Therapeutically harnessing extracellular vesicles.

The field of extracellular vesicle (EV) research has developed rapidly over the last decade from the study of fundamental biology to a subject of significant clinical relevance. The potential of harnessing EVs in the diagnosis and treatment of diseases - including cancer and neurological and cardiovascular disorders - is now being recognized. Accordingly, the applications of EVs as therapeutic targets, biomarkers, novel drug delivery agents and standalone therapeutics are being actively explored. This Review provides a brief overview of the characteristics and physiological functions of the various classes of EV, focusing on their association with disease and emerging strategies for their therapeutic exploitation.

Oral administration of bovine milk-derived extracellular vesicles induces senescence in the primary tumor but accelerates cancer metastasis.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.

Molecular signaling pathways underlying schizophrenia.

The molecular pathophysiological mechanisms underlying schizophrenia have remained unknown, and no treatment exists for primary prevention. We used Ingenuity Pathway Analysis to analyze canonical and causal pathways in two different datasets, including patients from Finland and USA. The most significant findings in canonical pathway analysis were observed for glutamate receptor signaling, hepatic fibrosis, and glycoprotein 6 (GP6) pathways in the Finnish dataset, and GP6 and hepatic fibrosis pathways in the US dataset. In data-driven causal pathways, ADCYAP1, ADAMTS, and CACNA genes were involved in the majority of the top 10 pathways differentiating patients and controls in both Finnish and US datasets. Results from a Finnish nation-wide database showed that the risk of schizophrenia relapse was 41% lower among first-episode patients during the use of losartan, the master regulator of an ADCYAP1, ADAMTS, and CACNA-related pathway, compared to those time periods when the same individual did not use the drug. The results from the two independent datasets suggest that the GP6 signaling pathway, and the ADCYAP1, ADAMTS, and CACNA-related purine, oxidative stress, and glutamatergic signaling pathways are among primary pathophysiological alterations in schizophrenia among patients with European ancestry. While no reproducible dopaminergic alterations were observed, the results imply that agents such as losartan, and ADCYAP1/PACAP -deficit alleviators, such as metabotropic glutamate 2/3 agonist MGS0028 and 5-HT7 antagonists - which have shown beneficial effects in an experimental Adcyap1-/- mouse model for schizophrenia - could be potential treatments even before the full manifestation of illness involving dopaminergic abnormalities.

Mitochondrial RNA in Alzheimer's Disease Circulating Extracellular Vesicles.

Alzheimer's disease (AD) is the most common type of dementia. Amyloid ß (Aß) plaques, tau-containing neurofibrillary tangles, and neuronal loss leading to brain atrophy are pathologic hallmarks of AD. Given the importance of early diagnosis, extensive efforts have been undertaken to identify diagnostic and prognostic biomarkers for AD. Circulating extracellular vesicles (EVs) provide a platform for "liquid biopsy" biomarkers for AD. Here, we characterized the RNA contents of plasma EVs of age-matched individuals who were cognitively normal (healthy controls (HC)) or had mild cognitive impairment (MCI) due to AD or had mild AD dementia (AD). Using RNA sequencing analysis, we found that mitochondrial (mt)-RNAs, including MT-ND1-6 mRNAs and other protein-coding and non-coding mt-RNAs, were strikingly elevated in plasma EVs of MCI and AD individuals compared with HC. EVs secreted from cultured astrocytes, microglia, and neurons after exposure to toxic conditions relevant to AD pathogenesis (Aß aggregates and H2O2), contained mitochondrial structures (detected by electron microscopy) and mitochondrial RNA and protein. We propose that in the AD brain, toxicity-causing mitochondrial damage results in the packaging of mitochondrial components for export in EVs and further propose that mt-RNAs in plasma EVs can be diagnostic and prognostic biomarkers for MCI and AD.

Small RNA fingerprinting of Alzheimer's disease frontal cortex extracellular vesicles and their comparison with peripheral extracellular vesicles.

