Extracellular Vesicles Research Articles

ApoEVs Transfer Mitochondrial Component to Modulate Macrophages in Periodontal Regeneration.

Macrophages are key players in the host immune response to periodontal pathogens and tissue repair. The aim of this study was to explore the potential of apoptotic cell-derived extracellular vesicles (ApoEVs) in modulating the mitochondrial function of macrophages as a mean to enhance periodontal tissue regeneration. ApoEVs were extracted from periodontal ligament stem cells (PDLSCs) and characterized to observe their effects on macrophage function. Invivo experiments, ApoEVs were mixed with hyaluronic acid and injected into the periodontal pockets of rats with periodontitis to observe their impact on periodontal tissue regeneration and the immune microenvironment. Functional assays were conducted to confirm whether ApoEVs contained mitochondrial components and which specific components were transferred to regulate macrophage function. The experimental findings showed that treatment of ApoEVs efficiently restored the homeostasis of macrophage and improved tissue regeneration in a periodontitis rat model. Mechanism investigation demonstrated that the efferocytosis of ApoEVs resulted in the transfer of mitochondrial components from PDLSCs to macrophage. The increased mitochondrial components within macrophages improved mitochondrial function and polarization of macrophages towards the anti-inflammatory M2 phenotype, resulting in the improvement of inflammatory environment in periodontal tissues. ApoEVs can transfer mtDNA to enhance mitochondrial function in macrophages, fostering their transition to an anti-inflammatory phenotype. Ultimately, this process improves the immune microenvironment in periodontitis and promotes periodontal tissue regeneration.

Abstract 4119680: First-in-Man Treatment of Heart Failure by Repeated Intravenous Infusions of a Cardiovascular Cell-Derived Extracellular Vesicle-Enriched Secretome

Background: Most of the effects of intramyocardially transplanted stem cells can be duplicated by the sole administration of their secretome but this approach has not yet been tested in heart failure (HF). Methods: The SECRET-HF phase I trial (NCT05774509) was designed according to a Bayesian optimal interval scheme to assess the effects of repeated intravenous infusions of a cellular secretome in 12 patients with a non-ischemic dilated cardiomyopathy. The main inclusion criteria are severe symptoms (NYHA Class III despite an optimal guideline-directed medical therapy), a reduced left ventricular (LV) ejection fraction (EF ≤40%) and ineligibility for a heart transplant. The Investigational Medicinal Product was derived from human induced pluripotent stem cells reprogrammed from a healthy donor and differentiated in cardiovascular progenitor cells (CPC). Following CPC culture in a dedicated "vesiculation" medium, the conditioned media were collected, filtered, enriched for extracellular vesicles, concentrated by tangential flow filtration and scaled out into glass vials stored at -80°C. Quality controls were implemented throughout the whole process which was conducted according to current Good Manufacturing Practices. Results: Three patients, out of the 4 of the initial low-dose cohort, have now been treated. All had been previously fitted with an automatic internal cardioverter defibrillator (AICD). The secretome was delivered intravenously 3 times, 3 weeks apart, at a dose of 20 x10 9 particles/kg for each infusion. By November, 2024, the follow-up will be 16, 9, 6 and 2 months for each of them (mean: 8 months). So far, none of the treated patients has experienced a dose-limiting toxicity; in particular, there have been no post-treatments ventricular arrhythmia detected by the AICD, no allo-immunization and no inflammation based on the enumeration of lymphocyte subpopulations by flow cytometry. At 6 months, the first patient improved to NYHA Class II with a decrease in echographic LVEDV and LVESV (from 108 mL/m 2 to 87 mL/m 2 and from 80 mL/m 2 to 60 mL/m 2 , respectively) and a concomitant increase in LVEF from 25% to 32%. At 1 month post-treatment, the second patient also reports a symptomatic improvement (NYHA Class II) with an increase in EF from 21% to 28%. Conclusions: This initial experience supports the safety of repeated intravenous administrations of a cardiovascular cellular secretome as a potential noninvasive and user-friendly treatment of severe HF.

