TCTP regulates genotoxic stress and tumorigenicity via intercellular vesicular signaling.

Oncogenic intercellular signaling is regulated by extracellular vesicles (EVs), but the underlying mechanisms remain mostly unclear. Since TCTP (translationally controlled tumor protein) is an EV component, we investigated whether it has a role in genotoxic stress signaling and malignant transformation. By generating a Tctp-inducible knockout mouse model (Tctp-/f-), we report that Tctp is required for genotoxic stress-induced apoptosis signaling via small EVs (sEVs). Human breast cancer cells knocked-down for TCTP show impaired spontaneous EV secretion, thereby reducing sEV-dependent malignant growth. Since Trp53-/- mice are prone to tumor formation, we derived tumor cells from Trp53-/-;Tctp-/f- double mutant mice and describe a drastic decrease in tumori-genicity with concomitant decrease in sEV secretion and content. Remarkably, Trp53-/-;Tctp-/f- mice show highly prolonged survival. Treatment of Trp53-/- mice with sertraline, which inhibits TCTP function, increases their survival. Mechanistically, TCTP binds DDX3, recruiting RNAs, including miRNAs, to sEVs. Our findings establish TCTP as an essential protagonist in the regulation of sEV-signaling in the context of apoptosis and tumorigenicity.

Identification and functional analysis of circulating small extracellular vesicle lncRNA signatures in children with fulminant myocarditis.

Fulminant myocarditis (FM) is the most serious type of myocarditis. However, the molecular mechanism underlying the pathogenesis of FM has not been fully elucidated. Small extracellular vesicles (sEVs) play important roles in many diseases, but any potential role in paediatric FM has not been reported. Here, the differential signatures of lncRNAs in plasma sEVs were studied in FM children and healthy children using transcriptome sequencing followed by functional analysis. Then immune-related lncRNAs were screened to study their role in immune mechanisms, the levels and clinical relevance of core immune-related lncRNAs were verified by qRT-PCR in a large sample size. Sixty-eight lncRNAs had increased levels of plasma sEVs in children with FM and 11 had decreased levels. Functional analysis showed that the sEVs-lncRNAs with different levels were mainly related to immunity, apoptosis and protein efflux. Seventeen core immune-related sEVs-lncRNAs were screened, functional enrichment analysis showed that these lncRNAs were closely related to immune activation, immune cell migration and cytokine pathway signal transduction. The results of the study show that sEVs-lncRNAs may play an important role in the pathogenesis of fulminant myocarditis in children, especially in the mechanism of immune regulation.

Sialyl Lewis X decorated integrin α3 on small extracellular vesicles promotes metastasis of bladder cancer via enhancing vascular permeability.

The permeability of blood vessels plays a crucial role in the spread of cancer cells, facilitating their metastasis at distant sites. Small extracellular vesicles (sEVs) are known to contribute to the metastasis of various cancers by crossing the blood vessel wall. However, the role of abnormal glycoconjugates on sEVs in tumor blood vessels remains unclear. Our study found elevated levels of fucosyltransferase VII (FUT7) and its product sialyl Lewis X (sLeX) in muscle-invasive bladder cancer (BLCA), with high levels of sLeX promoting the growth and invasion of BLCA cells. Further investigation revealed that sLeX was enriched in sEVs derived from BLCA. sLeX-decorated sEVs increased blood vessel permeability by disrupting the tight junctions of human umbilical vein endothelial cells (HUVECs). Using the glycoproteomics approach, we identified integrin α3 (ITGA3) as a sLeX-bearing glycoprotein in BLCA cells and their sEVs. Mechanically, sLeX modification stabilized ITGA3 by preventing its degradation in lysosomes. sEVs carrying sLeX-modified ITGA3 can be effectively internalized by HUVECs, leading to a decrease in the expression of tight junction protein. Conversely, silencing ITGA3 in sLeX-decorated sEVs restored tight junction proteins and reduced blood vessel permeability by inhibiting the MAPK pathway. Moreover, sLeX-modification of ITGA3 at Asn 265 in HUVECs promoted occludin dephosphorylation at Ser/Thr residues, followed by inducing its importin α1-mediated nuclear translocation, which resulted in the disruption of tight junctions. Our findings suggest a potential strategy for disrupting the formation of a metastatic microenvironment and preventing the spread of malignant bladder cancer.

