Publications

Niemann-Pick type C (NPC) disease, sometimes called childhood Alzheimer's, is a rare neurovisceral lipid storage disease with progressive neurodegeneration leading to premature death. The disease is caused by loss-of-function mutations in the Npc1 or Npc2 gene which both result into lipid accumulation in the late endosomes and lysosomes. Since the disease presents with a broad heterogenous clinical spectrum, the involved disease mechanisms are still incompletely understood and this hampers finding an effective treatment. As NPC patients, who carry NPC1 mutations, have shown to share several pathological features with Alzheimer's disease (AD) and we and others have previously shown that AD is associated with a dysfunctionality of the blood-cerebrospinal fluid (CSF) barrier located at choroid plexus, we investigated the functionality of this latter barrier in NPC1 pathology. Using NPC1-/- mice, we show that despite an increase in inflammatory gene expression in choroid plexus epithelial (CPE) cells, the blood-CSF barrier integrity is not dramatically affected. Interestingly, we did observe a massive increase in autophagosomes in CPE cells and enlarged extracellular vesicles (EVs) in CSF upon NPC1 pathology. Additionally, we revealed that these EVs exert toxic effects on brain tissue, in vitro as well as in vivo. Moreover, we observed that EVs derived from the supernatant of NPC1-/- choroid plexus explants are able to induce typical brain pathology characteristics of NPC1-/-, more specifically microgliosis and astrogliosis. Taken together, our data reveal for the first time that the choroid plexus and CSF EVs might play a role in the brain-related pathogenesis of NPC1.

Prostate cancer (PCa) is the most commonly diagnosed malignancy among men in developed countries. The five-year survival rate for men diagnosed with early-stage PCa is approximately 100%, while it is less than 30% for castration-resistant PCa (CRPC). Currently, the detection of prostate-specific antigens as biomarkers for the prognosis of CRPC is criticized because of its low accuracy, high invasiveness, and high false-positive rate. Therefore, it is important to identify new biomarkers for prediction of CRPC progression. Extracellular vesicles (EVs) derived from tumors have been highlighted as potential markers for cancer diagnosis and prognosis. Specifically, urinary EVs directly reflect changes in the pathophysiological conditions of the urogenital system because it is exposed to prostatic secretions. Thus, detecting biomarkers in urinary EVs provides a promising approach for performing an accurate and non-invasive liquid biopsy for CPRC. In this study, we effectively isolated urinary EVs with low protein impurities using size-exclusion chromatography combined with ultrafiltration. After EV isolation and characterization, we evaluated the miRNAs in urinary EVs from healthy donors and patients with CRPC. The results indicated that miRNAs (miR-21-5p, miR-574-3p, and miR-6880-5p) could be used as potential biomarkers for the prognosis of CRPC. This analysis of urinary EVs contributes to the fast and convenient prognosis of diseases, including CRPC, in the clinical setting.

In the biomedical field, there has been a requirement for developing theranostic nanomaterials with higher biosafety, leading to both diagnosis and therapy. Methylene blue (MB+) is an organic dye with both photoluminescence (PL) and photosensitization abilities to generate singlet oxygen (1O2). However, MB+ easily loses its generation ability by hydrogen reduction in vivo or by forming aggregates. In this study, MB+ immobilized on biocompatible hydroxyapatite (HA) nanoparticles was applied for the bifunctions of efficient PL and photosensitization. The MB+-immobilized HA nanoparticles (MH) formed aggregates with sizes of 80–100 nm in phosphate buffer (PB). The generation amount and efficiency of 1O2 from the nanoparticles in PB seem to depend on the immobilized MB+ amount and the percentage of the monomer, respectively. Considering the larger immobilized amount and percentage of the MB+ monomer, it was found that there was MH with the lower generation amount and efficiency of 1O2 to exhibit the highest PL intensity. The photofunctional measurement of MB+ revealed the state of MB+ molecules on the HA surface, and it was suggested that the MB+ molecules immobilized on the MH surface would form more hydrogen bonds to change their excitation states. In the cellular experiments, the Hela cancer cells reacted with the nanoparticles and showed red-color PL, indicating cellular imaging. Furthermore, the adherent cell coverage decreased by 1O2 generation, indicating the importance of the immobilization amount of the MB+ monomer. Therefore, theranostic nanomaterials with biosafety were successfully synthesized to show two photofunctions, which provide both cellular imaging and photodynamic therapy by the nanohybrid system between HA and MB+.