Inpp5e Is Critical for Photoreceptor Outer Segment Maintenance.

In humans, inositol polyphosphate-5-phosphatase e (INPP5E) mutations cause retinal degeneration as part of Joubert and MORM syndromes and can also cause non-syndromic blindness. In mice, mutations cause a spectrum of brain, kidney, and other anomalies and prevent the formation of photoreceptor outer segments. To further explore the function of Inpp5e in photoreceptors, we generated conditional and inducible knockouts of mouse Inpp5e where the gene was deleted either during outer segment formation or after outer segments were fully formed. In both cases, the loss of Inpp5e led to severe defects in photoreceptor outer segment morphology and ultimately photoreceptor cell loss. The primary morphological defect consisted of outer segment shortening and reduction in the number of newly forming discs at the outer segment base. This was accompanied by structural abnormalities of the Golgi apparatus, mislocalized rhodopsin, and an accumulation of extracellular vesicles. In addition, knockout cells showed a reduction in the size and prevalence of the actin network at the site of new disc morphogenesis and the occasional formation of membrane whorls instead of discs in a subset of cells. Together, these data demonstrate that Inpp5e plays a critical role in maintaining the outer segment and the normal process of outer segment renewal depends on the activity of this enzyme.

Abstract 4129709: Aspirin-Nanoparticle for Dual Therapies: Targeted Anti-Inflammatory and Prolonged Anti-Platelet Effects for Atherosclerosis

Background: The current unmet needs for aspirin usage in atherosclerosis lie in its short half-life and narrow indication for anti-platelet effects. Daily aspirin intake is mandatory, and the anti-inflammatory effects of aspirin for atherosclerosis have not successfully translated to clinical practice. Nanoparticles remain in circulation for 2-3 days, with a large portion being cleared by splenic monocytes, which are known to inherently target inflamed sites. Hypothesis: By altering the pharmacokinetics of aspirin through loading into nanoparticles, aspirin-nanoparticles can exert prolonged anti-platelet effects and target atherosclerosis sites via monocyte carriers for anti-inflammatory effects, resulting in dual therapies. Methods: Splenic monocytes were loaded with aspirin-liposomes and co-cultured with endothelial cells or platelets to examine the interactions between them using high-resolution time-series microscopy. Prolonged anti-platelet effects and targeted anti-inflammatory effects of aspirin were validated in intact mice and hindlimb ischemia models, respectively. Furthermore, the dual therapies of aspirin-liposomes were validated in an atherosclerotic mouse model created by partial carotid ligation and a western diet in apoE gene knock-out mice. Results: When splenic monocytes were loaded with aspirin-liposomes, they emitted extracellular vesicles (EVs) loaded with aspirin towards endothelial cells or platelets. As inflamed cells upregulate caveolin expression, they uptake an increased amount of transferred EVs compared to non-inflamed cells. Additionally, aspirin-liposomes showed prolonged circulation time and increased splenic accumulation compared to aspirin itself, resulting in prolonged anti-platelet effects (>7 days) and targeted anti-inflammatory effects at inflamed sites. Compared to the daily oral aspirin group in the atherosclerosis model, the weekly intravenous aspirin-liposome group showed superior therapeutic effects, including attenuated systemic and local inflammation and patent lumen in atherosclerotic sites. Conclusion: Aspirin-nanoparticles can exert prolonged anti-platelet effects combined with targeted anti-inflammatory effects, resulting in superior therapeutic effects on atherosclerosis.