Alzheimer's disease is a progressive neurodegenerative disorder, with the strongest disease-associated changes observed at clinical or end-stage disease. Transcriptomic deregulation of miRNA expression can spread via "horizontal" RNA transfer through extracellular vesicles (EVs) to act in conjunction with proteins, leading to changes in mRNA, which can provide early signals to indicate forthcoming neuropathological changes in the brain. Here, we analysed the small RNA content, in particular, miRNA, contained in brain-derived EVs isolated from the frontal cortex of Alzheimer's subjects (n = 8) and neurological control subjects (n = 9). Brain-derived EVs were found to contain an upregulation of disease-associated miRNA. RNA species from brain-derived EVs were correlated with miRNA profiles obtained from matching total brain homogenate. These results provide a blueprint into the biological pathways potentially effected during disease that may be assisted by brain-derived EV RNA horizontal transfer.We also correlated the miRNA changes in the brain with those detected in peripheral EVs collected from serum of Alzheimer's disease patients (n = 23, and healthy controls, n = 23) and revealed a panel of miRNA that could be used as a liquid brain biopsy. Overall, our study provides the first interrogation of the small RNA contents in brain-derived EVs and how they could be used to understand the early pathological changes in Alzheimer's disease which will benefit the development of an early diagnostic blood test.

Methods for loading therapeutics into extracellular vesicles and generating extracellular vesicles mimetic-nanovesicles.

Extracellular vesicles (EVs) are membrane bound vesicles released into the extracellular environment by eukaryotic and prokaryotic cells. EVs are enriched in active biomolecules and they can horizontally transfer cargo to recipient cells. In recent years EVs have demonstrated promising clinical applications due to their theragnostic potential. Although EVs have promising therapeutic potential, there are several challenges associated with using EVs before transition from the laboratory to clinical use. Some of these challenges include issues around low yield, isolation and purification methodologies, and efficient engineering (loading) of EVs with therapeutic cargo. Also, to achieve higher therapeutic efficiency, EV architecture and cargo may need to be manipulated prior to clinical application. Some of these issues have been addressed by developing biomimetic EVs. EV mimetic-nanovesicles (M-NVs) are a type of artificial EVs which can be generated from all cell types with comparable characteristics as EVs for an alternative therapeutic modality. In this review, we will discuss current techniques for modifying EVs and methodology used to generate and customize EV mimetic-nanovesicles.

Novel miR-29b target regulation patterns are revealed in two different cell lines.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene or protein expression by targeting mRNAs and triggering either translational repression or mRNA degradation. Distinct expression levels of miRNAs, including miR-29b, have been detected in various biological fluids and tissues from a large variety of disease models. However, how miRNAs "react" and function in different cellular environments is still largely unknown. In this study, the regulation patterns of miR-29b between human and mouse cell lines were compared for the first time. CRISPR/Cas9 gene editing was used to stably knockdown miR-29b in human cancer HeLa cells and mouse fibroblast NIH/3T3 cells with minimum off-targets. Genome editing revealed mir-29b-1, other than mir-29b-2, to be the main source of generating mature miR-29b. The editing of miR-29b decreased expression levels of its family members miR-29a/c via changing the tertiary structures of surrounding nucleotides. Comparing transcriptome profiles of human and mouse cell lines, miR-29b displayed common regulation pathways involving distinct downstream targets in macromolecular complex assembly, cell cycle regulation, and Wnt and PI3K-Akt signalling pathways; miR-29b also demonstrated specific functions reflecting cell characteristics, including fibrosis and neuronal regulations in NIH/3T3 cells and tumorigenesis and cellular senescence in HeLa cells.

Amyloid Precursor Protein Dimerisation Reduces Neurite Outgrowth.

The amyloid precursor protein (APP) undergoes extensive metabolism, and its transport and proteolytic processing can be modulated by its ability to form a homodimer. We have investigated the functional consequences of stabilised APP dimer expression in cells by studying the engineered dimerisation of the APPL17C (residue 17 in Aß sequence) construct, which is associated with a 30% increase in APP dimer expression, on APP's neurite outgrowth promoting activity. Overexpression of APPL17C in SH-SY5Y cells decreased neurite outgrowth upon retinoic acid differentiation as compared to overexpressing APPWT cells. The APPL17C phenotype was rescued by replacing the APPL17C media with conditioned media from APPWT cells, indicating that the APPL17C mutant is impairing the secretion of a neuritogenic promoting factor. APPL17C had altered transport and was localised in the endoplasmic reticulum. Defining the molecular basis of the APPL17C phenotype showed that RhoA GTPase activity, a negative regulator of neurite outgrowth, was increased in APPL17C cells. RhoA activity was decreased after APPWT conditioned media rescue. Moreover, treatment with the RhoA inhibitor, Y27632, restored a wild-type morphology to the APPL17C cells. Small RNAseq analysis of APPL17C and APPWT cells identified several differentially expressed miRNAs relating to neurite outgrowth. Of these, miR-34a showed the greatest decrease in expression. Lentiviral-mediated overexpression of miR-34a rescued neurite outgrowth in APPL17C cells to APPWT levels and changed RhoA activation. This study has identified a novel link between APP dimerisation and its neuritogenic activity which is mediated by miR-34a expression.

