Extracellular vesicles (EVs) may contain a variety of molecular cargo, including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated that vault RNA (vtRNA) and major vault protein (MVP) were highly abundant in EV pellets enriched by differential centrifugation (DC) by qPCR and western blotting, respectively. RNase and proteinase treatment of DC-derived pellets demonstrated that most vtRNA and MVP were not enclosed and protected within an EV membrane. Vault-like particles were visualised in 100k DC pellets by cryo-transmission electron microscopy. EVs were enriched by size exclusion chromatography, and western blotting of individual fractions showed co-elution of EV markers and vault particle proteins. Immunocapture of EVs post-ultracentrifugation (100k DC pellet) showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.

3D in vitro systems offer advantages over the shortcomings of two-dimensional models by simulating the morphological and functional features of in vivo-like environments, such as cell-cell and cell-extracellular matrix interactions, as well as the co-culture of different cell types. Nevertheless, these systems present technical challenges that limit their potential in cancer research requiring cell line- and culture-dependent standardization. This protocol details the use of a magnetic 3D bioprinting method and other associated techniques (cytotoxicity assay and histological analysis) using oral squamous cell carcinoma cell line, HSC3, which offer advantages compared to existing widely used approaches. This protocol is particularly timely, as it validates magnetic bioprinting as a method for the rapid deployment of 3D cultures as a tool for compound screening and development of heterotypic cultures such as co-culture of oral squamous cell carcinoma cells with cancer-associated fibroblasts (HSC3/CAFs).

IntroductionLinking olfactory epithelium to the central nervous system are cranial nerve 1, the olfactory nerve, and cranial nerve “0,” and the nervus terminalis (NT). Since there is minimal expression of angiotensin-converting enzyme-2 (ACE-2) in the olfactory nerve, it is unclear how SARS-CoV-2 causes anosmia (loss of smell) and hypogeusia (reduction of taste). In animal models, NT expresses ACE-2 receptors, suggesting a possible SARS-CoV-2 viral entry site in humans. The purpose of this study was to determine whether ultra-high-field 7 T magnetic resonance imaging (MRI) could visualize the NT, olfactory bulbs (OB), and olfactory tract (OT) in healthy controls and COVID-19 anosmia or hypogeusia and to qualitatively assess for volume loss and T2 alterations.MethodsIn this study, 7 T MRI was used to evaluate the brain and olfactory regions in 45 COVID-19 patients and 29 healthy controls. Neuroimaging was qualitatively assessed by four board-certified neuroradiologists who were blinded to outcome assignments: for the presence or absence of NT; for OB, OT, and brain volume loss; and altered T2 signal, white matter T2 hyperintensities, microhemorrhages, enlarged perivascular spaces, and brainstem involvement.ResultsNT was identifiable in all COVID-19 patients and controls. T2 hyperintensity in the NT, OB, and OT in COVID-19 patients with anosmia or hypogeusia was statistically significant compared to controls and COVID-19 patients without anosmia or hypogeusia.DiscussionOn 7 T MRI, NT was radiographically identifiable, adjacent to OB and OT. In COVID-19 anosmia and hypogeusia, T2 hyperintensity of NT, OB, and OT was statistically significant compared to COVID-19 patients without anosmia or hypogeusia and controls. The NT may be a potential entry site for SARs-CoV-2 and may play a role in the pathophysiology of COVID-19 anosmia.