Tfh cell-derived small extracellular vesicles exacerbate the severity of collagen-induced arthritis by enhancing B-cell responses.

Soluble components secreted by Tfh cells are critical for the germinal center responses. In this study, we investigated whether Tfh cells could regulate the B-cell response by releasing small extracellular vesicles (sEVs). Our results showed that Tfh cells promote B-cell differentiation and antibody production through sEVs and that CD40L plays a crucial role in Tfh-sEVs function. In addition, increased Tfh-sEVs were found in mice with collagen-induced arthritis (CIA). Adoptive transfer of Tfh cells significantly exacerbated the severity of CIA; however, the effect of Tfh cells on exacerbating the CIA process was significantly diminished after inhibiting sEVs secretion. Moreover, the levels of plasma Tfh-like-sEVs and CD40L expression on Tfh-like-sEVs in RA patients were significantly higher than those in healthy subjects. In summary, Tfh cell-derived sEVs can enhance the B-cell response, and exacerbate the procession of autoimmune arthritis.

Integrated proteomic, phosphoproteomic, and N-glycoproteomic analyses of small extracellular vesicles from C2C12 myoblasts identify specific PTM patterns in ligand-receptor interactions.

Small extracellular vesicles (sEVs) are important mediators of intercellular communication by transferring of functional components (proteins, RNAs, and lipids) to recipient cells. Some PTMs, including phosphorylation and N-glycosylation, have been reported to play important role in EV biology, such as biogenesis, protein sorting and uptake of sEVs. MS-based proteomic technology has been applied to identify proteins and PTM modifications in sEVs. Previous proteomic studies of sEVs from C2C12 myoblasts, an important skeletal muscle cell line, focused on identification of proteins, but no PTM information on sEVs proteins is available.In this study, we systematically analyzed the proteome, phosphoproteome, and N-glycoproteome of sEVs from C2C12 myoblasts with LC-MS/MS. In-depth analyses of the three proteomic datasets revealed that the three proteomes identified different catalogues of proteins, and PTMomic analysis could expand the identification of cargos in sEVs. At the proteomic level, a high percentage of membrane proteins, especially tetraspanins, was identified. The sEVs-derived phosphoproteome had a remarkably high level of tyrosine-phosphorylated sites. The tyrosine-phosphorylated proteins might be involved with EPH-Ephrin signaling pathway. At the level of N-glycoproteomics, several glycoforms, such as complex N-linked glycans and sialic acids on glycans, were enriched in sEVs. Retrieving of the ligand-receptor interaction in sEVs revealed that extracellular matrix (ECM) and cell adhesion molecule (CAM) represented the most abundant ligand-receptor pairs in sEVs. Mapping the PTM information on the ligands and receptors revealed that N-glycosylation mainly occurred on ECM and CAM proteins, while phosphorylation occurred on different categories of receptors and ligands. A comprehensive PTM map of ECM-receptor interaction and their components is also provided.In summary, we conducted a comprehensive proteomic and PTMomic analysis of sEVs of C2C12 myoblasts. Integrated proteomic, phosphoproteomic, and N-glycoproteomic analysis of sEVs might provide some insights about their specific uptake mechanism.

Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO3 axis.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEVs) are attractive candidates for ovarian function restoration and folliculogenesis for POF due to their safety and efficacy, however, the key mediator in MSCs-sEVs that modulates this response and underlying mechanisms remains elusive. Herein, we reported that YB-1 protein was markedly downregulated in vitro and in vivo models of POF induced with H2O2 and CTX respectively, accompanied by granulosa cells (GCs) senescence phenotype. Notably, BMSCs-sEVs transplantation upregulated YB-1, attenuated oxidative damage-induced cellular senescence in GCs, and significantly improved the ovarian function of POF rats, but that was reversed by YB-1 depletion. Moreover, YB-1 showed an obvious decline in serum and GCs in POF patients. Mechanistically, YB-1 as an RNA-binding protein (RBP) physically interacted with a long non-coding RNA, MALAT1, and increased its stability, further, MALAT1 acted as a competing endogenous RNA (ceRNA) to elevate FOXO3 levels by sequestering miR-211-5p to prevent its degradation, leading to repair of ovarian function. In summary, we demonstrated that BMSCs-sEVs improve ovarian function by releasing YB-1, which mediates MALAT1/miR-211-5p/FOXO3 axis regulation, providing a possible therapeutic target for patients with POF.

