Hypoxia is a pathologic condition characterized by a tissue oxygen deficiency due to either decreased oxygen intake from outside and/or disruption of oxygen utilization in cells. This condition may arise when the oxygen demand exceeds its supply or the partial pressure of oxygen is below 10 mmHg. This situation poses a significant problem for glioblastoma (GBM) patients as it can activate angiogenesis, increase invasiveness and metastatic risk, prolong tumor survival, and suppress anti-tumor immunity, making hypoxic cells resistant to radiotherapy and chemotherapy. Low oxygen levels in tumors can cause severe cellular changes that can affect the release of extracellular vesicles (EVs), especially exosomes (EXOs), altering their proteomic profile both qualitatively and quantitatively. EXOs represent an adaptive response to hypoxic stress; therefore, they can be used to determine oxygen levels in cancer and assess its aggressiveness. They not only release signaling molecules to attract cells that promote the formation of small vessel walls but also send signals to other tumor cells that trigger their migration, which in turn plays a crucial role in the formation of metastases under hypoxia. This review investigates how the molecular profile of GBM-derived exosomes changes under hypoxic conditions, offering future possibilities for noninvasive diagnosis and monitoring of brain tumor patients.
Aim: This study aims to elucidate the involvement of triple-negative breast cancer (TNBC)-derived extracellular vesicles in metastasis. The loss of components in the type 1 interferon (IFN1) signaling pathway has been linked to the promotion of metastasis. However, IFN1 signaling induces immunological dormancy and promotes tumorigenesis. Our hypothesis was that TNBC cells release tumor-derived extracellular vesicles (TEVs) that promote metastasis in an IFN1-independent manner.
Methods: Two murine TNBC models and transgenic mice were used to examine the role of IFN1 in TNBC progression to metastasis. Reserpine was employed to determine the effect of TEV education on TNBC progression and overall survival. EVs from cancer cells treated with vehicle and reserpine and from the serum of tumor-bearing mice receiving reserpine were examined to determine changes in EV release and EV content.
Results: TNBC cells progress to metastasis in mice lacking the IFN1-induced gene cholesterol-25 hydroxylase (CH25H) or expressing the IFNAR1S526 knock-in that cannot be downregulated. Reserpine suppresses EV release from TNBC cells in vitro and in vivo. Western blot analysis demonstrated reserpine decreased NUPR1 protein levels in EVs. RNAseq analysis demonstrated that endothelial cells lacking CH25H treated with TEVs exhibited increased NUPR1 expression that was decreased by adding reserpine with the TEVs. NUPR1 overexpression upregulated genes that mediate TEV biogenesis and incorporation. Knockdown of NUPR1 with shRNA decreased the release of TEVs.
Conclusion: In conclusion, our study suggests that TNBC is driven by aberrant packaging of NUPR1 into TEVs which were transferred into recipient cells to activate pro-metastatic transcription driven by NUPR1.
Cirrhotic patients can present hepatic encephalopathy (HE), showing motor and cognitive deficits. Hyperammonemia and peripheral inflammation are known to induce neuroinflammation and alter neurotransmission, which finally induces neurological impairment in HE. However, the mechanisms by which the deleterious effects of peripheral inflammation are transmitted to the brain are not well understood. Extracellular vesicles (EVs) have recently emerged as a new mediator between the periphery and the brain, particularly in pathologies associated with sustained inflammation and in neurological disorders. In this work, we summarized the main findings on the role of plasma EVs in hyperammonemia and HE and discussed its potential implication in the pathogenesis of hepatic encephalopathy.
