The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

a b s t r a c tAlterations in the levels of molecules which interact with the extracellular matrix, such as integrins, are associated with invasion of oral squamous cell carcinomas (OSCC). The molecular mechanisms underlying dysregulation of integrin expression in OSCC, however, remain unclear. Here, we show that microRNA-124, a small non-coding RNA down-regulated in OSCC, is able to downregulate expression of integrin beta-1 (ITGB1) by interacting with its 3 0 untranslated region. Over-expression of miR-124 attenuates endogenous ITGB1 expression and reduces the adherence and motility of OSCC cells, suggesting disruption of miR-124-mediated repression of ITGB1 may be a key factor in OSCC progression.

The efficacy of current anti-cancer gene therapies is limited by the inability of gene vectors to penetrate the poorly vascularized, hypoxic regions of tumors, leaving these sites untreated. We describe a new approach for targeting gene therapy to these sites, which employs an attenuated strain of the non-pathogenic bacterium, Salmonella typhimurium, carrying an exogenous (that is, reporter or therapeutic) gene under the regulation of a new, highly hypoxia-inducible promoter (FF+20 *). This bacterial vector was seen to rapidly migrate into, and thrive in, hypoxic areas of both mammary tumor spheroids grown in vitro and orthotopic mammary tumors after systemic injection. Using the reporter gene construct, FF+20 *-lacZ, we show that bacterial expression of high levels of b-galactosidase occurred only in hypoxic/necrotic sites of spheroids and tumors. We then replaced the reporter gene with one encoding a novel cytotoxic protein (HlyE) and showed that this was also expressed by bacteria only in hypoxic regions of murine mammary tumors. This resulted in a marked increase in tumor necrosis and reduced tumor growth. Our system represents a promising new strategy for delivering gene therapy to poorly vascularized regions of tumors and shows, for the first time, the efficacy of HlyE as an anti-tumor agent.

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The role of extracellular vesicles (EVs) as vehicles for cell-to-cell communication between a tumour and its environment is a relatively new concept. The hypothesis that EVs may be critical in co-opting tissues by tumours to generate distant metastatic niches is particularly pertinent to prostate cancer (PCa), where metastatic-tropism to bone predominates over other tissue types. The potential role of EVs as a means of communication between PCa cells and cells of the bone stroma such as osteoblasts, is yet to be fully explored. In this study, we demonstrate that PCa cell EVs both enhance osteoblast viability and produce a significantly more supportive growth environment for PCa cells when grown in co-culture with EV-treated osteoblasts ( p < 0.005). Characterisation of the RNA cargo of EVs produced by the bone-metastatic PCa cell line PC3, highlights the EV-RNA cargo is significantly enriched in genes relating to cell surface signalling, cell–cell interaction, and protein translation ( p < 0.01). Using novel techniques to track RNA, we demonstrate the delivery of a set of PCa-RNAs to osteoblast via PCa-EVs and show the effect on osteoblast endogenous transcript abundance. Taken together, by using proof-of-concept studies we demonstrate for the first time the contribution of the RNA element of the PCa EV cargo, providing evidence to support PCa EV communication via RNA molecules as a potential novel route to mediate bone metastasis. We propose targeting PCa EVs could offer a potentially important preventative therapy for men at risk of metastatic PCa.