Youthful small extracellular vesicles restore the function and reparative capacity of inflammatory-impaired periodontal ligament stem cells via delivering protein biglycan for bone regeneration.

Regenerating inflamed bone defects represents a severe clinical challenge due to the undesirable inflammatory microenvironment. The inflammatory stimulus poses a weighty threat to the regenerative capacity of endogenously derived mesenchymal stem cells (MSCs), which are mainly responsible for osteogenic differentiation, thereby resulting in compromised endogenous bone formation. Consequently, alleviating the biological characteristics of inflammatory-impaired MSCs is crucial for promoting inflamed bone regeneration. Nano-sized small extracellular vesicles (sEVs) have emerged as promising therapeutic tools to orchestrate MSCs fate due to their intrinsic biocompatibility and encapsulated bioactive contents. In the present study, we extracted sEVs from youthful and adult dental pulp MSCs and explored their ability to recover inflammation-compromised periodontal ligament stem cells (IPDLSCs). The results indicated that both types of sEVs were capable of facilitating IPDLSCs osteogenesis. However, young sEVs exhibited a more robust potential at a lower concentration compared to adult sEVs. Mechanically, young sEVs enhanced the expression of bone morphogenetic protein 4 (BMP4) via delivering the protein Biglycan, which correspondingly promoted the osteogenic capability of IPDLSCs. Collectively, our findings emphasized that young sEVs hold enormous potential to rescue the inherent function and regenerative competence of inflammation-impaired MSCs, shedding light on their promising therapeutic prospects for infected bone regeneration.

Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

Immobilization coupling with aptamer assisted dual cycle amplification for sensitive sEVs isolation and analysis.

Precise identification of small extracellular vesicles (sEVs) is crucial for improving disease diagnosis and treatments, such as bladder cancer. However, accurate isolation and simultaneously quantification of sEVs remain a huge challenge. We have introduced a new technique that combines immobilization with aptamer-assisted dual cycle amplification to isolate and analyze sEVs with high sensitivity. In this method, the CD9 protein antibody is attached to the plate's surface for the initial identification of sEVs, while an aptamer probe is used to detect the exosomal surface protein CD63. We have created an sEVs-surface method that combines target recognition initiated signal recycling and rolling circle amplification (RCA) for signal amplification. This approach allows for the "AND" logic analysis of dual biomarkers, enabling both sEVs quantification and tracing. The proposed approach has a broad detection range and a low limit of detection. Moreover, the established method showed good stability in detecting sEVs with a low coefficient of variation. Our method can effectively isolate certain sEVs and accurately identify them, making it suitable for many uses in biological science, biomedical engineering, and personalized medicine.

Hepatic steatosis aggravates atherosclerosis via small extracellular vesicle-mediated inhibition of cellular cholesterol efflux.