Predicting the Presence of Oral Squamous Cell Carcinoma Using Commonly Dysregulated MicroRNA in Oral Swirls.

Oral swirls are a noninvasive, rapidly collected source of salivary microRNA (miRNA) potentially useful in the early detection of disease states, particularly oral squamous cell carcinoma (OSCC). The aim of this study was to predict the presence of OSCC using a panel of OSCC-related dysregulated miRNA found in oral swirls, identified jointly in data from formalin-fixed paraffin-embedded (FFPE) and fresh-frozen specimens. Next-generation sequencing (NGS) was used to determine miRNA fold changes in FFPE OSCC specimens relative to histologically normal epithelium. These data were placed with NGS of fresh-frozen tissue data of The Cancer Genome Atlas database to select a panel of commonly dysregulated miRNA. This panel was then analyzed by RT-qPCR in RNA extracted from oral swirls collected from 30 patients with OSCC and 30 controls. Upregulation of miR-31 and miR-21 and downregulation of miR-99a, let-7c, miR-125b, and miR-100 were found between OSCC and controls in both FFPE and fresh-frozen samples. These miRNAs were studied in a training set of 15 OSCC versus 15 control oral swirls to develop a dysregulation score [AUC, 0.95; 95% confidence interval (CI), 0.88-1.03] and classification tree. A test cohort of 15 OSCC versus 15 control oral swirls yielded a dysregulation score AUC of 0.86 (95% CI, 0.79-1.00) with the classification tree identifying 100% (15/15) of OSCC and 67% (10/15) of controls. This study debuts the use of OSCC-associated miRNA, commonly dysregulated in both FFPE and frozen specimens, in oral swirls to indicate the presence of OSCC with high accuracy. Cancer Prev Res; 11(8); 491-502. ©2018 AACR.

Tight Junction Protein Claudin-2 Promotes Self-Renewal of Human Colorectal Cancer Stem-like Cells.

Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance in vitro While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil-based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal in vitro and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDHHigh CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDHHigh phenotype. Next-generation sequencing in ALDHHigh cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer.Significance: Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. Cancer Res; 78(11); 2925-38. ©2018 AACR.

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017.

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

BRAFV600 inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells.

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAFV600 mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.

Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells--Evidence of unique microRNA cargos.

Melanoma cells release different types of extracellular vesicles (EVs) into the extracellular milieu that are involved with communication and signaling in the tumor microenvironment. Subsets of EVs include exosomes, microvesicles, and apoptotic bodies that carry protein and genetic (RNA) cargos. To define the contribution of the RNA cargo of melanoma cell derived EVs we performed small RNA sequencing to identify different small RNAs in the EV subsets. Using validated centrifugation protocols, we separated these EV subsets released by the melanoma cell line MML-1, and performed RNA sequencing with the Ion Torrent platform. Various, but different, non-coding RNAs were detected in the EV subsets, including microRNA, mitochondrial associated tRNA, small nucleolar RNA, small nuclear RNA, Ro associated Y-RNA, vault RNA and Y-RNA. We identified in total 1041 miRNAs in cells and EV subsets. Hierarchical clustering showed enrichment of specific miRNAs in exosomes, including hsa-miR-214-3p, hsa-miR-199a-3p and hsa-miR-155-5p, all being associated with melanoma progression. Comparison of exosomal miRNAs with miRNAs in clinical melanoma samples indicate that multiple miRNAs in exosomes also are expressed specifically in melanoma tissues, but not in benign naevi. This study shows for the first time the presence of distinct small RNAs in subsets of EVs released by melanoma cells, with significant similarities to clinical melanoma tissue, and provides unique insights into the contribution of EV associated extracellular RNA in cancer.