Extracellular vesicle (EV) research has grown rapidly in recent years, largely due to the potential use of EVs as liquid biopsy biomarkers or therapeutics. However, in‐depth characterisation and validation of EVs produced using conventional in vitro cultures can be challenging due to the large area of cell monolayers and volumes of culture media required. To overcome this obstacle, multiple bioreactor designs have been tested for EV production with varying success, but the consistency of EVs produced over time in these systems has not been reported previously. In this study, we demonstrate that several breast cancer cell lines of different subtypes can be cultured simultaneously in space, resource, and time efficient manner using CELLine AD 1000 systems, allowing the consistent production of vast amounts of EVs for downstream experimentation. We report an improved workflow used for inoculating, maintaining, and monitoring the bioreactors, their EV production, and the characterisation of the EVs produced. Lastly, our proteomic analyses of the EVs produced throughout the lifetime of the bioreactors show that core EV‐associated proteins are relatively consistent, with few minor variations over time, but that tracking the production of EVs is a convenient method to indirectly monitor the bioreactor and consistency of the yielded EVs. These findings will aid future studies requiring the simultaneous production of large amounts of EVs from several cell lines of different subtypes of a disease and other EV biomanufacturing applications.

The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products. In this study, we evaluated the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process. Bind-elute chromatography, such as HAC, is an attractive method of purification because it is highly scalable, robust and can be automated. Our findings support an interaction between EVs and heparin. The recovery of EVs using HAC based on particle counts was a minimum of 68.7%. We found HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively. In addition to EV purification, HAC was used to separate EVs into three populations based on their affinity to the heparin column. Within these populations, we detected differences in the expression of the exosome-associated protein TSG101 and the tetraspanin immunophenotype. However, the significance of these observations is not clear. Overall HAC shows promise as a potential purification method to capture EVs and this study proposes a novel application of HAC for EV fractionation. Moving forward, a better understanding of the heparin-EV interaction would be required before HAC can be more widely adopted for these applications.

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35–100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.

The treatment of Parkinson’s disease (PD) has been hindered by the complex pathologies and multiple membrane barriers during drug delivery. Although exosomes derived from mesenchymal stem cells (MSCs) have great potential for PD, MSC-derived exosomes alone could not fully meet the therapeutic requirements due to their limitation in therapy and delivery. Here, we develop a self-oriented nanocarrier called PR-EXO/PP@Cur that combines therapeutic MSC-derived exosomes with curcumin. PR-EXO/PP@Cur can be self-oriented across the multiple membrane barriers and directly release drugs into the cytoplasm of target cells after intranasal administration. With enhanced accumulation of drugs in the action site, PR-EXO/PP@Cur achieves three-pronged synergistic treatment to deal with the complex pathologies of PD by reducing α-synuclein aggregates, promoting neuron function recovery, and alleviating the neuroinflammation. After treatment with PR-EXO/PP@Cur, the movement and coordination ability of PD model mice are significantly improved. These results show that PR-EXO/PP@Cur has great prospects in treatment of PD or other neurodegenerative diseases.

Recent studies have investigated the composition and functional effects of extracellular vesicles (EVs) secreted by a variety of cell types. However, the mechanisms underlying the impact of these vesicles on neurotransmission remain unclear. Here, we isolated EVs secreted by rat and mouse hippocampal neurons and found that they contain synaptic-vesicle-associated proteins, in particular the vesicular SNARE (soluble N-ethylmaleimide-sensitive factor [NSF]-attachment protein receptor) synaptobrevin (also called VAMP). Using a combination of electrophysiology and live-fluorescence imaging, we demonstrate that this extracellular pool of synaptobrevins can rapidly integrate into the synaptic vesicle cycle of host neurons via a CD81-dependent process and selectively augment inhibitory neurotransmission as well as specifically rescue neurotransmission in synapses deficient in synaptobrevin. These findings uncover a novel means of interneuronal communication and functional coupling via exchange of vesicular SNAREs.

SARS-CoV-2 cell entry is completed after viral spike (S) protein-mediated membrane fusion between viral and host cell membranes. Stable prefusion and postfusion S structures have been resolved by cryo-electron microscopy and cryo-electron tomography, but the refolding intermediates on the fusion pathway are transient and have not been examined. We used an antiviral lipopeptide entry inhibitor to arrest S protein refolding and thereby capture intermediates as S proteins interact with hACE2 and fusion-activating proteases on cell-derived target membranes. Cryo-electron tomography imaged both extended and partially folded intermediate states of S2, as well as a novel late-stage conformation on the pathway to membrane fusion. The intermediates now identified in this dynamic S protein-directed fusion provide mechanistic insights that may guide the design of CoV entry inhibitors.