IMMU-57. METHYL-B-CYCLODEXTRIN PARTIALLY REVERSES THE IMMUNOSUPPRESSIVE EFFECTS OF GLIOBLASTOMA DERIVED EXTRACELLULAR VESICLES ON LYMPHOCYTES

Abstract Extracellular vesicles (EVs) derived from glioblastoma (GBM) have been demonstrated in multiple studies to contribute to tumorigenesis, invasion, and immunosuppression. Our prior results demonstrate that EVs can induce the differentiation of myeloid-derived suppressor cells (MDSCs), which in turn impair T cell activation and proliferation in vitro. Methyl-B-Cyclodextrin (MBCD), a cholesterol depleting agent, can interfere with lipid rafts, which are cholesterol rich microdomains on the cellular membrane that act as a docking mechanism for EV uptake. Herein we demonstrate that MBCD can inhibit EV uptake and partially reverse the immunosuppressive effects of EVs on both monocytes and T cells. METHODS EVs were isolated using stepwise ultracentrifugation from GBM cultures. GBM derived EVs were then added to extracted monocytes from donor samples with or without MBCD for 72 hours under hypoxic conditions. The EV exposed monocytes were then co-cultured with T lymphocytes and cell proliferation was measured via flow cytometry. RESULTS Exposure to GBM-derived EVs induced MDSC differentiation in monocytes by at least 50% while the addition of MBCD reduced MDSC levels similar to that of the control group. Moreover, MBCD exposure resulted in increased T cell proliferation in the presence of EV treated monocytes compared to untreated samples. CONCLUSION MBCD can be used as a therapeutic agent to reverse MDSC induction by GBM derived EVs and can partially rescue T cell activation and proliferation.

Therapeutically Harnessing Tumor Cell-Derived Extracellular Vesicles for Multiple Myeloma: Recent Advances and Future Perspectives

Extracellular vesicles (EVs) have emerged as pivotal regulators for extensive intercellular crosstalk owing to capsuled diverse bioactive substances such as proteins, nucleic acids, and lipids. Recent studies have shown that tumor-derived EVs significantly influence the bone marrow microenvironment, contributing to the progression of multiple myeloma (MM). This highlights the robust potential of EVs as a promising avenue for developing more effective and precise diagnostic and therapeutic strategies for MM. In this review, we briefly discuss the multifaceted roles of EVs in MM progression, as well as the diagnostic and therapeutic value in MM management. Specifically, we focus on the latest research progress regarding the therapeutic potential of EVs for MM, particularly tumor cell-derived EVs, as we elaborate on three main aspects: (i) EVs as therapeutic targets, including the targeted inhibition of EV biogenesis and uptake, and the possibility of eliminating tumor-derived EVs; (ii) EVs as delivery nanovectors, where we discuss the latest anti-MM candidates and potential ways to optimize therapeutic efficiency; and (iii) engineered EVs as antitumor vaccines, focusing on the use of tumor cell-derived EVs in immunotherapy. Finally, we address the prospects and challenges of harnessing the therapeutic potential of EVs in clinical transformation.

TMIC-21. PYK2/MAPK/ERK SIGNALING PATHWAY REGULATES EXTRACELLULAR VESICLE RELEASE IN GLIOBLASTOMA

Abstract Glioblastoma (GBM), the deadliest brain cancer, typically proves fatal within a year despite treatment. Tumor-associated myeloid cells (TAMs) support GBM growth, treatment resistance, and immune surveillance. Extracellular vesicles (EVs) facilitate communication between tumor and TAMs, exchanging proteins, lipids, RNA, and genetic material. As EVs can travel through the bloodstream to distant tissues, they trigger signaling pathways and metabolic changes in remote organs while also recruiting bone marrow-derived cells and modulating immune responses. Therefore, EVs serve as a crucial mechanism for the distant regulation of the tumor’s immune microenvironment. We hypothesize that Pyk2/MAPK/Erk signaling regulates EV release by modulating the actin cytoskeleton, impacting TAM infiltration and activation in tumors. This study aims to investigate the role of Pyk2/MAPK/Erk in specific EV release in GBM cells, potentially offering insights into therapeutic targets. Using confocal imaging and flow cytometric analysis of EVs, purified from medium conditioned from human primary GBM cell lines with and without Pyk2 CRISPR/Cas9 knock-out (Pyk2KO), the study identified that Pyk2KO cells do not release the population of EVs with diameter bigger then 600nm and up-regulate release of smaller EVs. Additionally, flow cytometric analysis of EVs, released from cells, fluorescently labeled with membrane dye carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), detected 14% of CFSE+ events in EV’s population, purified from Pyk2WT, vs. to 2.5% from Pyk2KO cells. Membrane labeling of cells enables the tracking of microvesicles (MV) shedding and was used to differentiate between MV and ES based on the process of their biogenesis. Considering that larger size and the transition of fluorescence from the plasma membrane are indicative of MVs, our data indicate that Pyk2 is involved in the regulation of MV release. FUNDING: PRSTRT2022 and NIH Grant 1R15CA287203