Cell membrane-derived vesicles (CMVs) are particles generated from living cells, including extracellular vesicles (EVs) and artificial extracellular vesicles (aEVs) prepared from cell membranes. CMVs possess considerable potential in drug delivery, regenerative medicine, immunomodulation, disease diagnosis, etc. owing to their stable lipid bilayer structure, favorable biocompatibility, and low toxicity. Although the majority of CMVs inherit certain attributes from the original cells, it is still difficult to execute distinct therapeutic functions, such as organ targeting, signal regulation, and exogenous biotherapeutic supplementation. Hence, engineering CMVs by genetic engineering, chemical modification, and hybridization is a promising way to endow CMVs with specific functions and open up novel vistas for applications. In particular, there is a growing interest in genetically engineered CMVs harnessed to exhibit biotherapeutics. Herein, we outline the preparation strategies and their characteristics for purifying CMVs. Additionally, we review the advances of genetically engineered CMVs utilized to target organs, regulate signal transduction, and deliver biomacromolecules and chemical drugs. Furthermore, we also summarize the emerging therapeutic applications of genetically engineered CMVs in addressing tumors, diabetes, systemic lupus erythematosus, and cardiovascular diseases.
Stem cell therapy is a novel approach for treating various severe and intractable diseases, including autoimmune disorders, organ transplants, tumors, and neurodegenerative diseases. Nevertheless, the extensive utilization of stem cells is constrained by potential tumorigenicity, challenges in precise differentiation, rejection concerns, and ethical considerations. Extracellular vesicles possess the ability to carry diverse bioactive factors from stem cells and deliver them to specific target cells or tissues. Moreover, they offer the advantage of low immunogenicity. Consequently, they have the potential to facilitate the therapeutic potential of stem cells, mitigating the risks associated with direct stem cell application. Therefore, the use of stem cell extracellular vesicles in clinical diseases has received increasing attention. This review summarizes advances in the use of extracellular vesicles from mesenchymal stem cells (MSC). MSC extracellular vesicles are used in the treatment of inflammatory diseases such as rheumatoid arthritis, liver injury, COVID-19, and allergies; in the repair of tissue damage in heart disease, kidney injury, and osteoarthritic diseases; as a carrier in the treatment of tumors; and as a regenerative agent in neurodegenerative disorders such as Alzheimer's and Parkinson's.
Background: Vascular cell adhesion molecule-1 (VCAM-1+) endothelial cell-derived extracellular vesicles (EC-EVs) are augmented in cardiovascular disease, where they can signal the deployment of immune cells from the splenic reserve. Endothelial cells in culture activated with pro-inflammatory tumor necrosis factor-α (TNF-a) also release VCAM-1+ EC-EVs. However, isolating VCAM-1+ EC-EVs from conditioned cell culture media for subsequent in-depth analysis remains challenging.
Aim: We utilized the extracellular vesicles (EV) microfluidics herringbone chip (EVHB-Chip), coated with anti-VCAM-1 antibodies, for selective capture of VCAM-1+ cells and EC-EVs.
Methods and Results: Engineered EA.hy926 endothelial cells overexpressing VCAM-1 (P < 0.001 versus control) showed increased binding to the VCAM-1- EVHB-Chip versus an IgG device. TNF-α-stimulated human umbilical cord vein endothelial cells (HUVECs) exhibited elevated VCAM-1 protein levels (P < 0.001) and preferential binding to the VCAM-1- EVHB-Chip versus the IgG device. HUVECs stimulated with TNF-α showed differential gene expression of intercellular adhesion molecule-1 (ICAM-1) (P < 0.001) and VCAM-1 (P < 0.001) by digital droplet PCR versus control cells. HUVEC-derived EC-EVs were positive for CD9, CD63, HSP70, and ALIX and had a modal size of 83.5 nm. Control and TNF-α-stimulated HUVEC-derived EC-EV cultures were captured on the VCAM-1- EVHB-Chip, demonstrating selective capture. VCAM-1+ EC-EV were significantly enriched for ICAM-1 (P < 0.001) mRNA transcripts.
Conclusion: This study presents a novel approach using the EVHB-Chip, coated with anti-VCAM-1 antibodies and digital droplet PCR for the study of VCAM-1+ EC-EVs. Isolation of VCAM-1+ EC-EV from heterogeneous sources such as conditioned cell culture media holds promise for subsequent detailed characterization, and may facilitate the study of VCAM-1+ EC-EVs in cardiovascular and metabolic diseases, for disease monitoring and therapeutic insights.
