Cellular Responses to Extracellular Vesicles as Potential Markers of Colorectal Cancer Progression.

The development of novel screening tests aims to support early asymptomatic diagnosis and subtyping patients according to similar traits in the heterogeneous cancer cohort. Extracellular vesicles (EVs) are promising candidates for the detection of disease markers from bodily fluids, but limitations in the standardisation of isolation methods and the intrinsic EV heterogeneity obtained from liquid biopsies are currently obstacles to clinical adoption. Here, cellular responses to cancer EVs were initially explored as potential complementary biomarkers for stage separation using colorectal cancer (CRC) SW480 and SW620 cell line models. A pilot study on a small cohort of CRC patients and controls was then developed by performing a multivariate analysis of cellular responses to plasma-derived EVs. Several cell activities and markers involved in tumour microenvironment pathways were influenced by the treatment of cell line EVs in a stage-dependent manner. The multivariate analysis combining plasma EV markers and cellular responses to plasma EVs was able to separate patients according to disease stage. This preliminary study offers the potential of considering cellular responses to EVs in combination with EV biomarkers in the development of screening methods.

Myofibroblast transdifferentiation is associated with changes in cellular and extracellular vesicle miRNA abundance.

Transforming growth factor-beta 1 (TGF-ß1), a pro-fibrotic tumour-derived factor promotes fibroblast differentiation in the tumour microenvironment and is thought to contribute to the development of pro-tumourigenic cancer-associated fibroblasts (CAFs) by promoting myofibroblast differentiation. miRNA dysregulation has been demonstrated in myofibroblast transdifferentiation and CAF activation, however, their expression varies among cell types and with the method of fibroblast induction. Here, the expression profile of miRNA in human primary oral fibroblasts treated with TGF-ß1, to derive a myofibroblastic, CAF-like phenotype, was determined compared to untreated fibroblasts. Myofibroblast transdifferentiation was determined by the expression of alpha-smooth muscle actin (α-SMA) and fibronectin-1 extra domain A (FN-EDA1) using quantitative real-time PCR (qRT-PCR) and western blot. The formation of stress fibres was assessed by fluorescence microscopy, and associated changes in contractility were assessed using collagen contraction assays. Extracellular vesicles (EVs) were purified by using size exclusion chromatography and ultracentrifugation and their size and concentration were determined by nanoparticle tracking analysis. miRNA expression profiling in oral fibroblasts treated with TGF-ß1 and their extracellular vesicles was carried out using tiling low-density array cards. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform functional and pathway enrichment analysis of target genes. In this study, TGF-ß1 induced a myofibroblastic phenotype in normal oral fibroblasts as assessed by expression of molecular markers, the formation of stress fibres and increased contractility. TaqMan Low-Density Array (TLDA) analysis demonstrated that miR-503 and miR-708 were significantly upregulated, while miR-1276 was significantly downregulated in TGF-ß1-treated oral fibroblasts (henceforth termed experimentally-derived CAF, eCAF). The gene functional enrichment analysis showed that the candidate miRNAs have the potential to modulate various pathways; including the Ras associated protein 1 (Rap1), PI3K-Akt, and tumour necrosis factor (TNF) signalling pathways. In addition, altered levels of several miRNAs were detected in eCAF EV, including miR-142 and miR-222. No differences in size or abundance of EV were detected between eCAF and normal oral fibroblast (NOF). Little overlap was observed between changes in cellular and EV miRNA profiles, suggesting the possibility of selective loading of EV miRNA. The study reveals miRNA expression signature could be involved in myofibroblast transdifferentiation and the miRNA cargo of their EV, providing novel insight into the involvement of miRNA in CAF development and function.

Extracellular vesicles and the extracellular matrix: a new paradigm or old news?

Extracellular vesicles (EV) are implicated in a variety of functions affecting the extracellular matrix (ECM), including matrix degradation, cross-linking of matrix proteins and matrix calcification. These processes are important in many physiological contexts such as angiogenesis and wound healing, and dysregulation of ECM homeostasis contributes to a wide range of diseases including fibrosis, cancer and arthritis. Most studies of EV have focussed on their roles in cell:cell communication, but EV can exist as integral components of the ECM. By far the most well-characterised ECM-resident EV are matrix vesicles (MV) in bone, but the broader role of EV in the ECM is not well understood. This review will explore what is known of the roles of EV in the ECM and will also highlight the similarities and differences between MV and other EV.

Efficient isolation, biophysical characterisation and molecular composition of extracellular vesicles secreted by primary and immortalised cells of reproductive origin.