Proteoglycans are proteins that are modified with glycosaminoglycan chains. Chondroitin sulfate proteoglycans (CSPGs) are currently being exploited as targets for drug-delivery in various cancer indications, however basic knowledge on how CSPGs are internalized in tumor cells is lacking. In this study we took advantage of a recombinant CSPG-binding lectin VAR2CSA (rVAR2) to track internalization and cell fate of CSPGs in tumor cells. We found that rVAR2 is internalized into cancer cells via multiple internalization mechanisms after initial docking on cell surface CSPGs. Regardless of the internalization pathway used, CSPG-bound rVAR2 was trafficked to the early endosomes in an energy-dependent manner but not further transported to the lysosomal compartment. Instead, internalized CSPG-bound rVAR2 proteins were secreted with exosomes to the extracellular environment in a strictly chondroitin sulfate-dependent manner. In summary, our work describes the cell fate of rVAR2 proteins in tumor cells after initial binding to CSPGs, which can be further used to inform development of rVAR2-drug conjugates and other therapeutics targeting CSPGs.

Senescent cells produce chronic inflammation that contributes to the diseases and debilities of aging. How this process is orchestrated in epithelial cells, the origin of human carcinomas, is poorly understood. We used human normal oral keratinocytes (NOKs) to elucidate senescence programs in a prototype primary mucosal epithelial cell that senesces spontaneously. While NOKs exhibit several typical facets of senescence, they also display distinct characteristics. These include expression of p21WAF1/CIP1 at early passages, making this common marker of senescence unreliable in NOKs. Transcriptome analysis by RNA-seq revealed specific commonalities with and differences from cancer cells, explicating the tumor avoidance role of senescence. Repression of DNA repair genes that correlated with downregulation of E2F1 mRNA and protein was observed for two donors; a divergent result was seen for the third. Using proteomic profiling of soluble (non-vesicular) and extracellular vesicle (EV) associated secretions, we propose additions to the senescence associated secretory phenotype, including HSP60, which localizes to the surface of EVs. Finally, EVs from senescent NOKs activate interferon pathway signaling in THP-1 monocytes in a STING-dependent manner and associate with mitochondrial and nuclear DNA. Our results highlight senescence changes in epithelial cells and how they might contribute to chronic inflammation and age-related diseases.

Indium tin oxide (ITO) nanoparticles triggered the release of IL-1β from macrophages, followed by the significant induction of epithelial-mesenchymal transition (EMT) in alveolar epithelial cells. Epithelial–mesenchymal transition (EMT) is a crucial process by which epithelial cells lose polarity and acquire migratory mesenchymal properties, eventually leading to tissue fibrosis and cancer. Indium tin oxide (ITO) is one of the most widely manufactured materials with broad applications, such as flat panel displays, touch panels, and solar panels. Whereas cases of indium-related lung disease have been reported worldwide, the effects of ITO on the progression of EMT are completely unknown. In the current study, we explored whether ITO nanoparticles (NPs) induce EMT in human alveolar epithelial cells (A549 cells). We found that although ITO NPs did not directly induce EMT in A549 cells, a conditioned medium (CM) obtained from THP-1-derived macrophages (dTHP-1 cells) stimulated with ITO NPs induced morphological changes, high motility, and EMT progression in A549 cells. After co-culture with ITO NP-treated dTHP-1 cells, A549 cells exhibited morphological and molecular signatures of EMT. Furthermore, we identified that interleukin-1β (IL-1β) produced via the activation of nod-like receptor protein 3 (NLRP3) inflammasome is an ITO NP-mediated EMT inducer based on the results of cytokine array as well as cellular physiological and biochemical analysis. Our results also indicated that the IL-1β-mediated EMT occurs not only in A549 cells, but also in bronchial epithelial cells (BEAS-2B cells) and primary human alveolar epithelial cells (hAEC). In addition, a neutralizing antibody against IL-1 receptor can effectively inhibit the induction of EMT caused by CM from ITO NP-treated dTHP-1 cells. Taken together, these findings suggest that IL-1β is released from macrophages stimulated with ITO NPs and is able to induce EMT progression in A549 cells, thereby potentially triggering the genesis and development of pulmonary fibrosis.

