Manufacturing Therapeutic Exosomes: from Bench to Industry

There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag–liquid chromatography–tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers.

Sonication is a suitable method for loading nanobody into glioblastoma small extracellular vesicles

Identifying biomarkers is essential for early diagnosis of neurodegenerative diseases (NDs). Large (LEVs) and small extracellular vesicles (SEVs) are extracellular vesicles (EVs) of different sizes and biological functions transported in blood and they may be valid biomarkers for NDs. The aim of our study was to investigate common and different miRNA signatures in plasma derived LEVs and SEVs of Alzheimer’s disease (AD), Parkinson’s disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Fronto-Temporal Dementia (FTD) patients. LEVs and SEVs were isolated from plasma of patients and healthy volunteers (CTR) by filtration and differential centrifugation and RNA was extracted. Small RNAs libraries were carried out by Next Generation Sequencing (NGS). MiRNAs discriminate all NDs diseases from CTRs and they can provide a signature for each NDs. Common enriched pathways for SEVs were instead linked to ubiquitin mediated proteolysis and Toll-like receptor signaling pathways and for LEVs to neurotrophin signaling and Glycosphingolipid biosynthesis pathway. LEVs and SEVs are involved in different pathways and this might give a specificity to their role in the spreading of the disease. The study of common and different miRNAs transported by LEVs and SEVs can be of great interest for biomarker discovery and for pathogenesis studies in neurodegeneration.

Plasma small‐extracellular vesicles enriched in miR‐122‐5p promote disease aggressiveness in pediatric anaplastic large‐cell lymphoma

Extracellular vesicles (EVs) are important mediators of cell-cell communication due to their cargo content of proteins, lipids, and RNAs. We previously reported that small EVs (SEVs) called exosomes promote directed and random cell motility, invasion, and serum-independent growth. In contrast, larger EVs (LEVs) were not active in those assays, but might have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Bioinformatics analyses revealed that SEVs are enriched in proteins associated with cell-cell junctions, cell-matrix adhesion, exosome biogenesis machinery, and various signaling pathways. In contrast, LEVs are enriched in proteins associated with ribosome and RNA biogenesis, processing, and metabolism. Western blot analysis of EVs purified from two different cancer cell types confirmed the enrichment of cell-matrix and cell-cell adhesion proteins in SEVs. Consistent with those data, we found that cells exhibit enhanced adhesion to surfaces coated with SEVs compared to an equal protein concentration of LEVs. These data suggest that a major function of SEVs is to promote cellular adhesion.

The market offers a wide range of extracellular vesicles (EVs) isolation products, but their lack of standardization is a concern. Therefore, it is important to carefully assess the quality of the EVs obtained using these products to patent the ideal method. In this study, we compared the EXOCIB kit with the ultracentrifuge method, which is considered the gold standard for small EV isolation. After overnight fasting, small plasma EVs were extracted from four individuals using both the ultracentrifuge and the EXOCIB kit methods. The pooled EVs were then compared for the presence of the cluster of differentiation 63 (CD63) protein using the western blot analysis, and their size and zeta potential were performed by Dynamic Light Scattering (DLS). In addition, the size and morphology of small EVs were determined by using the Transmission Electron Microscopy (TEM) technique. An average hydrodynamic size of 135.7 nm and a zeta potential of -6.33 Mv at 25°C was found for small EVs isolated by the ultracentrifuge, whereas the kit method resulted in small EVs with a hydrodynamic size of 102.8 nm and a zeta potential of -0.907. Notably, the size of the particles in the kit samples was smaller compared to those obtained through the ultracentrifuge (P < 0.001). The western blot method confirmed the expression of CD63 in both methods, so the ultracentrifuge yielded small EVs with a higher level of purity compared to the kit-based approach (P = 0.036). The DLS findings revealed the existence of vesicles within the appropriate size range for small EVs like exosomes in both isolation techniques. The results of the western blot analysis, in conjunction with DLS, displayed that the ultracentrifuge method extracted small EVs with a greater degree of purity than the kit-based approach.