Effective communication between the maternal reproductive tract, gametes and the pre-implantation embryo is essential for the successful establishment of pregnancy. Recent studies have recognised extracellular vesicles (EVs) as potent vehicles for intercellular communication, potentially via their transport of microRNAs (miRNAs). The aim of the current investigation was to determine the size, concentration and electrical surface properties (zeta potential) of EVs secreted by; (1) primary cultures of porcine oviductal epithelial cells (POECs) from the isthmus and ampullary regions of the female reproductive tract; (2) Ishikawa and RL95-2 human endometrial epithelial cell line cultures; and (3) the non-reproductive epithelial cell line HEK293T. In addition, this study investigated whether EVs secreted by POECs contained miRNAs. All cell types were cultured in EV-depleted medium for 24 or 48 h. EVs were successfully isolated from conditioned culture media using size exclusion chromatography. Nanoparticle tracking analysis (NTA) was performed to evaluate EV size, concentration and zeta potential. QRT-PCR was performed to quantify the expression of candidate miRNAs (miR-103, let-7a, miR-19a, miR-203, miR-126, miR-19b, RNU44, miR-92, miR-196a, miR-326 and miR-23a). NTA confirmed the presence of EVs with diameters of 50-150 nm in all cell types. EV size distribution was significantly different between cell types after 24 and 48 h of cell culture and the concentration of EVs secreted by POECs and Ishikawa cells was also time dependent. The distribution of EVs with specific electrokinetic potential measurements varied between cell types, indicating that EVs of differing cellular origin have varied membrane components. In addition, EVs secreted by POECs exhibited significantly different time dependant changes in zeta potential. QRT-PCR confirmed the presence of miR-103, let-7a, miR-19a, miR-203, miR-126, and miR-19b in EVs secreted by POECs (CT ≥ 29). Bioinformatics analysis suggests that these miRNAs are involved in cell proliferation, innate immune responses, apoptosis and cellular migration. In conclusion, reproductive epithelial cells secrete distinct populations of EVs containing miRNAs, which potentially act in intercellular communication in order to modulate the periconception events leading to successful establishment of pregnancy.

Extracellular vesicle microRNA cargo is correlated with HPV status in oropharyngeal carcinoma.

BACKGROUND: The incidence of human papilloma virus positive (HPV+ ) oropharyngeal squamous cell carcinoma (OPSCC) has increased rapidly in recent decades. These tumours have a favourable outcome compared to HPV-negative (HPV- ) OPSCC. However, HPV+ tumours are more likely to metastasise to distant sites, suggesting a difference in how these tumour subtypes interact with the metastatic niche. Extracellular vesicles (EVs) have emerged as important players in cell-to-cell communication and are a potential source of biomarkers for cancer diagnosis. This study aims to characterise the microRNA cargo of small EVs released by HPV+ and HPV- OPSCC cell lines. METHODS: Extracellular vesicles produced by HPV+ (SCC2 and SCC90) and HPV- (SCC72 an SCC89) OPSCC cells were characterised by tunable resistive pulse sensing (TRPS) and western blotting. RNA was extracted from EVs and analysed by small RNA sequencing. A bioinformatics approach was used to identify EV miRNA signatures associated with HPV status. RESULTS: HPV- OPSCC cells produced more EVs than HPV+ OPSCC cells. EVs were positive for the common EV markers CD63, CD9 and TSG101. Unbiased hierarchical clustering analysis of EV miRNA cargo revealed that samples clustered based on HPV status. 14 miRNA were enriched in HPV+ cell-derived EVs, whereas 19 miRNA were enriched in EVs derived from HPV- cell lines. CONCLUSIONS: Here, we identify EV miRNA signatures indicative of the HPV status of the parent cell. This may provide a platform from which to validate salivary or blood-based biomarkers with utility for early detection and stratifying risk in OPSCC patients.

A miRNA-145/TGF-ß1 negative feedback loop regulates the cancer-associated fibroblast phenotype.

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-ß1 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-ß1 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-ß signalling pathway. miR-145 was also overexpressed in CAF derived from oral cancers. Overexpression of miR-145 blocked TGF-ß1-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.

Role of OmpA2 surface regions of Porphyromonas gingivalis in host-pathogen interactions with oral epithelial cells.

Outer membrane protein A (OmpA) is a key outer membrane protein found in Gram-negative bacteria that contributes to several crucial processes in bacterial virulence. In Porphyromonas gingivalis, OmpA is predicted as a heterotrimer of OmpA1 and OmpA2 subunits encoded by adjacent genes. Here we describe the role of OmpA and its individual subunits in the interaction of P. gingivalis with oral cells. Using knockout mutagenesis, we show that OmpA2 plays a significant role in biofilm formation and interaction with human epithelial cells. We used protein structure prediction software to identify extracellular loops of OmpA2, and determined these are involved in interactions with epithelial cells as evidenced by inhibition of adherence and invasion of P. gingivalis by synthetic extracellular loop peptides and the ability of the peptides to mediate interaction of latex beads with human cells. In particular, we observe that OmpA2-loop 4 plays an important role in the interaction with host cells. These data demonstrate for the first time the important role of P. gingivalis OmpA2 extracellular loops in interaction with epithelial cells, which may help design novel peptide-based antimicrobial therapies for periodontal disease.

