A physiologically relevant, estradiol‐17β [E2]‐responsive in vitro tissue‐engineered model of the vaginal epithelium for vaginal tissue research

Aims There are many situations where preclinical models of the human vagina would be valuable for in vitro studies into the pathophysiology of vaginally transmitted diseases, microbicide efficacy, irritability testing, and particularly, for assessing materials to be inserted in the vagina for support of the pelvic floor. The aim of this study is to develop a physiologically relevant, low cost, and ethically suitable model of the vagina using sheep vaginal tissue (SVT) to reduce the need for animal testing in gynecological research. Methods Tissue-engineered (TE) vaginal models were developed by culturing primary vaginal epithelial cells and vaginal fibroblasts, isolated from the native SVTs on decellularized sheep vaginal matrices at an air–liquid interface. Morphological analyses of the models were conducted by performing hematoxylin and eosin staining and further characterization was done by immunohistofluorescence (IHF) of structural proteins and cytokeratins. Results Histological analysis of the models revealed a gradual formation of a stratified epithelium on our decellularized matrices and cell metabolic activity remained high for 21 days as measured by the resazurin assay. Our models showed a dose-dependent response to estradiol-17β [E2] with an increase in the vaginal epithelium thickness and cellular proliferation under higher E2 concentrations (100–400 pg/ml). The physiological relevance of these results was confirmed by the IHF analysis of Ki67, and cytokeratins 10 and 19 expression. Conclusion In this study, we have developed an estradiol-responsive TE vaginal model that closely mimics the structural and physiological properties of the native SVT

PAUSED for thought? Using verbal protocol analysis to understand the situational and temporal cues in the decision-making of residential burglars

Using verbal protocol analysis (VPA) alongside semi-structured interviews, this research aimed to explicate the situational dynamics that inform the decision-making and target selection of residential burglars. Focusing on the VPA method, novel to criminological research, the paper considers the contribution of this empirical approach for studying the decision-making of offenders in situ. The findings reveal a series of cues, encapsulated in the ‘PAUSED’ model, that are drawn upon by residential burglars to assess the suitability of a target; determining whether it is profitable, accessible, uninterruptible, surveillable, escapable and/or dishonourable. The PAUSED model is unpacked to articulate a collection of visual stimuli that serve to disrupt and suspend the otherwise rapid flow of target appraisal. Discussion of the strengths and limitations of the VPA method, and how it can compliment other approaches to understanding the decision-making of residential burglars, is provided

Polymersome-Mediated Delivery of Combination Anticancer Therapy to Head and Neck Cancer Cells: 2D and 3D in Vitro Evaluation

Polymersomes have the potential to encapsulate and deliver chemotherapeutic drugs into tumor cells, reducing off-target toxicity that often compromises anticancer treatment. Here, we assess the ability of the pH-sensitive poly 2-(methacryloyloxy)ethyl phosphorylcholine (PMPC)- poly 2-(diisopropylamino)ethyl methacrylate (PDPA) polymersomes to encapsulate chemotherapeutic agents for effective combinational anticancer therapy. Polymersome uptake and ability to deliver encapsulated drugs into healthy normal oral cells and oral head and neck squamous cell carcinoma (HNSCC) cells was measured in two and three-dimensional culture systems. PMPC-PDPA polymersomes were more rapidly internalized by HNSCC cells compared to normal oral cells. Polymersome cellular uptake was found to be mediated by class B scavenger receptors. We also observed that these receptors are more highly expressed by cancer cells compared to normal oral cells, enabling polymersome-mediated targeting. Doxorubicin and paclitaxel were encapsulated into pH-sensitive PMPC-PDPA polymersomes with high efficiencies either in isolation or as a dual-load for both singular and combinational delivery. In monolayer culture, only a short exposure to drug-loaded polymersomes was required to elicit a strong cytotoxic effect. When delivered to three-dimensional tumor models, PMPC-PDPA polymersomes were able to penetrate deep into the center of the spheroid resulting in extensive cell damage when loaded with both singular and dual-loaded chemotherapeutics. PMPC-PDPA polymersomes offer a novel system for the effective delivery of chemotherapeutics for the treatment of HNSCC. Moreover, the preferential internalization of PMPC polymersomes by exploiting elevated scavenger receptor expression on cancer cells opens up the opportunity to target polymersomes to tumors

Methyl-donor depletion of head and neck cancer cells in vitro establishes a less aggressive tumour cell phenotype