Changes in the cellular microRNA (miRNA) expression profile in response to HIV infection, replication or latency have been reported. Nevertheless, little is known concerning the abundance of miRNA in extracellular vesicles (EVs). In the search for a reliable predictor of viral rebound, we quantified the amount of miR-29a, miR-146a, and miR-155 in two types of plasma extracellular vesicles. Venous blood was collected from 235 ART-treated and ART-naive persons living with HIV (85 with ongoing viral replication, ≥20 copies/mL) and 60 HIV-negative participants at five HIV testing or treatment centers in Burkina Faso. Large and small plasma EVs were purified and counted, and mature miRNA miR-29a, miR-146a, and miR-155 were measured by RT-qPCR. Diagnostic performance of miRNA levels in large and small EVs was evaluated by a receiver operating characteristic curve analysis. The median duration of HIV infection was 36 months (IQR 14–117). The median duration of ART was 34 months (IQR 13–85). The virus was undetectable in 63.8% of these persons. In the others, viral load ranged from 108 to 33,978 copies/mL (median = 30,032). Large EVs were more abundant in viremic participants than aviremic. All three miRNAs were significantly more abundant in small EVs in persons with detectable HIV RNA, and their expression levels in copies per vesicle were a more reliable indicator of viral replication in ART-treated patients with low viremia (20–1000 copies/mL). HIV replication increased the production of large EVs more than small EVs. Combined with viral load measurement, quantifying EV-associated miRNA abundance relative to the number of vesicles provides a more reliable marker of the viral status. The expression level as copies per small vesicle could predict the viral rebound in ART-treated patients with undetectable viral loads.

MYC genes are amplified/overexpressed in 20% of SCLCs, showing that Myc and Myc-dependent cellular mechanisms are strong candidates as therapeutic targets in SCLC. Small extracellular vesicles support the carcinogenesis process by acting as messengers delivering nucleic acids and proteins-moreover, no reports associate Myc and the functional effect of small extracellular vesicles in small cell lung cancer. After the effects of small extracellular vesicles (sEVs) obtained from H82 and H209 cells on HUVEC and MRC-5 cells were observed, the Myc-dependent effect of the sEVs on oncogenic potentials was further evaluated by manipulating Myc expression via lentiviral vectors in H82 and H209 cells. Then, small extracellular vesicles of Myc-manipulated SCLC cells were isolated using sEVs isolation reagents. Finally, HUVEC and MRC5 cells were treated with SCLC-derived small extracellular vesicles. Cellular activity of recipient normal lung cells was investigated by cell growth assay, wound healing assay, and transwell assay. miRNA composition changes in small extracellular vesicles and SCLC cells were investigated using miRNA microarray and QRT-PCR assay. Our results indicated that normal lung cells treated with SCLC-derived small extracellular vesicles had higher proliferation, migration capability than non-treated counterparts. Additionally, after investigating the potential effects of small extracellular vesicles derived from Myc-dysregulated SCLC cell lines, we further evaluated the Myc-dependent miRNA composition in the small extracellular vesicles. The present study revealed that Myc regulates hsa-miR-7, hsa-miR-9, hsa-miR-125b, hsa-miR-181a_2, hsa-miR-455, hsa-miR-642, and hsa-miR-4417 expressions in SCLC cell lines, not only in cellular but also in exosomal content. Small extracellular vesicles and MYC are essential targets for therapeutic strategy in SCLC. Our study revealed that the expression level of MYC can affect the function of sEVs and encapsulate the miRNA composition in SCLC. Besides, small extracellular vesicles derived from SCLC cells can modulate normal lung cells.