Proteomics analysis of extracellular vesicles for biomarkers of autism spectrum disorder

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by symptoms that include social interaction deficits, language difficulties and restricted, repetitive behavior. Early intervention through medication and behavioral therapy can eliminate some ASD-related symptoms and significantly improve the life-quality of the affected individuals. Currently, the diagnosis of ASD is highly limited.MethodsTo investigate the feasibility of early diagnosis of ASD, we tested extracellular vesicles (EVs) proteins obtained from ASD cases. First, plasma EVs were isolated from healthy controls (HCs) and ASD individuals and were analyzed using proximity extension assay (PEA) technology to quantify 1,196 protein expression level. Second, machine learning analysis and bioinformatic approaches were applied to explore how a combination of EV proteins could serve as biomarkers for ASD diagnosis.ResultsNo significant differences in the EV morphology and EV size distribution between HCs and ASD were observed, but the EV number was slightly lower in ASD plasma. We identified the top five downregulated proteins in plasma EVs isolated from ASD individuals: WW domain-containing protein 2 (WWP2), Heat shock protein 27 (HSP27), C-type lectin domain family 1 member B (CLEC1B), Cluster of differentiation 40 (CD40), and folate receptor alpha (FRalpha). Machine learning analysis and correlation analysis support the idea that these five EV proteins can be potential biomarkers for ASD.ConclusionWe identified the top five downregulated proteins in ASD EVs and examined that a combination of EV proteins could serve as biomarkers for ASD diagnosis.

Small and Long Non-Coding RNA Analysis for Human Trophoblast-Derived Extracellular Vesicles and Their Effect on the Transcriptome Profile of Human Neural Progenitor Cells

In mice, the fetal brain is dependent upon the placenta for factors that guide its early development. This linkage between the two organs has given rise to the term, the placenta–brain axis. A similar interrelationship between the two organs may exist in humans. We hypothesize that extracellular vesicles (EVs) released from placental trophoblast (TB) cells transport small RNA and other informational biomolecules from the placenta to the brain where their contents have pleiotropic effects. Here, EVs were isolated from the medium in which human trophoblasts (TBs) had been differentiated in vitro from induced pluripotent stem cells (iPSC) and from cultured iPSC themselves, and their small RNA content analyzed by bulk RNA-seq. EVs derived from human TB cells possess unique profiles of miRs, including hsa-miR-0149-3p, hsa-302a-5p, and many long non-coding RNAs (lncRNAs) relative to EVs isolated from parental iPSC. These miRs and their mRNA targets are enriched in neural tissue. Human neural progenitor cells (NPCs), generated from the same iPSC, were exposed to EVs from either TB or iPSC controls. Both sets of EVs were readily internalized. EVs from TB cells upregulate several transcripts in NPCs associated with forebrain formation and neurogenesis; those from control iPSC upregulated a transcriptional phenotype that resembled glial cells more closely than neurons. These results shed light on the possible workings of the placenta–brain axis. Understanding how the contents of small RNA within TB-derived EVs affect NPCs might yield new insights, possible biomarkers, and potential treatment strategies for neurobehavioral disorders that originate in utero, such as autism spectrum disorders (ASDs).