The invasion and migration of cancer cells is increasingly recognised to be influenced by factors derived from adjacent tumour-associated stroma. The contextual signals regulating stromal-tumour interactions, however, remain poorly understood. Here, we identify a role for endothelin-1 (ET-1), a mitogenic peptide elevated in a number of malignancies, in promoting pro-metastatic cross-talk between head and neck cancer cells and adjacent fibroblasts. We demonstrate that treatment of oral fibroblasts with ET-1 activates ADAM17-mediated release of epidermal growth factor receptor (EGFR) ligands, triggering EGFR signalling and increased motility in neighbouring head and neck cancer cells. ET-1-mediated paracrine transactivation of EGFR also increased cyclo-oxygenase-2 levels in the cancer cells, providing a molecular insight into the mechanisms by which the elevated levels of ET-1 observed in head and neck cancers may contribute to disease progression.It is increasingly apparent that cancer cell migration, invasion and subsequent metastasis are the result of complex interactions between the epithelial tumour cells and the surrounding stroma. 1 Within the tumour-associated stroma, a variety of different cell types are thought to provide contextual signals which promote cancer cell motility, including endothelial cells, inflammatory cells and fibroblasts. 2 Of these, fibroblasts are the most numerous and play a critical role in maintaining homeostasis of the extracellular matrix (ECM). 3 It is proposed that within tumour-associated stroma, fibroblasts become aberrantly activated and contribute to tumour invasion by modifying the ECM and secreting factors which promote cancer cell motility. 3 The factors promoting tumourstromal interactions, however, remain to be fully elucidated.Head and neck cancer (predominantly comprising head and neck squamous cell carcinomas, HNSCC) is the sixth most common cancer worldwide and is increasing in prevalence. It carries a poor prognosis and survival rates have improved little over the last three decades. 4 In comparison with other common cancers, the mechanisms underlying the progression of head and neck cancer are poorly understood, hampering the development of novel therapeutic strategies. The motility of head and neck cancer cells is known to be influenced by soluble factors that bind to specific G-protein coupled receptors (GPCRs) such as bradykinin (BK) 5 and gastrin releasing peptide. 6 These act in an autocrine manner, directly binding to receptors on cancer cells to promote invasion and migration by transactivating epidermal growth factor receptor (EGFR) signalling. 5 EGFR signalling is an important regulator of cellular processes such as proliferation, differentiation, apoptosis and migration. 7 Aberrant EGFR activation is found in a wide variety of malignancies, including HNSCC, resulting from mutation, overexpression or the elevation of local levels of soluble ligands such as TGFa, amphiregulin and HB-EGF. 7 Binding of these ligands induces dimerisation and autophosphorylat...

The dissemination of cancer cells to local and distant sites depends on a complex and poorly understood interplay between malignant cells and the cellular and non-cellular components surrounding them, collectively termed the tumour microenvironment. One of the most abundant cell types of the tumour microenvironment is the fibroblast, which becomes corrupted by locally derived cues such as TGF-β1 and acquires an altered, heterogeneous phenotype (cancer-associated fibroblasts, CAF) supportive of tumour cell invasion and metastasis. Efforts to develop new treatments targeting the tumour mesenchyme are hampered by a poor understanding of the mechanisms underlying the development of CAF. Here, we examine the contribution of microRNA to the development of experimentally-derived CAF and correlate this with changes observed in CAF derived from tumours. Exposure of primary normal human fibroblasts to TGF-β1 resulted in the acquisition of a myofibroblastic CAF-like phenotype. This was associated with increased expression of miR-145, a miRNA predicted in silico to target multiple components of the TGF-β signalling pathway. miR-145 was also overexpressed in CAF derived from oral cancers. Overexpression of miR-145 blocked TGF-β1-induced myofibroblastic differentiation and reverted CAF towards a normal fibroblast phenotype. We conclude that miR-145 is a key regulator of the CAF phenotype, acting in a negative feedback loop to dampen acquisition of myofibroblastic traits, a key feature of CAF associated with poor disease outcome.

The glutathione/cysteine exporter CydDC maintains redox balance in Escherichia coli. A cydD mutant strain was used to probe the influence of CydDC upon reduced thiol export, gene expression, metabolic perturbations, intracellular pH homoeostasis and tolerance to nitric oxide (NO). Loss of CydDC was found to decrease extracytoplasmic thiol levels, whereas overexpression diminished the cytoplasmic thiol content. Transcriptomic analysis revealed a dramatic up-regulation of protein chaperones, protein degradation (via phenylpropionate/phenylacetate catabolism), β-oxidation of fatty acids and genes involved in nitrate/nitrite reduction. 1 H NMR metabolomics revealed elevated methionine and betaine and diminished acetate and NAD + in cydD cells, which was consistent with the transcriptomics-based metabolic model. The growth rate and pH, however, were unaffected, although the cydD strain did exhibit sensitivity to the NO-releasing compound NOC-12. These observations are consistent with the hypothesis that the loss of CydDC-mediated reductant export promotes protein misfolding, adaptations to energy metabolism and sensitivity to NO. The addition of both glutathione and cysteine to the medium was found to complement the loss of bd-type cytochrome synthesis in a cydD strain (a key component of the pleiotropic cydDC phenotype), providing the first direct evidence that CydDC substrates are able to restore the correct assembly of this respiratory oxidase. These data provide an insight into the metabolic flexibility of E. coli, highlight the importance of bacterial redox homoeostasis during nitrosative stress, and report for the first time the ability of periplasmic low molecular weight thiols to restore haem incorporation into a cytochrome complex.