SARS-CoV-2 continues to evolve into variants of concern (VOC), with greatest variability in the multidomain, entry-facilitating spike proteins. To recognize the significance of adaptive spike protein changes, we compare variant SARS-CoV-2 virus particles in several assays reflecting authentic virus-cell entry. Virus particles with adaptive changes in spike amino-terminal domains (NTDs) are hypersensitive to proteolytic activation of membrane fusion, an essential step in virus-cell entry. Proteolysis is within fusion domains (FDs), at sites over 10 nm from the VOC-specific NTD changes, indicating allosteric inter-domain control of fusion activation. In addition, NTD-specific antibodies block FD cleavage, membrane fusion, and virus-cell entry, suggesting restriction of inter-domain communication as a neutralization mechanism. Finally, using structure-guided mutagenesis, we identify an inter-monomer β sheet structure that facilitates NTD-to-FD transmissions and subsequent fusion activation. This NTD-to-FD axis that sensitizes viruses to infection and to NTD-specific antibody neutralization provides new context for understanding selective forces driving SARS-CoV-2 evolution.

Extracellular vesicles (EVs) are lipid-based nanosized particles that convey biological material from donor to recipient cells. EVs play key roles in glioblastoma progression because glioblastoma stem-like cells (GSCs) release pro-oncogenic, pro-angiogenic, and pro-inflammatory EVs. However, the molecular basis of EV release remains poorly understood. Here, we report the identification of the pseudokinase MLKL, a crucial effector of cell death by necroptosis, as a regulator of the constitutive secretion of EVs in GSCs. We find that genetic, protein, and pharmacological targeting of MLKL alters intracellular trafficking and EV release, and reduces GSC expansion. Nevertheless, this function ascribed to MLKL appears independent of its role during necroptosis. In vivo, pharmacological inhibition of MLKL reduces the tumor burden and the level of plasmatic EVs. This work highlights the necroptosis-independent role of MLKL in vesicle release and suggests that interfering with EVs is a promising therapeutic option to sensitize glioblastoma cells.

Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1β injection and two hours later plasma and brain were collected. IL-1β injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1β-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1β or PDDC. IL-1β injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1β-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.

An increasing number of individuals are suffering from lower back and neck pain caused by intervertebral disc degeneration each year. Although the application of mesenchymal stem cells (MSCs) has provided desirable results in the treatment of intervertebral disc degeneration, there are multiple risks associated with the directed application of MSCs. An increasing number of studies have suggested that stem cells, through the release of extracellular nanovesicles, have vital functions in tissue regeneration and repair with low risk. The present study investigated the effect of extracellular nanovesicles derived from adipose-derived stem cells (ADSCs) on nucleus pulposus (NP) cells from patients with intervertebral disc degeneration. Human NP cells were obtained from patients with intervertebral disc degeneration undergoing surgical procedures in addition to ADSCs from liposuction patients. ADSC-derived extracellular nanovesicles were isolated and characterized. The differentiation and biological activity of NP cells cultured with or without ADSC-derived extracellular nanovesicles were assessed and inflammatory factors and intervertebral disc degeneration-associated markers were also measured. The results indicated that extracellular nanovesicles derived from ADSCs increased the migration and proliferation of NP cells and inhibited inflammatory activity, suggesting their utility for the treatment of intervertebral disc degeneration.

BACKGROUND: Extracellular vesicles (EVs) are capable of manipulating cellular functions for the maintenance of biological homeostasis and disease progression, such as in glaucoma disease. These nano-particles carry a net negative surface charge under physiological conditions that can contribute to EVs:EVs interaction and their uptake by target cells. PURPOSE: To investigate the effect of glaucoma drugs on EVs physicochemical characters and the implications for their uptake by trabecular meshwork (TM) cells. METHODS: TM or non-pigmented ciliary epithelium (NPCE) cells derived EVs were incubated with commercial anti-glaucoma formulation, Timolol maleate, Brinzolamide or Benzalkonium Cl and their size and zeta potential (ZP) and physical interactions of EVs derived from NPCE cells and TM cells were evaluated. The contribution of EVs interactions to up-take by TM cells was examined using fluorescence-activated cell sorting. RESULTS: EVs size and ZP were affected by the ionic strength of the buffer rather than EVs type. Commercial glaucoma eye drops, including β-blocker, α-2-agonist and prostaglandin analogs, reduced NPCE EVs ZP, whereas exposure of EVs to carbonic anhydrase inhibitor caused an increase in the ZP. A correlation was found between increased ZP values and increased NPCE EVs uptake by TM cells. We were able to show that Benzalkonium chloride stands behind this ZP effect and not Timolol or Brinzolamide. CONCLUSION: Altogether, our findings demonstrate that EVs size, surface membrane charge, and ionic strength of the surrounding have an impact on EVs:EVs interactions, which affect the uptake of NPCE EVs by TM cells.