Hypertrophic scarring results from myofibroblast differentiation and persistence during wound healing. Currently no effective treatment for hypertrophic scarring exists however, autologous fat grafting has been shown to improve scar elasticity, appearance, and function. The aim of this study was to understand how paracrine factors from adipose tissues and adipose-derived stromal cells (ADSC) affect fibroblast to myofibroblast differentiation. The transforming growth factor-β1 (TGF-β1) induced model of myofibroblast differentiation was used to test the effect of conditioned media from adipose tissue, ADSC or lipid on the proportion of fibroblasts and myofibroblasts. Adipose tissue conditioned media inhibited the differentiation of fibroblasts to myofibroblasts but this inhibition was not observed following treatment with ADSC or lipid conditioned media. Hepatocyte growth factor (HGF) was readily detected in the conditioned medium from adipose tissue but not ADSC. Cells treated with HGF, or fortinib to block HGF, demonstrated that HGF was not responsible for the inhibition of myofibroblast differentiation. Conditioned media from adipose tissue was shown to reduce the proportion of myofibroblasts when added to fibroblasts previously treated with TGF-β1, however, conditioned media treatment was unable to significantly reduce the proportion of myofibroblasts in cell populations isolated from scar tissue. Cultured ADSC or adipocytes have been the focus of most studies, however, this work highlights the importance of considering whole adipose tissue to further our understanding of fat grafting. This study supports the use of autologous fat grafts for scar treatment and highlights the need for further investigation to determine the mechanism.

IntroductionHPV-associated oropharyngeal squamous cell carcinoma (OPSCC) shows distinct biological and clinical behaviour when compared to HPV-negative OPSCC. The overall role of the tumour microenvironment (TME) in head and neck cancer progression and metastasis has been studied intensively, but differences in HPV-negative and HPV-positive OPSCCs are less understood.ObjectiveTo investigate the role of cancer-associated fibroblasts (CAFs) and the functional interactions of normal tonsil fibroblasts (NTFs) and OP CAFs with HPV+ and HPV− OPSCC cells and explore novel candidates in tumour-fibroblast crosstalk.Materials and methodsA retrospective cohort of 143 primary OPSCCs was characterised using HPV16/18 RNAScope assay, p16 IHC and ɑ-SMA. Four OPSCC, three NTF and 2 new OPSCC CAF cultures were used to assess the cytokine-based interactions using cytokine arrays on conditioned media (CM), followed by co-culture approaches to identify the role of individual cell types and the role of OPN (SPP1) and IL-6 in SCC/fibroblast communication.ResultsHPV status was associated with better overall survival. Although ɑ-SMA expression was observed in both OPSCC subtypes, it provided survival stratification only in the HPV−positive group (Log-Rank p = 0.02). Three normal tonsillar fibroblast cultures (NTFs) were characterised by induction of myofibroblastic and senescent phenotypes with similar reactivity to our published NOF phenotype. The OPSCC-derived CAF cultures were characterised and their baseline myofibroblastic and senescence phenotypes varied. Cytokine array analysis of CM to identify novel candidates in the crosstalk between OPSCC tumour cells and NTFs/CAFs identified differences in the cytokine profiles on comparison of HPV+ and HPV− OPSCC cells. Osteopontin (OPN/SPP1) was identified, particularly in HPV-negative OPSCC cell analyses. We have demonstrated that OPN was produced by the OPSCC cells and revealed an associated upregulation of IL-6 in fibroblasts. Treatment of NTFs with rOPN showed alteration in phenotype, including increased contraction and IL-6 production. Antibody-mediated inhibition of CD44v6 attenuated the production of IL-6 by OPN in NTFs.ConclusionThis investigation with OPSCC fibroblasts provides novel insights into the role of CAFs in OPSCC mediated by IL-6 stimulated release of OPN from HPV negative OPSCC cells. The details of HPV-positive SCC cell/fibroblast cytokine crosstalk remain elusive.