ABSTRACTAntiretroviral therapy (ART) suppresses viral replication in most people living with HIV‐1 (PLWH). However, PLWH remain at risk of viral rebound. HIV‐1 infection modifies the content of extracellular vesicles (EVs). The changes in microRNA content in EVs are biomarkers of immune activation and viral replication in PLWH. Moreover, viral molecules are enclosed in EVs produced from infected cells. Our objective was to assess the value of EV‐associated HIV‐1 RNA as a biomarker of immune activation and viral replication in PLWH. Plasma samples were obtained from a cohort of 53 PLWH with a detectable viremia. Large and small EVs were respectively purified by plasma centrifugation at 17 000g and by precipitation with ExoQuick. HIV‐1 RNA and microRNAs were quantified in the EV subtypes by RT‐qPCR. HIV‐1 RNA content was higher in large EVs of ART‐naive PLWH. Small EVs HIV‐1 RNA was equivalent in ART‐naive and ART‐treated PLWH and positively correlated with the CD4/CD8 T cell ratio. In ART‐naive PLWH, HIV‐1 RNA content of large EVs correlated with small EV‐associated miR‐29a, miR‐146a, and miR‐155, biomarkers of viral replication and immune activation. A receiver operating characteristic analysis showed that HIV‐1 RNA in large EVs discriminated PLWH with a high CD8 T cell count. HIV‐1 RNA in large EVs was associated with viral replication and immune activation biomarkers. Inversely, HIV‐1 RNA in small EVs was related to immune restoration. Overall, these results suggest that HIV‐1 RNA quantification in purified EVs could be a useful parameter to monitor HIV‐1 infection.

Filopodia are dynamic adhesive cytoskeletal structures that are critical for directional sensing, polarization, cell-cell adhesion, and migration of diverse cell types. Filopodia are also critical for neuronal synapse formation. While dynamic rearrangement of the actin cytoskeleton is known to be critical for filopodia biogenesis, little is known about the upstream extracellular signals. Here, we identify secreted exosomes as potent regulators of filopodia formation. Inhibition of exosome secretion inhibited the formation and stabilization of filopodia in both cancer cells and neurons and inhibited subsequent synapse formation by neurons. Rescue experiments with purified small and large extracellular vesicles (EVs) identified exosome-enriched small EVs (SEVs) as having potent filopodia-inducing activity. Proteomic analyses of cancer cell-derived SEVs identified the TGF-β family coreceptor endoglin as a key SEV-enriched cargo that regulates filopodia. Cancer cell endoglin levels also affected filopodia-dependent behaviors, including metastasis of cancer cells in chick embryos and 3D migration in collagen gels. As neurons do not express endoglin, we performed a second proteomics experiment to identify SEV cargoes regulated by endoglin that might promote filopodia in both cell types. We discovered a single SEV cargo that was altered in endoglin-KD cancer SEVs, the transmembrane protein Thrombospondin Type 1 Domain Containing 7A (THSD7A). We further found that both cancer cell and neuronal SEVs carry THSD7A and that add-back of purified THSD7A is sufficient to rescue filopodia defects of both endoglin-KD cancer cells and exosome-inhibited neurons. We also find that THSD7A induces filopodia formation through activation of the Rho GTPase, Cdc42. These findings suggest a new model for filopodia formation, triggered by exosomes carrying THSD7A.

