Challenges and directions in studying cell-cell communication by extracellular vesicles.

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these.

The role of icIL-1RA in keratinocyte senescence and development of the senescence-associated secretory phenotype.

There is compelling evidence that senescent cells, through the senescence-associated secretory phenotype (SASP), can promote malignant transformation and invasion. Interleukin-1 (IL-1) is a key mediator of this cytokine network, but the control of its activity in the senescence programme has not been elucidated. IL-1 signalling is regulated by IL-1RA, which has four variants. Here, we show that expression of intracellular IL-1RA type 1 (icIL-1RA1), which competitively inhibits binding of IL-1 to its receptor, is progressively lost during oral carcinogenesis ex vivo and that the pattern of expression is associated with keratinocyte replicative fate in vitro We demonstrate that icIL-1RA1 is an important regulator of the SASP in mortal cells, as CRISPR/Cas9-mediated icIL-1RA1 knockdown in normal and mortal dysplastic oral keratinocytes is followed by increased IL-6 and IL-8 secretion, and rapid senescence following release from RhoA-activated kinase inhibition. Thus, we suggest that downregulation of icIL-1RA1 in early stages of the carcinogenesis process can enable the development of a premature and deregulated SASP, creating a pro-inflammatory state in which cancer is more likely to arise.

Oral cancer stem cells drive tumourigenesis through activation of stromal fibroblasts.

BACKGROUND: Cancer stem cells are responsible for tumour progression and chemoresistance. Fibroblasts surrounding a tumour also promote progression and fibroblast "activation" is an independent prognostic marker in oral cancer. Cancer stem cells may therefore promote tumourigenesis through communication with stromal fibroblasts. METHODS: Cancer stem cells were isolated from oral cancer cell lines by adherence to fibronectin or cisplatin resistance. Fibroblasts were exposed to conditioned medium from these cells, and the activation markers, alpha smooth muscle actin and interleukin-6, were assessed using qPCR and immunofluorescence. Stem cell markers and smooth muscle actin were examined in oral cancer tissue using immunohistochemistry. RESULTS: Adherent and chemoresistant cells expressed increased levels of stem cell markers CD24, CD44 and CD29 compared with unsorted cells. Adherent cells exhibited lower growth rate, higher colony forming efficiency and increased cisplatin resistance than unsorted cells. Smooth muscle actin and Interleukin-6 expression were increased in fibroblasts exposed to conditioned medium. In oral cancer tissue, there was a positive correlation between expression of αSMA and stem cell markers. CONCLUSIONS: Adherence to fibronectin and chemoresistance isolates stem-like cells that can activate fibroblasts, which together with a correlation between markers of both in vivo, provides a mechanism by which such cells drive tumourigenesis.

RETRACTED: Proof of concept efficacy study of intranasal stabilized isoamyl nitrite (SIAN) in rhesus monkeys against acute cyanide poisoning.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editors-in-Chief as the authors were unable to provide documentation of approval for the interinstitutional assurance /vertebrate animal section of the paper by the relevant authority, Public Health Service (PHS) Office of Laboratory Animal Welfare (OLAW) in the time that was provided.

Retraction notice to "Proof of concept efficacy study of intranasal stabilized isoamyl nitrite (SIAN) in Rhesus monkeys against acute cyanide poisoning" [Regul. Toxicol. Pharmacol. 123 (2021) 104927].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editors-in-Chief as the authors were unable to provide documentation of approval for the interinstitutional assurance/vertebrate animal section of the paper by the relevant authority, Public Health Service (PHS) Office of Laboratory Animal Welfare (OLAW) in the time that was provided.

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.

Discovery and characterization of ACE2 - a 20-year journey of surprises from vasopeptidase to COVID-19.