PATH-10. UTILIZATION OF TUMOR-DERIVED EXTRACELLULAR VESICLES FOR PATIENT STRATIFICATION AND BIOMARKER DISCOVERY

Abstract Liquid biopsy is poised to play an increasingly important role in clinical decision making by enabling earlier cancer detection, diagnosis, patient stratification, and treatment monitoring. Most approaches rely on detection of cell-free circulating tumor DNA or circulating tumor cells in the peripheral blood of cancer patients. However, there are several limitations to these approaches, including miniscule abundance, unfavorable signal to noise ratios, and limited insight into mechanisms driving disease. Extracellular vesicles (EVs) are an alternative liquid biopsy analyte comprising lipid membrane encapsulated particles carrying diverse protein, nucleic acid, and metabolite cargos. Blood plasma of cancer patients contains EVs derived directly from tumor tissue that serve as rich sources of insight into tumor biology and disease pathology. We have developed a clinically applicable, multi-omic EV subpopulation interrogation pipeline that robustly profiles tumor-derived EVs in biofluids. We have demonstrated the ability of the platform to enrich cancer markers and detect activation of pathways driving oncogenesis across several different cancers, including brain cancer. There is a particularly compelling need for liquid biopsy-based solutions in neuro-oncology. We have applied our EV-omic (EVO) pipeline in combination with machine learning to distinguish adult high-grade gliomas from benign brain tumor and healthy patients, using blood-based EV proteomic and transcriptomic signatures. Marker profiles found to be most important for patient stratification combine markers known to be relevant in tumor tissue along with novel features. Furthermore, IDH status and MGMT methylation metadata derived from matched patient tissue for select cases was used to generate unique blood-based profiles for glioma subtypes. Lastly, we show preliminary longitudinal data demonstrating the utility of EVs to track tumor changes post-surgical resection and post-treatment. This work demonstrates that EV-omics can add significant value in the neuro-oncology space.

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

Influencing immunity: role of extracellular vesicles in tumor immune checkpoint dynamics.

Immune checkpoint proteins (ICPs) serve as critical regulators of the immune system, ensuring protection against damage due to overly activated immune responses. However, within the tumor environment, excessive ICP activation weakens antitumor immunity. Despite the development of numerous immune checkpoint blockade (ICB) drugs in recent years, their broad application has been inhibited by uncertainties about their clinical efficacy. A thorough understanding of ICP regulation in the tumor microenvironment is essential for advancing the development of more effective and safer ICB therapies. Extracellular vesicles (EVs), which are pivotal mediators of cell-cell communication, have been extensively studied and found to play key roles in the functionality of ICPs. Nonetheless, a comprehensive review summarizing the current knowledge about the crosstalk between EVs and ICPs in the tumor environment is lacking. In this review, we summarize the interactions between EVs and several widely studied ICPs as well as their potential clinical implications, providing a theoretical basis for further investigation of EV-related ICB therapeutic approaches.

MiR-24-3p secreted as extracellular vesicle cargo by cardiomyocytes inhibits fibrosis in human cardiac microtissues.

