Ectopic ATP synthase stimulates the secretion of extracellular vesicles in cancer cells.

ABSTARCT: Ectopic ATP synthase on the plasma membrane (eATP synthase) has been found in various cancer types and is a potential target for cancer therapy. However, whether it provides a functional role in tumor progression remains unclear. Here, quantitative proteomics reveals that cancer cells under starvation stress express higher eATP synthase and enhance the production of extracellular vesicles (EVs), which are vital regulators within the tumor microenvironment. Further results show that eATP synthase generates extracellular ATP to stimulate EV secretion by enhancing P2X7 receptor-triggered Ca2+ influx. Surprisingly, eATP synthase is also located on the surface of tumor-secreted EVs. The EVs-surface eATP synthase increases the uptake of tumor-secreted EVs in Jurkat T-cells via association with Fyn, a plasma membrane protein found in immune cells. The eATP synthase-coated EVs uptake subsequently represses the proliferation and cytokine secretion of Jurkat T-cells. This study clarifies the role of eATP synthase on EV secretion and its influence on immune cells.

Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina.

Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration, and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. EV association with PBMCs, especially B-cells, was observed as early as one minute post-administration. EVs were detected in liver and spleen within one hour of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

The power of imaging to understand extracellular vesicle biology in vivo.

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles.

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50-200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex "work-in-progress" MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.

Glioblastoma hijacks microglial gene expression to support tumor growth.

BACKGROUND: Glioblastomas are the most common and lethal primary brain tumors. Microglia, the resident immune cells of the brain, survey their environment and respond to pathogens, toxins, and tumors. Glioblastoma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Despite the presence of large numbers of microglia in glioblastoma, the tumors continue to grow, and these neuroimmune cells appear incapable of keeping the tumor in check. To understand this process, we analyzed gene expression in microglia interacting with glioblastoma cells. METHODS: We used RNASeq of isolated microglia to analyze the expression patterns of genes involved in key microglial functions in mice with glioblastoma. We focused on microglia that had taken up tumor-derived EVs and therefore were within and immediately adjacent to the tumor. RESULTS: We show that these microglia have downregulated expression of genes involved in sensing tumor cells and tumor-derived danger signals, as well as genes used for tumor killing. In contrast, expression of genes involved in facilitating tumor spread was upregulated. These changes appear to be in part EV-mediated, since intracranial injection of EVs in normal mice led to similar transcriptional changes in microglia. We observed a similar microglial transcriptomic signature when we analyzed datasets from human patients with glioblastoma. CONCLUSION: Our data define a microgliaGlioblastoma specific phenotype, whereby glioblastomas have hijacked gene expression in the neuroimmune system to favor avoiding tumor sensing, suppressing the immune response, clearing a path for invasion, and enhancing tumor propagation. For further exploration, we developed an interactive online tool at http://www.glioma-microglia.com with all expression data and additional functional and pathway information for each gene.

Combined delivery of sorafenib and a MEK inhibitor using CXCR4-targeted nanoparticles reduces hepatic fibrosis and prevents tumor development.

Liver damage and fibrosis are precursors of hepatocellular carcinoma (HCC). In HCC patients, sorafenib-a multikinase inhibitor drug-has been reported to exert anti-fibrotic activity. However, incomplete inhibition of RAF activity by sorafenib may also induce paradoxical activation of the mitogen-activated protein kinase (MAPK) pathway in malignant cells. The consequence of this effect in non-malignant disease (hepatic fibrosis) remains unknown. This study aimed to examine the effects of sorafenib on activated hepatic stellate cells (HSCs), and develop effective therapeutic approaches to treat liver fibrosis and prevent cancer development. Methods: We first examined the effects of sorafenib in combination with MEK inhibitors on fibrosis pathogenesis in vitro and in vivo. To improve the bioavailability and absorption by activated HSCs, we developed CXCR4-targeted nanoparticles (NPs) to co-deliver sorafenib and a MEK inhibitor to mice with liver damage. Results: We found that sorafenib induced MAPK activation in HSCs, and promoted their myofibroblast differentiation. Combining sorafenib with a MEK inhibitor suppressed both paradoxical MAPK activation and HSC activation in vitro, and alleviated liver fibrosis in a CCl4-induced murine model of liver damage. Furthermore, treatment with sorafenib/MEK inhibitor-loaded CXCR4-targeted NPs significantly suppressed hepatic fibrosis progression and further prevented fibrosis-associated HCC development and liver metastasis. Conclusions: Our results show that combined delivery of sorafenib and a MEK inhibitor via CXCR4-targeted NPs can prevent activation of ERK in activated HSCs and has anti-fibrotic effects in the CCl4-induced murine model. Targeting HSCs represents a promising strategy to prevent the development and progression of fibrosis-associated HCC.

Engineered nanointerfaces for microfluidic isolation and molecular profiling of tumor-specific extracellular vesicles.