BACKGROUND & AIMS: While it is recognized that non-alcoholic fatty liver disease (NAFLD) is associated with cardiovascular disease (CVD), how NAFLD affects the development and progression of CVD remains unclear and debatable. Hence, we aimed to determine the role of steatotic hepatocyte-derived small extracellular vesicles (sEVs) in foam cell formation and atherosclerosis progression. METHODS: sEVs from steatotic hepatocytes were isolated and characterized. MicroRNA (miRNA) deep sequencing was utilized to identify functional miRNA in sEVs. Lastly, we conducted a cross-sectional study on patients with NAFLD to validate these findings. RESULTS: Treatment of sEVs from steatotic hepatocytes promoted macrophage-derived foam cell formation and atherosclerosis progression via inhibition of ABCA1-mediated cholesterol efflux. Macrophage-specific deletion of Abca1 in ApoE-/- mice abolished the role of steatotic hepatocyte-derived sEVs in atherosclerosis progression. In addition, hepatocyte-specific deletion of Rab27a, which is the key GTPase regulating sEV release, significantly ameliorated high-fat, high-cholesterol diet-induced atherosclerosis progression in ApoE-/- mice. The miRNA deep sequencing results showed that miR-30a-3p was enriched in sEVs from steatotic hepatocytes. miR-30a-3p directly targeted the 3' untranslated region of ABCA1 to inhibit ABCA1 expression and cholesterol efflux. Treatment with antagomiR-30a-3p significantly attenuated atherosclerosis progression in high-fat, high-cholesterol diet-fed ApoE-/- mice. Moreover, serum sEVs from patients with NAFLD and sEV-miR-30a-3p expression were associated with decreased cholesterol efflux levels in foam cells. CONCLUSION: Steatotic hepatocyte-derived sEVs promote foam cell formation and facilitate atherogenesis via the miR-30a-3p/ABCA1 axis. Reducing sEV secretion by steatotic hepatocytes or targeting miR-30a-3p may be potential therapeutic approaches to slow the progression of NAFLD-driven atherosclerosis. IMPACT AND IMPLICATIONS: The presence of hepatic steatosis is strongly correlated with the risk of cardiovascular disease and cardiovascular events, yet the molecular mechanisms linking steatosis to progression of atherosclerosis are unclear. Herein, we identified small extracellular vesicles from steatotic hepatocytes as a trigger that accelerated the progression of atherosclerosis. Steatotic hepatocyte-derived small extracellular vesicles promoted foam cell formation via the miR-30a-3p/ABCA1 axis. Our findings not only provide mechanistic insight into non-alcoholic fatty liver disease-driven atherosclerosis but also provide potential therapeutic targets for patients with atherosclerosis.

LILRB2-containing small extracellular vesicles from glioblastoma promote tumor progression by promoting the formation and expansion of myeloid-derived suppressor cells.

BACKGROUND: Leukocyte immunoglobulin-like receptor subfamily B2 (LILRB2) was reported to be an inhibitory molecule with suppressive functions. sEVs mediate communication between cancer cells and other cells. However, the existence of LILRB2 on sEVs in circulation and the function of sEVs-LILRB2 are still unknown. This study aims to investigate the role of LILRB2 in GBM and determine how LILRB2 in sEVs regulates tumor immunity. METHODS: LILRB2 expression in normal brain and GBM tissues was detected by immunohistochemistry, and the effect of LILRB2 on prognosis was evaluated in an orthotopic brain tumor model. Next, a subcutaneous tumor model was constructed to evaluate the function of pirb in vivo. The immune cells in the tumor sites and spleen were detected by immunofluorescence staining and flow cytometry. Then, the presence of pirb in sEVs was confirmed by WB. The percentage of immune cells after incubation with sEVs from GL261 (GL261-sEVs) or sEVs from GL261-pirb+ (GL261-sEVs-pirb) was detected by flow cytometry. Then, the effect of pirb on sEVs was evaluated by a tumor-killing assay and proliferation assay. Finally, subcutaneous tumor models were constructed to evaluate the function of pirb on sEVs. RESULTS: LILRB2 was overexpressed in human GBM tissue and was closely related to an immunosuppressive TME in GBM. Then, a protumor ability of LILRB2 was observed in subcutaneous tumor models, which was related to lower CD8 + T cells and higher MDSCs (myeloid-derived suppressor cells) in the tumor and spleen compared to those of the control group. Next, we found that pirb on sEVs (sEVs-pirb) inhibits the function of CD8 + T cells by promoting the formation and expansion of MDSCs. Furthermore, the protumor function of sEVs-pirb was demonstrated in subcutaneous tumor models. CONCLUSION: We discovered that LILRB2/pirb can be transmitted between GBM cells via sEVs and that pirb on sEVs induces the formation and expansion of MDSCs. The induced MDSCs facilitate the formation of an immunosuppressive TME.

Enterovirus-71 utilizes small extracellular vesicles to cross the blood-brain barrier for infecting the central nervous system via transcytosis.