Oral squamous cell carcinoma (OSCC) is the most common and lethal type of head and neck cancer in the world. Variable response and acquisition of resistance to traditional therapies show that it is essential to develop novel strategies that can provide better outcomes for the patient. Understanding of cellular and molecular mechanisms of cell death control has increased rapidly in recent years. Activation of cell death pathways, such as the emerging forms of non-apoptotic programmed cell death, including ferroptosis, pyroptosis, necroptosis, NETosis, parthanatos, mitoptosis and paraptosis, may represent clinically relevant novel therapeutic opportunities. This systematic review summarizes the recently described forms of cell death in OSCC, highlighting their potential for informing diagnosis, prognosis and treatment. Original studies that explored any of the selected cell deaths in OSCC were included. Electronic search, study selection, data collection and risk of bias assessment tools were realized. The literature search was carried out in four databases, and the extracted data from 79 articles were categorized and grouped by type of cell death. Ferroptosis, pyroptosis, and necroptosis represented the main forms of cell death in the selected studies, with links to cancer immunity and inflammatory responses, progression and prognosis of OSCC. Harnessing the potential of these pathways may be useful in patient-specific prognosis and individualized therapy. We provide perspectives on how these different cell death types can be integrated to develop decision tools for diagnosis, prognosis, and treatment of OSCC.

Cell-in-cell (CIC) structures contribute to tumor aggressiveness and poor prognosis in oral squamous cell carcinoma (OSCC). In vitro 3D models may contribute to the understanding of the underlying molecular mechanisms of these events. We employed a spheroid model to study the CIC structures in OSCC. Spheroids were obtained from OSCC (HSC3) and cancer-associated fibroblast (CAF) lines using the Nanoshuttle-PLTM bioprinting system (Greiner Bio-One). Spheroid form, size, and reproducibility were evaluated over time (EvosTM XL; ImageJ version 1.8). Slides were assembled, stained (hematoxylin and eosin), and scanned (Axio Imager Z2/VSLIDE) using the OlyVIA System (Olympus Life Science) and ImageJ software (NIH) for cellular morphology and tumor zone formation (hypoxia and/or proliferative zones) analysis. CIC occurrence, complexity, and morphology were assessed considering the spheroid regions. Well-formed spheroids were observed within 6 h of incubation, showing the morphological aspects of the tumor microenvironment, such as hypoxic (core) and proliferative zone (periphery) formation. CIC structures were found in both homotypic and heterotypic groups, predominantly in the proliferative zone of the mixed HSC3/CAF spheroids. "Complex cannibalism" events were also noted. These results showcase the potential of this model in further studies on CIC morphology, formation, and relationship with tumor prognosis.

Extracellular vesicles (EVs) may contain a variety of molecular cargo including proteins and nucleic acids. Vault particle components have been repeatedly reported in the literature as EV cargo. Here, we demonstrated by small RNA sequencing that vault RNA (vtRNA) were highly abundant in EV pellets enriched by differential centrifugation. EVs were prepared by commonly used enrichment methods and biochemical assays used to determine whether vault particle components were bona fide EV cargo. EVs were isolated by differential centrifugation, size exclusion chromatography (SEC) and Dynabead immunocapture. RNase and proteinase treatment of EV preparations demonstrated that most vtRNA and major vault protein (MVP) were not enclosed and protected within the EV membrane. Vault-like particles were visualised in differential centrifugation pellets by cryo-transmission electron microscopy. EVs enriched by size exclusion chromatography and those isolated by immunocapture post-ultracentrifugation showed co-purification of MVP, whereas EVs isolated by direct immunocapture from conditioned medium were MVP-negative. Taken together, commonly used isolation techniques, such as differential centrifugation and SEC, can lead to contamination of EVs with vault particles. The current study highlights the importance of determining the topology of putative EV-associated components to determine if they are EV cargo or contaminants that have been co-purified.