Filopodia are dynamic adhesive cytoskeletal structures that are critical for directional sensing, polarization, cell-cell adhesion, and migration of diverse cell types. Filopodia are also critical for neuronal synapse formation. While dynamic rearrangement of the actin cytoskeleton is known to be critical for filopodia biogenesis, little is known about the upstream extracellular signals. Here, we identify secreted exosomes as potent regulators of filopodia formation. Inhibition of exosome secretion inhibited the formation and stabilization of filopodia in both cancer cells and neurons and inhibited subsequent synapse formation by neurons. Rescue experiments with purified small and large extracellular vesicles (EVs) identified exosome-enriched small EVs (SEVs) as having potent filopodia-inducing activity. Proteomic analyses of cancer cell-derived SEVs identified the TGF-β family coreceptor endoglin as a key SEV-enriched cargo that regulates filopodia. Cancer cell endoglin levels also affected filopodia-dependent behaviors, including metastasis of cancer cells in chick embryos and 3D migration in collagen gels. As neurons do not express endoglin, we performed a second proteomics experiment to identify SEV cargoes regulated by endoglin that might promote filopodia in both cell types. We discovered a single SEV cargo that was altered in endoglin-KD cancer SEVs, the transmembrane protein Thrombospondin Type 1 Domain Containing 7A (THSD7A). We further found that both cancer cell and neuronal SEVs carry THSD7A and that add-back of purified THSD7A is sufficient to rescue filopodia defects of both endoglin-KD cancer cells and exosome-inhibited neurons. We also find that THSD7A induces filopodia formation through activation of the Rho GTPase, Cdc42. These findings suggest a new model for filopodia formation, triggered by exosomes carrying THSD7A.

Filopodia are dynamic adhesive cytoskeletal structures that are critical for directional sensing, polarization, cell-cell adhesion, and migration of diverse cell types. Filopodia are also critical for neuronal synapse formation. While dynamic rearrangement of the actin cytoskeleton is known to be critical for filopodia biogenesis, little is known about the upstream extracellular signals. Here, we identify secreted exosomes as potent regulators of filopodia formation. Inhibition of exosome secretion inhibited the formation and stabilization of filopodia in both cancer cells and neurons and inhibited subsequent synapse formation by neurons. Rescue experiments with purified small and large extracellular vesicles (EVs) identified exosome-enriched small EVs (SEVs) as having potent filopodia-inducing activity. Proteomic analyses of cancer cell-derived SEVs identified the TGF-β family coreceptor endoglin as a key SEV-enriched cargo that regulates filopodia. Cancer cell endoglin levels also affected filopodia-dependent behaviors, including metastasis of cancer cells in chick embryos and 3D migration in collagen gels. As neurons do not express endoglin, we performed a second proteomics experiment to identify SEV cargoes regulated by endoglin that might promote filopodia in both cell types. We discovered a single SEV cargo that was altered in endoglin-KD cancer SEVs, the transmembrane protein Thrombospondin Type 1 Domain Containing 7A (THSD7A). We further found that both cancer cell and neuronal SEVs carry THSD7A and that add-back of purified THSD7A is sufficient to rescue filopodia defects of both endoglin-KD cancer cells and exosome-inhibited neurons. We also find that THSD7A induces filopodia formation through activation of the Rho GTPase, Cdc42. These findings suggest a new model for filopodia formation, triggered by exosomes carrying THSD7A.

Filopodia are dynamic adhesive cytoskeletal structures that are critical for directional sensing, polarization, cell-cell adhesion, and migration of diverse cell types. Filopodia are also critical for neuronal synapse formation. While dynamic rearrangement of the actin cytoskeleton is known to be critical for filopodia biogenesis, little is known about the upstream extracellular signals. Here, we identify secreted exosomes as potent regulators of filopodia formation. Inhibition of exosome secretion inhibited the formation and stabilization of filopodia in both cancer cells and neurons and inhibited subsequent synapse formation by neurons. Rescue experiments with purified small and large extracellular vesicles (EVs) identified exosome-enriched small EVs (SEVs) as having potent filopodia-inducing activity. Proteomic analyses of cancer cell-derived SEVs identified the TGF-β family coreceptor endoglin as a key SEV-enriched cargo that regulates filopodia. Cancer cell endoglin levels also affected filopodia-dependent behaviors, including metastasis of cancer cells in chick embryos and 3D migration in collagen gels. As neurons do not express endoglin, we performed a second proteomics experiment to identify SEV cargoes regulated by endoglin that might promote filopodia in both cell types. We discovered a single SEV cargo that was altered in endoglin-KD cancer SEVs, the transmembrane protein Thrombospondin Type 1 Domain Containing 7A (THSD7A). We further found that both cancer cell and neuronal SEVs carry THSD7A and that add-back of purified THSD7A is sufficient to rescue filopodia defects of both endoglin-KD cancer cells and exosome-inhibited neurons. We also find that THSD7A induces filopodia formation through activation of the Rho GTPase, Cdc42. These findings suggest a new model for filopodia formation, triggered by exosomes carrying THSD7A.