BACKGROUND: Central nervous system (CNS) infections caused by Enterovirus 71 (EV71) pose a serious threat to children, causing severe neurogenic complications and even fatality in some patients. However, the pathogenesis of EV71 infections in the CNS remains unclear. METHODS: An in vitro blood-brain barrier (BBB) model was constructed by coculturing brain microvascular endothelial cells (BMECs) and astrocytes in transwell inserts for simulating CNS infections. EV71 virions and small extracellular vesicles (sEVs) derived from EV71-infected cells (EV71-sEVs) were isolated from the cell culture supernatant by density gradient centrifugation. The BBB model was separately infected with EV71 virions and EV71-sEVs. The mechanism of crossing the BBB was determined by inhibiting the different endocytic modes. A murine model of EV71 infection was constructed for confirming the results of in vitro experiments. RESULTS: The EV71-sEVs containing viral components were endocytosed by BMECs and released on the abluminal side of the BBB model, where they infected the astrocytes without disrupting the BBB in the early stages of infection. The integrity of the tight junctions (TJs) between BMECs was breached via downregulation of PI3K/Akt signaling in the late stages of infection. CONCLUSIONS: EV71 utilized the circulating sEVs for infecting the CNS by crossing the BBB.

Small extracellular vesicle-mediated metabolic reprogramming: from tumors to pre-metastatic niche formation.

Metastasis, the spread of a tumor or cancer from the primary site of the body to a secondary site, is a multi-step process in cancer progression, accounting for various obstacles in cancer treatment and most cancer-related deaths. Metabolic reprogramming refers to adaptive metabolic changes that occur in cancer cells in the tumor microenvironment (TME) to enhance their survival ability and metastatic potential. Stromal cell metabolism also changes to stimulate tumor proliferation and metastasis. Metabolic adaptations of tumor and non-tumor cells exist not only in the TME but also in the pre-metastatic niche (PMN), a remote TME conducive for tumor metastasis. As a novel mediator in cell-to-cell communication, small extracellular vesicles (sEVs), which have a diameter of 30-150 nm, reprogram metabolism in stromal and cancer cells within the TME by transferring bioactive substances including proteins, mRNAs and miRNAs (microRNAs). sEVs can be delivered from the primary TME to PMN, affecting PMN formation in stroma rewriting, angiogenesis, immunological suppression and matrix cell metabolism by mediating metabolic reprogramming. Herein, we review the functions of sEVs in cancer cells and the TME, how sEVs facilitate PMN establishment to trigger metastasis via metabolic reprogramming, and the prospective applications of sEVs in tumor diagnosis and treatment. Video Abstract.

Small extracellular vesicles derived from human adipose-derived stem cells regulate energetic metabolism through the activation of YAP/TAZ pathway facilitating angiogenesis.

Recent studies have found small extracellular vesicles (sEVs) that are secreted from human adipose tissue-derived stem cells (hADSCs-sEVs) and contribute to angiogenesis. Glycolysis, the primary energetic pathway of vascular endothelial cells, plays a key role in the process of angiogenesis. However, hADSCs-sEVs' effects on energy metabolism within endothelial cells remain unclear. In our study, we found that hADSCs-sEVs restored glycolytic metabolism suppressed by 3-(pyridinyl)-1-(4-pyridinyl)-2-propen-1-one(3PO), a unique glycolytic inhibitor increasing the extracellular acidification rate (ECAR), oxygen consumption rate (OCR), glycolytic gene expression as well as pyruvate, lactate, and ATP production in HUVEC cells. In contrast, hADSCs-sEVs decreased PDH-E1α expression and acetyl-CoA production. The above results indicate that hADSCs-sEVs promote HUVEC angiogenesis via enhancing glycolysis and suppressing mitochondrial oxidative phosphorylation. Furthermore, we found that the YAP/TAZ pathway may play a key role in the effects hADSCs-sEVs have on HUVECs, thus, providing a promising approach for pro-angiogenesis-related regeneration.

sEVsRVG selectively delivers antiviral siRNA to fetus brain, inhibits ZIKV infection and mitigates ZIKV-induced microcephaly in mouse model.