Cardiac fibrosis in response to injury leads to myocardial stiffness and heart failure. At the cellular level, fibrosis is triggered by the conversion of cardiac fibroblasts (CF) into extracellular matrix-producing myofibroblasts. miR-24-3p regulates this process in animal models. Here, we investigated whether miR-24-3p plays similar roles in human models. Gain- and loss-of-function experiments were performed using human induced pluripotent stem cell-derived cardiomyocytes (hCM) and primary hCF under normoxic or ischaemia-simulating conditions. hCM-derived extracellular vesicles (EVs) were added to hCF. Similar experiments were performed using three-dimensional human cardiac microtissues and ex vivo-cultured human cardiac slices.hCF transfection with miR-24-3p mimic prevented TGFβ1-mediated induction of FURIN, CCND1 and SMAD4-miR-24-3p target genes participating in TGFβ1-dependent fibrinogenesis -, regulating hCF-to-myofibroblast conversion. hCM secreted miR-24-3p as EV cargo. hCM-derived EVs modulated hCF activation. Ischaemia-simulating conditions induced miR-24-3p depletion in hCM-EVs and microtissues. Similarly, hypoxia downregulated miR-24-3p in cardiac slices. Analyses of clinical samples revealed decreased miR-24-3p levels in circulating EVs in acute myocardial infarction (AMI) patients, compared with healthy subjects. Post-mortem RNAScope analysis showed miR-24-3p downregulation in myocardium from AMI patients, compared with patients who died from noncardiac diseases. Berberin, a plant-derived agent with miR-24-3p-stimulatory activity, increased miR-24-3p contents in hCM-EVs, downregulated FURIN, CCND1 and SMAD4, and inhibited fibrosis in cardiac microtissues. These findings suggest that hCM may control hCF activation through miR-24-3p secreted as EV cargo. Ischaemia impairs this mechanism, favouring fibrosis.

TMIC-87. ICAM-1 REGULATE EXTRACELLULAR VESICLE-MEDIATED IMMUNOSUPPRESSIVE MONOCYTE IN HUMAN GLIOBLASTOMA CELL

Abstract Glioblastoma is the most common malignant primary brain tumor and is universally fatal. Treatment involves maximal safe resection with adjuvant chemoradiation. Poor prognosis in glioblastoma is multifactorial, and local and systemic immunosuppression is an important factor. Extracellular vesicles (EVs) are small, membrane-bound particles released by cells into the external environment. They play a key role in intercellular communication. In glioblastomas, EVs regulate immunosuppression through induction of myeloid-derived suppressor cells (MDSCs) in a process dependent on the PD-1/PD-L1 and IL-6 axes. Primary cilia are microtubule-based organelles that play crucial roles in various cellular processes and signal transduction cascades. We recently showed that primary cilia modulate IL-6 expression and consequently, MDSC induction. Comparative proteomic analysis of EVs derived from human glioblastoma cells devoid of cilia showed loss of ICAM-1 expression compared to control glioblastoma cells. ICAM-1 (Intercellular Adhesion Molecule-1) is a cell surface glycoprotein that mediates cell-cell adhesion and immune responses. In this study, we showed that depletion of primary cilia in human glioblastoma cells result in increased PD-L1 but loss of IL-6 and ICAM-1. RNAi-meidated loss of ICAM-1 was synergistic with primary cilia depletion in preventing MDSC inductionn normal human monocytes exposed to glioblastoma-derived EVs. Exogenous IFN-g was sufficient to restore IL-6 levels and MDSC induction despite loss of primary cilia and ICAM-1 suggesting that primary cilia act upstream of IFN-g, IL-6 and ICAM-1 in glioblastoma-mediated MDSC induction. This study is the first to connect primary cilia signal transduction to IL-6 and ICAM-1 expression and to MDSC induction. The evidence suggests a potential parallel primary cilia regulatory cascade independent of the PD-L1 axis that controls tumor-mediated immunosuppression. These results may inform novel therapeutic strategies aimed at increasing efficacy of immunotherapy in the glioblastoma and other neoplastic processes.