This commentary provides an in-depth analysis and perspective on the pioneering research article titled ‘Extracellular Vesicle-Encapsulated Adeno-Associated Viruses for Therapeutic Gene Delivery to the Heart’. The original study explores the innovative use of extracellular vesicle-encapsulated AAVs (EV-AAV-6 and -9) as a superior gene-delivery approach for cardiomyocytes (CMs), which not only provides increased AAV neutralizing antibody (NAb) resistance but also has implications for increased gene delivery efficacy to ischemic hearts. This study examined the efficacy of EVs isolated from the conditioned medium of AAV-6 and -9 producing HEK293T cells in combinatorial in vitro and in vivo model systems in comparison to free AAVs in the presence of the NAbs. This commentary highlights the key findings, discusses potential implications, limitations, and suggests future directions for research in this evolving field.
Aims: Analysis of miRNA (18-23nt) encapsulated in small extracellular vesicles (sEVs) (diameter ~30-200 nm) is critical in understanding the diagnostic and therapeutic value of sEV miRNA. However, various sEV enrichment techniques yield different quantities and qualities of sEV miRNA. Here, we compare the efficacy of three sEV isolation techniques in four combinations for miRNA next-generation sequencing.
Methods: Blood plasma from four Holstein-Friesian dairy cows (Bos taurus) (n = 4) with similar genetic traits and physical characteristics were pooled to isolate sEV. Ultracentrifugation (UC) (100,000 × g, 2 h at 4 °C), size-exclusion chromatography (SEC) and ultrafiltration (UF) were used to design four groups of sEV isolations (UC+SEC, SEC+UC, SEC+UF and UC+SEC+UF). sEV miRNAs were isolated using a combination of TRIzol, Chloroform and miRNeasy mini kit (n = 4/each), later sequenced utilizing Novaseq S1 platform (single-end 100 bp sequencing).
Results: All four sEV methods yielded > 1,700 miRNAs and sEV miRNAs demonstrated a clear separation from control blood plasma circulating miRNA (PCA analysis). MiR-381-3p, miR-23-3p, and miR-18b-3p are among the 25 miRNAs unique to sEV, indicating potential sEV-specific miRNA markers. Further, those 25 miRNAs mostly regulate immune-related functions, indicating the value of sEV miRNA cargo in immunology.
Conclusion: The four sEV miRNA isolation methods employed in this study are valid techniques. The choice of method depends on the research question and study design. If purity is of concern, the UC+SEC method resulted in the best particles/µg protein ratio, which is often used as an indication of sample purity. These results could eventually establish sEV miRNAs as effective diagnostic and therapeutic tools of immunology.
Aim: The lung is the second most frequent site of metastatic dissemination. Early detection is key to improving survival. Given that the lung interfaces with the external environment, the collection of exhaled breath condensate (EBC) provides the opportunity to obtain biological material including exhaled miRNAs that originate from the lung.
Methods: In this proof-of-principal study, we used the highly metastatic MDA-MB-231 subline 3475 breast cancer cell line (LM-3475) to establish an orthotopic lung tumor-bearing mouse model and investigate non-invasive detection of lung tumors by analysis of exhaled miRNAs. We initially conducted miRNA NGS and qPCR validation analyses on condensates collected from unrestrained animals and identified significant miRNA expression differences between the condensates of lung tumor-bearing and control mice. To focus our purification of EBC and evaluate the origin of these differentially expressed miRNAs, we developed a system to collect EBC directly from the nose and mouth of our mice.
Results: Using nanoparticle distribution analyses, TEM, and ONi super-resolution nanoimaging, we determined that human tumor EVs could be increasingly detected in mouse EBC during the progression of secondary lung tumors. Using our customizable EV-CATCHER assay, we purified human tumor EVs from mouse EBC and demonstrated that the bulk of differentially expressed exhaled miRNAs originate from lung tumors, which could be detected by qPCR within 1 to 2 weeks after tail vein injection of the metastatic cells.
Conclusion: This study is the first of its kind and demonstrates that lung tumor EVs are exhaled in mice and provide non-invasive biomarkers for detection of lung tumors.