In dairy cows, Staphylococcus aureus (S. aureus) is among the most prevalent microorganisms worldwide, causing mastitis, an inflammation of the mammary gland. Production of extracellular vesicles (EVs) is a common feature of S. aureus strains, which contributes to its pathogenesis by delivering bacterial effector molecules to host cells. In the current study, we evaluated the differences between five S. aureus mastitis isolates regarding their EV production. We found that different mastitis-related S. aureus strains differ in their behaviour of shedding EVs, with M5512VL producing the largest amount of EVs containing alpha-haemolysin, a strong cytotoxic agent. We stimulated primary cultured bovine mammary epithelial cells (pbMECs) with EVs from the S. aureus strain M5512VL. After 24 h of incubation, we observed a moderate increase in gene expression of tumour necrosis factor-alpha (TNF-α) but, surprisingly, a lack of an associated pronounced pro-inflammatory response. Our results contribute to understanding the damaging nature of S. aureus in its capacity to effectively affect mammary epithelial cells.

Infectious organisms and damage of cells can activate inflammasomes, which mediate tissue inflammation and adaptive immunity. These mechanisms evolved to curb the spread of microbes and to induce repair of the damaged tissue. Chronic activation of inflammasomes, however, contributes to non-resolving inflammatory responses that lead to immuno-pathologies. Inflammasome-activated cells undergo an inflammatory cell death associated with the release of potent pro-inflammatory cytokines and poorly characterized extracellular vesicles (EVs). Since inflammasome-induced EVs could signal inflammasome pathway activation in patients with chronic inflammation and modulate bystander cell activation, we performed a systems analysis of the ribonucleic acid (RNA) content and function of two EV classes. We show that EVs released from inflammasome-activated macrophages carry a specific RNA signature and contain interferon β (IFNβ). EV-associated IFNβ induces an interferon signature in bystander cells and results in dampening of NLRP3 inflammasome responses. EVs could, therefore, serve as biomarkers for inflammasome activation and act to prevent systemic hyper-inflammatory states by restricting NLRP3 activation in bystander cells.

Tumor peptides associated with MHC class I molecules or their synthetic variants have attracted great attention for their potential use as vaccines to induce tumor-specific CTLs. However, the outcome of clinical trials of peptide-based tumor vaccines has been disappointing. There are various reasons for this lack of success, such as difficulties in delivering the peptides specifically to professional Ag-presenting cells, short peptide half-life in vivo, and limited peptide immunogenicity. We report here a novel peptide vaccination strategy that efficiently induces peptide-specific CTLs. Nanoparticles (NPs) were fabricated from a biodegradable polymer, poly(D,L-lactic-co-glycolic acid), attached to H-2Kb molecules, and then the natural peptide epitopes associated with the H-2Kb molecules were exchanged with a model tumor peptide, SIINFEKL (OVA257-268). These NPs were efficiently phagocytosed by immature dendritic cells (DCs), inducing DC maturation and activation. In addition, the DCs that phagocytosed SIINFEKL-pulsed NPs potently activated SIINFEKL-H-2Kb complex-specific CD8+ T cells via cross-presentation of SIINFEKL. In vivo studies showed that intravenous administration of SIINFEKL-pulsed NPs effectively generated SIINFEKL-specific CD8+ T cells in both normal and tumor-bearing mice. Furthermore, intravenous administration of SIINFEKL-pulsed NPs into EG7.OVA tumor-bearing mice almost completely inhibited the tumor growth. These results demonstrate that vaccination with polymeric NPs coated with tumor peptide-MHC-I complexes is a novel strategy for efficient induction of tumor-specific CTLs.