The hypoxia imaging agent CuII(atsm) is neuroprotective and improves motor and cognitive functions in multiple animal models of Parkinson's disease.

Parkinson's disease (PD) is a progressive, chronic disease characterized by dyskinesia, rigidity, instability, and tremors. The disease is defined by the presence of Lewy bodies, which primarily consist of aggregated α-synuclein protein, and is accompanied by the loss of monoaminergic neurons. Current therapeutic strategies only give symptomatic relief of motor impairment and do not address the underlying neurodegeneration. Hence, we have identified Cu(II)(atsm) as a potential therapeutic for PD. Drug administration to four different animal models of PD resulted in improved motor and cognition function, rescued nigral cell loss, and improved dopamine metabolism. In vitro, this compound is able to inhibit the effects of peroxynitrite-driven toxicity, including the formation of nitrated α-synuclein oligomers. Our results show that Cu(II)(atsm) is effective in reversing parkinsonian defects in animal models and has the potential to be a successful treatment of PD.

Modulation of cellular Hsp72 levels in undifferentiated and neuron-like SH-SY5Y cells determines resistance to staurosporine-induced apoptosis.

Increased expression of Hsp72 accompanies differentiation of human neuroblastoma SH-SY5Y cells to neuron-like cells. By modulating cellular levels of Hsp72, we demonstrate here its anti-apoptotic activity both in undifferentiated and neuron-like cells. Thermal preconditioning (43°C for 30 min) induced Hsp72, leading to cellular protection against apoptosis induced by a subsequent treatment with staurosporine. Preconditioned staurosporine-treated cells displayed decreased Bax recruitment to mitochondria and subsequent activation, as well as reduced cytochrome c redistribution from mitochondria. The data are consistent with Hsp72 blocking apoptosis upstream of Bax recruitment to mitochondria. Neuron-like cells (with elevated Hsp72) were more resistant to staurosporine by all measured indices of apoptotic signaling. Use of stable transfectants ectopically expressing moderately elevated levels of Hsp72 revealed that such cells in the undifferentiated state showed enhanced resistance to staurosporine-induced apoptosis, which was even more robust after differentiation to neuron-like cells. Overall, the protective effects of differentiation, thermal preconditioning and ectopic Hsp72 expression were additive. The strong inverse correlation between cellular Hsp72 levels and susceptibility to apoptosis support the notion that Hsp72 acts as a significant neuroprotective factor, enabling post-mitotic neurons to withstand potentially lethal stress that induces apoptosis.

Human neuroblastoma SH-SY5Y cells show increased resistance to hyperthermic stress after differentiation, associated with elevated levels of Hsp72.

PURPOSE: Terminally differentiated neurones in the central nervous system need to be protected from stress. We ask here whether differentiation of progenitor cells to neurones is accompanied by up-regulation of Hsp72, with acquisition of enhanced thermotolerance. MATERIALS AND METHODS: Human neuroblastoma SH-SY5Y cells were propagated in an undifferentiated form and subsequently differentiated into neurone-like cells. Thermotolerance tests were carried out by exposure of cells to various temperatures, monitoring nuclear morphology as index of cell death. Abundance of Hsp72 was measured in cell lysates by western immunoblotting. RESULTS: The differentiation of SH-SY5Y cells was accompanied by increased expression of Hsp72. Further, in both cell states, exposure to mild hyperthermic stress (43°C for 30 min) increased Hsp72 expression. After differentiation, SH-SY5Y cells were more resistant to hyperthermic stress compared to their undifferentiated state, correlating with levels of Hsp72. Stable exogenous expression of Hsp72 in SH-SY5Y cells (transfected line 5YHSP72.1, containing mildly elevated levels of Hsp72), led to enhanced resistance to hyperthermic stress. Hsp72 was found to be inducible in undifferentiated 5YHSP72.1 cells; such heat-treated cells displayed enhanced thermotolerance. Treatment of cells with KNK437, a suppressor of Hsp72 induction, resulted in acute thermosensitisation of all cell types tested here. CONCLUSIONS: Hsp72 has a major role in the enhanced hyperthermic resistance acquired during neuronal differentiation of SH-SY5Y cells. These findings model the requirement in intact organisms for highly differentiated neurones to be specially protected against thermal stress.