Extracellular vesicles (EVs) and their cargo have been studied intensively as potential sources of biomarkers in HIV infection; however, their DNA content, particularly the mitochondrial portion (mtDNA), remains largely unexplored. It is well known that human immunodeficiency virus (HIV) infection and prolonged antiretroviral therapy (ART) lead to mitochondrial dysfunction and reduced mtDNA copy in cells and tissues. Moreover, mtDNA is a well-known damage-associated molecular pattern molecule that could potentially contribute to increased immune activation, oxidative stress, and inflammatory response. We investigated the mtDNA content of large and small plasma EVs in persons living with HIV (PLWH) and its implications for viral replication, ART use, and immune status. Venous blood was collected from 196 PLWH, ART-treated or ART-naïve (66 with ongoing viral replication, ≥20 copies/mL), and from 53 HIV-negative persons, all recruited at five HIV testing or treatment centers in Burkina Faso. Large and small plasma EVs were purified and counted, and mtDNA level was measured by RT-qPCR. Regardless of HIV status, mtDNA was more abundant in large than small EVs. It was more abundant in EVs of viremic than aviremic and control participants and tended to be more abundant in participants treated with Tenofovir compared with Zidovudine. When ART treatment was longer than six months and viremia was undetectable, no variation in EV mtDNA content versus CD4 and CD8 count or CD4/CD8 ratio was observed. However, mtDNA in large and small EVs decreased with years of HIV infection and ART. Our results highlight the impact of viral replication and ART on large and small EVs’ mtDNA content. The mechanisms underlying the differential incorporation of mtDNA into EVs and their effects on the surrounding cells warrant further investigation.

Epidemiological studies have identified the role of periodontitis in the pathogenesis of type 2 diabetes, but the underlying mechanism is poorly understood. It is well-known that small extracellular vesicles are lipid bilayer vesicles derived from cells with a diameter around 30 to 200nm. The purpose of this study was to investigate whether periodontitis induced or exacerbated insulin resistance via circulating small extracellular vesicles. Plasma small extracellular vesicles from control and periodontitis rats were intravenously injected into type 2 diabetic rats. Insulin tolerance tests, glucose tolerance tests, and the activation of the insulin signaling pathway were measured to detect the effect of the plasma small extracellular vesicles on insulin sensitivity. In addition, circulating small extracellular vesicles from patients with periodontitis with or without diabetes were isolated and co-cultured with HepG2 cells. The ability of glucose uptake was assessed using the fluorescence of 2-NBDG via flow cytometry. The activation of insulin signaling pathway was examined via Western blotting. Real time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of enzyme related to glycolysis and gluconeogenesis. Small extracellular vesicles derived from the plasma of periodontitis rats further impaired glucose tolerance and insulin tolerance in diabetic rats and significantly reduced the activation of the insulin signaling pathway in liver tissues, as evidenced by the decreased levels of p-AKT and p-GSK3β and the reduced hepatic glycogen content. For small extracellular vesicles isolated from human plasma, the concentration of small extracellular vesicles in patients with type 2 diabetes combined with periodontitis was higher than that of the healthy control and periodontitis alone. Moreover, circulating small extracellular vesicles from patients with periodontitis significantly inhibited the glucose uptake capacity and inhibited insulin signaling of HepG2 cells. Periodontitis acted as a contributing factor to exacerbate insulin resistance of type 2 diabetic rats. Plasma small extracellular vesicles played a critical role in periodontitis aggravating insulin resistance.