Angiotensin-converting enzyme (ACE) is a zinc membrane metallopeptidase that plays a key role in regulating vasoactive peptide levels and hence cardiovascular activity through its conversion of angiotensin I (Ang I) to Ang II and its metabolism of bradykinin. The discovery of its homologue, ACE2, 20 years ago has led to intensive comparisons of these two enzymes revealing surprising structural, catalytic and functional distinctions between them. ACE2 plays multiple roles not only as a vasopeptidase but also as a regulator of amino acid transport and serendipitously as a viral receptor, mediating the cellular entry of the coronaviruses causing severe acute respiratory syndrome (SARS) and, very recently, COVID-19. Catalytically, ACE2 functions as a monocarboxypeptidase principally converting the vasoconstrictor angiotensin II to the vasodilatory peptide Ang-(1-7) thereby counterbalancing the action of ACE on the renin-angiotensin system (RAS) and providing a cardioprotective role. Unlike ACE, ACE2 does not metabolise bradykinin nor is it inhibited by classical ACE inhibitors. However, it does convert a number of other regulatory peptides in vitro and in vivo. Interest in ACE2 biology and its potential as a possible therapeutic target has surged in recent months as the COVID-19 pandemic rages worldwide. This review highlights the surprising discoveries of ACE2 biology during the last 20 years, its distinctions from classical ACE and the therapeutic opportunities arising from its multiple biological roles.

Neonatal and Juvenile Ocular Development in Göttingen Minipigs and Domestic Pigs: A Histomorphological and Immunohistochemical Study.

Pigs are considered one of the relevant animal models for ocular research as they share several histological and anatomical similarities with the human eye. With the increasing interest in juvenile animal models, this study aimed to describe the postnatal development of ocular structures in 16 Göttingen minipigs and 25 F2 domestic pigs, between birth and 6 months of age, using histopathology and immunohistochemistry against Ki-67, caspase-3, calbindin, glial fibrillary acidic protein, rhodopsin, and synaptophysin. All ocular structures in both pig breeds were incompletely developed at birth and for variable periods postnatally. Noteworthy histological features of immaturity included vascularization in the corneal stroma in neonatal Göttingen minipigs, increased cellularity in different substructures, remnants of the hyaloid vasculature, short and poorly ramified ciliary body processes, and a poorly developed cone inner segment. Increased cellular proliferation, highlighted by abundant Ki-67 immunolabeling, was observed in almost all developing structures of the pig eye for variable periods postnatally. Apoptosis, highlighted with caspase-3 immunolabeling, was observed in the retinal inner nuclear layer at birth and in the regressing hyaloid vasculature remnants. Immunohistochemistry against rhodopsin, synaptophysin, and calbindin demonstrated the short size of the developing photoreceptors and the immature cone inner segment morphology. Calbindin labeling revealed significant differences in the amount of positively labeled cone nuclei between the retinal area centralis and the non-area centralis regions. The elongation of Müller cell processes in the developing retina was shown with glial fibrillary acidic protein. In both pig breeds, the eyes reached histomorphological and immunohistochemical maturity at 6 months of age.

Comprehensive functional profiling of long non-coding RNAs through a novel pan-cancer integration approach and modular analysis of their protein-coding gene association networks.

BACKGROUND: Long non-coding RNAs (lncRNAs) are emerging as crucial regulators of cellular processes in diseases such as cancer, although the functions of most remain poorly understood. To address this, here we apply a novel strategy to integrate gene expression profiles across 32 cancer types, and cluster human lncRNAs based on their pan-cancer protein-coding gene associations. By doing so, we derive 16 lncRNA modules whose unique properties allow simultaneous inference of function, disease specificity and regulation for over 800 lncRNAs. RESULTS: Remarkably, modules could be grouped into just four functional themes: transcription regulation, immunological, extracellular, and neurological, with module generation frequently driven by lncRNA tissue specificity. Notably, three modules associated with the extracellular matrix represented potential networks of lncRNAs regulating key events in tumour progression. These included a tumour-specific signature of 33 lncRNAs that may play a role in inducing epithelial-mesenchymal transition through modulation of TGFß signalling, and two stromal-specific modules comprising 26 lncRNAs linked to a tumour suppressive microenvironment and 12 lncRNAs related to cancer-associated fibroblasts. One member of the 12-lncRNA signature was experimentally supported by siRNA knockdown, which resulted in attenuated differentiation of quiescent fibroblasts to a cancer-associated phenotype. CONCLUSIONS: Overall, the study provides a unique pan-cancer perspective on the lncRNA functional landscape, acting as a global source of novel hypotheses on lncRNA contribution to tumour progression.

