Development of a Novel Orthotopic Gastric Cancer Mouse Model.

BACKGROUND: Gastric cancer metastasis is a highly fatal disease with a five-year survival rate of less than 5%. One major obstacle in studying gastric cancer metastasis is the lack of faithful models available. The cancer xenograft mouse models are widely used to elucidate the mechanisms of cancer development and progression. Current procedures for creating cancer xenografts include both heterotopic (i.e., subcutaneous) and orthotopic transplantation methods. Compared to the heterotopic model, the orthotopic model has been shown to be the more clinically relevant design as it enables the development of cancer metastasis. Although there are several methods in use to develop the orthotopic gastric cancer model, there is not a model which uses various types of tumor materials, such as soft tissues, semi-liquid tissues, or culture derivatives, due to the technical challenges. Thus, developing the applicable orthotopic model which can utilize various tumor materials is essential. RESULTS: To overcome the known limitations of the current orthotopic gastric cancer models, such as exposure of tumor fragments to the neighboring organs or only using firm tissues for the orthotopic implantation, we have developed a new method allowing for the complete insertion of soft tissue fragments or homogeneously minced tissues into the stomach submucosa layer of the immunodeficient NOD.Cg-Prkdc CONCLUSION: Here we describe a new protocol, step by step, for the establishment of orthotopic xenograft of gastric cancer. This novel technique will be able to increase the use of orthotopic models in broader applications for not only gastric cancer research but also any research related to the stomach microenvironment

Growth and Viability of Cutaneous Squamous Cell Carcinoma Cell Lines Display Different Sensitivities to Isoform-Specific Phosphoinositide 3-Kinase Inhibitors.

Cutaneous squamous cell carcinomas (cSCCs) account for about 20% of keratinocyte carcinomas, the most common cancer in the UK. Therapeutic options for cSCC patients who develop metastasis are limited and a better understanding of the biochemical pathways involved in cSCC development/progression is crucial to identify novel therapeutic targets. Evidence indicates that the phosphoinositide 3-kinases (PI3Ks)/Akt pathway plays an important role, in particular in advanced cSCC. Questions remain of whether all four PI3K isoforms able to activate Akt are involved and whether selective inhibition of specific isoform(s) might represent a more targeted strategy. Here we determined the sensitivity of four patient-derived cSCC cell lines to isoform-specific PI3K inhibitors to start investigating their potential therapeutic value in cSCC. Parallel experiments were performed in immortalized keratinocyte cell lines. We observed that pan PI3Ks inhibition reduced the growth/viability of all tested cell lines, confirming the crucial role of this pathway. Selective inhibition of the PI3K isoform p110α reduced growth/viability of keratinocytes and of two cSCC cell lines while affecting the other two only slightly. Importantly, p110α inhibition reduced Akt phosphorylation in all cSCC cell lines. These data indicate that growth and viability of the investigated cSCC cells display differential sensitivity to isoform-specific PI3K inhibitors

Barotrauma developed during intra-hospital transfer -A case report-

A 74-year-old male patient receiving ventilatory support due to aspiration pneumonia developed bilateral pneumothorax, pneumopericardium, pneumomediastinum, pneumo-retroperitoneum, and subcutaneous emphysema, after manual ventilation while being transferred from the intensive care unit (ICU) to the operating room (OR). These complications were assumed to be secondary to inappropriate manual ventilation of the intubated patient. In addition, it is likely that the possible migration of an already marginally acceptable endotracheal tube (ETT) position during transport was the cause of these complications. Finally, aggravation of a latent pneumothorax might have contributed to these complications

Rhomboid family member 2 regulates cytoskeletal stress-associated Keratin 16.

Keratin 16 (K16) is a cytoskeletal scaffolding protein highly expressed at pressure-bearing sites of the mammalian footpad. It can be induced in hyperproliferative states such as wound healing, inflammation and cancer. Here we show that the inactive rhomboid protease RHBDF2 (iRHOM2) regulates thickening of the footpad epidermis through its interaction with K16. K16 expression is absent in the thinned footpads of irhom2-/- mice compared with irhom2+/+mice, due to reduced keratinocyte proliferation. Gain-of-function mutations in iRHOM2 underlie Tylosis with oesophageal cancer (TOC), characterized by palmoplantar thickening, upregulate K16 with robust downregulation of its type II keratin binding partner, K6. By orchestrating the remodelling and turnover of K16, and uncoupling it from K6, iRHOM2 regulates the epithelial response to physical stress. These findings contribute to our understanding of the molecular mechanisms underlying hyperproliferation of the palmoplantar epidermis in both physiological and disease states, and how this 'stress' keratin is regulated

Transcriptomic analysis of cutaneous squamous cell carcinoma reveals a multi-gene prognostic signature associated with metastasis.