Extracellular Vesicle microRNAs as Possible Liquid Biopsy Markers in HNSCC—A Longitudinal, Monocentric Study

Background: Biomarkers for HNSCC are still lacking. Biomolecules obtained via liquid biopsy are being investigated for diagnosis, prognosis, and therapy monitoring, including extracellular vesicles (EVs) and EV-cargo, e.g., proteins, RNA, and microRNA. This study aims to understand localization-dependent EV-microRNA expression in blood sera, their dynamics over time (12 months FU), and insights into their potential in diagnostics and therapy monitoring. Methods: Via liquid biopsy, blood serum was taken from 50 patients with HNSCC and 16 controls. Extracellular vesicles were isolated from serum by precipitation, and the contained microRNA-21, -1246, -200c, -let-7a, -181a, and -26a were amplified by reverse transcription and determined with real-time PCR. Expression ratios (HNSCC to healthy controls) were collated with the patients’ clinical parameters. A second liquid biopsy was carried out avg. 12 months later in the tumor aftercare. A sub-analysis with the Oropharynx subsite was implemented. Results: EV-mir-21, -let-7a, and -181a were 2.5–3-fold higher expressed in HPV/p16+ than in HPV/p16- HNSCC. Different expressions of EV-mir-181a and -26a could be demonstrated depending on the therapy modality. Conclusions: EV-microRNA could be a promising biomarker in the diagnosis and therapy monitoring of HNSCC. A systematic comparison of EV- and tissue microRNA expression in different HNSCC-subsites is needed.

Formulation and characterization of nanofibrous scaffolds incorporating extracellular vesicles loaded with curcumin.

Due to their small size, flexibility, and adhesive properties, extracellular vesicles (EVs) hold promises as effective drug delivery systems. However, challenges such as the variability in vesicle types and the need to maintain their integrity for medical applications exist. Curcumin, a compound found in turmeric and known for its diverse health benefits, including anti-cancer and anti-inflammatory properties, faces obstacles in clinical use due to issues like low solubility, limited absorption, and rapid breakdown in the body. This study aimed to incorporate large-sized curcumin-loaded extracellular vesicles (lEVs) into fast-dissolving nanofibers made of poly(vinyl alcohol) (PVA) by electrospinning. By using aqueous PVA-based solutions for electrospinning, the presence of curcumin-loaded lEVs in the nanofibers was confirmed by confocal laser scanning microscopy. Furthermore, the release study demonstrated high concentrations of the drug in nanofibers containing lEVs. These findings are significant for advancing the development and utilization of active ingredient-loaded EV systems within nanofibrous formulations, potentially leading to improved patient outcomes.

MSCs-derived EVs protect against chemotherapy-induced ovarian toxicity: role of PI3K/AKT/mTOR axis.

Chemotherapy detrimentally impacts fertility via depletion of follicular reserves in the ovaries leading to ovarian failure (OF) and development of estrogen deficiency-related complications. The currently proposed options to preserve fertility such as Oocyte or ovarian cortex cryopreservation are faced with many technical obstacles that limit their effective implementation. Therefore, developing new modalities to protect ovarian function remains a pending target. Exosomes are nano-sized cell-derived extracellular vesicles (EVs) with documented efficacy in the field of regenerative medicine. The current study sought to determine the potential beneficial effects of mesenchymal stem cells (MSCs)-derived EVs in experimentally induced OF. Female albino rats were randomly allocated to four groups: control, OF group, OF + MSCs-EVs group, OF + Rapamycin (mTOR inhibitor) group, and OF + Quercetin (PI3K/AKT inhibitor) group. Follicular development was assessed via histopathological and immunohistochemical examination, and ovarian function was evaluated by hormonal assay. PI3K/Akt/mTOR signaling pathway as a key modulator of ovarian follicular activation was also assessed. MSCs-EVs administration to OF rats resulted in restored serum hormonal levels, preserved primordial follicles and oocytes, suppressed ovarian PI3K/AKT axis and downstream effectors (mTOR and FOXO3), modulated miRNA that target this axis, decreased expression of ovarian apoptotic markers (BAX, BCl2) and increased expression of proliferation marker Ki67. The present study validated the effectiveness of MSCs-EVs therapy in preventing ovarian insufficiency induced by chemotherapy. Concomitant MSCs-EVs treatment during chemotherapy could significantly preserve ovarian function and fertility by suppressing the PI3K/Akt axis, preventing follicular overactivation, maintaining normal ovarian cellular proliferation, and inhibiting granulosa cell apoptosis.