Extranodal extension in oral cancer: A role for the nodal microenvironment?

Oral squamous cell carcinoma (OSCC) is a significant cause of morbidity and mortality worldwide and accounts for the majority of head and neck cancers. Metastasis of primary tumours, primarily to cervical lymph nodes in the neck, is associated with worsening prognosis. Furthermore, the prognosis of patients with extranodal extension of metastatic tumour from the lymph nodes into the neck tissues is particularly poor. The factors affecting this process are poorly understood, and detection is difficult pre-surgery. Mounting evidence shows that components of the tumour microenvironment including cancer-associated fibroblasts, vascular and lymphatic endothelial cells, the extracellular matrix and inflammatory immune cells, are important modulators of tumour behaviour in primary OSCC and other cancers. However, little is known about the lymph node microenvironment, its response to tumour presence and role in extranodal extension. In addition, there are many lymph node-specific cell types and structures, such as fibroblast reticular cells and high endothelial venules, making the lymph node microenvironment distinct from that found at primary tumour sites, and which contribute to the nodal response to tumour presence. This review details the current knowledge regarding the lymph node tumour microenvironment in OSCC and its role in lymph node metastasis and extranodal extension and relates this to features of the primary tumour. Understanding the role that the lymph node microenvironment plays in promoting tumour development and extranodal extension may aid the identification of novel biomarkers and alternative treatment strategies to improve the prognosis of patients with advanced OSCC.

HPV-negative, but not HPV-positive, oropharyngeal carcinomas induce fibroblasts to support tumour invasion through micro-environmental release of HGF and IL-6.

Human papillomavirus (HPV) infection is causally related to a subset of oropharyngeal carcinomas (OPC) and is linked to a more favourable prognosis compared to HPV-negative OPC. The mechanisms underlying this effect on prognosis are not fully understood, but interactions with the tumour microenvironment may be pivotal. Here, we investigated the role of the tumour microenvironment in HPV-positive compared to HPV-negative cancer using 2D and 3D modelling of OPC interactions with stromal fibroblasts. HPV-negative, but not HPV-positive, OPC-derived cell lines induced a rapid fibroblast secretory response that supported 2D cancer cell migration and invasion in vitro. Array profiling of this HPV-negative induced fibroblast secretome identified hepatocyte growth factor (HGF) as the principal secreted factor that promoted cancer cell migration. The interaction between HPV-negative cell lines and fibroblasts in 2D was prevented using c-Met (HGF receptor) inhibitors, which further restricted both HPV-negative and positive cell invasion in 3D co-culture models. Furthermore, we discovered a synergistic relationship between HGF and IL-6 in the support of migration that relates JAK activation to HGF responsiveness in HPV-negative lines. In summary, our data show significant differences in the interactions between HPV-positive and HPV-negative OPC cells and stromal fibroblasts. In addition, we, provide in vitro evidence to support the clinical application of c-MET inhibitors in the control of early HPV-negative OPC.

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.

Extracellular vesicles: translational challenges and opportunities.

Extracellular vesicles (EVs) are a heterogeneous group of small lipid-enclosed structures with myriad roles in physiology and disease. The recent surge of interest in EVs has led to greater understanding of their biology and appreciation of how they might be utilised as diagnostic and therapeutic tools. There remain, however, a number of challenges that must be overcome before EVs may be used routinely in the clinic. In this review we will discuss the translational potential of EVs and the current technologies available to isolate, purify and analyse EVs and their contents.

Targeting HOX-PBX interactions causes death in oral potentially malignant and squamous carcinoma cells but not normal oral keratinocytes.

