression, metastasis and the maintenance of an immunosuppressive microenvironment. LMTK3 overexpression in BC promotes therapy resistance, disease progression and invasion and metastasis, leading to a poorer prognosis in patients. Analysis of deconvoluted TCGA data indicated that LMTK3 overexpression also correlates with exclusion of M1 macrophages from the tumour microenvironment. This study aimed to explore the role of LMTK3 in EV-mediated communication between BC and immune cells. Methods EVs were collected from conditioned media of wild-type and LMTK3-overexpressing BC cells through differential ultracentrifugation, then characterised by transmission electron microscopy, nanoparticle tracking analysis, quantitative mass spectrometry and Western blotting. Monocytes were treated with EVs and proteomic and phosphoproteomic changes were analysed. 3D coculture models of BC and immune cells were used to assess monocyte and macrophage infiltration, polarisation and anti-cancer activity. Immunofluorescence and flow cytometry was used to assess macrophage polarisation. Bioinformatic analysis of TCGA datasets validated the findings in a clinical setting and the data was also validated in vivo, using syngeneic immunocompetent mouse models. Results LMTK3 overexpression in BC cells promotes the upregulation of a subpopulation of large (80-150nm) CD81+ EVs which are significantly enriched in oncogenic and immunoregulatory proteins, including LDHB, ARHGEF15, PSAT1 and HIC2. EVs from LMTK3-overexpressing but not control BC cells strongly reduce the ability of monocytes to infiltrate BC tumour spheroids and migrate through Matrigel. Proteomics analysis of the monocytes showed an enrichment in M2 polarisation proteins and a downregulation of proteins essential for migration, including S100A8, KLF13 and TRPC3. LMTK3 overexpression in BC also promotes M2 polarisation and exclusion of M1 macrophages in vivo. Summary LMTK3 overexpression in BC correlates with poor prognosis and reduced overall survival. We hypothesise that this is, in part, due to monocyte M2 polarisation and exclusion of M1 macrophages from the TME, mediated by LMTK3-dependent alterations in EV cargo, contributing to tumour immune evasion. Citation Format: Mark Samuels, William Jones, Charlotte Turner, Georgios Giamas. LMTK3 promotes macrophage M2 polarisation and suppresses monocyte infiltration in breast cancer by altering the proteomic cargo of exosomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-01.

Plasma exosomes impair microglial degradation of α-synuclein through V-ATPase subunit V1G1.

Microglia are the main phagocytes in the brain and can induce neuroinflammation. Moreover, they are critical to alpha-synuclein (α-syn) aggregation and propagation. Plasma exosomes derived from patients diagnosed with Parkinson's disease (PD-exo) reportedly evoked α-syn aggregation and inflammation in microglia. In turn, microglia internalized and released exosomal α-syn, enhancing α-syn propagation. However, the specific mechanism through which PD-exo influences α-syn degradation remains unknown. Exosomes were extracted from the plasma of patients with PD by differential ultracentrifugation, analyzed using electron microscopy (EM) and nanoparticle flow cytometry, and stereotaxically injected into the unilateral striatum of the mice. Transmission EM was employed to visualize lysosomes and autophagosomes in BV2 cells, and lysosome pH was measured with LysoSensor Yellow/Blue DND-160. Cathepsin B and D, lysosomal-associated membrane protein 1 (LAMP1), ATP6V1G1, tumor susceptibility gene 101 protein, calnexin, α-syn, ionized calcium binding adaptor molecule 1, and NLR family pyrin domain containing 3 were evaluated using quantitative polymerase chain reaction or western blotting, and α-syn, LAMP1, and ATP6V1G1 were also observed by immunofluorescence. Small interfering ribonucleic acid against V1G1 was transfected into BV2 cells and primary microglia using Lipofectamine® 3000. A PD mouse model was established via injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into mice. A lentiviral-mediated strategy to overexpress ATP6V1G1 in the brain of MPTP-treated mice was employed. Motor coordination was assessed using rotarod and pole tests, and neurodegeneration in the mouse substantia nigra and striatum tissues was determined using immunofluorescence histochemical and western blotting of tyrosine hydroxylase. PD-exo decreased the expression of V1G1, responsible for the acidification of intra- and extracellular milieu. This impairment of lysosomal acidification resulted in the accumulation of abnormally swollen lysosomes and decreased lysosomal enzyme activities, impairing lysosomal protein degradation and causing α-syn accumulation. Additionally, V1G1 overexpression conferred the mice neuroprotection during MPTP exposure. Pathogenic protein accumulation is a key feature of PD, and compromised V-type ATPase dysfunction might participate in PD pathogenesis. Moreover, V1G1 overexpression protects against neuronal toxicity in an MPTP-based PD mouse model, which may provide opportunities to develop novel therapeutic interventions for PD treatment.