Probiotic-derived extracellular vesicles alleviate AFB1-induced intestinal injury by modulating the gut microbiota and AHR activation.

Aflatoxin B1 (AFB1) is a mycotoxin that widely found in the environment and mouldy foods. AFB1 initially targets the intestine, and AFB1-induced intestinal injury cannot be ignored. Lactobacillus amylovorus (LA), a predominant species of Lactobacillus, plays a role in carbohydrate metabolism. Extracellular vesicles (EVs), small lipid membrane vesicles, are widely involved in diverse cellular processes. However, the mechanism by which Lactobacillus amylovorus-QC1H-derived EVs (LA.EVs) protect against AFB1-induced intestinal injury remains unclear. In our study, a new strain named Lactobacillus amylovorus-QC1H (LA-QC1H) was isolated from pig faeces. Then, EVs derived from LA-QC1H were extracted via ultracentrifugation. Our results showed that LA.EVs significantly alleviated AFB1-induced intestinal injury by inhibiting the production of proinflammatory cytokines, decreasing intestinal permeability and increasing the expression of tight junction proteins. Moreover, 16S rRNA analysis revealed that LA.EVs modulated AFB1-induced gut dysbiosis in mice. However, LA.EVs did not exert beneficial effects in antibiotic-treated mice. LA.EVs treatment increased intestinal levels of indole-3-acetic acid (IAA) and activated intestinal aryl hydrocarbon receptor (AHR)/interleukin-22 (IL-22) signalling in AFB1-exposed mice. Inhibition of intestinal AHR signalling markedly weakened the protective effect of LA.EVs in AFB1-exposed mice. LA.EVs alleviated AFB1-induced intestinal injury by modulating the gut microbiota, activating the intestinal AHR/IL-22 signalling, reducing the inflammatory response and promoting intestinal barrier repair in mice.

EXTH-52. SIMULTANEOUS TARGETING OF TUMOR CELLS AND TUMOR-ASSOCIATED MACROPHAGES TO REPROGRAM GLIOBLASTOMA BY TUMOR-SUPPRESSIVE MICRORNA

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumors due in part to cell-intrinsic and cell-extrinsic mechanisms. The ability of GBM tumor to not only resist conventional therapies but also induce an immunosuppressive tumor microenvironment (TME) is thought to underlie the failure of immunotherapies. Thus, strategies to target both tumor cells and immunosuppressive immune cells, such as tumor-associated macrophages (TAMs), are of great interest. The ability of microRNA (miRNA) to target multiple genes is favorable to alter the character of the TME and cancer cells. Previously, we have shown that tumor suppressive miRNA, miR-138, can effectively regulate the genes in GBM cells relating to cell proliferation and viability. The exogenous delivery of miRNAs, however, has been limited due to the lack of delivery mechanisms that minimize off target effects. We argue that extracellular vesicles (EVs) are an ideal modality of targeted delivery for therapeutic miRNA, which we can accomplish by decorating the EV surface with cancer specific ligands, such as folate. Here, we report that trypsin digestion of the EV surface can increase the surface labeling efficiency for cancer targeting ligands. Folate-labeled trypsinized EVs effectively targeted folate receptor (FR) positive GBM cells and TAMs, delivering miR-138 in in vitro and in vivo orthotopic intracranial murine models of GBM. This simultaneous delivery decreased cancer cell proliferation and altered macrophage polarization through the inhibition of known target genes, phenotypically reprogramming both cancer cells and TAMs. To our knowledge, this is the first preclinical study attempting to target both tumor cells and innate immunity by delivering tumor suppressive miR-138 using a trypsinized EV-based RNA delivery system. These findings can be rapidly translated into a novel therapeutic option for GBM patients.