BACKGROUND: High HOX gene expression has been described in many cancers, including oral squamous cell carcinoma and the functional roles of these genes are gradually being understood. The pattern of overexpression suggests that inhibition may be useful therapeutically. Inhibition of HOX protein binding to PBX cofactors by the use of synthetic peptides, such as HXR9, results in apoptosis in multiple cancers. METHODS: Activity of the HOX-PBX inhibiting peptide HXR9 was tested in immortalised normal oral (NOK), potentially-malignant (PMOL) and squamous cell carcinoma (OSCC) cells, compared to the inactive peptide CXR9. Cytotoxicity was assessed by LDH assay. Expression of PBX1/2 and c-Fos was assessed by qPCR and western blotting. Apoptosis was assessed by Annexin-V assay. RESULTS: PMOL and OSCC cells expressed PBX1/2. HOX-PBX inhibition by HXR9 caused death of PMOL and OSCC cells, but not NOKs. HXR9 treatment resulted in apoptosis and increased expression of c-Fos in some cells, whereas CXR9 did not. A correlation was observed between HOX expression and resistance to HXR9. CONCLUSION: Inhibition of HOX-PBX interactions causes selective apoptosis of OSCC/PMOL, indicating selective toxicity that may be useful clinically.

Prognostic value of the immunohistochemical detection of cancer-associated fibroblasts in oral cancer: A systematic review and meta-analysis.

AIM: To perform a meta-analysis to assess whether the presence of cancer-associated fibroblasts (CAF) is a prognostic marker of oral squamous cell carcinomas (OSCC). METHODS: Immunohistochemical studies assessing the prognostic relevance of CAF (alpha smooth muscle actin (α-SMA)-positive fibroblasts) in patients with OSCC were systematically reviewed using Cochrane, Lilacs, PubMed, Scopus, and Web of Science databases. The outcomes assessed were overall survival (OS) and disease-free survival (DFS). The meta-analysis was performed using the random- and fixed-effects model with adjusted hazard ratio (HR) and 95% confidence intervals (95% CI) as effect measures. The methodological quality of the included studies was assessed using the Meta-Analysis of Statistics Assessment and Review Instrument (MAStARI) tool, and the evidence quality was assessed by the Grading of Recommendation, Assessment, Development, and Evaluation (GRADE) system. RESULTS: The presence of high levels of CAF in the stroma of OSCC predicted shortened time to DFS (HR = 3.32, 95% CI: 2.09-5.26, P < .00001) and an overall decrease in survival (HR: 2.16, 95% CI: 1.60-2.92, P < .00001). Moreover, high presence of CAF was frequently reported in association with parameters that worsen the prognosis in OSCC, including advanced disease stage (TNM classification), recurrence, tumor grade, depth of invasion, vascular, lymphatic and neural invasion, and extranodal metastatic spread. CONCLUSION: The presence of CAF, as assessed by α-SMA-positive fibroblasts in the stroma, indicates poor prognosis in patients with OSCC.

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.

Continuous-Phase Control of a Powered Knee-Ankle Prosthesis: Amputee Experiments Across Speeds and Inclines.

Control systems for powered prosthetic legs typically divide the gait cycle into several periods with distinct controllers, resulting in dozens of control parameters that must be tuned across users and activities. To address this challenge, this paper presents a control approach that unifies the gait cycle of a powered knee-ankle prosthesis using a continuous, user-synchronized sense of phase. Virtual constraints characterize the desired periodic joint trajectories as functions of a phase variable across the entire stride. The phase variable is computed from residual thigh motion, giving the amputee control over the timing of the prosthetic joint patterns. This continuous sense of phase enabled three transfemoral amputee subjects to walk at speeds from 0.67 to 1.21 m/s and slopes from -2.5 to +9.0 deg. Virtual constraints based on task-specific kinematics facilitated normative adjustments in joint work across walking speeds. A fixed set of control gains generalized across these activities and users, which minimized the configuration time of the prosthesis.

Physiological Fluid Flow Moderates Fibroblast Responses to TGF-ß1.