Zika virus (ZIKV), a flavivirus associated with neurological disorders, constitutes a global health threat. During pregnancy, ZIKV traverses the placenta and causes congenital disease such as microcephaly and Guillain-Barré syndrome in newborns. To develop a specific antiviral therapy against ZIKV-induced microcephaly that could cross placental and blood-brain barriers, we designed targeted small extracellular vesicles (sEVs) encapsulating antiviral siRNA (small interfering RNA) to inhibit ZIKV. The neuro-specific targeting was achieved by engineering EVs membrane protein lamp2b fused with a neuron-specific rabies virus glycoprotein derived peptide (RVG). Intravenous administration of the RVG-engineered sEVs loaded with siRNA (ZIKV-specific siRNA) protected pregnant AG6 mice against vertical transmission of ZIKV. Particularly, sEVsRVG-siRNA traversed placental and blood-brain barriers and suppressed ZIKV infection in fetal brains. Moreover, sEVsRVG-siRNA alleviated the neuroinflammation and neurological damage caused by ZIKV in the fetal mouse model. In general, we developed a sEVs-based targeted system of antiviral therapy for brain and fetal brain infections.

RIP1 Mediates Manzamine-A-Induced Secretory Autophagy in Breast Cancer.

Cancer-derived small extracellular vesicles (sEVs) serve as critical mediators of cell-to-cell communication. Manzamine A (MA), a unique marine-derived alkaloid with various bioactivities, exerts anticancer effects against several kinds of tumors, but it remains unclear whether it has the same activity against breast cancer. Here, we proved that MA inhibits MDA-MB-231 and MCF-7 cell proliferation, migration, and invasion in a time- and dose-dependent manner. In addition, MA promotes autophagosome formation but suppresses autophagosome degradation in breast cancer cells. Importantly, we also found that MA stimulates sEVs secretion and increases autophagy-related protein accumulation in secreted sEVs, further potentiated by autophagy inhibitor chloroquine (CQ). Mechanistically, MA decreases the expression level of RIP1, the key upstream regulator of the autophagic pathway, and reduces the acidity of lysosome. Overexpression of RIP1 activated AKT/mTOR signaling, thus attenuating MA-induced autophagy and the corresponding secretion of autophagy-associated sEVs. Collectively, these data suggested that MA is a potential inhibitor of autophagy by preventing autophagosome turnover, and RIP1 mediates MA-induced secretory autophagy, which may be efficacious for breast cancer treatment.

miR-124 as a Liquid Biopsy Prognostic Biomarker in Small Extracellular Vesicles from NSCLC Patients.

Despite advances in non-small cell lung cancer (NSCLC) research, this is still the most common cancer type that has been diagnosed up to date. microRNAs have emerged as useful clinical biomarkers in both tissue and liquid biopsy. However, there are no reliable predictive biomarkers for clinical use. We evaluated the preclinical use of seven candidate miRNAs previously identified by our group. We collected a total of 120 prospective samples from 88 NSCLC patients. miRNA levels were analyzed via qRT-PCR from tissue and blood samples. miR-124 gene target prediction was performed using RNA sequencing data from our group and interrogating data from 2952 NSCLC patients from two public databases. We found higher levels of all seven miRNAs in tissue compared to plasma samples, except for miR-124. Our findings indicate that levels of miR-124, both free-circulating and within exosomes, are increased throughout the progression of the disease, suggesting its potential as a marker of disease progression in both advanced and early stages. Our bioinformatics approach identified KPNA4 and SPOCK1 as potential miR-124 targets in NSCLC. miR-124 levels can be used to identify early-stage NSCLC patients at higher risk of relapse.

CA-IX-Expressing Small Extracellular Vesicles (sEVs) Are Released by Melanoma Cells under Hypoxia and in the Blood of Advanced Melanoma Patients.