In vivo, quiescent fibroblasts reside in three-dimensional connective tissues and are activated in response to tissue injury before proliferating rapidly and becoming migratory and contractile myofibroblasts. When deregulated, chronic activation drives fibrotic disease. Fibroblasts cultured on stiff 2D surfaces display a partially activated phenotype, whilst many 3D environments limit fibroblast activation. Cell mechanotransduction, spreading, polarity, and integrin expression are controlled by material mechanical properties and micro-architecture. Between 3D culture systems, these features are highly variable, and the challenge of controlling individual properties without altering others has led to an inconsistent picture of fibroblast behaviour. Electrospinning offers greater control of mechanical properties and microarchitecture making it a valuable model to study fibroblast activation behaviour in vitro. Here, we present a comprehensive characterisation of the activation traits of human oral fibroblasts grown on a microfibrous scaffold composed of electrospun polycaprolactone. After over 7 days in the culture, we observed a reduction in proliferation rates compared to cells cultured in 2D, with low KI67 expression and no evidence of cellular senescence. A-SMA mRNA levels fell, and the expression of ECM protein-coding genes also decreased. Electrospun fibrous scaffolds, therefore, represent a tuneable platform to investigate the mechanisms of fibroblast activation and their roles in fibrotic disease.

Neuropathic pain is a debilitating condition affecting around 8% of the adult population in the UK. The pathophysiology is complex and involves a wide range of processes, including alteration of neuronal excitability and synaptic transmission, dysregulated intracellular signalling and activation of pro-inflammatory immune and glial cells. In the past 15 years, multiple miRNAs–small non-coding RNA–have emerged as regulators of neuropathic pain development. They act by binding to target mRNAs and preventing the translation into proteins. Due to their short sequence (around 22 nucleotides in length), they can have hundreds of targets and regulate several pathways. Several studies on animal models have highlighted numerous miRNAs that play a role in neuropathic pain development at various stages of the nociceptive pathways, including neuronal excitability, synaptic transmission, intracellular signalling and communication with non-neuronal cells. Studies on animal models do not always translate in the clinic; fewer studies on miRNAs have been performed involving human subjects with neuropathic pain, with differing results depending on the specific aetiology underlying neuropathic pain. Further studies using human tissue and liquid samples (serum, plasma, saliva) will help highlight miRNAs that are relevant to neuropathic pain diagnosis or treatment, as biomarkers or potential drug targets.

One of the ways in which cancer associated fibroblasts (CAF) influence the tumour-microenvironment (TME) is by releasing soluble factors to elicit responses in nearby cells. These factors may be released by modification of the extracellular matrix (ECM), secretion from intracellular compartments, or proteolytic cleavage from the cell surface. A major mediator of proteolytic processing of cell surface proteins is the ‘a disintegrin and metalloproteinase’ (ADAM) family, commonly ADAM17. The role of ADAM17 in CAF, however, remains largely unknown. Here, we show that expression of ADAM17 was induced in normal oral fibroblasts (NOF) and CAF by exposure to oral cancer cell-derived conditioned medium and depletion of ADAM17 reduced the ability of CAF to promote cancer cell migration by negatively regulating cancer cell-associated N-cadherin. Proteomic analysis of ADAM17-depleted CAF revealed changes in the expression of pro-tumorigenic proteins, including fibroblast growth factor 2 (FGF2). Inhibition of FGF2/FGFR1 signalling abrogated the pro-migratory effects of CAF by reducing cancer cell-associated N-cadherin, an effect rescued by addition of recombinant FGF2. Taken together, these results indicate a novel molecular mechanism underpinning cancer cell migration in which tumour-derived factors induce ADAM17 expression in CAF, thus initiating a feed-forward loop wherein CAF release FGF2 to stimulate N-cadherin-dependent cancer cell migration.

Extracellular vesicles (EVs) are small, membrane-enclosed vesicles released by cells into the extracellular milieu. They are found in all body fluids and contain a variety of functional cargo including DNA, RNA, proteins, glycoproteins and lipids, able to provoke phenotypic responses in cells, both locally and at distant sites. They are implicated in a wide array of physiological and pathological processes and hence have attracted considerable attention in recent years as potential therapeutic targets, drug delivery vehicles and biomarkers of disease. In this review we summarise the major functions of EVs in health and disease and discuss their translational potential, highlighting opportunities of - and challenges to - capitalising on our rapidly increasing understanding of EV biology for patient benefit.image Abstract figure legend Schematic illustration of the clinical translational potential of extracellular vesicles from body fluids as biomarkers (top panel) and therapeutic agents (lower panel). Created with BioRender.com.image

