Development of a cell based chronic wound bioassay

Impaired wound healing affects 3% of the population over 60 and costs over £1 billion annually in the UK. Chronic wounds are characterised by prolonged inflammation, impaired re-epithelialisation and defective extracellular matrix (ECM) remodelling. Fibroblasts play an important role in the closure of skin wounds, they replace and remodel the lost tissue and also influence both re-epithelialisation and angiogenesis. Numerous in vivo chronic wound animal models have been reported however, they fail to accurately model human chronic wounds. This investigation aims to develop an in vitro chronic wound bioassay. Chronic wound fibroblast (CWF) and patient matched normal fibroblast (NF) cell strains (n=3) were retrovirally infected with a hTERT/puromycin construct. Population doubling levels (PDLs) were determined over an extended time in culture. RNA at defined time points was extracted and analysed using Asymetrix U133A microarrays. A cohort of gene expression changes were confirmed by QRT-PCR and the promoter regions of these genes used to construct fluorescent reporter human NF and CWF cell lines. Population doubling levels indicated that, compared to primary (non-immortalised) CWF and NF, the hTERT infected fibroblasts had escaped replicative senescence and formed an immortalised cell line. Affymetrix microarray analysis of the CWF-hTERT with the NF-hTERT identified 247 genes that were significantly up- or down-regulated in CWF (validated by QRT-PCR). Upstream promoter regions of 13 genes of interest and a housekeeping gene have been identified by database searches/sequence analysis, amplified and cloned into the promoterless reporter vector pZsGreen1-DR. Reporter constructs have been transiently transfected into human NF and CWF cell lines and are currently being tested for efficacy. CWF and NF cell lines have been created which demonstrated distinct gene expression profiles. The disease marker genes will form the basis of a low cost, highly reproducible fluorescent reporter cell-based bioassay which will be utilised in the discovery of novel therapeutics which have the potential to alter the chronic wound healing and, in turn, reduce unnecessary animal experimentation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of a cell based chronic wound bioassay

Impaired wound healing affects 3% of the population over 60 and costs over 1 billion annually in the UK. Chronic wounds are characterised by prolonged inflammation, impaired re-epithelialisation and defective extracellular matrix (ECM) remodelling. Fibroblasts play an important role in the closure of skin wounds, they replace and remodel the lost tissue and also influence both re-epithelialisation and angiogenesis. Numerous in vivo chronic wound animal models have been reported however, they fail to accurately model human chronic wounds. This investigation aims to develop an in vitro chronic wound bioassay. Chronic wound fibroblast (CWF) and patient matched normal fibroblast (NF) cell strains (n=3) were retrovirally infected with a hTERT/puromycin construct. Population doubling levels (PDLs) were determined over an extended time in culture. RNA at defined time points was extracted and analysed using Asymetrix U133A microarrays. A cohort of gene expression changes were confirmed by QRT-PCR and the promoter regions of these genes used to construct fluorescent reporter human NF and CWF cell lines. Population doubling levels indicated that, compared to primary (non-immortalised) CWF and NF, the hTERT infected fibroblasts had escaped replicative senescence and formed an immortalised cell line. Affymetrix microarray analysis of the CWF-hTERT with the NF-hTERT identified 247 genes that were significantly up- or down-regulated in CWF (validated by QRT-PCR). Upstream promoter regions of 13 genes of interest and a housekeeping gene have been identified by database searches/sequence analysis, amplified and cloned into the promoterless reporter vector pZsGreen1-DR. Reporter constructs have been transiently transfected into human NF and CWF cell lines and are currently being tested for efficacy. CWF and NF cell lines have been created which demonstrated distinct gene expression profiles. The disease marker genes will form the basis of a low cost, highly reproducible fluorescent reporter cell-based bioassay which will be utilised in the discovery of novel therapeutics which have the potential to alter the chronic wound healing and, in turn, reduce unnecessary animal experimentation

Tumor-associated Endo180 requires stromal-derived LOX to promote metastatic prostate cancer cell migration on human ECM surfaces

The diverse composition and structure of extracellular matrix (ECM) interfaces encountered by tumor cells at secondary tissue sites can influence metastatic progression. Extensive in vitro and in vivo data has confirmed that metastasizing tumor cells can adopt different migratory modes in response to their microenvironment. Here we present a model that uses human stromal cell-derived matrices to demonstrate that plasticity in tumor cell movement is controlled by the tumor-associated collagen receptor Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) and the crosslinking of collagen fibers by stromal-derived lysyl oxidase (LOX). Human osteoblast-derived and fibroblast-derived ECM supported a rounded ‘amoeboid-like’ mode of cell migration and enhanced Endo180 expression in three prostate cancer cell lines (PC3, VCaP, DU145). Genetic silencing of Endo180 reverted PC3 cells from their rounded mode of migration towards a bipolar ‘mesenchymal-like’ mode of migration and blocked their translocation on human fibroblast-derived and osteoblast-derived matrices. The concomitant decrease in PC3 cell migration and increase in Endo180 expression induced by stromal LOX inhibition indicates that the Endo180-dependent rounded mode of prostate cancer cell migration requires ECM crosslinking. In conclusion, this study introduces a realistic in vitro model for the study of metastatic prostate cancer cell plasticity and pinpoints the cooperation between tumor-associated Endo180 and the stiff microenvironment imposed by stromal-derived LOX as a potential target for limiting metastatic progression in prostate cancer