Colorectal cancer is the leading cause of cancer-related deaths worldwide, warranting the urgent need for a new treatment option. Plant-derived nanovesicles containing bioactive compounds represent new therapeutic avenues due to their unique characteristics as natural nanocarriers for bioactive molecules with therapeutic effects. Recent evidence has revealed potential anticancer activity of bioactive compounds from Boesenbergia rotunda (L.) Mansf. (fingerroot). However, the effect and the underlying mechanisms of fingerroot-derived nanovesicles (FDNVs) against colorectal cancer are still unknown. We isolated the nanovesicles from fingerroot and demonstrated their anticancer activity against two colorectal cancer cell lines, HT-29 and HCT116. The IC50 values were 63.9 ± 2.4, 57.8 ± 4.1, 47.8 ± 7.6 μg/ml for HT-29 cells and 57.7 ± 6.6, 47.2 ± 5.2, 34 ± 2.9 μg/ml for HCT116 cells at 24, 48, and 72 h, respectively. Interestingly, FDNVs were not toxic to a normal colon epithelial cell line, CCD 841 CoN. FDNVs exhibited selective uptake by the colorectal cancer cell lines but not the normal colon epithelial cell line. Moreover, dose- and time-dependent FDNV-induced apoptosis was only observed in the colorectal cancer cell lines. In addition, reactive oxygen species levels were substantially increased in colorectal cancer cells, but total glutathione decreased after treatment with FDNVs. Our results show that FDNVs exhibited selective anticancer activity in colorectal cancer cell lines via the disruption of intracellular redox homeostasis and induction of apoptosis, suggesting the utility of FDNVs as a novel intervention for colorectal cancer patients.

Extracellular vesicles (EVs) are involved in a multitude of physiological functions and play important roles in health and disease. The largest proportion of studies on EVs is based on the analysis and characterization of EVs secreted in the cell culture medium. These studies remain challenging due to the small size of the EV particles, a lack of universal EV markers, and sample loss or technical artifacts that are often associated with EV labeling for single particle tracking and/or separation techniques. To address these problems, we characterized and validated a method for in-cell EV labeling with fluorescent lipids coupled with direct analysis of lipid-labeled EVs in the conditioned medium by imaging flow cytometry (IFC). This approach significantly reduces sample processing and loss compared to established methods for EV separation and labeling in vitro, resulting in improved detection of quantitative changes in EV secretion and subpopulations compared to protocols that rely on EV separation by size-exclusion chromatography and ultracentrifugation. Our optimized protocol for in-cell EV labeling and analysis of the conditioned medium reduces EV sample processing and loss, and is well-suited for cell biology studies that focus on modulation of EV secretion by cells in culture.

Increasing evidence indicates that extracellular vesicles (EVs) play an important role in the pathogenesis of Alzheimer’s disease (AD). We previously reported that the blood–cerebrospinal fluid (CSF) interface, formed by the choroid plexus epithelial (CPE) cells, releases an increased amount of EVs into the CSF in response to peripheral inflammation. Here, we studied the importance of CP-mediated EV release in AD pathogenesis. We observed increased EV levels in the CSF of young transgenic APP/PS1 mice which correlated with high amyloid beta (Aβ) CSF levels at this age. The intracerebroventricular (icv) injection of Aβ oligomers (AβO) in wild-type mice revealed a significant increase of EVs in the CSF, signifying that the presence of CSF-AβO is sufficient to induce increased EV secretion. Using in vivo, in vitro and ex vivo approaches, we identified the CP as a major source of the CSF-EVs. Interestingly, AβO-induced, CP-derived EVs induced pro-inflammatory effects in mixed cortical cultures. Proteome analysis of these EVs revealed the presence of several pro-inflammatory proteins, including the complement protein C3. Strikingly, inhibition of EV production using GW4869 resulted in protection against acute AβO-induced cognitive decline. Further research into the underlying mechanisms of this EV secretion might open up novel therapeutic strategies to impact the pathogenesis and progression of AD.

Small extracellular vesicles (sEV) have emerged as a potential rich source of biomarkers in human blood and present the intriguing potential for a 'liquid biopsy' to track disease and the effectiveness of interventions. Recently, we have further demonstrated the potential for EV derived biomarkers to account for variability in drug exposure. This study sought to evaluate the variability in abundance and cargo of global and liver-specific circulating sEV, within (diurnal) and between individuals in a cohort of healthy subjects (n = 10). We present normal ranges for EV concentration and size and expression of generic EV protein markers and the liver-specific asialoglycoprotein receptor 1 (ASGR1) in samples collected in the morning and afternoon. EV abundance and cargo was generally not affected by fasting, except CD9 which exhibited a statistically significant increase (p = 0.018). Diurnal variability was observed in the expression of CD81 and ASGR1, which significantly decreased (p = 0.011) and increased (p = 0.009), respectively. These results have potential implications for study sampling protocols and normalisation of biomarker data when considering the expression of sEV derived cargo as a biomarker strategy. Specifically, the novel finding that liver-specific EVs exhibit diurnal variability in healthy subjects should have broad implications in the study of drug metabolism and development of minimally invasive biomarkers for liver disease.