Objective: The study aimed to investigate the role of the PGN2012 gene of the periodontitis contributing pathobiont Porphyromonas gingivalis. PGN2012 is a homolgue of TolC and is a gene our group previously showed was overexpressed in hyperinvasive cells. Methods: The study used a combination of bioinformatics, knockout mutagenesis, growth experiments, biofilm assays and human cell invation assays to investigate PGN2012 function.Results: Bioinformatics identified that PGN2012 is part of one of four TolC containing gene loci in P. gingivalis that we predicted may encode a metal resistance RND family tripartite pump, similar to those present in other Gram-negative bacteria, but which are not well understood in anaerobic bacteria. A DPGN2012 deletion displayed slightly reduced growth in liquid culture but did not effect biofilm for-mation or human cell invasion. When metal ions were included in the medium the mutant displayed significantly increased sensitivity to the divalent metal ions Zn2 thorn (500 mM), Co2 thorn (2 mM), and Cd2 thorn (0.1 mM) but not Cu2 thorn .Conclusions: We propose to rename the PGN2012-2014 genes czcCBA, which we suggest plays a role in intracellular stress resistance where zinc is often employed by host cells in antibacterial defence with implications for chronic infection in humans.(c) 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

For over a century, cells within other cells have been detected by pathologists as common histopathological findings in tumors, being generally identified as "cell-in-cell" structures. Despite their characteristic morphology, these structures can originate from various processes, such as cannibalism, entosis and emperipolesis. However, only in the last few decades has more attention been given to these events due to their importance in tumor development. In cancers such as oral squamous cell carcinoma, cell-in-cell events have been linked to aggressiveness, metastasis, and therapeutic resistance. This review aims to summarize relevant information about the occurrence of various cell-in-cell phenomena in the context of oral squamous cell carcinoma, addressing their causes and consequences in cancer. The lack of a standard terminology in diagnosing these events makes it difficult to classify the existing cases and to map the behavior and impacts of these structures. Despite being frequently reported in oral squamous cell carcinoma and other cancers, their impacts on carcinogenesis aren't fully understood. Cell-in-cell formation is seen as a survival mechanism in the face of a lack of nutritional availability, an acid microenvironment and potential harm from immune cell defense. In this deadly form of competition, cells that engulf other cells establish themselves as winners, taking over as the predominant and more malignant cell population. Understanding the link between these structures and more aggressive behavior in oral squamous cell carcinoma is of paramount importance for their incorporation as part of a therapeutic strategy.

The periodontal pathogen Tannerella forsythia is associated with severe periodontitis, and expresses NanH sialidases that cleave sialic acids by hydrolyzing the glycosidic bonds to underlying sugars. Palmatine and berberine chloride are plant-derived alkaloids with pharmacological effects, including anti-inflammatory and anti-bacterial properties. Recombinant NanH sialidase was purified using HisTag affinity chromatography while sialidase activity was determined using 4-methylumbelliferyl N-acetyl-α-D-neuraminic acid (MUNANA) as a substrate. The individual and synergistic effects of palmatine and berberine chloride on NanH sialidase inhibition was determined as well as their antimicrobial effects. The IC50 values of palmatine and berberine chloride were found to be 0.143 and 0.474 mM respectively. A significant synergistic effect was observed when a 0.20 mM:0.50 mM Palmatine:Berberine chloride mixture was used, inhibiting NanH sialidase by almost 100%, as compared to 0.2 mM palmatine and 0.5 mM berberine chloride invidually, which inhibit sialidase activityby 60.33 and 55.94%, respectively. Additionally, an antimicrobial viability assay was conducted and, 0.5 mM palmatine and 0.45 mM berberine showed a significant antimicrobial activity against Tannerella forsythia. Lastly, to examine potential toxicity to host cells, thecytotoxic effects of palmatine (0.15 mM) on H357 oral squamous carcinoma cells was investigated using a trypan blue assay and palmatine was found not to be toxic. In summary, a combination of palmatine and berberine display significant synergistic inhibitory effects on NanH with minimal cytotoxic effects as well as potential antimicrobialeffects on the oral pathogen T. forsythia. Suggesting thatthese compounds may have potential for future development.