AGE-modified basement membrane cooperates with Endo180 to promote epithelial cell invasiveness and decrease prostate cancer survival

Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell–cell junctions, luminal infiltration and basal invasion induced by AGE-modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180ΔEx2–6/ΔEx2–6 mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors

Development and Characterisation of a Human Chronic Skin Wound Cell Line:Towards an Alternative for Animal Experimentation

Background: Chronic skin wounds are a growing financial burden for healthcare providers, causing discomfort/immobility to patients. Whilst animal chronic wound models have been developed to allow for mechanistic studies and to develop/test potential therapies, such systems are not good representations of the human chronic wound state. As an alternative, human chronic wound fibroblasts (CWFs) have permitted an insight into the dysfunctional cellular mechanisms that are associated with these wounds. However, such cells strains have a limited replicative lifespan and therefore a limited reproducibility/usefulness. Objectives: To develop/characterise immortalised cell lines of CWF and patient-matched normal fibroblasts (NFs). Methods and Results: Immortalisation with human telomerase resulted in both CWF and NF proliferating well beyond their replicative senescence end-point (respective cell strains senesced as normal). Gene expression analysis demonstrated that, whilst proliferation-associated genes were up-regulated in the cell lines (as would be expected), the immortalisation process did not significantly affect the disease-specific genotype. Immortalised CWF (as compared to NF) also retained a distinct impairment in their wound repopulation potential (in line with CWF cell strains). Conclusions: These novel CWF cell lines are a credible animal alternative and could be a valuable research tool for understanding both the aetiology of chronic skin wounds and for therapeutic pre-screening

A Unique Panel of Patient-Derived Cutaneous Squamous Cell Carcinoma Cell Lines Provides a Preclinical Pathway for Therapeutic Testing

Background: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. Methods: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. Results: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. Conclusions: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation

Effects of dredging-related pressures on critical ecological processes for organisms other than fish or coral. Report of Theme 9 - Project 9.1 prepared for the Dredging Science Node

In November 2013 a workshop was held at CSIRO Floreat, which brought together national and international marine scientists. The workshop addressed two primary objectives: • identify the timing of critical ecological processes in tropical and temperate ecosystems with a focus on non-coral and non-fish biota (seagrass, seaweed, sponges, ascidians, bryozoans, molluscs, echinoderms, crustaceans and non-coral cnidarians); and • identify environmental windows for critical ecological processes identified in Objective 1. This will be achieved by compiling information on the timing of reproduction, release of propagules and recruitment for these organisms, as well as the temporal and spatial scales of reproduction and recruitment events. During Workshop 1 a conceptual diagram was developed to illustrate and guide the decision process behind the selection of environmental windows (EWs) (see Figure 1). The life histories of the biota investigated were then identified and listed in detailed tables with specific reference to potential effects of dredging at each life history stage..

RNA sequencing and lipidomics uncovers novel pathomechanisms in recessive X-linked ichthyosis

Recessive X-linked ichthyosis (RXLI), a genetic disorder caused by deletion or point mutations of the steroid sulfatase (STS) gene, is the second most common form of ichthyosis. It is a disorder of keratinocyte cholesterol sulfate retention and the mechanism of extracutaneous phenotypes such as corneal opacities and attention deficit hyperactivity disorder are poorly understood. To understand the pathomechanisms of RXLI, the transcriptome of differentiated primary keratinocytes with STS knockdown was sequenced. The results were validated in a stable knockdown model of STS, to confirm STS specificity, and in RXLI skin. The results show that there was significantly reduced expression of genes related to epidermal differentiation and lipid metabolism, including ceramide and sphingolipid synthesis. In addition, there was significant downregulation of aldehyde dehydrogenase family members and the oxytocin receptor which have been linked to corneal transparency and behavioural disorders respectively, both of which are extracutaneous phenotypes of RXLI. These data provide a greater understanding of the causative mechanisms of RXLI’s cutaneous phenotype, and show that the keratinocyte transcriptome and lipidomics can give novel insights into the phenotype of patients with RXLI