BackgroundMetastasis of cutaneous squamous cell carcinoma (cSCC) is uncommon. Current staging methods are reported to have sub-optimal performances in metastasis prediction. Accurate identification of patients with tumours at high risk of metastasis would have a significant impact on management.ObjectiveTo develop a robust and validated gene expression profile (GEP) signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach.MethodsArchival formalin-fixed paraffin-embedded primary cSCC with perilesional normal tissue from 237 immunocompetent patients (151 non-metastasising and 86 metastasising) were collected retrospectively from four centres. TempO-seq was used to probe the whole transcriptome and machine learning algorithms were applied to derive predictive signatures, with a 3:1 split for training and testing datasets.ResultsA 20-gene prognostic model was developed and validated, with an accuracy of 86.0%, sensitivity of 85.7%, specificity of 86.1%, and positive predictive value of 78.3% in the testing set, providing more stable, accurate prediction than pathological staging systems. A linear predictor was also developed, significantly correlating with metastatic risk.LimitationsThis was a retrospective 4-centre study and larger prospective multicentre studies are now required.ConclusionThe 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC

Transcriptomic analysis of cutaneous squamous cell carcinoma reveals a multi-gene prognostic signature associated with metastasis.

BACKGROUND: Metastasis of cutaneous squamous cell carcinoma (cSCC) is uncommon. Current staging methods are reported to have sub-optimal performances in metastasis prediction. Accurate identification of patients with tumours at high risk of metastasis would have a significant impact on management. OBJECTIVE: To develop a robust and validated gene expression profile (GEP) signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach. METHODS: Archival formalin-fixed paraffin-embedded primary cSCC with perilesional normal tissue from 237 immunocompetent patients (151 non-metastasising and 86 metastasising) were collected retrospectively from four centres. TempO-seq was used to probe the whole transcriptome and machine learning algorithms were applied to derive predictive signatures, with a 3:1 split for training and testing datasets. RESULTS: A 20-gene prognostic model was developed and validated, with an accuracy of 86.0%, sensitivity of 85.7%, specificity of 86.1%, and positive predictive value of 78.3% in the testing set, providing more stable, accurate prediction than pathological staging systems. A linear predictor was also developed, significantly correlating with metastatic risk. LIMITATIONS: This was a retrospective 4-centre study and larger prospective multicentre studies are now required. CONCLUSION: The 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC

Correction to: Development of a Novel Orthotopic Gastric Cancer Mouse Model

An amendment to this paper has been published and can be accessed via the original article

Analytic philosophy for biomedical research: the imperative of applying yesterday's timeless messages to today's impasses

The mantra that "the best way to predict the future is to invent it" (attributed to the computer scientist Alan Kay) exemplifies some of the expectations from the technical and innovative sides of biomedical research at present. However, for technical advancements to make real impacts both on patient health and genuine scientific understanding, quite a number of lingering challenges facing the entire spectrum from protein biology all the way to randomized controlled trials should start to be overcome. The proposal in this chapter is that philosophy is essential in this process. By reviewing select examples from the history of science and philosophy, disciplines which were indistinguishable until the mid-nineteenth century, I argue that progress toward the many impasses in biomedicine can be achieved by emphasizing theoretical work (in the true sense of the word 'theory') as a vital foundation for experimental biology. Furthermore, a philosophical biology program that could provide a framework for theoretical investigations is outlined

Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling

<p>Abstract</p> <p>Background</p> <p>Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes.</p> <p>Methods</p> <p>We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods.</p> <p>Results</p> <p>COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional <it>in vitro </it>models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays <it>in vivo</it>, and do not develop tumorigenic ability.</p> <p>Conclusions</p> <p>We propose that COCA constitutes a good experimental system for <it>in vitro </it>and <it>in vivo </it>skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.</p

Crystallographic and cellular characterisation of two mechanisms stabilising the native fold of α1-Antitrypsin: implications for disease and drug design

The common Z mutant (Glu342Lys) of α1-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native α1-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 Å structure of Thr114Phe α1-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F–β-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z α1-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of α1-antitrypsin and increase secretion of monomeric protein in cell models of disease