Fibroblasts are the major cellular component of connective tissue and experience mechanical perturbations due to matrix remodelling and interstitial fluid movement. Transforming growth factor ß1 (TGF-ß1) can promote differentiation of fibroblasts in vitro to a contractile myofibroblastic phenotype characterised by the presence of α-smooth muscle actin (α-SMA) rich stress fibres. To study the role of mechanical stimulation in this process, we examined the response of primary human fibroblasts to physiological levels of fluid movement and its influence on fibroblast differentiation and responses to TGF-ß1. We reported that in both oral and dermal fibroblasts, physiological levels of fluid flow induced widespread changes in gene expression compared to static cultures, including up-regulation of genes associated with TGFß signalling and endocytosis. TGF-ß1, activin A and markers of myofibroblast differentiation including α-SMA and collagen IA1 were also increased by flow but surprisingly the combination of flow and exogenous TGF-ß1 resulted in reduced differentiation. Our findings suggest this may result from enhanced internalisation of caveolin and TGF-ß receptor II. These findings suggest that a) low levels of fluid flow induce myofibroblast differentiation and b) fluid flow antagonises the fibroblast response to pro-differentiation signals such as TGF-ß1. We propose that this may be a novel mechanism by which mechanical forces buffer responses to chemical signals in vivo, maintaining a context-specific fibroblast phenotype. J. Cell. Biochem. 118: 878-890, 2017. © 2016 Wiley Periodicals, Inc.

Effects of Src-kinase inhibition in cancer-induced bone pain.

BACKGROUND: Bone metastases occur frequently in advanced breast, lung, and prostate cancer, with approximately 70% of patients affected. Pain is a major symptom of bone metastases, and current treatments may be inadequate or have unacceptable side effects. The mechanisms that drive cancer-induced bone pain are not fully understood; however, it is known that there is sensitization of both peripheral bone afferents and central spinal circuits. It is well established that the N-methyl-D-aspartate receptor plays a major role in the pathophysiology of pain hypersensitivity. Inhibition of the non-receptor tyrosine kinase Src controls N-methyl-D-aspartate receptor activity and inhibiting Src reduces the hypersensitivity associated with neuropathic and inflammatory pains. As Src is also implicated in osteoclastic bone resorption, we have investigated if inhibiting Src ameliorates cancer-induced bone pain. We have tested this hypothesis using an orally bioavailable Src inhibitor (saracatinib) in a rat model of cancer-induced bone pain. RESULTS: Intra-tibial injection of rat mammary cancer cells (Mammary rat metastasis tumor cells -1), but not vehicle, in rats produced hindpaw hypersensitivity to thermal and mechanical stimuli that was maximal after six days and persisted for at least 13 days postinjection. Daily oral gavage with saracatinib (20 mg/kg) beginning seven days after intra-tibial injection reversed the thermal hyperalgesia but not the mechanical allodynia. The analgesic mechanisms of saracatinib appear to be due to an effect on the nervous system as immunoblotting of L2-5 spinal segments showed that mammary rat metastasis tumor cells-1 injection induced phosphorylation of the GluN1 subunit of the N-methyl-D-aspartate receptor, indicative of receptor activation, and this was reduced by saracatinib. Additionally, histology showed no anti-tumor effect of saracatinib at any dose and no significant effect on bone preservation. CONCLUSIONS: This is the first demonstration that Src plays a role in the development of cancer-induced bone pain and that Src inhibition represents a possible new analgesic strategy for patients with bone metastases.

Characterization of the pH and Temperature in the Rabbit, Pig, and Monkey Eye: Key Parameters for the Development of Long-Acting Delivery Ocular Strategies.

Many long-acting delivery strategies for ocular indications rely on pH- and/or temperature-driven release of the therapeutic agent and degradation of the drug carrier. Yet, these physiological parameters are poorly characterized in ocular animal models. These strategies aim at reducing the frequency of dosing, which is of particular interest for the treatment of chronic disorders affecting the posterior segment of the eye, such as macular degeneration that warrants monthly or every other month intravitreal injections. We used anesthetized white New Zealand rabbits, Yucatan mini pigs, and cynomolgus monkeys to characterize pH and temperature in several vitreous locations and the central aqueous location. We also established post mortem pH changes in the vitreous. Our data showed regional and species differences, which need to be factored into strategies for developing biodegradable long-acting delivery systems.