Skeletal Muscle Extracellular Vesicles Enhance Mitochondrial Movement and have a Dose-dependent Effect on Increasing Mitochondrial Respiration

Chronic exercise leads to systemic health benefits across tissues, including an increase in mitochondrial function in skeletal muscle. One potential mechanism is via the release of extracellular vesicles (EVs), which can transfer functional cargo to recipient cells. Previously, we have shown that using an in vitro model of exercise, chronic contractile activity (CCA)-derived EVs (CCA-EV) increased mitochondrial biogenesis in healthy myoblasts. Here, we hypothesize that CCA-EVs will have a concentration-dependent effect on mitochondrial respiration, and also regulate mitochondrial dynamics in myoblasts. C2C12 myoblasts were differentiated into myotubes, and electrically paced (3hrs/day, 4 days, 14V, C-PACE EM, IonOptix). EVs from control and CCA-stimulated myotubes were isolated from conditioned media using differential ultracentrifugation and biophysically characterized by size and concentration using TRPS (Izon). C2C12 myoblasts were treated daily for 4 days using three different concentrations: 1.0E4; 2.0E4 and 4.0E4 EVs/cell (N=4-5). Basal and maximal oxygen consumption rates (OCR) were measured using the Seahorse XFe24 (Agilent). We used live-cell fluorescence microscopy and Mitometer software to access mitochondrial movement: area/volume, displacement, distance, length, perimeter, speed and velocity. Average size was unchanged between control-EVs (123±10.8 nm) and CCA-EVs (123±11.9 nm) (p=0.9137, N=3), and 2.3-fold more CCA-EVs were released vs. control-EVs (p=0.2072, N=3). Lowest EV concentration (1.0E+4 EVs/cell, N=5) had no effect on basal (p=0.4226) and maximal (p=0.1835) OCR. Intermediate dosage of CCA-EVs (2.0E+4 EVs/cell, N=5) increased basal OCR by 20% (p=0.0037) and maximal OCR by 18% (p=0.0095), while the highest concentration (4.0E+4 EVs/cell, N=4) decreased basal OCR by 9% (p=0.0001) and maximal OCR by 29% (p=0.0009), compared to control-EVs. Mitochondrial motility (N=484-976) in myoblasts treated daily after each day of CCA showed enhanced mitochondrial dynamics with CCA-EVs: increased area/volume (p<0.0001), displacement (p<0.0001), distance (p<0.0001), mean intensity (p<0.0001), perimeter/surface area (p=0.0137) and speed (p<0.0001), compared to control-EVs. In summary, CCA-EVs induced concentration-dependent improvements in mitochondrial respiration in healthy myoblasts, concomitant with enhanced mitochondrial movement. These results open exciting avenues for the therapeutic potential of CCA-EVs in rescuing mitochondrial dysfunction. TFGS is funded by a Research Manitoba Postdoctoral Fellowship. Grants from CHRIM, CHF, DREAM and NSERC to AS funded the research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of In Vitro Enzyme Digestion and Bile Treatment on Milk Extracellular Vesicles Stability.

Milk extracellular vesicles (EVs) are nanosized particles with potential immune bioactivities. This study examines their fate during in vitro infant gastrointestinal digestion (GI). Bovine milk is digested using the in vitro INFOGEST method, adjusted for the infant. To unravel the contribution of digestive enzymes from bile, milk is treated with digestive enzymes, bile, or a combination of both. EVs are collected posttreatment using differential ultracentrifugation. EVs characterization includes electrophoresis, immunoblotting, nanoparticle tracking analysis, and atomic force microscopy. EVs protein markers programmed cell death 6-interacting protein (ALIX), tumor susceptibility gene 101 (TSG101), cluster of differentiation 9 (CD9), and xanthine dehydrogenase (XDH) are detected after gastric digestion (G60), but their signal intensity is significantly reduced by intestinal conditions (p<0.05). Enzyme digestion, compared to bile treatment (I60+bile), results in a significant reduction of signal intensities for TSG101 and CD9 (p<0.05). Nanoparticle tracking analysis shows a significant reduction (p<0.05) of EV numbers at the end of the intestinal phase. EVs are detected by atomic force microscopy at the end of the intestinal phase, showing that intact EVs can survive upper gut digestion. Intact EVs can be found at the end of the intestinal phase. However, digestive enzymes and bile reduce the quantity and characteristics of EVs, with digestive enzymes playing a larger role.

Extracellular vesicles isolated from Arabidopsis thaliana leaves reveal characteristics of mammalian exosomes.