Extracellular vesicles (EVs) carry RNA, DNA, proteins, and lipids. Specifically, tumor-derived EVs have the potential to be utilized as disease-specific biomarkers. However, a lack of methods to isolate tumor-specific EVs has limited their use in clinical settings. Here we report a sensitive analytical microfluidic platform (EVHB-Chip) that enables tumor-specific EV-RNA isolation within 3 h. Using the EVHB-Chip, we achieve 94% tumor-EV specificity, a limit of detection of 100 EVs per µL, and a 10-fold increase in tumor RNA enrichment in comparison to other methods. Our approach allows for the subsequent release of captured tumor EVs, enabling downstream characterization and functional studies. Processing serum and plasma samples from glioblastoma multiforme (GBM) patients, we can detect the mutant EGFRvIII mRNA. Moreover, using next-generation RNA sequencing, we identify genes specific to GBM as well as transcripts that are hallmarks for the four genetic subtypes of the disease.

Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles.

Growing interest in extracellular vesicles (EVs, including exosomes and microvesicles) as therapeutic entities, particularly in stem cell-related approaches, has underlined the need for standardization and coordination of development efforts. Members of the International Society for Extracellular Vesicles and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore convened a Workshop on this topic to discuss the opportunities and challenges associated with development of EV-based therapeutics at the preclinical and clinical levels. This review outlines topic-specific action items that, if addressed, will enhance the development of best-practice models for EV therapies. Stem Cells Translational Medicine 2017;6:1730-1739.

Obstacles and opportunities in the functional analysis of extracellular vesicle RNA - an ISEV position paper.

The release of RNA-containing extracellular vesicles (EV) into the extracellular milieu has been demonstrated in a multitude of different in vitro cell systems and in a variety of body fluids. RNA-containing EV are in the limelight for their capacity to communicate genetically encoded messages to other cells, their suitability as candidate biomarkers for diseases, and their use as therapeutic agents. Although EV-RNA has attracted enormous interest from basic researchers, clinicians, and industry, we currently have limited knowledge on which mechanisms drive and regulate RNA incorporation into EV and on how RNA-encoded messages affect signalling processes in EV-targeted cells. Moreover, EV-RNA research faces various technical challenges, such as standardisation of EV isolation methods, optimisation of methodologies to isolate and characterise minute quantities of RNA found in EV, and development of approaches to demonstrate functional transfer of EV-RNA in vivo. These topics were discussed at the 2015 EV-RNA workshop of the International Society for Extracellular Vesicles. This position paper was written by the participants of the workshop not only to give an overview of the current state of knowledge in the field, but also to clarify that our incomplete knowledge - of the nature of EV(-RNA)s and of how to effectively and reliably study them - currently prohibits the implementation of gold standards in EV-RNA research. In addition, this paper creates awareness of possibilities and limitations of currently used strategies to investigate EV-RNA and calls for caution in interpretation of the obtained data.

Delivery of Therapeutic Proteins via Extracellular Vesicles: Review and Potential Treatments for Parkinson's Disease, Glioma, and Schwannoma.

Extracellular vesicles present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids, and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson's disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases.

SCS macrophages suppress melanoma by restricting tumor-derived vesicle-B cell interactions.

Tumor-derived extracellular vesicles (tEVs) are important signals in tumor-host cell communication, yet it remains unclear how endogenously produced tEVs affect the host in different areas of the body. We combined imaging and genetic analysis to track melanoma-derived vesicles at organismal, cellular, and molecular scales to show that endogenous tEVs efficiently disseminate via lymphatics and preferentially bind subcapsular sinus (SCS) CD169(+) macrophages in tumor-draining lymph nodes (tdLNs) in mice and humans. The CD169(+) macrophage layer physically blocks tEV dissemination but is undermined during tumor progression and by therapeutic agents. A disrupted SCS macrophage barrier enables tEVs to enter the lymph node cortex, interact with B cells, and foster tumor-promoting humoral immunity. Thus, CD169(+) macrophages may act as tumor suppressors by containing tEV spread and ensuing cancer-enhancing immunity.

Biogenesis, delivery, and function of extracellular RNA.

The Extracellular RNA (exRNA) Communication Consortium was launched by the National Institutes of Health to focus on the extent to which RNA might function in a non-cell-autonomous manner. With the availability of increasingly sensitive tools, small amounts of RNA can be detected in serum, plasma, and other bodily fluids. The exact mechanism(s) by which RNA can be secreted from cells and the mechanisms for the delivery and uptake by recipient cells remain to be determined. This review will summarize current knowledge about the biogenesis and delivery of exRNA and outline projects seeking to understand the functional impact of exRNA.

Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters.

Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo. Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH2-termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA.

Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study.

The release of extracellular vesicles (EVs), including exosomes and microvesicles, is a phenomenon shared by many cell types as a means of communicating with other cells and also potentially removing cell contents. The cargo of EVs includes the proteins, lipids, nucleic acids, and membrane receptors of the cells from which they originate. EVs released into the extracellular space can enter body fluids and potentially reach distant tissues. Once taken up by neighboring and/or distal cells, EVs can transfer functional cargo that may alter the status of recipient cells, thereby contributing to both physiological and pathological processes. In this article, we will focus on EV composition, mechanisms of uptake, and their biological effects on recipient cells. We will also discuss established and recently developed methods used to study EVs, including isolation, quantification, labeling and imaging protocols, as well as RNA analysis.