Cutaneous melanoma is a highly aggressive skin cancer, with poor prognosis. The tumor microenvironment is characterized by areas of hypoxia. Carbonic anhydrase IX (CA-IX) is a marker of tumor hypoxia and its expression is regulated by hypoxia-inducible factor-1 (HIF-1). CA-IX has been found to be highly expressed in invasive melanomas. In this study, we investigated the effects of hypoxia on the release of small extracellular vesicles (sEVs) in two melanoma in vitro models. We demonstrated that melanoma cells release sEVs under both normoxic and hypoxic conditions, but only hypoxia-induced sEVs express CA-IX mRNA and protein. Moreover, we optimized an ELISA assay to provide evidence for CA-IX protein expression on the membranes of the sEVs. These CA-IX-positive sEVs may be exploited as potential biomarkers for liquid biopsy.

MSC-sEV Treatment Polarizes Pro-Fibrotic M2 Macrophages without Exacerbating Liver Fibrosis in NASH.

Mesenchymal stem/stromal cell small extracellular vesicles (MSC-sEVs) have shown promise in treating a wide range of animal models of various human diseases, which has led to their consideration for clinical translation. However, the possibility of contraindication for MSC-sEV use is an important consideration. One concern is that MSC-sEVs have been shown to induce M2 macrophage polarization, which is known to be pro-fibrotic, potentially indicating contraindication in fibrotic diseases such as liver fibrosis. Despite this concern, previous studies have shown that MSC-sEVs alleviate high-fat diet (HFD)-induced non-alcoholic steatohepatitis (NASH). To assess whether the pro-fibrotic M2 macrophage polarization induced by MSC-sEVs could worsen liver fibrosis, we first verified that our MSC-sEV preparations could promote M2 polarization in vitro prior to their administration in a mouse model of NASH. Our results showed that treatment with MSC-sEVs reduced or had comparable NAFLD Activity Scores and liver fibrosis compared to vehicle- and Telmisartan-treated animals, respectively. Although CD163+ M2 macrophages were increased in the liver, and serum IL-6 levels were reduced in MSC-sEV treated animals, our data suggests that MSC-sEV treatment was efficacious in reducing liver fibrosis in a mouse model of NASH despite an increase in pro-fibrotic M2 macrophage polarization.

αvß3-targeted sEVs for efficient intracellular delivery of proteins using MFG-E8.

BACKGROUND: Small extracellular vesicles (sEVs) are nanometer-sized membranous particles shed by many types of cells and can transfer a multitude of cargos between cells. Recent studies of sEVs have been focusing on their potential to be novel drug carriers due to natural composition and other promising characteristics. However, there are challenges in sEVs-based drug delivery, one of which is the inefficient loading of drugs into sEVs, especially for large biomolecules. RESULTS: In this study, we proposed a membrane-associated protein, milk fat globule-epidermal growth factor 8 protein (MFG-E8), to produce αvß3-targeted sEVs with high delivery efficiency of interested protein. MFG-E8 is a secreted protein with NH2-terminal epidermal growth factor (EGF)-like domains, containing an Arg-Gly-Asp(RGD) sequence that binds αvß3 and αvß5 integrins, and COOH terminal domains C1 and C2, which can bind to lipid membrane with strong affinity. Firstly, we transiently expressed MFG-E8 in HEK293F cells and found that this protein could be secreted and adhere to the cell membrane. The recombinant MFG-E8 is also found to locate at the outer membrane of sEVs. Then we generated engineered sEVs by expressing high levels of the EGFP fused to MFG-E8 in HEK293F cells and showed that MFG-E8 could increase the delivery efficiency of EGFP into sEVs. Further delivery of Gaussia luciferase (GL) by fusion expression with MFG-E8 in donor cells demonstrated that target proteins fused with MFG-E8 still kept their activity. Finally, we identified the sEVs' target to integrin αvß3 by comparing the transfection efficiency with MFG-E8 loaded sEVs (MFG-E8-sEVs) in αvß3 positive cells and αvß3 negative cells. Analysis showed higher target protein could transfect into αvß3 positive cells with MFG-E8-sEVs than with EGFP loaded sEVs (EGFP-sEVs), meaning the engineered sEVs with MFG-E8 not only could increase the delivery of target protein into sEVs, but also could target the αvß3 positive cells. CONCLUSION: This study suggests that recombinant MFG-E8 is an ideal protein to increasingly deliver the drug into sEVs and give sEVs the ability to target the αvß3 positive cells.