Plant-derived extracellular vesicles (EVs), containing a myriad of bioactive proteins, microRNAs, lipids, and secondary metabolites, have recently become the focus of rising interest due to their important roles in various applications. The widely accepted method for isolating plant EVs is differential ultracentrifugation plus density gradient centrifugation. However, the combination of differential ultracentrifugation and density gradient centrifugation for the isolation of plant EVs is time-consuming and labor-intensive. Hence, there is a need for more efficient methods to perform the separation of plant EVs. In this study, EVs were separated from Arabidopsis thaliana leaves by a cost-effective polyethylene glycol (PEG)-based precipitation approach. The mean size of purified Arabidopsis thaliana EVs determined by dynamic light scattering was 266nm, which is consistent with nanoparticle tracking analysis. The size was also confirmed via transmission electron microscopy with morphology of a cup-shaped appearance which is the typical mammalian exosome's morphology. Additionally, Western blotting of the purified Arabidopsis thaliana EVs, using commercially available mammalian exosomal kits, displayed surface marker tetraspanin proteins (CD9, CD63, and CD81), and endosomal sorting complexes required for transport (ESCRT)-associated proteins (TSG101 and ALIX). This demonstrates that the purified Arabidopsis thaliana EVs reveal the typical proteins reported in mammalian exosomes.

Optimization of protocols for blood-derived extracellular vesicles for studies in liver diseases

Aim: Extracellular vesicles (EVs) have gained significant attention for their diagnostic and therapeutic potential in various diseases, including liver disorders. This study focuses on optimizing the isolation and characterization of small EVs from plasma and serum samples in patients with liver diseases, aiming to advance our understanding and potential clinical applications of EVs. Methods: Blood samples were collected from patients with end-stage liver disease (ESLD) enlisted in the TransplantLines Cohort and Biobank Study, and healthy donors. We employed differential ultracentrifugation (DUC) to evaluate three distinct protocols: a 3-step DUC, a washing step omitted [samples without washing (WW)], and a contaminant-depleted plasma (CDP) protocol. RNA isolation methodologies were compared, involving the use of TRI-reagent or the commercial AllPrep DNA/RNA kit. Further insights into EV composition were obtained through proteomic analyses, comparing samples subjected to traditional cell lysis (L) with those processed without lysis (NL). Results: We successfully isolated EVs from both plasma and serum samples as confirmed by the presence of specific EV markers, including CD9, CD63, CD81, and tumor susceptibility gene 101 (TSG-101). While some contaminants remained, such as albumin and lipoproteins, the protocol selected to continue EVs analysis was the 3-step protocol. Transmission electron microscopy (TEM) and nanotracking analysis (NTA) further confirmed EVs presence. RNA extraction was achieved using TRI-reagent, but not with the commercial kit highlighting the importance of selecting an appropriate method for RNA isolation. Finally, proteomics analysis showed that lysed samples were significantly more enriched in proteins compared to non-lysed samples, although protein variability was still present in both groups. Conclusions: Optimizing EV isolation techniques is essential for harnessing their potential in liver disease diagnosis and therapy. Further refinement of purification methods, a deep characterization of our cohort and understanding the variability and cargo within EVs will be crucial for future biomarker discovery and therapeutic applications in liver-related diseases.

Mass Production of Rg1-Loaded Small Extracellular Vesicles Using a 3D Bioreactor System for Enhanced Cardioprotective Efficacy of Doxorubicin-Induced Cardiotoxicity.

Small extracellular vesicles (sEVs) obtained from human umbilical cord mesenchymal stromal cells (MSCs) have shown cardioprotective efficacy in doxorubicin-induced cardiotoxicity (DIC). However, their clinical application is limited due to the low yield and high consumption. This study aims to achieve large-scale production of sEVs using a three-dimensional (3D) bioreactor system. In addition, sEVs were developed to deliver Ginsenoside Rg1 (Rg1), a compound derived from traditional Chinese medicine, Ginseng, that has cardioprotective properties but limited bioavailability, to enhance the treatment of DIC. The 3D bioreactor system with spinner flasks was used to expand human umbilical cord MSCs and collect MSC-conditioned medium. Subsequently, sEVs were isolated from the conditioned medium using differential ultra-centrifugation (dUC). The sEVs were loaded with Ginsenoside Rg1 by electroporation and evaluated for cardioprotective efficacy using Cell Counting Kit-8 (CCK-8) analysis, Annexin V/PI staining and live cell count of H9c2 cells under DIC. Using the 3D bioreactor system with spinner flasks, the expansion of MSCs reached ~600 million, and the production of sEVs was up to 2.2 × 1012 particles in five days with significantly reduced bench work compared to traditional 2D flasks. With the optimized protocol, the Ginsenoside Rg1 loading efficiency of sEVs by electroporation was ~21%, higher than sonication or co-incubation. Moreover, Rg1-loaded sEVs had attenuated DOX-induced cardiotoxicity with reduced apoptosis compared to free Ginsenoside Rg1 or sEVs. The 3D culture system scaled up the production of sEVs, which facilitated the Rg1 delivery and attenuated cardiomyocyte apoptosis, suggesting a potential treatment of DOX-induced cardiotoxicity.