CydDC-mediated reductant export in Escherichia coli controls the transcriptional wiring of energy metabolism and combats nitrosative stress.

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), ß-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 ΔpH, 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.

Endothelin-1 stimulates motility of head and neck squamous carcinoma cells by promoting stromal-epithelial interactions.

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.

MicroRNA-124 suppresses oral squamous cell carcinoma motility by targeting ITGB1.

Alterations 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' 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.

Hemolysin E (HlyE, ClyA, SheA) and related toxins.

Certain strains of Escherichia coli, Salmonella enterica and Shigella flexneri produce a pore-forming toxin hemolysin E (HlyE), also known as cytolysin A (ClyA) and silent hemolysin, locus A (SheA). HlyE lyses erythrocytes and mammalian cells, forming transmembrane pores with a minimum internal diameter of-25 A. We review the current knowledge of HlyE structure and function in its solution and pore forms, models for membrane insertion, its potential use in biotechnology applications and its relationship to a wider superfamily of toxins.

Severe zinc depletion of Escherichia coli: roles for high affinity zinc binding by ZinT, zinc transport and zinc-independent proteins.

Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn(2+) from the environment, making it exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive understanding of the importance of Zn(2+) has not been attained. Reduction of the Zn(2+) content of Escherichia coli growth medium to 60 nm or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn(2+)-deficient medium had a reduced growth rate and contained up to five times less cellular Zn(2+). To understand global responses to Zn(2+) deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn(2+) or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+) finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn(2+) than was originally added to the culture, presumably because of leaching from the culture vessel. Zn(2+) elimination is shown to be a more precise method of depleting Zn(2+) than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

The formation and structure of Escherichia coli K-12 haemolysin E pores.

Some enteric bacteria synthesize a pore-forming toxin, HlyE, which is cytolytic and cytotoxic to host cells. Measurement of HlyE binding to erythrocyte ghosts and the kinetics of HlyE-mediated erythrocyte lysis suggests that interaction with target membranes is not the rate-limiting step in the formation of HlyE pores, but that there is a temperature-dependent lag phase before a functional pore is formed. Circular dichroism and fluorescence energy transfer analyses show that HlyE protomers retain an alpha-helical structure when oligomerized to form a pore consisting of parallel HlyE protomers. Comparison of the proteolytic sensitivities of the water-soluble and oligomeric forms of HlyE identifies inner and outer surfaces of the pore. This new information has been used to constrain a model of the HlyE pore, which allows a more detailed interpretation of previous low-resolution 3D reconstructions and suggests a novel mechanism for insertion of HlyE into target membranes.

Drug loading and elution from a phosphorylcholine polymer-coated coronary stent does not affect long-term stability of the coating in vivo.

A drug eluting coronary stent was developed for use in preclinical and clinical trial evaluation. The stent was coated with a phosphorylcholine (PC)-based polymer coating containing the cell migration inhibitor batimastat. A pharmacokinetic study was conducted in a rabbit iliac model using (14)C-radiolabeled version of the drug; this showed the drug release to be first order with 94% of it being released within 28 days. Unloaded and drug-loaded stents were implanted in a porcine coronary artery model; a number were explanted at 5 days and scanning electron microscopy was used to show that the presence of the drug did not affect the rate of stent endothelialization. The remainder of the stents were removed after 6 months and the stents carefully removed from the arterial tissue. Fourier-transform infrared (FT-IR) spectroscopy (both attenuated total reflectance and microscopic imaging) was used to show the presence of the PC coating on control unloaded, drug-loaded and explanted stents, providing evidence that the coating was still present. This was further confirmed by use of atomic force microscopy (AFM) amplitude-phase, distance (a-p,d) curves which generated the characteristic traces of the PC coating. Further AFM depth-profiling techniques found that the thicknesses of the PC coatings on an control unloaded stent was 252+/-19 nm, on an control batimastat-loaded stent 906+/-224 nm and on an explanted stent 405+/-224 nm. The increase in thickness after the drug-loading process was a consequence of drug incorporation in the film, and the return to the unloaded dimensions for the explanted sample indicative of elution of the drug from the coating. The drug delivery PC coating was therefore found to be stable following elution of the drug and after 6 months implantation in vivo.