PURPOSE: DNA methylation plays a fundamental role in the epigenetic control of carcinogenesis and is, in part, influenced by the availability of methyl donors obtained from the diet. In this study, we developed an in-vitro model to investigate whether methyl donor depletion affects the phenotype and gene expression in head and neck squamous cell carcinoma (HNSCC) cells. METHODS: HNSCC cell lines (UD-SCC2 and UPCI-SCC72) were cultured in medium deficient in methionine, folate, and choline or methyl donor complete medium. Cell doubling-time, proliferation, migration, and apoptosis were analysed. The effects of methyl donor depletion on enzymes controlling DNA methylation and the pro-apoptotic factors death-associated protein kinase-1 (DAPK1) and p53 upregulated modulator of apoptosis (PUMA) were examined by quantitative-PCR or immunoblotting. RESULTS: HNSCC cells cultured in methyl donor deplete conditions showed significantly increased cell doubling times, reduced cell proliferation, impaired cell migration, and a dose-dependent increase in apoptosis when compared to cells cultured in complete medium. Methyl donor depletion significantly increased the gene expression of DNMT3a and TET-1, an effect that was reversed upon methyl donor repletion in UD-SCC2 cells. In addition, expression of DAPK1 and PUMA was increased in UD-SCC2 cells cultured in methyl donor deplete compared to complete medium, possibly explaining the observed increase in apoptosis in these cells. CONCLUSION: Taken together, these data show that depleting HNSCC cells of methyl donors reduces the growth and mobility of HNSCC cells, while increasing rates of apoptosis, suggesting that a methyl donor depleted diet may significantly affect the growth of established HNSCC

Cancer-associated fibroblasts promote bone invasion in oral squamous cell carcinoma

Background: The molecular mechanisms involved in the invasion of bone by oral squamous cell carcinomas (OSCC) are poorly understood, and little is known about the role of cancer-associated fibroblasts (CAF), the presence of which confers a poor prognosis. Methods: Clinicopathological data from 277 OSCC cases involving bone resections were reviewed, and 32 cases thoroughly analysed histologically. Immunohistochemistry was used to examine αSMA, RANKL and OPG. Western blotting and qPCR were used to assess myofibroblast (CAF-like) differentiation, RANKL and OPG expression in vitro, and RANKL secretion was analysed by ELISA. Osteoclastogenesis was examined using TRAP staining, multinucleation and pit forming assays. Results: Fibrous stroma intervened between tumour and bone in the majority of cases, with no direct contact between cancer cells and bone. RANKL and OPG, two proteins key to regulating bone resorption, were expressed in tumour cells as well as fibrous stroma adjacent to bone and αSMA-positive myofibroblastic CAF were consistently seen infiltrating into bone ahead of tumour cells. Human primary osteoblasts cultured with conditioned media from human OSCC-derived cells and human primary CAF showed a significant increase in RANKL and a decline in OPG mRNA expression. RANKL secretion was significantly increased in primary oral fibroblasts induced to differentiate into a CAF-like phenotype by transforming growth factor-β1 (TGF-β1) treatment and in primary CAF. Indirect co-culture of murine macrophages with conditioned media from CAF (experimentally derived and isolated from OSCCs) resulted in a marked increase in osteoclastogenesis (in excess of that provoked by cancer cells) determined by tartrate-resistant acid phosphatase activity, multinucleation and resorption pit formation. Conclusions: This study is the first to describe a functional role for CAFs in bone invasion and turnover, identifying a novel potential therapeutic target and diagnostic indicator in this difficult to treat bone invasive malignancy

Development of tissue-engineered models of oral dysplasia and early invasive oral squamous cell carcinoma

BACKGROUND: Current organotypic models of dysplasia and oral squamous cell carcinoma (OSCC) lack the complexity that mimics in vivo tissue. Here we describe a three-dimensional in vitro model of the oral epithelium that replicates tumour progression from dysplasia to an invasive phenotype. METHODS: The OSCC cell lines were seeded as a cell suspension (D20, Cal27) or as multicellular tumour spheroids (FaDu) with oral fibroblasts on to a de-epidermised acellular dermis to generate tissue-engineered models and compared with patient biopsies. RESULTS: The D20 and Cal27 cells generated a model of epithelial dysplasia. Overtime Cal27 cells traversed the basement membrane and invaded the connective tissue to reproduce features of early invasive OSCC. When seeded onto a model of the normal oral mucosa, FaDu spheroids produced a histological picture mimicking carcinoma in situ with severe cellular atypia juxtaposed to normal epithelium. CONCLUSION: It is possible to culture in vitro models with the morphological appearance and histological characteristics of dysplasia and tumour cell invasion seen in vivo using native dermis. Such models could facilitate study of the molecular processes involved in malignant transformation, invasion and tumour growth as well as in vitro testing of new treatments, diagnostic tests and drug delivery systems for OSCC