Dynamic biodistribution of extracellular vesicles in vivo using a multimodal imaging reporter.

Extracellular vesicles (EVs) are nanosized vesicles released by normal and diseased cells as a novel form of intercellular communication and can serve as an effective therapeutic vehicle for genes and drugs. Yet, much remains unknown about the in vivo properties of EVs such as tissue distribution, blood levels, and urine clearance, important parameters that will define their therapeutic effectiveness and potential toxicity. Here we combined Gaussia luciferase and metabolic biotinylation to create a sensitive EV reporter (EV-GlucB) for multimodal imaging in vivo, as well as monitoring of EV levels in the organs and biofluids ex vivo after administration of EVs. Bioluminescence and fluorescence-mediated tomography imaging on mice displayed a predominant localization of intravenously administered EVs in the spleen followed by the liver. Monitoring EV signal in the organs, blood, and urine further revealed that the EVs first undergo a rapid distribution phase followed by a longer elimination phase via hepatic and renal routes within six hours, which are both faster than previously reported using dye-labeled EVs. Moreover, we demonstrate systemically injected EVs can be delivered to tumor sites within an hour following injection. Altogether, we show the EVs are dynamically processed in vivo with accurate spatiotemporal resolution and target a number of normal organs as well as tumors with implications for disease pathology and therapeutic design.

Noninvasive in vivo monitoring of extracellular vesicles.

Extracellular vesicles (EVs) including exosomes and microvesicles are nanometer-sized vesicles released by cells to deliver lipids, cellular proteins, mRNAs, and noncoding RNAs, thereby facilitating intercellular communication without direct cell-to-cell contacts. Due to their nanoscale size, EVs have been visualized under microscopy in vitro. We here describe a strategy to label EVs with Gaussia luciferase for noninvasive bioluminescence imaging and monitoring of systemically administered EVs in vivo.

Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury.

Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell-cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT-CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.

Pannexin1 drives multicellular aggregate compaction via a signaling cascade that remodels the actin cytoskeleton.

Pannexin 1 (Panx1) is a novel gap junction protein shown to have tumor-suppressive properties. To model its in vivo role in the intratumor biomechanical environment, we investigated whether Panx1 channels modulate the dynamic assembly of multicellular C6 glioma aggregates. Treatment with carbenoxolone and probenecid, which directly and specifically block Panx1 channels, respectively, showed that Panx1 is involved in accelerating aggregate assembly. Experiments further showed that exogenous ATP can reverse the inhibitive effects of carbenoxolone and that aggregate compaction is sensitive to the purinergic antagonist suramin. With a close examination of the F-actin microfilament network, these findings show that Panx1 channels act as conduits for ATP release that stimulate the P(2)X(7) purinergic receptor pathway, in turn up-regulating actomyosin function. Using a unique three-dimensional scaffold-free method to quantify multicellular interactions, this study shows that Panx1 is intimately involved in regulating intercellular biomechanical interactions pivotal in the progression of cancer.

Tumor-suppressive effects of pannexin 1 in C6 glioma cells.

Mammalian gap junction proteins, connexins, have long been implicated in tumor suppression. Recently, a novel family of proteins named pannexins has been identified as the mammalian counterpart of the invertebrate gap junction proteins, innexins. To date, pannexin 1 (Panx1) and pannexin 2 (Panx2) mRNAs are reported to be expressed in the brain. Most neoplastic cells, including rat C6 gliomas, exhibit reduced connexin expression, aberrant gap junctional intercellular communication (GJIC), and an increased proliferation rate. When gap junctions are up-regulated by transfecting C6 cells with connexin43, GJIC is restored and the proliferation is reduced. In this study, we examined the tumor-suppressive effects of Panx1 expression in C6 cells. Reverse transcription-PCR analysis revealed that C6 cells do not express any of the pannexin transcripts, whereas its nontumorigenic counterpart, rat primary astrocytes, exhibited mRNAs for all three pannexins. On generation of stable C6 transfectants with tagged Panx1 [myc or enhanced green fluorescent protein (EGFP)], a localization of Panx1 expression to the Golgi apparatus and plasma membrane was observed. In addition, Panx1 transfectants exhibited a flattened morphology, which differs greatly from the spindle-shaped control cells (EGFP only). Moreover, Panx1 expression increased gap junctional coupling as shown by the passage of sulforhodamine 101. Finally, we showed that stable expression of Panx1 in C6 cells significantly reduced cell proliferation in monolayers, cell motility, anchorage-independent growth, and in vivo tumor growth in athymic nude mice. Altogether, we conclude that the loss of pannexin expression may participate in the development of C6 gliomas, whereas restoration of Panx1 plays a tumor-suppressive role.