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.

Traditional monolayer culture fails to fully recapitulate the in vivo environment of connective tissue cells such as the fibroblast. When cultured on stiff two-dimensional (2D) plastic, fibroblasts become highly proliferative forming broad lamellipodia and stress fibers. Conversely, in different three-dimensional (3D) culture systems, fibroblasts have displayed a diverse array of features; from an "activated" phenotype like that observed in 2D cultures and by myofibroblasts, to a quiescent state that likely better represents in vivo fibroblasts at rest. Today, a plethora of microfabrication techniques have made 3D culture commonplace, for both tissue engineering purposes and in the study of basic biological interactions. However, establishing the in vivo mimetic credentials of different biomimetic materials is not always straightforward, particularly in the context of fibroblast responses. Fibroblast behavior is governed by the complex interplay of biological features such as integrin binding sites, material mechanical properties that influence cellular mechanotransduction, and microarchitectural features like pore and fiber size, as well as chemical cues. Furthermore, fibroblasts are a heterogeneous group of cells with specific phenotypic traits dependent on their tissue of origin. These features have made understanding the influence of biomaterials on fibroblast behavior a challenging task. In this study, we present a review of the strategies used to investigate fibroblast behavior with a focus on the material properties that influence fibroblast activation, a process that becomes pathological in fibrotic diseases and certain cancers. Impact statement This review considers the range of materials that have been used to investigate fibroblast behavior in three-dimensional (3D) culture. It evaluates the merits of each material, the results gained, and the evolving rationale behind the presented studies. We highlight aspects of 3D culture from porosity to polarity and their varied impact on fibroblast behavior. Understanding fibroblast behavior and mechanisms of control in vitro not only serves as a direct therapeutic opportunity in diseases from cancer to fibrosis but also can facilitate progress for future regenerative therapies.

Objectives. In the progression of cancer, interactions between cancer cells and cancer-associated fibroblasts (CAFs) play important roles. Cancer cell invasion is facilitated by filamentous actin (F-actin)-rich membrane protrusions called invadopodia, and the relationship between CAFs and invadopodia has been unclear. We used oral squamous cell carcinoma (OSCC) to investigate CAFs’ effects on the formation of invadopodia, and we assessed the expressions of invadopodia markers and CAF markers ex vivo and their relationship with clinical parameters and survival. Materials and Methods. We examined the effect of culture with normal oral fibroblast (NOF)-derived and CAF-derived conditioned medium on the migration and invasion of two OSCC-derived cell lines using Transwells in the absence/presence of Matrigel. Immunoblotting and immunocytochemistry were conducted to assess the expressions of the invadopodia markers tyrosine kinase substrate 5 (Tks5) and membrane type 1 matrix metalloproteinase (MT1-MMP). We also used immunohistochemistry to examine patients with OSCC for an evaluation of the relationship between the CAF marker alpha smooth muscle actin (αSMA) and the expression of Tks5. The patients’ survival was also assessed. Results. Compared to the use of culture medium alone, NOF-CM and CAF-CM both significantly increased the OSCC cells’ migration and invasion ( p < 0.05 ), and they significantly increased the expressions of both Tks5 and MT1-MMP. After the depletion of Tks5, the OSCC cells’ migration and invasion abilities decreased. The expression of Tks5 and that of αSMA were correlated with poor survival, and a high expression of both markers was associated with an especially poor prognosis. Conclusions. These results indicate that the formation of invadopodia is (i) important for OSCC cells’ migration and invasion and (ii) regulated by the interaction of OSCC cells and stromal fibroblasts.